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EEnneerrggeettiiccss ooff
mmoouussee ppaappiillllaarryy mmuussccllee
Cecilia Widén
MSc. Biomedicine
A thesis submitted in fulfillment of the requirements of the degree of
Doctor of Philosophy
Heart Foundation Research Centre
School of Physiotherapy & Exercise Science
Faculty of Health
Griffith University
Queensland
AUSTRALIA April 2006
i
SSuummmmaarryy
The overall aim of this Thesis was to characterise the energetic properties of the mouse
papillary muscle as this preparation could become a useful model to study alterations of
energetic aspects of cardiac pathologies and heart-focussed genetic changes.
Measurements of resting and active metabolism of the papillary muscles were made in
vitro using the myothermic technique.
In the first study the mechanism underlying impaired contractility of post-ischaemic rat
papillary muscle was investigated. The rat preparation is well established and was used
to develop protocols and approaches that could later be used as the basis for studies with
mouse papillary muscle. The muscles were exposed to simulated ischaemia for 60 min
and change in energetics was studied 30 min into the reperfusion phase. The work
output was reduced to 66 ± 3% of the pre-ischaemia value and the enthalpy output
decreased to 71 ± 3% of pre-ischaemia value. However, there was no change in either
initial, 19 ± 3%, or net mechanical efficiency, 9.0 ± 0.9%. These data, in combination
with studies of Ca2+
handling, suggests that the reduced work output was caused by
attachment of fewer cross-bridges in each twitch, but with no change in work generated
by each cross-bridge.
The following two studies involved characterisation of the energetics of the mouse
papillary muscle and included measurements of resting and active metabolism. The
resting metabolic rate varied with muscle size but the mean initial value was ∼25 mW
ii
g-1
and the estimated steady value ∼5 mW g-1
. The resting metabolic rate declined
exponentially with time towards a steady value, with a time constant of 18 ± 2 min.
There was no alteration in isometric force output during this time. The magnitude of
resting metabolism depended inversely on muscle mass, more than doubled following a
change in substrate from glucose to pyruvate and was increased 2.5-fold when the
osmolarity of the bathing solution was increased by addition of 300 mM sucrose.
Addition of 30 mM BDM affected neither the time course of the decline in metabolic
rate nor the eventual steady value.
The energy requirements associated with contractile activity were ∼7 mJ g-1
twitch-1
at a
contraction frequency of 1 Hz. The enthalpy output was not affected by changing
substrate from glucose to pyruvate but did decrease with an increase in temperature. The
enthalpy output was partitioned into force-dependent and force-independent
components using BDM to selectively inhibit cross-bridge cycling. The force-
independent enthalpy output was 18.6 ± 1.9% of the initial enthalpy output. Muscle
initial efficiency was ∼32% and net efficiency ∼17% when shortening at a realistic
velocity. The enthalpy output decreased with increased contraction frequency but was
independent of shortening velocity. On the basis of these values, it was calculated that
the twitch energetics were consistent with ATP splitting by half the cross-bridges and
the pumping of one Ca2+
into the SR for every three cross-bridge cycles. The lack of
influence of shortening velocity on energy cost supports the idea that the amount of
energy to be used is determined early in a twitch and is not greatly influenced by events
that occur during the contraction.
The suitability of the mouse papillary muscle as a model to study ischaemia and
reperfusion damage was also assessed. This preparation is excellent for studying muscle
specific changes in work and enthalpy output; however, due to the long-term instability
and variability amongst preparations, the suitability of this preparation in prolonged
experiments remains uncertain.
iii
DDeeccllaarraattiioonn
This work has previously not been submitted for a degree or diploma in any university.
To all the best of my knowledge and belief, the thesis contains no material previously
published or written by another person except where due reference is made in the thesis
itself.
Cecilia Widén
iv
v
TTaabbllee ooff ccoonntteennttss
LIST OF FIGURES...........................................................................................IX
LIST OF TABLES ............................................................................................XI
LIST OF PUBLICATIONS.............................................................................. XIII
ACKNOWLEDGMENTS..................................................................................XV
CHAPTER 1: INTRODUCTION......................................................................... 1
CHAPTER 2: BRIEF BACKGROUND ................................................................ 3
2.1 Cardiac energetics..................................................................................................3 2.1.1 Initial biochemical reactions.................................................................................4
2.1.1.1 ATP hydrolysis ................................................................................................................4 2.1.1.2 Creatine kinase reaction...................................................................................................8
2.1.2 Recovery biochemical reactions ...........................................................................8
2.2 Papillary muscles as a model of ventricular muscle energetics .......................10
2.3 Isolated mouse papillary model ..........................................................................11
CHAPTER 3: METHODS ............................................................................... 13
3.1 Papillary muscle preparation and dissection.....................................................13
vi
3.2 Overview of experimental apparatus .................................................................14 3.2.1 Measurement of muscle force production ..........................................................15
3.2.2 Measurement of muscle length changes .............................................................16
3.2.3 Principles of measuring muscle heat output using a thermopile ........................16 3.2.3.1 Calibration of thermopile...............................................................................................16 3.2.3.2 Conversion of thermopile signals into heat production .................................................19
3.2.4 Determination of stimulus heat...........................................................................20
3.3 Data recording......................................................................................................21
3.4 Partitioning of initial and recovery metabolism................................................22 3.4.1 Partitioning initial metabolism into force-dependent and force-independent
components .........................................................................................................24
3.5 Calculations of efficiency.....................................................................................24 3.5.1 Mechanical efficiency.........................................................................................24
3.5.1.1 Initial and net mechanical efficiency .............................................................................24 3.5.2 Mitochondrial efficiency.....................................................................................25
3.6 Ratio of recovery heat output to initial heat output .........................................25
3.7 Oxygenation of papillary muscles.......................................................................26
3.8 Data normalisation...............................................................................................27
CHAPTER 4: MECHANISM OF DEPRESSED WORK OUTPUT IN POST-ISCHAEMIC CARDIAC MUSCLE................................................ 29
4.1 Introduction..........................................................................................................29
4.2 Methods.................................................................................................................30 4.2.1 Initial and recovery metabolism .........................................................................31
4.2.2 Experimental protocols .......................................................................................32
4.2.3 Statistical analysis...............................................................................................33
4.2.4 Analysis of diffusive O2 supply..........................................................................33
4.3 Results .................................................................................................................34 4.3.1 Control experiments............................................................................................34
4.3.2 Effects of simulated ischaemia on force output..................................................36
4.3.3 Effects of simulated ischaemia and reperfusion on mechanical and energetic
parameters...........................................................................................................36
4.3.4 Partitioning energy cost between force-dependent and force-independent
components .........................................................................................................37
4.3.5 Time course of oxidative recovery metabolism..................................................39
4.4 Discussion..............................................................................................................40 4.4.1 Impaired work output due to fewer attached cross-bridges ................................41
4.4.2 Mitochondrial efficiency.....................................................................................44
4.4.3 Recovery time course..........................................................................................44
4.5 Conclusion.............................................................................................................45
vii
CHAPTER 5: RESTING METABOLISM OF MOUSE PAPILLARY MUSCLE ....... 47
5.1 Introduction..........................................................................................................47
5.2 Methods.................................................................................................................49 5.2.1 Heat measurements .............................................................................................49
5.2.2 Calculation of rate of resting heat production ....................................................50
5.2.3 Measurement of myocyte sarcomere length by diffraction of laser light ...........50
5.2.4 Experimental protocols .......................................................................................52
5.2.5 Analysis of diffusive O2 supply..........................................................................52
5.2.6 Statistical analysis...............................................................................................53
5.3 Results .................................................................................................................53 5.3.1 Resting metabolic rate depended on time and muscle mass...............................53
5.3.2 The effect of BDM on resting metabolic rate .....................................................56
5.3.3 The effect of hyperosmolarity on resting metabolic rate ....................................56
5.3.4 The effect of metabolic substrate on resting metabolism and force output ........57
5.4 Discussion..............................................................................................................58 5.4.1 Comparison with other studies ...........................................................................58
5.4.2 Adequacy of diffusive oxygen supply ................................................................61
5.5 Recommendations for performing experiments with isolated papillary
muscles .................................................................................................................63
CHAPTER 6: CHARACTERISATION OF ACTIVE METABOLISM..................... 65
6.1 Introduction..........................................................................................................65
6.2 Methods.................................................................................................................66 6.2.1 Mechanical output...............................................................................................67
6.2.2 Contribution of recovery heat to initial heat measurements ...............................67
6.2.3 Experimental protocols .......................................................................................67
6.2.4 Calculation of number of cross-bridge cycles ....................................................69
6.2.5 Analysis of diffusive O2 supply..........................................................................70
6.2.6 Statistical analysis...............................................................................................70
6.3 Results .................................................................................................................71 6.3.1 Force output of mouse papillary muscle.............................................................71
6.3.2 Energy cost of a twitch and effects of contraction frequency, substrate and
temperature .........................................................................................................72
6.3.3 Effects of shortening on twitch energy cost........................................................74
6.3.4 Mechanical efficiency.........................................................................................76
6.3.5 Ratio of recovery to initial enthalpy output ........................................................76
6.3.6 Partitioning energy cost between force-dependent and force-independent
components .........................................................................................................77
6.4 Discussion..............................................................................................................77 6.4.1 Number of cross-bridge cycles per twitch ..........................................................78
6.4.2 Amount of Ca2+
released from the SR in each twitch.........................................80
6.4.3 Partitioning of energy between force-dependent and force-independent
components .........................................................................................................82
viii
6.4.4 Mitochondrial efficiency.....................................................................................83
6.5 Conclusion.............................................................................................................83
CHAPTER 7: CAN MOUSE PAPILLARY MUSCLES BE USED IN PROLONGED
EXPERIMENTS? ....................................................................... 85
7.1 The observations ..................................................................................................86
7.2 Solution .................................................................................................................87
7.3 Experimental set-up .............................................................................................88
7.4 Adequate oxygenation..........................................................................................89
7.5 Method of euthanasia...........................................................................................89
7.6 Assessment of the suitability of isolated mouse papillary muscles for
investigating cardiac muscle physiology ............................................................89
CHAPTER 8: CONCLUDING COMMENTS ...................................................... 91
8.1 Resting metabolism ..............................................................................................91
8.2 Active metabolism ................................................................................................92
8.3 Ischaemia and reperfusion ..................................................................................93
8.4 Conclusion.............................................................................................................94
APPENDIX I ................................................................................................ 95
APPENDIX II ................................................................................................ 97
REFERENCES................................................................................................ 99
ix
LLiisstt ooff ffiigguurreess
Fig. 2.1. Initial and recovery reactions........................................................................................ 10
Fig. 3.1. Calibration of force transducer. .................................................................................... 15
Fig. 3.2. Time-course of cooling of silver blocks. ...................................................................... 18
Fig. 3.3. Calibration curve for the Seebeck coefficient............................................................... 18
Fig. 3.4. Heat loss correction....................................................................................................... 20
Fig. 3.5. Stimulus heat using a mouse papillary muscle. ............................................................ 21
Fig. 3.6. Example of the time course of force production and enthalpy output. ......................... 23
Fig. 4.1. Comparison of twitches recorded using isometric and realistic contraction protocols. 33
Fig. 4.2. Simulations of time courses of muscle oxygenation at the onset and end of a period of
simulated ischaemia. ................................................................................................. 35
Fig. 4.3. Changes in force output during ischaemia and reperfusion of rat papillary muscle. .... 38
Fig. 4.4. Effects of simulated ischaemia on relative work and relative enthalpy output............. 39
Fig. 4.5. Effect of simulated ischaemia on time course of rate of recovery heat output. ............ 40
Fig. 4.6. Effect of simulated ischaemia on cross-bridge-dependent and -independent energy cost.
................................................................................................................................... 43
Fig. 5.1. Example of myocyte sarcomere.................................................................................... 51
Fig. 5.2. Example of decline in resting heat rate with time during an experiment...................... 54
Fig. 5.3. Exponential decline in resting metabolism. .................................................................. 55
Fig. 5.4. Effect of muscle mass on resting metabolic rate........................................................... 55
Fig. 5.5. Example of the effect of hyperosmolarity on resting heat output and force production.
................................................................................................................................... 57
Fig. 5.6. The effect of glucose and pyruvate on the resting metabolic rate of mouse papillary
muscle. ...................................................................................................................... 58
x
Fig. 5.7. Prediction of in vivo resting metabolism....................................................................... 60
Fig. 5.8. Adequacy of diffusive O2 supply to resting mouse papillary muscle. .......................... 62
Fig. 6.1. Simulations of time course of PO2 at muscle centre during contraction series............. 71
Fig. 6.2. Contraction frequency dependence of normalised isometric force output.................... 72
Fig. 6.3. Enthalpy output per twitch in isometric contractions. .................................................. 73
Fig. 6.4. Effect of shortening velocity on work-loop. ................................................................. 75
Fig. 6.5. Enthalpy output per twitch in shortening contractions. ................................................ 75
Fig. 6.6. Initial and net mechanical efficiency. ........................................................................... 76
Fig. 6.7. Example of determining force-independent enthalpy output........................................ 77
Fig. 6.8. Dependence of Ca2+
released and ATP used on magnitude of force-independent
enthalpy output.......................................................................................................... 82
Fig. 7.1. Changes in mechanical performance with time. ........................................................... 86
xi
LLiisstt ooff ttaabblleess
Table 2.1. Reported values of free energy of ATP hydrolysis. ..................................................... 6
Table 3.1. Thermopile characteristics. ........................................................................................ 17
Table 3.2. Estimates of quantization noise and noise from other sources................................... 22
Table 4.1. Left ventricular rat papillary muscle characteristics. ................................................. 31
Table 4.2. Energetic variables before and after 60 min of simulated ischaemia......................... 37
Table 4.3. Recovery time constant before and after 60 min of simulated ischaemia.................. 40
Table 4.4. Theoretical changes in εI for different mechanisms underlying depressed work output.
................................................................................................................................... 42
Table 5.1. Characteristics of mouse papillary muscles. .............................................................. 55
Table 6.1. Characteristics of mouse papillary muscles. .............................................................. 67
Table 6.2 Effect of glucose and pyruvate on enthalpy output (n = 11). ..................................... 73
Table 6.3. Characteristics of mechanical and energetic properties of mouse papillary muscles
performing isometric contractions (2 Hz) at 22, 27 and 37°C using glucose as a
substrate..................................................................................................................... 74
xii
xiii
LLiisstt ooff ppuubblliiccaattiioonnss
The following publications are listed in support of this Thesis:
Papers Widén C. and Barclay, C.J. (2006). ATP-splitting by half the cross-bridges can explain
the twitch energetics of mouse papillary muscle. Journal of Physiology 573: 5-15.
Widén C. and Barclay, C.J. (2005). Resting metabolism of mouse papillary muscle.
Pflugers Archiv 450: 209-216.
Abstract Widén C. and Barclay, C.J. (2005). Active metabolism of mouse papillary muscle.
Proceedings of the Australian Physiological Society 36: 114P.
xiv
xv
AAcckknnoowwlleeddggmmeennttss
I would sincerely like to thank my supervisor, mentor and friend Dr Chris Barclay for
his guidance, patience and encouragement throughout this candidature. “I think you’re
brilliant”, said Cissi! Also, you can stop looking in your mailbox now because I am
awarding you this year’s Nobel Prize in physiology and medicine!
Many thanks also to the Heart Foundation Research Centre and Griffith University for
supporting my PhD candidature.
I would also like to express my gratitude to the many wonderful people I have met
during my stay here in Australia. A very special thank you to my office buddies Tracey
Norling and Andrew Petersen for keeping me from going insane and Luke de Beus for
his invaluable help, especially with presentations and computers. I would also like to
thank my fellow students and colleagues at the School of Physiotherapy & Exercise
Science and the School of Medical Sciences.
Thank you also to the people I have met outside of the university through beach
volleyball and the gym. I have truly enjoyed our Sunday morning boot camp sessions
and I will be forever grateful for the friendship I have formed with the Lancettes!
Thanks also to Carole Rushton for helping me with secret women’s business!
Finally, I couldn’t have said it better than A.V. Hill, but the “slavery of writing” is
finally over!
xvi
1
CChhaapptteerr 11:: IInnttrroodduuccttiioonn
Muscles are biological machines that convert the chemical energy obtained from
breakdown of metabolic substrates into mechanical work. Muscle energetics is the study
of the processes involved in this energy conversion. A muscle pathology that potentially
lends itself to investigation based on energetics is the damage to cardiac muscle that
results from prolonged ischaemia (insufficient blood supply) and subsequent
reperfusion. It seems likely that at least part of the cause of post-ischaemic dysfunction
relates to cellular elements involved in energy conversion, especially the mitochondria
(transfer energy from metabolic substrates to ATP) and myosin cross-bridges (convert
energy from ATP into work). Much current research on the causes of ischaemia and
reperfusion damage is carried out using hearts and cardiac muscle from genetically
modified mice. However, relatively little is known about mouse heart energetics and
nothing is known about specific mouse cardiac muscle energetics. The overall aim of
this study was to characterise the energetics of the mouse papillary muscle and to
develop a protocol to study alterations of energetic aspects of cardiac muscle during
ischaemia and reperfusion.
To lay the theoretical foundations for the work described in the Thesis, a brief
background section has been provided (Chapter 2). This describes the function of the
papillary muscle, the biochemical reactions that underlie muscle contraction and also
provides a review of published values of the change in free energy of ATP hydrolysis.
2
This section is followed by a description of the methods that are common to most of the
experimental chapters (Chapter 3). Details specific to particular investigations are given
in the appropriate chapters.
The first experimental chapter (Chapter 4) describes an initial set of experiments
designed to investigate the mechanism of impaired work output arising from ischaemia
and reperfusion. This work was done using rat papillary muscle. This preparation is well
established and was used to develop protocols and approaches that could later be used
as the basis for studies with mouse papillary muscle.
The following chapters describe the characterisation of the energetics of mouse
papillary muscle. In Chapter 5 experiments to measure the resting metabolism (energy
required to maintain cell structure and integrity) are described. An important aspect of
this study was that of diffusive O2 supply to ensure that the muscles had an adequate O2
supply to meet the metabolic demand. This is important because, at least when first
dissected, the resting metabolic rate of papillary muscles is very high. Active
metabolism (energy required for contractile activity) was measured using both isometric
and realistic contraction protocols (Chapter 6) and experiments were performed to
determine the effects of contraction frequency, substrate, temperature and shortening on
twitch energy use. From these measurements it was concluded that about half of the
cross-bridges cycle during one twitch and that one Ca2+
is pumped into the sarcoplasmic
reticulum for every three cross-bridge cycles. In the final experimental chapter, the
suitability of the mouse papillary muscle as a model to study prolonged ischaemia is
discussed (Chapter 7).
3
CChhaapptteerr 22:: BBrriieeff bbaacckkggrroouunndd
This chapter provides a brief background on cardiac energetics and introduces the
biochemical reactions that underlie muscle contraction. The importance of ATP as the
energy source for muscle activity is highlighted and a review of reported literature
values of the change in free energy of ATP hydrolysis is presented. The difficulty of
interpreting the performance of the heart in terms of muscle specific energy output is
explained and the advantages of using an isolated cardiac muscle preparation as a model
of ventricular muscle function are discussed.
2.1 Cardiac energetics
Muscles convert the chemical free energy obtained from ATP hydrolysis into heat, and
if allowed to shorten, work. The First Law of Thermodynamics states that (in a closed
system∗) energy can be converted from one form to another but cannot be created or
destroyed. In the context of muscle contraction all the energy, or more properly,
enthalpy produced by the biochemical reactions underlying muscle contraction appears
as either heat or work. The chemical reactions are made up of two sets of processes: (1)
the breakdown of high-energy phosphates, which occurs simultaneously with
contraction, and (2) the regeneration of high-energy phosphates. The first set of
∗ By definition, a system in which the total energy of the system remains constant.
4
processes is called initial processes and the second, recovery processes. When there is
an adequate supply of O2 and metabolic substrate, recovery reactions are primarily those
of oxidative phosphorylation (Crow & Kushmerick, 1982; Paul, 1983; Smith et al.,
2005).
2.1.1 Initial biochemical reactions
Two biochemical reactions, the breakdown of adenosine triphosphate (ATP) and the
creatine kinase (CK) reaction, are commonly referred to as the initial reactions since
they take place within the time course of a contraction.
2.1.1.1 ATP hydrolysis
Muscles convert the chemical free energy obtained from ATP breakdown into heat and
work. During hydrolysis, bonds of the molecule are broken and energy is released.
iATP ADP P→ + (1)
where ADP is adenosine diphospate and Pi is inorganic phosphate. The energy produced
is known as enthalpy (∆H) and a negative value is indicative of liberation of energy (or
a decrease in the energy content of the molecules) from the process. For convenience, in
the remainder of this Thesis, enthalpy and free energy values are expressed as absolute
values. The enthalpy change of this process is affected by factors such as pH,
temperature (T), ionic conditions (I) and the free magnesium (Mg2+
) concentration.
Alberty (2003) reported a molar enthalpy value of approximately 26 kJ mol-1
at pH 7,
[Mg2+] = 0.1 mM, T = 40°C and I = 0.25 M.
The enthalpy term consists of free energy (∆G) and entropy (T∆S; where T is the
absolute temperature and ∆S is the change of entropy).
H G T S∆ = ∆ + ∆ (2)
Only the free energy can potentially be converted to work. The entropy and any free
energy not converted to work is converted to heat. The free energy change associated
with ATP hydrolysis, under specified intracellular conditions, can be described as
follows.
[ ][ ]ln
[ ]
iATP ATP
ADP PG G RT
ATP
° ∆ = ∆ +
(3)
5
where R is the Universal gas constant (8.314 J mol-1
K-1
). °
ATP∆G is the standard energy
of ATP hydrolysis, which is the free energy change that occurs under specified
conditions of pH, free [Mg2+], temperature and ionic strength and with the ratio [ADP]
[Pi]/[ATP] = 1. Although there are several published values for °
ATP∆G , they are mostly
around 30 kJ mol-1
and the most commonly used value is 30.5 kJ mol-1
, which was
calculated using the equilibrium constant for CK under conditions approximating those
in muscle cells (Lawson & Veech, 1979). The second term on the right-hand side of
Equation (3) quantifies the difference in chemical potential between the products and
the reactants.
An estimate of ∆GATP can be obtained by substituting typical values into Equation (3).
For example, if [ADP] = 50 µM, [Pi] = 1 mM and [ATP] = 8 mM (Kammermeier et al.,
1982), then the change in free energy is:
∆GATP = 30.5 + (0.0083 × 310) × ln ((50 × 10-6
× 1 × 10-3
) / (8 × 10-3
)) = 61.4 kJ mol-1
In muscle ∆GATP is important because it is the fraction of energy from ATP hydrolysis
that can potentially be converted into work. A summary of published values for cardiac
muscle is shown in Table 2.1. Most studies used NMR to measure [ATP] and [Pi] in
isolated, perfused hearts. Determining [ADP] is difficult because its levels are usually
below detection threshold of NMR and it must be estimated from the CK equilibrium
constant. There is considerable variation in published values of the latter and its value is
particularly sensitive to pH (Golding et al., 1995). Even with chemical analysis of
muscle extracts, [ADP] is difficult to estimate as the relative amounts of bound and free
ADP (both of which are measured but only the free affects ∆GATP) are uncertain.
Reported values of ∆GATP range between 54 and 70 kJ mol-1
and the average of all
studies is ∼59 kJ mol-1
(see Table 2.1). It is notable that in contrast to the suggestion of
Dobson (Dobson & Headrick, 1995; Dobson & Himmelreich, 2002; Dobson, 2003)
there does not appear to be any systematic variation with animal size. Furthermore,
there is no clear difference between values from chemical analysis and NMR.
6
Species Technique ∆GATP References
(kJ mol-1
)
Rat Biochemical analysis ∼57a Hassinen & Hiltunen (1975)
Rat Biochemical analysis ∼53b Nishiki et al. (1978)
Rat HPLC 60.5 Kammermeier et al. (1982)
Hamster Biochemical analysis 61.2 Sievers et al. (1983)
Rat Biochemical analysis 55.2 Fiolet et al. (1984)
Ferret NMR ∼60c Allen et al. (1985)
Guinea pig Biochemical analysis ∼62 Zweier & Jacobus (1987)
Rat HPLC 56.6 Griese et al. (1988)
Rat Biochemical analysis ∼55 Masuda et al. (1990)
Rat Biochemical analysis 57.3d Siegmund et al. (1991)
Rat Biochemical analysis 58.2d Fiolet et al. (1991)
Rat NMR ∼58e Barbour et al. (1991)
Rat MRS 60.9 Figueredo et al. (1992)
Rat Biochemical analysis 58.8d Koop & Piper (1992)
Rat NMR 63.9f
Headrick et al. (1994)
Rat HPLC 59.9 Headrick et al. (1994)
Rat NMR 64.7g Dobson & Headrick (1995)
Rabbit NMR 63.2g Dobson & Headrick (1995)
Dog Estimated 61.9g Dobson & Headrick (1995)
Human Estimated 60.2g Dobson & Headrick (1995)
Rat Biochemical analysis ∼62 Headrick (1996)
Guinea pig NMR 61.8 Kelm et al. (1997)
Rat NMR ∼55h Balschi et al. (1997)
Mouse NMR ∼54 Saupe et al. (1998)
Mouse NMR 59.7 Spindler et al. (1998)
Rat NMR ∼59 Saupe et al. (1999)
Mouse NMR & Biochemical 54.4 Saupe et al. (2000)
Mouse MRI/MRS ∼60i Chacko et al. (2000)
Human MRI/MRS ∼59i Chacko et al. (2000)
Pig Biochemical analysis 57.8j Heusch et al. (2000)
Pig Biochemical analysis 58.6j Schulz et al. (2001)
Dog MR 56.3k Bottomley & Weiss (2001)
Mouse NMR 69.9l Dobson & Himmelreich (2002)
Rat NMR 67.5l Dobson & Himmelreich (2002)
Table 2.1. Reported values of free energy of ATP hydrolysis.
7
Guinea pig NMR 66.5l Dobson & Himmelreich (2002)
Mouse NMR 58.5 Spindler et al. (2002)
Mouse NMR & Biochemical 60.0 Weiss et al. (2002)
Mouse NMR & Biochemical 54.3 Javadpour et al. (2003)
Human MRS 59.7m Weiss et al. (2005)
Mouse NMR & Biochemical ∼59n Day et al. (2006)
a Average from reported ∆GATP for beating (55.6 kJ mol
-1) and arrested (57.7 kJ mol
-1)
heart.
b Average from five experimental conditions (different afterloads, ionotropic
stimulation and arrested heart).
c Average from reported values from measurements allowing glycolysis (61.5 kJmol
-1)
and where glycolysis had been prevented (58.9 kJ mol-1
).
d Ventricular myocytes.
e Average value from measurements made at two different Mg
2+ concentrations (56.7 kJ
mol-1
at [Mg2+
]o = 1.2 mM and 59.3 kJ mol-1
at [Mg2+
]o = 4.8 mM).
f Measurements made in situ. [Pi] was below NMR detection but estimated to be 0.83
mM.
g The phosphorylation ratio was determined in hearts under resting conditions for the
rat, rabbit and dog under anesthesia and in resting non-anesthetized healthy human
subjects. Cytosolic [ATP] for dog myocardium and [total creatine] was used from
other studies. Myocardial pH and [Pi] was not measured in the human heart because of
the low signal/noise ratio of Pi in the NMR spectra; resting human skeletal muscle
values of pH 7.2 and free [Mg2+
] and [Pi] of dog heart were used.
h Average ∆GATP from three different work states: (1) heart rate, 300 beats per minute
(bpm) (55.7 kJ mol-1
), (2) 450 bpm (54.5 kJ mol-1
), (3) 450 bpm and 80 µg L-1
dobutamine (53.2 kJ mol-1
).
i In mice: based on literature values of [total creatine] = 30 mM, [ATP] = 9.6 mM, pHi
= 7.2. [Pi] estimated at ≤2 mM, based on the inorganic phosphate peak relative to that
of ATP, when Pi was unambiguously identified. In humans: based on literature values
of [total creatine] = 43.3 mM, [ATP] = 12.08 mM, pHi = 7.2. [Pi] estimated at ≤2 mM.
The PCr/ATP ratio in the mouse heart measured in vivo, the human ratio determined
from previous measurements by the authors and other sources.
j Chemical analysis using biopsy samples.
k Unable to reliably quantify [Pi], referred to other sources.
l Concentrations of ATP, PCr and Pi, pH and [Mg
2+] determined from in situ
31P-NMR
spectra. Total creatine (PCr + Cr) measured enzymatically on freeze-clamped tissue
and intracellular creatine concentration calculated from subtracting the NMR derived
PCr from the total creatine concentration.
m Unable to reliably quantify [Pi], assumed Pi of ∼1 µmol g
-1.
n Offered no explanation on how the changes in free energy were calculated.
8
2.1.1.2 Creatine kinase reaction
The ADP formed in Equation (1) is rapidly regenerated into ATP at the expense of
phosphocreatine (PCr), a reaction catalysed by CK.
ADP PCr ATP Cr+ → + ∆H = 12 kJ mol-1
(Teague & Dobson, 1992) (4)
where Cr is creatine. When pH = 7.3, [Mg2+] = 0.4 mM, T = 38°C and I = 0.25 M
(conditions approximating those in muscle cells) the equilibrium constant for this
reaction is
' [ ] [ ]62
[ ] [ ]CK
ATP CrK
PCr ADP= = (Golding et al., 1995) (5)
The large equilibrium constant indicates that this reaction maintains ATP at a relatively
constant level in the cell. The net chemical reaction of Equations (1) and (4) is the
breakdown of PCr.
iPCr Cr P→ + (6)
where ∆H ≈ 34 kJ mol-1
when pH = 7, [Mg2+
] = 0.1 mM (see Figure 2, Woledge &
Reilly, 1988).
2.1.2 Recovery biochemical reactions
The initial biochemical processes occurring in muscle contraction are reversed by
oxidative phosphorylation. Creatine and Pi diffuse or are shuttled to the mitochondria
where ATP rephosphorylates free Cr to PCr, a reaction catalysed by the mitochondrial
isoenzyme of CK (Fig. 2.1).
Cr ATP PCr ADP+ → + (7)
The PCr can then diffuse back to the sites of ATP consumption and can be used again
by the ATPases that fuel ion pumps and cross-bridge cycling. ATP is formed during
oxidative phosphorylation by the breakdown of metabolic substrates such as
carbohydrates and fats.
2 2 2iSubstrate ADP P ATP CO H O+ + + Ο → + + (8)
The net recovery chemical reaction is PCr resynthesis and substrate utilization.
2 2 2iSubstrate P Cr CO H O PCr+ Ο + + → + + (9)
9
By combining net initial and net recovery reactions, Equations (6) and (9), the net
overall reaction of muscle contraction is thus the oxidation of substrates.
2 2 2Substrate CO H O+ Ο → + (10)
The heart normally utilises both carbohydrates and fats as metabolic substrates. The
molar enthalpy change for substrate oxidation can be used to convert enthalpy output to
equivalent O2 consumption. The molar enthalpy change for glucose oxidation is 2820 kJ
mol-1
(Crabtree & Nicholson, 1988). Breakdown of 1mole of glucose requires six moles
of O2, so the energetic equivalent of the O2 consumed is 2820/6 = 470 kJ (mol O2)-1
.
This can be converted into units of mJ µL-1
with the Ideal Gas Law.
PV nRT= (11)
where P is pressure (atm), V is volume (L), n is quantity of gas (mol), R is gas constant
(0.0821 L atm mol-1
K-1
) and T is temperature (K). By rearranging the formula, the
molar gas volume at a given temperature, for instance 27°C as used in most of the
experiments in the current project, can be calculated:
-1nRT 1 mol × 0.0821 L atm mol × 300 KV = = = 24.6 L
P 1 atm
Thus, the energetic equivalent of the O2 consumed when temperature is 27°C, and
glucose is the substrate, is 470 kJ mol-1
/24.6 L mol-1
= 19.1 mJ µL-1
.
Interestingly, the breakdown of lipids gives a similar energy yield per mol of O2
consumed. The oxidation of palmitate (∆Hpalmitate = 9790 kJ mol-1
), the most abundant
fatty acid in the body, requires 23 moles of O2, so that the energy yield is 9790/23 = 426
kJ (mol O2)-1
. This corresponds to 426 kJ mol-1
/24.6 mol L-1
= 17.3 mJ µL-1
.
10
2.2 Papillary muscles as a model of ventricular muscle energetics
The structure of the musculature of the heart is complex (for a review, see Stevens &
Hunter, 2003). Ventricular pressure development arises from the actions of layers of
myocytes (i.e. cardiac muscle cells) with different fibre alignments relative to the long
axis of the heart. The loading experienced by myocytes is likely to differ among regions
of the heart with the consequence that it is difficult to precisely measure the work and
the energy used to perform that work by any specific section of the cardiac muscle. This
Fig. 2.1. Initial and recovery reactions.
Two groups of biochemical reactions take place in muscle contraction, initial
and recovery reactions. The initial reactions shown in the upper half of the
diagram involve consumption of high-energy phosphates whereas recovery
reactions shown in the lower half of the diagram involve the regeneration of
high-energy phosphates. In the presence of adequate supplies of metabolic
substrate and O2, recovery reactions are primarily those of mitochondrial
oxidative phosphorylation.
11
can be overcome by using a small section of ventricular tissue where the myocytes are
aligned from end-to-end, such as the papillary muscle.
The papillary muscle is of cylindrical shape with its fibres aligned relatively parallel to
the long axis of the muscle and has tissue at either end that can be tied or clipped for
attaching to experimental apparatus. Papillary muscles are discrete bundles of fibres
projecting from the wall of the ventricle to the mitral or bicuspid valve in the left
ventricle and the tricuspid valve in the right ventricle. Their function is to prevent the
valves protruding into the atrium during ventricular contraction. Papillary muscles
(from cat, rabbit, ferret, guinea pig and rat), have been used in a number of studies
(Chapman, 1972; Loiselle & Gibbs, 1979; Allen et al., 1989) and are a good model of
ventricular muscle function (Rayhill et al., 1994; Gibbs & Barclay, 1998). In fact, in
1998 Professor Gibbs declared that that there is little evidence suggesting that the
mechanical or energetic results obtained with papillary muscles are different from the
results obtained from the in vivo, whole heart situation (Gibbs & Barclay, 1998).
Examples of indices of cardiac function that have direct correlates with muscle function
include the pressure−volume index and isovolumic pressure development. The
pressure−volume diagram of the heart has a two-dimensional analogue in the
force−length diagram for isolated papillary muscle (Hisano & Cooper, 1987; Mast &
Elzinga, 1990; Baxi et al., 2000; Mellors & Barclay, 2001). The area enclosed by a
pressure−volume loop for a heart and a force−length loop for a papillary muscle is
closely related to the energy (or O2) consumption (Hisano & Cooper, 1987; Mast &
Elzinga, 1990; Suga, 1990, 2003a, b). The papillary muscle equivalent of isovolumic
pressure development is isometric force development. In addition, isolated papillary
muscle preparations can provide information about myocyte force generation, the
relationship between energy supply and demand and kinetics of mitochondrial
metabolism (Barclay et al., 2003).
2.3 Isolated mouse papillary model
With the development of genetically modified mice, there is a need for a cardiac muscle
model for determining the physiological and functional consequences of the various
genetic manipulations. A few studies have used the mouse papillary muscle to
investigate consequences of heart-focussed genetic changes (He et al., 1997; Meyer et
al., 1999; Bluhm et al., 2000; Pyle et al., 2002) but all of them have used an isometric
12
contraction protocol and none has attempted a more realistic contraction protocol or
performed energetic measurements. The suitability of isometric contractions as a model
of cardiac function has been questioned (Sonnenblick, 1962; Mellors & Barclay, 2001;
Redel et al., 2002) as no mechanical work is done and the protocol thus bears little
resemblance to the in situ situation of papillary or ventricular muscles. Therefore, a
protocol designed to closely simulate the reported changes in muscle shortening
(Semafuko & Bowie, 1975) will be used in most sections of this study (Mellors &
Barclay, 2001).
13
CChhaapptteerr 33:: MMeetthhooddss
The general experimental approach used in the experiments described in this Thesis is
measurement of the changes in muscle enthalpy content that accompany contraction.
The enthalpy output comes from the biochemical reactions that underlie contraction (i.e.
the hydrolysis and regeneration of ATP). Enthalpy changes appear as both heat and
mechanical work. The thermal changes can be measured using the myothermic
technique, an elegant and ingenious system (Warshaw, 2005) that allows partitioning of
energy used for processes that require and supply ATP and that has excellent temporal
and chemical resolution (Woledge, 1998). In this chapter, methods common to most of
the experiments are described. Where particular methods were used for a section of the
study, these are described in the relevant section.
3.1 Papillary muscle preparation and dissection
Papillary muscles were dissected from the left ventricle of hearts from six to twelve
week old male Swiss mice or Wistar rats. The animals were rendered unconscious by
inhalation of 80% CO2–20% O2 gas mixture and killed by cervical dislocation.
Euthanasia by short exposure to this gas mixture has previously been shown not to stop
the heart beating (Kohler et al., 1999). All animal-handling procedures were approved
by the Griffith University Animal Ethics Committee. The chest was opened and the
heart rapidly excised and transferred to oxygenated (95% O2–5% CO2) Krebs-Henseleit
14
solution of the following composition (mM): 118 NaCl, 4.75 KCl, 1.18 KH2PO4, 1.18
MgSO4, 24.8 NaHCO3, 2.5/1.5 (mice and rats, respectively) CaCl2, 10 glucose. The
value for ionized [Ca2+
] measured in the blood of mice is ∼1.4 mM (Sutherland et al.,
2003), a concentration that was used in the Krebs buffer in preliminary experiments
using the mouse papillary muscle. However, in those experiments it was found difficult
to reliably obtain preparations that contracted consistently and that continued to contract
for the duration of an experiment. When 2.5 mM Ca2+
was used muscles contracted
vigorously and continued to do so for at least 90 min after dissection.
The heart was gently massaged in the oxygenated saline buffer, with the apex removed
to facilitate removal of blood, and then placed in Krebs solution containing 30 mM 2,3–
butanedione monoxime (BDM) (Sigma, St. Louis, MO, USA) to prevent the myocytes
from contracting. BDM was included in the solution only during dissection to avoid
contracture upon freeing the muscle from its in situ length constraints; it does not alter
energetic properties once washed out (Kiriazis & Gibbs, 1995). The heart remained
immersed in oxygenated BDM-Krebs-Henseleit solution throughout the dissection
period. The right ventricular free wall was removed and the inter-ventricular septum
was bisected and pinned open, exposing the papillary muscles in the left ventricle.
Papillary muscles were dissected free from the wall of the heart and T-shaped platinum
clips (Ford et al., 1977; Donald et al., 1980) were attached to the tendon at one end of
the muscle and to a piece of ventricular wall at the other.
Muscles were stimulated using rectangular electric pulses (amplitude, 4–6 V; duration,
1–2 ms) that were passed along thin platinum wires, which were wound around the rods
up to the hook so that the platinum clips were in direct contact with the platinum wire.
The thin diameter (15 or 25 µm) prevented the wires affecting movement of the rod
connected to the motor or recording of force output.
3.2 Overview of experimental apparatus
In the experimental chamber used in most experiments (for an exception, see Chapter
5), muscles were mounted between a semi-conductor force transducer (AE801,
SensorOne, CA, USA) and a servo-controlled motor (322B, Aurora Scientific Inc.,
Ontario, Canada) via fine stainless steel wires that provided a low compliance linkage
between the preparation and the recording equipment. The muscle lay along the active
15
thermocouples of a thin-film, antimony–bismuth thermopile (Mulieri et al., 1977;
Barclay et al., 1995).
3.2.1 Measurement of muscle force production
The force produced was measured using a silicon strain gauge force transducer. A
stainless steel pin was glued on to the transducer using a fast-setting adhesive (Prism
406, Loctite, Welwyn Garden City, UK) and the stainless steel wire connecting the
muscle to the transducer was attached to the pin by a drop of wax.
A set of weights, the masses of which were determined using an analytical balance
(Scout II, Ohaus Corporation, NJ, USA), was used to calibrate the force transducer. The
masses of the weights were chosen to cover the range of expected muscle force outputs.
The force transducer was positioned vertically and the weights were hung from the pin,
thus exerting force in the same direction as the force applied by a muscle preparation in
an experiment.
The relationship between force output and applied load was linear and the data were
fitted with a straight line (Fig. 3.1). The calibration factor, which corresponds to the
inverse slope of the fitted line, was 782.9 mN V-1
for the force transducer used in most
of the experiments described in this Thesis. The force transducer output was amplified
by a factor of 50, 100 or 250.
0 10 20 30 40Force (mN)
0.00
0.01
0.02
0.03
0.04
0.05
0.06
Fo
rce
tra
nsd
uce
r o
utp
ut
(V)
Fig. 3.1. Calibration of force transducer. The relationship between transducer output and applied force for the force
transducer used in this project. The output has been adjusted to account for the
amplifier gain so the values shown are the true transducer output. The straight
line was fitted using linear regression; its slope is 1.28 mV mN-1
.
16
3.2.2 Measurement of muscle length changes
The remaining end of the muscle was attached to an aluminium lever via a stainless
steel rod. The lever was attached to the servo-controlled motor. The motor system
allowed muscle length to be controlled and measured simultaneously. Lever position,
and thus muscle length, was controlled using the output of a 12-bit digital-to-analogue
converter (DAS-1802AO, Keithley Instruments, Cleveland, OH, USA). The patterns of
muscle length changes were generated using software.
3.2.3 Principles of measuring muscle heat output using a thermopile
Muscle heat production was determined by measuring the change in muscle temperature
using a thermopile. A thermopile consists of a number of thermocouples connected in
series. The thermocouples are arranged so that every second thermocouple lies along the
centre of the thermopile (the “active” thermocouples) and the alternate thermocouples
(the reference thermocouples) are positioned on the edges of the thermopile. The edges
were clamped between the jaws of the aluminium frame that supported the thermopile
so that the reference thermocouples were maintained at close to the temperature of the
frame. Frame temperature was kept constant by circulating water through channels in
the frame. The muscle preparation was positioned along the active thermocouples, thus
the muscle regulated the temperature of the active region. Upon stimulation, the muscle
contracted and released heat, increasing the temperature of the active thermocouples
relative to the reference thermocouples. The temperature difference between the active
and reference junctions generated a signal with an amplitude proportional to the change
in muscle temperature.
3.2.3.1 Calibration of thermopile
To convert the thermopile output into temperature units, the Seebeck coefficient of the
thermopile must be known. This can be determined using the Peltier effect: when a
current is passed through a thermopile, heat is absorbed at one set of junctions and
evolved at the other. The Seebeck coefficient can be calculated from the relationship
between the heat capacity of the thermopile and the rate at which its output changes
upon starting or stopping the current (Kretzschmar & Wilkie, 1972; Woledge et al.,
1985).
In practice, a series of small silver blocks (12−60 mg; heat capacity 0.234 mJ K-1
mg-1
),
with a drop of glycerol to aid thermal transfer with the thermopile (89% glycerol, 11%
17
water; heat capacity 2.59 mJ K-1
mg-1
), were placed over the active junctions of the
thermopile. A current was passed through the thermopile to heat the active junctions and
the silver block. Once a steady temperature had been reached, the current was turned off
and the time-course of the cool-off measured (Equation (12) Fig. 3.2). The relationship
between the initial rate of cooling and the added heat capacity is described as follows.
2 2
0
( )thermopile Ag glycerol
dV ITn
dt C C
α
+
=+
(p.188, Woledge et al., 1985) (12)
where dV0/dt is the initial rate of change of the thermopile output (V s-1
) , I is the
heating current (A), T is the absolute temperature (K), n is the number of active
thermocouples, α is the Seebeck coefficient (V K-1
couple-1
) and CAg+glycerol (J K-1
) is the
sum of the heat capacities of the silver block and the glycerol and Cthermopile the heat
capacity of the region of the thermopile beneath the silver block.
If the current passed through the thermopile is small, heating by the Peltier effect
(proportional to the current) is much greater than that by the Joule effect (proportional
to the square of the current). For example, the heating current used was 123 µA, the
temperature 300 K, and typically the resistance of 16 thermocouples was ∼180 Ω, which
gives a rate of heating due to the Peltier effect (= ITnα) of 5.0 × 10-5
W and due to the
Joule effect (= I2R) of 2.8 × 10
-6 W. That is, heating due to the Peltier effect was ∼18
times greater than that due to the Joule effect. This would result in the Seebeck
coefficient being overestimated by ∼2%. No correction was made for this effect.
Knowing the cool-off rate constant, the initial thermopile output and the added heat
capacity of the silver block and the glycerol, the Seebeck coefficient was determined by
fitting Equation (12) using a non-linear least squares regression (Fig. 3.3). This was
performed using Mathcad’s (version 11.0, Mathsoft Inc., USA) “genfit” function.
Values for the Seebeck coefficient for each thermopile used in this Thesis are presented
in Table 3.1.
Thermopile 1 2 3
Active region (mm) 4 5 2
n (couple) 16 20 8
α (µV K-1
couple-1
) 65.0 75.6 78.3
Output (mV °C-1
) 1.30 1.21 0.63
Table 3.1. Thermopile characteristics.
18
Fig. 3.2. Time-course of cooling of
silver blocks. An example of the time-course with
which the silver blocks cooled
following a period of Peltier heating.
Records from two silver blocks
weighing 12 and 60 mg are shown. The
bigger silver block took longer to cool
than the smaller block. The time-course
of cooling could be described by a
single exponential curve. The black
lines show the measured thermopile
output and the white lines show fitted
exponential curves. Data from the first
1 s were excluded from the fitting
because that section was largely due to
the change in temperature at the
reference junctions. This was rapidly
reversed due to the proximity of the
frame.
0 10 20 30 40 50 60
Time (s)
0.0
0.2
0.4
0.6
0.8
1.0
Th
erm
op
ile o
utp
ut
(V)
12 mg 60 mg
Fig. 3.3. Calibration curve for the
Seebeck coefficient. Data from one thermopile showing the
relationship between the initial rate of
cooling of the thermopile and the added
heat capacity. Initial rate of cooling
was calculated as the product of the
initial value of the thermopile output
and the rate constant for the heat loss.
Both these values were determined
from the exponential curves fitted to
the cool-off data (Fig. 3.2). The dashed
line is the line of best fit described by
Equation (12) and fitted through the
data using non-linear regression (pp
683-688, Press, 1992). The Seebeck
coefficient for this example was 78.3
µV K-1
couple-1
.
0.000 0.005 0.010 0.015 0.020
Added heat capacity (J K-1)
0.0
0.5
1.0
1.5
2.0
Initia
l co
olin
g r
ate
(V
s-1
)
10-5
19
3.2.3.2 Conversion of thermopile signals into heat production
To calculate heat production from the measured change in muscle temperature it is
necessary to (1) convert the thermopile output into temperature units, (2) correct the
records for heat lost from the active thermocouples to the frame while recording and (3)
multiply the corrected temperature change by the heat capacity with which the heat
produced by the muscle is shared.
The output of the thermopile (∆V) can be converted into units of temperature change
(∆T) by dividing the output by the product of the number of thermocouples and the
Seebeck coefficient:
VT
nα
∆∆ = (13)
Due to the temperature difference between the muscle and the frame, heat is constantly
lost from a preparation. The corrected temperature at time t (Tc(t)) is given by the
integral of the measured temperature (Tm) with respect to time multiplied by the rate of
heat loss (k) (Equation (14), Fig. 3.4).
0( ) t
c mT t k T dt= ∫ (14)
The cooling after Peltier heating was used not only to measure the rate of heat loss but
also to calculate muscle heat capacity (pp 187-188, Woledge et al., 1985).
0 /muscle
ITnC
dT dt
α= (15)
where dT0/dt is the initial rate of cooling (in temperature units) after Peltier heating.
Note that from Equation (13) dT0/dt = (dV0/dt)/nα. Finally, by rearranging Equations
(13) and (15) an expression for calculating the heat (∆Q) produced by the muscle is
derived.
0 0/ /muscle
V ITn ITnQ TC V
n dT dt dV dt
α α
α
∆∆ = ∆ = ⋅ = ∆ ⋅ (16)
20
3.2.4 Determination of stimulus heat
When a muscle is stimulated, the current running through the muscle and any adhering
solution produces heat by the Joule effect. The amount of heat produced by the stimulus
current was measured by stimulating an artificial muscle created from agar gel made
with Krebs solution and with the same dimensions as a muscle. The stimulus heat was
∼0.1 µJ pulse-1
using the artificial muscle (amplitude 6 V, duration 2 ms). This typically
corresponded to less than ~2% of the net heat produced by a muscle in response to a
stimulus pulse. Another way of illustrating the magnitude of the stimulus heat relative to
muscle heat production is shown in Fig. 3.5. This muscle failed to respond to the first
four stimulus pulses (for unknown reasons) but then contracted upon delivery of the
fifth pulse. The first four pulses produced very small increments in the cumulative heat
record. When the muscle contracted there was a large, rapid increase in heat output. In
that example the heat per 1 ms stimulus pulse was ∼0.08 mJ g-1
twitch-1
which was
∼1.5% of the initial heat produced in the twitch. Given that the net heat is approximately
twice the initial heat (see Section 3.6), this estimate of stimulus heat corresponds to
0 5 10 15 20
Time (s)
0
50
100
150
Mu
scle
te
mp
era
ture
(m
°C)
Measured temperature
Corrected temperature
0 40 80
Time (s)
0
50
100
150
Te
mp
era
ture
(m
°C)
Fig. 3.4. Heat loss correction. Temperature signals were corrected for heat lost from the preparation due to the
temperature difference between the muscle and the frame. The recording shows
the measured (dotted line) and corrected (solid line) temperature from a
contracting mouse papillary muscle stimulated at a contraction frequency of 2
Hz lasting 20 s. The inset shows the complete time course of change in muscle
temperature. The vertical dashed line indicates the time at which contractions
ended and the recorded muscle temperature was then ∼10 m°C. Heat was
produced at a rate greater than the resting rate for ∼60 s after the end of the
contraction series, indicating the ongoing recovery metabolism. Muscle mass:
1.57 mg; length: 3.8 mm.
21
∼0.8% of the likely net heat. If this value were doubled to allow comparison with 2 ms
pulses, as used for the artificial muscle, the result is consistent with the estimated
stimulus heat measured using the agar “muscles”.
3.3 Data recording
The thermopile output was low pass filtered (cut-off frequency, 100 Hz) and amplified
using a series arrangement of two low-noise amplifiers (15C-3A, Ancom Instruments,
Cheltenham, UK; SR560, Stanford Research, CA, USA). The output of the thermopile
was sampled at 220 Hz. Software developed using TestPoint (Capital Equipment
Corporation, Middleborough, MA, USA) was used to control data recording and to
analyse the data.
All signals were recorded using a 12-bit A/D converter with an input range of ±10 V
(DAS-1802AO, Keithley Instruments, Cleveland, OH, USA). The resolution of the A/D
converter (i.e. the voltage change giving a change of 1 digit) is 20/212
= 20/4096 = 4.88
mV. The force, length and temperature signals were amplified prior to recording to
reduce the quantization noise (4.88 mV/signal amplitude) to an acceptable level; that is,
to <1% of the signal amplitude or to less than the amplitude of noise from other sources,
whichever was larger. Estimates of the relative amplitude of quantization noise and
noise from other sources are provided in Table 3.2.
Fig. 3.5. Stimulus heat using a mouse
papillary muscle. Records from a mouse papillary muscle
that was stimulated five times at a
contraction frequency of 2 Hz (amplitude
6 V, duration 1 ms). The muscle did not
respond to the first four stimuli but did
respond to the fifth, producing an active
force of ∼18 mN mm-2
. The amount of
stimulus heat was estimated by measuring
the cumulative heat produced from the
first four stimulus pulses (dashed line).
Muscle mass; 1.50 mg, muscle length; 3.7
mm, temperature; 37°C. 0.0 0.5 1.0 1.5 2.0 2.5
Time (s)
0
5
10
15
20
25
Fo
rce
ou
tpu
t (m
N m
m-2
)0
2
4
6
8
10
12
He
at o
utp
ut (m
J g
-1)
Stimulus
Heat
Force
22
Force Length Temperature
mN 5 mm 0.3 m°C 5
V 0.6 V 0.9 V∗ 0.5
Nq (%) 0.8 Nq (%) 0.5 Nq (%) 1
N0 (%) <1 N0 (%) <0.5 N0 (%) 2
3.4 Partitioning of initial and recovery metabolism
The heat and the work liberated from a contracting muscle arise from enthalpy changes
associated with the biochemical reactions that underlie contraction. Enthalpy output
associated with contractile activity can be separated into initial enthalpy output and
recovery enthalpy output because of the difference in the time courses with which they
are produced during the transition from rest to steady activity. Initial enthalpy is
produced simultaneously with contractile activity and thus its production commences in
synchrony with the activity (see Fig. 3.6A & Fig. 3.6B). In contrast, recovery enthalpy
output increases exponentially from its resting value towards its steady state value with
a time constant, in the rat and mouse papillary muscle at 27°C, of about 10 and 12 s,
respectively. In this study, the net enthalpy output produced during and after a short set
of contractions (lasting 20 s) was measured (Fig. 3.6C & Fig. 3.6D) and the initial
enthalpy output was determined from the enthalpy produced during the first three
contraction cycles, before the rate of recovery enthalpy production became significant
(Fig. 3.6B).
The relative contributions of initial and recovery processes to enthalpy output in the first
three cycles of a contraction protocol were estimated as follows. An exponential
increase in the rate of recovery heat production ( RQg
) can be described by:
-
,( ) 1-t
R R SSQ t Q eτ
=
g g
(17)
where R,SSQg
is the steady-state recovery heat rate.
Table 3.2. Estimates of quantization noise and noise from other sources.
Nq = relative amplitude of quantization error.
N0 = typical relative amplitude of electrical noise. ∗ Typical gain 100 000.
23
0
5
10
15
20
25
30
Fo
rce
ou
tpu
t (m
N m
m-2
)
1 s
A
0
5
10
15
En
tha
lpy o
utp
ut (m
J g
-1)
IH1
IH2
IH3
1 s
B
0 20 40 60 80
Time (s)
0
5
10
15
20
25
30
Fo
rce
ou
tpu
t (m
N m
m-2
)
C
0 20 40 60 80
Time (s)
Net
enthalpy
0
50
100
150
200
En
tha
lpy o
utp
ut (m
J g
-1)
D
Fig. 3.6. Example of the time course of force production and enthalpy
output. An example of force production and enthalpy output of a mouse papillary
muscle. The muscle performed 20 isometric contractions at a frequency of 1 Hz.
A. Force output for the first three twitches. B. Cumulative enthalpy production
during the first three twitches. Because the muscle was not shortening, there was
no work production so enthalpy output was equal to the heat output. The solid
line indicates the measured heat output and the dashed line indicates the
estimated time course of recovery heat production (from Equation (18)). The
quantities indicated by the arrows labelled IH1, IH2 and IH3 represent the
cumulative initial heat production up to the end of each of the first three
contraction cycles. C. The time course of force output for the whole contraction
series. D. The cumulative enthalpy output during and after the 20 contractions.
The vertical dashed line indicates the time at which contractions ended. The rate
of heat production remained above the resting heat rate for ~60 s, indicating the
ongoing recovery metabolism. Muscle mass 1.72 mg; length 3.4 mm.
24
The cumulative recovery enthalpy production associated with contractile activity (QR) is
given by the integral of Equation (17) and assuming that RQ (0)g
= 0:
( )-
, ,( ) -t
R R SS R SSQ t Q t e Qττ τ
= + ⋅ ⋅
g g
(18)
Recovery heat production starts quite quickly in cardiac muscle so a detailed assessment
of the likely contribution of recovery heat to initial heat measurements was made. This
is described in Section 4.2.1 for rat papillary muscles and 6.2.2 for mouse papillary
muscle.
3.4.1 Partitioning initial metabolism into force-dependent and force-independent components
Initial enthalpy output was partitioned using an isometric contraction protocol (2 Hz)
into a force-dependent component (i.e. enthalpy output associated with cross-bridge
activity) and a force-independent, or activation, component (i.e. enthalpy output
associated primarily with ion pumping) by selectively inhibiting cross-bridge cycling
with BDM and/or exposure to hyperosmotic solution (Alpert et al., 1989). The
relationship between initial enthalpy output and force-time integral (FTI) was
determined by making incremental reductions in FTI by step-wise increases in BDM
concentration from 2 to 10 mM and using 5 mM BDM in combination with 150 mM
sucrose. The magnitude of force-independent enthalpy output was determined by
extrapolating the enthalpy output−FTI relationship to zero FTI.
3.5 Calculations of efficiency
3.5.1 Mechanical efficiency
In general terms, efficiency is the ratio of the work performed to the energy expended to
do that work. Mechanical efficiency is defined as the ratio of the work output to the
enthalpy change accompanying the work performance. The precise definition depends
on whether just the enthalpy from initial reactions is used or whether that from recovery
reactions is also included.
3.5.1.1 Initial and net mechanical efficiency
The initial mechanical efficiency (εI) is the ratio of the work output to the initial
enthalpy output (∆HI). In this project ∆HI was defined to be the enthalpy produced
25
between the start of a series of contractions and the end of the third contraction cycle
(for details see Fig. 3.6).
I
I
W
Hε =
∆ (19)
In isometric contractions, ∆HI is equivalent to the heat produced in the three cycles and
in shortening contractions it is the sum of the heat and work produced. Contraction
frequency was typically 2 Hz, so ∆HI was measured over the first 1.5 s of activity.
Net mechanical efficiency (εNet) was defined as the ratio of the work output produced in
a series of contractions to the total enthalpy produced, in excess of that due to resting
metabolism, during and after the contractions series .
Net
Net
W
Hε =
∆ (20)
3.5.2 Mitochondrial efficiency
An index of mitochondrial function is the efficiency (ηR) with which the mitochondria
transfer free energy from metabolic substrate to ATP (Barclay et al., 2003; Smith et al.,
2005). This can be calculated from the ratio of εNet and εI.
Net ATPR
I PCr
G
H
εη
ε
∆= ⋅
∆ (21)
3.6 Ratio of recovery heat output to initial heat output
The ratio of the amount of recovery heat relative to the amount of initial enthalpy output
(R/I) quantifies the coupling between energy supply and energy demand and its value
reflects the cost of oxidative recovery metabolism relative to the amount of ATP used
during contraction. R/I can be derived from the initial and net mechanical efficiency (for
details, see Barclay & Weber, 2004).
1I
Net
R
I
ε
ε= − (22)
The initial and net values are not strictly comparable as they come from different sets of
contractions. However, Mast & Elzinga (1988) showed that initial and recovery heat
ratios are the same for whole series of contractions as for sub-sets. Moreover, the values
obtained for rat and mouse papillary muscles are similar to those reported previously in
26
isometric contractions for rabbit papillary muscles (1.14 and 1.18; Mast & Elzinga,
1988; 1.10; Mast et al., 1990) as well as for rat papillary muscles (1.16, Barclay et al.,
2003) using a shortening protocol. In the studies by Mast et al. (1990) and Barclay et al.
(2003) a mathematical approach was used to partition the initial and recovery
components.
3.7 Oxygenation of papillary muscles
The isolated muscle preparation lacks normal circulation and thus the sole source of O2
is diffusion from the outer surface of the muscle. In a thick piece of muscle or a muscle
with a high metabolic rate, O2 diffusion may be insufficient to match the muscle’s
metabolic needs, resulting in formation of an anoxic core. In this Thesis, the adequacy
of oxygenation of the papillary muscles was assessed using a mathematical model of
diffusion of O2 into muscles as described by A.V. Hill (1928; 1965). In this analysis it
was assumed that the papillary muscle was cylindrical, of uniform radius and that
negligible O2 diffusion occurs through the ends of the cylinder.
The adequacy of diffusive O2 supply to papillary muscles during protocols in which
muscles performed a short series of contractions was assessed by numerical solution of
Equation (23) describing diffusion of O2 into muscles of cylindrical geometry with a
rate of O2 consumption that varied with time (p. 229, Hill, 1965) as described in detail
by Barclay (2005).
2 2 2
2
2
1( )
O O OP P PK A t
t r r r
δ δ δ
δ δ δ
= + ⋅ −
(Barclay, 2005) (23)
where t is time, PO2 is the partial pressure of O2, r is the radial distance from the
muscle’s centre and A(t) is the time-dependent rate of O2 consumption. The equation
was solved using a programme written using Mathcad. The contribution of myoglobin-
facilitated O2 diffusion to O2 supply was not included in the model because myoglobin
contributes little to total O2 flux within isolated papillary muscles when the external PO2
is >0.2 atm (Loiselle, 1987). It was assumed that the rate of mitochondrial O2
consumption was PO2-dependent such that it varied sigmoidally with PO2 at values
below ∼10 mm Hg, as observed experimentally (Wittenberg & Wittenberg, 1985).
27
3.8 Data normalisation
At the end of experiments, the platinum clips were cut off the preparation, the muscle
was lightly blotted and its mass determined using an electronic balance (Cahn 25, Cahn
Instruments, Cerritos, CA, USA). Some of the mouse papillary muscles were kept after
the end of the experiment and later placed to dry in an oven with the temperature set at
80°C. The preparations were re-weighed and the wet-to-dry mass ratio determined. The
mean ratio of wet mass to dry mass was 4.9 ± 0.2 (n = 22). The average cross-sectional
area was calculated by dividing mass by length, and assuming muscle density was 1.06
g cm-3
. Active force was normalised by cross-sectional area. Work and enthalpy output
were normalised by muscle wet mass. All data are presented as the mean ± S.E.M.
28
29
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oouuttppuutt iinn ppoosstt--iisscchhaaeemmiicc ccaarrddiiaacc mmuussccllee
4.1 Introduction
Exposure of cardiac muscle to ischaemia lasting more than a few minutes results in a
prolonged depression of cardiac contractile function even after blood flow is restored
(for a review, see Bolli & Marban, 1999). Hearts in this state are referred to as
“stunned” and are characterised by reduced cardiac output (Heyndrickx et al., 1975;
Braunwald & Kloner, 1982). The mechanism underlying the impaired contractility is
unclear but must ultimately reflect changes in work generation by myosin cross-bridges
during their cyclic interactions with actin filament. Decreased cardiac muscle work
output can potentially arise from either of two mechanisms (or a combination of both): a
decrease in the number of cross-bridges contributing to muscle work output or a
decrease in the work generated by each cross-bridge. In the first case, the amount of
work produced in each cross-bridge cycle would be the same as that before ischaemia
but a smaller proportion of cross-bridges are attached and generating force at any instant
during contraction so that the total work output is decreased. In the second case, the
number of attached cross-bridges does not alter but the work produced in each cross-
bridge cycle is decreased.
30
The likely effects of these two mechanisms on muscle efficiency potentially provide a
means of distinguishing between the mechanisms. Efficiency is the ratio of the work
produced to the energy used to produce that work. If, as seems likely in most conditions,
each cross-bridge cycle is associated with splitting of one ATP molecule, then the
efficiency of a population of cross-bridges is the ratio of the average cross-bridge work
output to the number of cross-bridge cycles that produced that work. In the first
mechanism described above, the work produced in each cross-bridge cycle would be
unaffected by ischaemia, so efficiency would also be unaltered. For the second
mechanism, however, less work would be performed in each ATP splitting cross-bridge
cycle, so cross-bridge efficiency would be decreased.
The aim of the current study was to identify the mechanism underlying the impaired
work output of post-ischaemic cardiac muscle by comparing the efficiency of cardiac
muscle measured before exposure to ischaemia with that measured following
reperfusion. The experiments were performed using an isolated rat papillary muscle
preparation so that precise measurements could be made of both work output and
energy use. Work output of a papillary muscle can be accurately measured because,
unlike the ventricular wall, the myocytes are aligned parallel to the long axis of the
muscle. Energy use was determined by measuring the energy liberated during
contraction as heat and work. This enthalpy output arises from the enthalpy changes
accompanying the biochemical reactions that underlie contraction and the amount of
enthalpy produced is proportional to the amount of ATP consumed. Another advantage
of an isolated muscle preparation is that it is possible to closely specify the “ischaemic”
conditions. In this study, ischaemia was simulated by removing superfusate from the
muscles and by replacing the O2 supply with N2.
4.2 Methods
The materials and methods used for the experiments reported in this chapter have been
described in Chapter 3. Only those methods specific to these experiments will be
described here. Characteristics of the rat papillary muscles are given in Table 4.1.
31
Number of preparations 15
Wet muscle mass (mg) 3.7 ± 0.2
Muscle length (mm) 6.2 ± 0.3
Cross-sectional area (mm2) 0.57 ± 0.02
Muscle radius (mm) 0.43 ± 0.01
4.2.1 Initial and recovery metabolism
The enthalpy output produced by the contracting muscle was measured in the form of
net enthalpy output (∆HNet) and initial enthalpy output (∆HI). The former was the total,
suprabasal enthalpy output produced in response to a 20 s series of contractions and the
latter was the cumulative enthalpy produced during the first three contraction cycles.
The initial and recovery enthalpy output were separated as described in Section 3.4 and
used to measure force-independent enthalpy output (∆HP). The relative contribution of
recovery heat to the measured initial heat was calculated using Equation (18). The
steady state of recovery heat output ( R,SSQg
in Equation (18)) was estimated on the basis
that the amount of recovery heat required to completely reverse the initial heat was 1.1
× the amount of initial heat (i.e. R/I ratio, Table 4.2) and that in an energetic steady state
this amount of recovery heat would be produced within each contraction cycle (i.e. in
0.5 s when contracting at 2 Hz). The value of the R/I ratio was calculated as described
previously (see Section 3.6, Equation (22)) using data obtained from rat papillary
muscles in the current study.
The initial enthalpy output was ~3.2 mJ g-1
twitch-1
, at a contraction frequency of 2 Hz,
so in one cycle R,SSQg
= 1.1 × 3.2 × 2 cycles s-1
= 7 mW g-1
. Substituting this value and
the time-constant for recovery metabolism (~10 s; see Table 4.3) into Equation (18), the
amounts of recovery heat produced between the start of recording and the ends of the
first, second and third contraction cycles were 0.05, 0.20, and 0.44 mJ g-1
, respectively,
which was 1.6, 3.1 and 4.6% of the total heat produced by the end of those cycles (Fig.
3.6B). The post-ischaemic recovery time constant was ∼6 s but the initial enthalpy
output was smaller and the R/I unaltered so the contribution of recovery heat remained
the same.
The initial enthalpy output was used to partition energy use into force-dependent and
force-independent components (see Section 3.4.1) and to calculate initial efficiency.
Table 4.1. Left ventricular rat papillary muscle characteristics.
32
Recordings of net enthalpy output were used to calculate the overall energetic cost of
contractile activity, the net efficiency and the time course of recovery heat output.
4.2.2 Experimental protocols
After mounting in the experimental chamber, the length of the muscles was adjusted to
that at which twitch force was maximal (Lmax) and they were then allowed to equilibrate
for 60 min. The protocol consisted of pre-ischaemia measurements, followed by either
30 or 60 min of simulated ischaemia and 30 min of simulated reperfusion. Ischaemia
was simulated by withdrawing the bathing solution from around the muscle and by
replacing the 95% O2–5% CO2 gas flow with 95% N2–5% CO2. Gas was supplied to the
muscle chamber via a humidifier that saturated the gas with water vapour before it
reached the muscles. This prevented muscles from dehydrating when the solution was
removed from the chamber (e.g. during simulated ischaemia). At the end of either 30 or
60 min, solution aerated with 95% O2 was returned to the muscle.
Two control experiments were performed. In the first, muscles remained in solution
throughout the protocol and were continuously aerated with 95% O2 whereas in the
second the solution was removed from the muscle but aeration with 95% O2 was
maintained. This enabled the effect of removing the saline from the muscle to be
distinguished from effects due to the withdrawal of O2.
A total of five measurements were made: three to investigate changes in mechanical and
energetic output and two to determine the contribution of force-independent heat to total
enthalpy output. Muscle enthalpy output was measured before ischaemia and after 5 and
30 min of reperfusion using separate series of isometric and more realistic contractions.
The realistic contraction protocol (Mellors & Barclay, 2001), illustrated in Fig. 4.1, was
designed to mimic the in situ pattern of length changes reported for papillary muscles
(Semafuko & Bowie, 1975). The isometric contraction protocol was used for
measurements of the force-independent enthalpy output. These measurements were
made at the end of the equilibration period and 40 min after the end of the ischaemic
phase. Force-independent enthalpy output was calculated by extrapolation of the two-
point, enthalpy output versus force-time integral plot constructed from measurements
made with the muscle in normal Krebs solution and in an hyperosmotic solution of
Krebs with 5 mM BDM and 150 mM sucrose added (Alpert et al., 1989). For all
measurements using both isometric and realistic protocols, muscles performed 40
contractions at a contraction frequency of 2 Hz. During simulated ischaemia muscles
33
remained isometric, were stimulated at 0.5 Hz and both total and passive force outputs
were recorded continuously. Passive force was defined as the force measured in the
absence of contractile activity and total force was the sum of the passive force and the
additional force produced in response to stimulation.
4.2.3 Statistical analysis
Statistical significance of variations in force output and energetic variables with time
were assessed using repeated measures, one-way analysis of variance. A paired t-test
was used to compare measurements of force-independent enthalpy output made before
ischaemia with those made after 40 min of reperfusion. Decisions concerning statistical
significance were made at the 95% level of confidence.
4.2.4 Analysis of diffusive O2 supply
A mathematical model of diffusion of O2 into cylindrical muscles (see Section 3.7) was
used to assess the diffusive O2 supply to the isolated preparations, both to ensure
adequacy of supply during control and reperfusion phases and also to estimate the
degree of anoxia during the ischaemic phase and the time course of changes in muscle
oxygenation at the start and end of that phase. The O2 partial pressure (PO2) at the
muscle surface was measured under conditions matching those used during
Fig. 4.1. Comparison of twitches
recorded using isometric and realistic
contraction protocols. An example of changes in muscle length (A)
and force output (B) during an isometric
(dashed line) and realistic (solid line)
contraction cycle. In the realistic contraction
protocol, muscle length was held constant for
110 ms, then shortened by 10% Lmax in 185 ms;
the velocity was 0.54 (Lmax s-1
). The vertical
dotted lines indicate the time for the start and
end of the shortening phase. Once the muscle
was relaxed, it was returned to its original
length. Muscle mass; 3.64 mg; muscle length
6.7 mm.
-0.8
-0.6
-0.4
-0.2
0.0L
en
gth
(m
m)
0.0 0.1 0.2 0.3 0.4 0.5
Time (s)
0
5
10
15
20
25
Fo
rce
(m
N)
A.
B.
34
experiments. An O2-sensitive microelectrode (OX500, Unisense, Aarhus, Denmark) was
placed above the thermopile, in the location normally occupied by a muscle and the bath
was sealed using microscope slides. When the system was supplied with 95% O2, PO2 in
the muscle chamber was 0.84 atm. When the gas mixture changed to 95% N2, PO2
decreased to ∼0.05 atm.
The analysis took account of resting metabolic rate, variations in mitochondrial activity
at low PO2 and the time course of changes in O2 consumption. The results of the
analysis for the transitions from high PO2 to low PO2, and back again, are shown in Fig.
4.2. The figure shows both the fraction of the muscle cross-section that is oxygenated
(Fig. 4.2A) and also the PO2 values at the surface and centre of the muscles (Fig. 4.2B).
When aerated with 95% O2, diffusive O2 supply would have been adequate to keep the
muscles fully oxygenated. Upon switching to 95% N2, PO2 within the muscle would
have fallen rapidly so that within 30 s almost the entire muscle would have been anoxic.
Similarly, when 95% O2 was returned to the muscle surface, O2 would have diffused
quickly into the muscle so that the preparation was likely to have been fully oxygenated
in less than 25 s (i.e. the time at which the calculated PO2 at the muscle centre became
>0).
4.3 Results
4.3.1 Control experiments
The total and passive forces recorded from twitches delivered at 0.5 Hz throughout the
60 min ischaemia protocol are illustrated in Fig. 4.3. One of the sets of data shown in
Fig. 4.3 is for “control” measurements (open symbols; n = 4) in which the solution was
drained from around the muscle for 60 min but aeration with 95% O2 was maintained.
In that case, there were no significant changes in either passive or total force during the
protocol. In addition, neither enthalpy output, work output nor efficiency, measured in
series of twitches, was altered significantly. In the other set of control experiments both
the solu tion surrounding the muscle and 95% O2 supply were maintained throughout. In
this protocol, too, there were no significant changes in any of the measured variables.
These results show that the measured variables were stable with time in the absence of
anoxia and were unaffected by just removal of the solution surrounding the muscle.
35
0
20
40
60
80
100F
ractio
n o
f m
uscle
with
PO
2>
0
A.
0.0
0.2
0.4
0.6
0.8
1.0
PO
2 (
atm
)
N2 O2
10 s
Muscle surface
Muscle centre
B.
Fig. 4.2. Simulations of time courses of muscle oxygenation at the
onset and end of a period of simulated ischaemia. A. Time-course of muscle oxygenation, quantified as fraction of muscle cross-
sectional area with PO2>0, after switching from 95% O2 to 95% N2 (left panel)
and upon returning to 95% O2. It was assumed that the muscle was contracting at
0.5 Hz throughout the protocol. When O2 was returned to the anoxic muscle
(arrow, right panel), the initial increase in area oxygenated was very rapid, due
to the very high PO2 gradient across the muscle surface and because small
increases in the radius of the oxygenated region produce large increases in
oxygenated area. B. Time course of changes in PO2 at the muscle surface (solid
lines) and at the centre of the muscle (dashed lines). Steady-state surface PO2
was set to be that measured in the chamber (0.84 atm with 95% O2 and 0.05 atm
with 95% N2). When aerated with 95% O2, PO2 at the muscle centre was
calculated to be 0.4 atm. This decreased to 0 within 25 s of switching to 95% N2.
The arrows indicate the time at which 95% O2 was reintroduced into the muscle
chamber.
36
4.3.2 Effects of simulated ischaemia on force output
The second set of data in Fig. 4.3 (closed symbols; n = 6) are for muscles that had both
solution removed and were exposed to 95% N2 for 60 min. In that case, active twitch
force (i.e. the difference between total force and passive force) declined in the first 20
min of N2 exposure until it was the same as the passive force; that is, active force
production became completely inhibited. Upon the return of O2, active force production
rapidly recovered so that total force had returned to pre-ischaemia values in 10 min.
Note, however, that the recovery of mechanical function was much slower than the
estimated time-course of return of O2 to the muscle (Fig. 4.2). After recovering, force
output then declined again and was significantly lower than its pre-ischaemia value after
30 min of re-oxygenation. Passive force also altered significantly with time but only
after O2 was returned to the experimental chamber, when it was transiently higher than
the pre-ischaemia value.
The contrast between the absence of changes in the measured variables in the two
control groups and the clear changes in force output when O2 was removed indicate
that, for this muscle preparation, the absence of solution surrounding the muscle per se
did not affect contractile function but that anoxia resulted in both an immediate and,
after re-oxygenation, delayed reduction in the ability of the papillary muscles to produce
force.
4.3.3 Effects of simulated ischaemia and reperfusion on mechanical and energetic parameters
In Fig. 4.4, the effects of both 30 and 60 min simulated ischaemia on the work and
enthalpy outputs measured after reperfusion are illustrated. In control muscles, there
were no significant changes in work or enthalpy output measured 5 min and 30 min
after re-oxygenation compared to that before the simulated ischaemia. Thirty min of
ischaemia had no significant effect on either work output or enthalpy output, regardless
of whether it was measured after 5 or 30 min of re-oxygenation. In contrast, after 60
min ischaemia both work output and enthalpy output were significantly decreased at
both the post-ischaemia recording times (Table 4.2). For instance, after 30 min re-
oxygenation, work output was 66 ± 3% of the pre-ischaemia value and enthalpy output
was 71 ± 3% of its pre-ischaemia value.
Prior to exposure to N2, εnet was ∼9%; that is, work accounted for 9% of the enthalpy
produced. This value did not alter significantly after either 30 or 60 min of simulated
37
ischaemia. Similarly, εI was ∼19% before the ischaemic period and there was no
significant variation over the time course of the experiment. Thus, neither initial nor net
mechanical efficiency was affected by a period of simulated ischaemia.
4.3.4 Partitioning energy cost between force-dependent and force-independent components
Initial enthalpy output, measured over the first three contraction cycles, was partitioned
into force-dependent and force-independent components by selectively inhibiting force
output using 5 mM BDM and 150 mM sucrose. Force-independent enthalpy output
accounted for 19 ± 5% (n = 6) of the initial enthalpy output measured before ischaemia.
Using a paired comparison, there was no significant difference between the values
before ischaemia and those measured after 40 min of reperfusion.
Table 4.2. Energetic variables before and after 60 min of simulated
ischaemia.
Pre-ischaemia
Post-ischaemia
5 min
Post-ischaemia
30 min
∆HNet (mJ g-1
twitch-1
) 10.7 ± 0.3 6.1 ± 1.0 7.6 ± 0.3
∆HI (mJ g-1
twitch-1
) 3.2 ± 0.5 2.3 ± 0.5 2.4 ± 0.3
∆HP (mJ g-1
twitch-1
) 0.6 ± 0.1 - 0.62 ± 0.08
W (mJ g-1
twitch-1
) 1.0 ± 0.1 0.6 ± 0.1 0.66 ± 0.03
εNet (%) 9.0 ± 0.9 9 ± 1 8.2 ± 0.5
εI (%) 19 ± 3 19 ± 2 16 ± 3
R/I 1.1 ± 0.2 1.1 ± 0.2 0.9 ± 0.2
38
0 20 40 60 80 100
Time (min)
0
50
100
Re
lative
fo
rce
ou
tpu
t (%
)
O2 N2 O2
Ischaemia
Control
Fig. 4.3. Changes in normalised force output during ischaemia and
reperfusion of rat papillary muscle. Force output is expressed relative to isometric twitch force measured at the start
of the experiment. Symbols represent the mean values and error bars the S.E.M.
The open symbols (, passive force; , total force) are data from control
experiments in which muscles (n = 4) were exposed to 95% O2 throughout the
experiment although solution was withdrawn for 60 min starting 15 min after the
beginning of the recordings. The solid symbols (, passive force; , total force)
are for data from muscles (n = 6) which were exposed to 95% N2 for 60 min. In
muscles exposed to N2, active force output was abolished after 20 min (i.e. total
force equalled passive force) while passive force output increased gradually and
remained elevated upon return of 95% O2. Active force recovered to control
levels within 10 min of the return of O2 but then decreased significantly over the
next 20 min.
39
4.3.5 Time course of oxidative recovery metabolism
The time course of oxidative recovery metabolism was determined from the time course
of the decline in rate of heat output after a series of contractions. The time course was
well described by a single exponential and could thus be quantified by the time constant
of an exponential function fitted through the post-contraction heat rate data (Fig. 4.5).
Prior to exposure to ischaemic conditions, the mean time constant was 9.5 ± 0.7 s (Table
4.3). Following 30 and 60 min ischaemia the time constant was significantly shorter
after both 5 min (5.6 ± 0.2 s) and 30 min of reperfusion (6.5 ± 0.3). The mean
differences between the pre-ischaemia values were 3.8 ± 0.7 s after 5 min reperfusion
and 3.0 ± 0.7 s after 30 min reperfusion. In other words, after simulated ischaemia
recovery metabolism was complete in about two-thirds of the time it took before
ischaemia. This was not simply an effect of time in vitro because there was no change in
the time constant for control muscles (Table 4.3). The amount of recovery metabolism
relative to the amount of initial metabolism was not significantly different (R/I ratio,
Table 4.2) so, although the time course was altered, the coupling between initial and
recovery metabolism was preserved.
0
50
100
150R
ela
tive
wo
rk o
utp
ut
(%)
Control N2 30 N2 60
A. Work
0
50
100
150
Re
lative
en
tha
lpy o
utp
ut
(%)
Control N2 30 N2 60
Equilibration
Reperfusion 30 min
Reperfusion 5 min
B. Enthalpy
Fig. 4.4. Effects of simulated ischaemia on relative work and relative
enthalpy output. Measurements of work (A) and enthalpy (B) output were made at three different
times: during the equilibration phase (black) and 5 min (dark grey) and 30 min
(light grey) min after re-oxygenation. There was no statistically significant
difference after 30 min simulated ischaemia but after 60 min of simulated
ischaemia both work and enthalpy output were significantly reduced at both
measurement times. N230 and N260 min represents 30 min and 60 min of
simulated ischaemia, respectively.
40
Fig. 4.5. Effect of simulated ischaemia on
time course of rate of recovery heat
output. The time course of rate of recovery enthalpy
output was calculated by differentiation of the
cumulative enthalpy record from rat papillary
muscles before and after exposure to N2. The
records were normalised by the value measured
1 s after the end of the last contraction cycle.
The time constant of the decline in rate of
recovery heat output was determined by fitting
an exponential through the records using non-
linear, least squares regression. In this example
the time constant decreased from 12.2 s to 6.6 s
after exposure to N2. Muscle mass, 3.66 mg;
muscle length, 5.5 mm.
0.0
0.2
0.4
0.6
0.8
1.0
Re
lative
en
tha
lpy r
ate
20 s
Pre-ischaemia
Post-
ischaemia
4.4 Discussion
The main finding of this study was that ischaemia resulted in a substantial decrease in
the work output of rat papillary muscles with no alteration in net or initial mechanical
efficiency. Thus, the transduction of energy from metabolic substrates, via the
mitochondria and myosin cross-bridges, to mechanical work was unaffected by a
preceding period of ischaemia. Consequently, the reduced work output of the muscles
cannot be attributed to a deficit in energy supply pathways, but rather must be
associated with the processes involved in either initiation or generation of force; that is,
with either excitation-contraction coupling or with the cycling of myosin cross-bridges.
Impaired excitation-contraction coupling would decrease the influx of Ca2+
into
myocytes at the start of contraction thereby decreasing the number of cross-bridges that
could attach to actin. Alternatively, the impaired work output could result from a deficit
in cross-bridge work output. This could come about in two ways: fewer attached cross-
Table 4.3. Recovery time constant before and after 60 min of simulated
ischaemia.
Recovery time constant (s) Pre-ischaemia
Post-ischaemia
5 min
Post-ischaemia
30 min
Control∗
9.5 ± 0.7 10.2 ± 0.7 9.8 ± 0.6
Ischaemia 60 min† 9.4 ± 0.6 5.6 ± 0.2 6.5 ± 0.3
* 16 observations, 4 muscles.
† 22 observations, 11 muscles.
41
bridges or less work per cross-bridge cycle. The overall aim of this study was to
distinguish among these possibilities.
4.4.1 Impaired work output due to fewer attached cross-bridges
This distinction can be made using the initial mechanical efficiency values and
partitioning of energy use between force-dependent and force-independent processes as
follows. εI is the ratio of the work output to the initial enthalpy output and the latter is
the sum of the initial heat (QI) and work produced. Cross-bridge cycling accounts for all
the work performed and for part of the heat produced (QCB). The remainder of the heat
arises from ion pumping (QP), in particular Ca2+
pumping, so Equation (19) can be re-
expressed as follows.
I
I CB P
W W
W Q W Q Qε = =
+ + + (24)
Equation (24) can be used to calculate the expected difference in εI in post-ischaemic
muscles for the mechanisms proposed to account for decreased work output. The
calculation was performed assuming that εI was 0.19 (Table 4.2), ion pumping
accounted for ~19% of ∆HI and that W declined to 0.66 of the pre-ischaemic value (Fig.
4.4). The results of the calculations are presented in Table 4.4, in which the predicted
post-ischaemic values of εI and the factors in Equation (24) expressed as percentages of
the pre-ischaemic values, are shown for the three following mechanisms. (1) The
decrease in work output was due to release of less Ca2+
. This would reduce QP, QCB and
W by the same proportion (i.e. to 0.66 of their control values). (2) Work output
decreased due to fewer cross-bridge cycles occurring but with no change in Ca2+
release. In this case, both W and QCB would be decreased to 0.66 of their control values
but QP would be unaltered. (3) The decline in work output was entirely due to a
decrease in the work performed in each cross-bridge cycle but with no change in either
the number of cross-bridge cycles or the amount of Ca2+
released (i.e. only W
decreased). A comparison between the data obtained in the current study and the
expected values for each mechanism can be made with reference to Table 4.2 and Fig.
4.6.
42
For Mechanisms 1 and 2, εI would be either unaltered or only slightly decreased
whereas for Mechanism 3, εI would be reduced to about two-thirds of its control value
(Table 4.4). However, there was no significant change in εI (Table 4.2, Fig. 4.6A). This
suggests that Mechanism 3 was the least likely mechanism to have accounted for the
change in work output.
Mechanisms 2 and 3 can be distinguished from Mechanism 1 on the basis of changes in
QP (i.e. force-independent heat output). For Mechanisms 2 and 3 QP would be unaltered
by ischaemia whereas for Mechanism 1 QP would be reduced to two-thirds of its pre-
ischaemic value. There was no significant change in QP (Fig. 4.6B), suggesting
Mechanism 1 was unlikely to have contributed. It should be noted that there was
considerable variation in post-ischaemic QP values. However, the notion that the amount
of Ca2+
released would not be altered after re-oxygenation is supported by previous
work in which the free Ca2+
transients in ferret papillary muscle were shown to rapidly
regain their pre-ischaemic characteristics upon re-oxygenation (Allen et al., 1989).
Note, however, that there are almost certainly changes in Ca2+
cycling at any time when
∆GATP (which determines the amount of Ca2+
that can be accumulated in the
sarcoplasmic reticulum) is decreased, such as during and immediately after ischaemia.
The final evidence to clarify the most likely of the three mechanisms to have accounted
for the impaired contractility is that Mechanism 3 would be expected to be associated
with an unaltered QCB (i.e. heat output associated with cross-bridge cycling) whereas
Mechanisms 1 and 2 would be expected to lead to a reduction in QCB. The calculated
QCB did decrease significantly and by the amount expected for Mechanisms 1 and 2
(Fig. 4.6C).
Table 4.4. Theoretical (expected) values of εI for different mechanisms
underlying depressed work output.
Relative values†
Mechanism∗ W QP QCB εI
1 66 66 66 100
2 66 100 66 90
3 66 100 100 66
* 1: decreased number of cross-bridges due to decreased Ca2+
release; 2: decreased
number of cross-bridges but no alteration in Ca2+
release; 3: decreased work per
cross-bridge cycle but unaltered number of cross-bridge cycles.
† Symbols refer to Equation (24).
43
To summarise, the effects of ischaemia on QCB and εI are not consistent with
Mechanism 3 and the changes in QP are probably inconsistent with predictions based on
Mechanism 1. Thus, the mechanism most likely to account for the depressed post-
ischaemic work output is that fewer cross-bridge cycles occur in each contraction,
despite little alteration in Ca2+
release. In other words, the sensitivity of the myofibrils
to Ca2+
is reduced. Several changes that occur in the intracellular environment after
ischaemia (e.g. decreased pH and accumulation of inorganic phosphate and reactive
oxygen species) can potentially reduce the sensitivity of the muscle to Ca2+
(Fabiato &
Fabiato, 1978; Allen & Orchard, 1983; Camara et al., 2004).
0
20
40
60
80
100
ε Ι (%
)
Mech 3
Pre Post
Mech 1 or 2
A.
0
20
40
60
80
100
QP (
% in
itia
l e
nth
alp
y)
Mech 1
Pre Post
Mech 2 or 3
B.
0
20
40
60
80
100
QC
B (
% in
itia
l e
nth
alp
y)
Mech 3
Pre Post
Mech 1 or 2
C.
Fig. 4.6. Effect of simulated ischaemia on cross-bridge-dependent and
-independent energy cost. The arrows indicate the values of the post-ischaemic data for the mechanisms
described in the text and Table 4.4. A. The initial efficiency expressed as a
percentage. The difference in initial efficiency between Mechanism 1 and 2 is
only 10%, which would be too small to be detected from the current data.
Mechanism 3 corresponds to an efficiency two-thirds of the pre-ischaemic value.
There was no significant change in efficiency, indicating Mechanism 1 or 2 were
likely to have caused the decreased in work. B. The measured force-independent
heat (QP), expressed as a percentage of the initial enthalpy. Sixty min of
simulated ischaemia had no significant effect on QP. It is most likely that
Mechanisms 2 or 3 are consistent with this result. C. Cross-bridge-dependent
heat output (QCB), expressed as a percentage of the initial enthalpy, calculated
for each muscle by subtracting from the measured initial enthalpy output the sum
of the work output and force-independent heat output. The cross-bridge-
dependent heat output was significantly lower after 60 min of simulated
ischaemia. The post-ischaemic data are consistent with the expected values for
Mechanisms 1 and 2. Data indicating pre-ischaemic and post-ischaemic values
are labelled Pre and Post, respectively.
44
4.4.2 Mitochondrial efficiency
It has been proposed that an important consequence of ischaemia and reperfusion
damage is impaired mitochondrial function. One index of mitochondrial function is the
efficiency (ηR) with which the mitochondria transfer free energy from metabolic
substrate to ATP (Barclay et al., 2003). This can be calculated from the ratio of εNet and
εI (Equation (21), Section 3.5.2).
Assuming that ∆GATP in rat cardiac muscle in the absence of ischaemia was ∼59 kJ
mol-1
(Table 2.1) and ∆HPCr was ∼35 kJ mol-1
(Woledge & Reilly, 1988), then the pre-
ischaemic value of ηR would have been (9/19) × (59/35) = 0.82, which indicates that
82% of the free energy produced by oxidation of the substrate was transferred to free
energy in ATP. Neither εNet nor εI changed significantly during the experiments, so if
the ratio of ∆GATP to ∆HPCr was also unaltered, then ηR would have been unaffected by
ischaemia. There is considerable evidence that, although ∆GATP is reduced during
ischaemia, its value returns to pre-ischaemic values quickly during reperfusion so that
after 30 min of reperfusion ∆GATP differs from pre-ischaemic value by <10% (Griese et
al., 1988; Headrick et al., 1990; Headrick, 1996; Schulz et al., 2001; Day et al., 2006).
Given that the R/I ratio was also unaltered after ischaemia, indicating that the coupling
between ATP use and ATP regeneration was preserved, then the only alteration in
mitochondrial function following the simulated ischaemia was the more rapid kinetics
(Fig. 4.5).
4.4.3 Recovery time course
The time course with which mitochondria respond to changes in energy demand due, for
example, to changes in heart rate or progressing from rest to steady activity is greatly
influenced by the function of creatine kinase (CK). When CK, either mitochondrial or
cytosolic, in rabbit and mouse hearts was inhibited pharmacologically or by genetic
modification, then the mitochondria responded more rapidly to changes in ATP demand
produced by the changes in heart rate (Harrison et al., 1999; Gustafson & Van Beek,
2002), supporting the idea that CK acts as an energetic buffer. The results in the current
study are, therefore, consistent with impaired CK activity following simulated
ischaemia. It has been shown before that ischaemia of even quite short duration (10−60
min), in rabbit hearts can cause loss of CK activity that persists for at least 30 min after
the end of ischaemia (Bittl et al., 1985). Therefore, our results are consistent with the
45
notion that the CK reaction in the rat papillary muscles became inhibited during
exposure to N2.
It should be noted that published reports of kinetics of mitochondrial oxidative
phosphorylation in response to changes in energy demand are somewhat contradictory.
The time course of changes in mitochondrial O2 consumption has been reported to be
prolonged in acidotic muscles (Mast & Elzinga, 1989) and to be unaltered in stunned
cardiac muscle following ischaemia (Zuurbier et al., 1997). However, interpretation of
the latter result was confounded by a substantial slowing of mitochondrial response over
the time course of an experiment in “control” muscles. In the current study, the time
constant of recovery heat production was stable in control muscles (Table 4.3), making
it easier to establish the nature of the response to simulated ischaemia.
4.5 Conclusion
In conclusion, the results of the current study, taken in concert with those of a previous
study of Ca2+
handling, indicate that the most likely cause of the depressed work output
of the post-ischaemic papillary muscles in this study was attachment of fewer cross-
bridges in each twitch but with no change in Ca2+
release or in work generated by each
attached cross-bridge. Furthermore, there were no signs of impairment of mitochondrial
function per se because neither the R/I ratio nor the estimated mitochondrial efficiency
were altered by ischaemia. However, the mitochondria responded more rapidly to
changes in energy demands, which may reflect impaired creatine kinase activity after
ischaemia and reperfusion.
46
47
CChhaapptteerr 55:: RReessttiinngg mmeettaabboolliissmm ooff mmoouussee
ppaappiillllaarryy mmuussccllee
5.1 Introduction
The isolated mouse papillary muscle is useful for determining the physiological
consequences of heart-focussed genetic manipulations. Papillary muscles are composed
of ventricular myocytes aligned along the long axis of the muscle, making them ideal
for measurement of ventricular muscle force generation, work output and energy
expenditure (Gibbs et al., 1967; Hisano & Cooper, 1987; Dietrich & Elzinga, 1993;
Baxi et al., 2000; Barclay et al., 2003). An important consideration for any isolated
muscle preparation is the adequacy of O2 supply. The only source of O2 for an isolated
muscle preparation is diffusive supply from the muscle surface. If the rate of O2
consumption is sufficiently high, diffusive O2 supply may be inadequate to oxygenate
the entire muscle cross-section, leaving an anoxic region in the centre of the muscle. An
anoxic region of muscle will eventually be unable to generate force or use energy, thus
reducing mass specific values for these variables, and, as observed in ischaemic cardiac
muscle, contracture is likely to develop.
The adequacy of diffusional O2 supply is governed by the balance between the rate at
which O2 diffuses into the muscle, quantified by the diffusivity, and the rate at which
the tissue consumes O2. The probability of an anoxic core forming is enhanced by
conditions that increase metabolic rate relative to O2 diffusivity. Muscles from small
48
animals, such as mice, have inherently high metabolic rates compared to muscles from
larger animals. Furthermore, it is common for investigations using mouse papillary
muscles to be performed at 37°C (He et al., 1997; Meyer et al., 1999; Bluhm et al.,
2000). Metabolic rate is more temperature sensitive than O2 diffusivity (Mahler et al.,
1985) so higher experimental temperatures increase the probability of anoxic core
formation.
In most studies that have used mouse papillary muscles there appears to have been a
poor appreciation of the potential limitations of diffusive O2 supply (He et al., 1997;
Meyer et al., 1999; Bluhm et al., 2000; Stull et al., 2002; Wang et al., 2002). In one
study in which the size of muscle required to avoid anoxia was assessed (Redel et al.,
2002), the assessment was based on published data for rat papillary muscles but the
metabolic rate of mouse cardiac muscle is likely to be greater than that of rat cardiac
muscle (Gibbs & Loiselle, 2001). Another study used metabolic data from isolated
beating mouse hearts to analyse O2 diffusion into isolated mouse trabeculae (Stuyvers et
al., 2002). However, the rate of O2 consumption used in those authors’ calculations is in
error through not appreciating that the original value (Gustafson & Van Beek, 2000)
was expressed per gram dry weight. Consequently, those authors (2002) overestimated
the rate of O2 consumption by a factor of ~5 and concluded, incorrectly, that even thin
mouse trabeculae (half-thickness ~0.11 mm) would become anoxic at metabolic rates
occurring during contractile activity. These two examples highlight the fundamental
problem for analysis of diffusional O2 supply to isolated mouse cardiac muscles: the
metabolic rate of these muscles has not been measured.
Metabolism can be divided into a resting component, corresponding to the metabolism
required to support cellular processes other than those related to contractile activity, and
a contractile or active component, representing the metabolism associated with
excitation, activation and contraction. In cardiac muscle, resting metabolism accounts
for 20 to 35% of the metabolism of a beating heart (Gibbs & Loiselle, 2001), a much
higher fraction than for other striated muscles. The first factor to be considered in
assessing the viability of in vitro preparations is the ability of diffusive O2 supply to
meet the metabolic demands of the resting muscle. The aims of the current study were
to measure the resting metabolic rate of mouse papillary muscles and to assess the
ability of O2 diffusion to meet the resting energy demands.
The resting metabolism of rat, cat and rabbit papillary muscles has been measured
previously. It was observed that resting metabolic rate declined exponentially with time
49
during an experiment (Loiselle & Gibbs, 1979, 1983; Loiselle, 1985c). For instance, the
resting metabolic rate of rat papillary muscles was ∼12 mW g-1
1 h after the heart was
removed from the animal but 2 h later it had decreased to ∼7 mW g-1
and subsequently
remained at that value. This characteristic could potentially result in a transient, central
anoxia in the muscles early in an experiment, when metabolic rate is high, with the
possibility that ischaemia and reperfusion damage then occurs as the resting metabolic
rate declines and O2 supply becomes adequate. Therefore, in the current study, the time
course of changes in resting metabolism was characterised.
5.2 Methods
The details of dissection of the preparations were given in Chapter 3. The following
sections describe methods specific to the experiments on resting metabolism.
In the experimental chamber, the clip at one end of the muscle was connected to a semi-
conductor force transducer (AE801, SensorOne, CA, USA) and the other end was held
in a clamp. Preparations were placed on the thermopile so that the section of ventricular
wall and the clip holding that end of the preparation were not on the recording region.
That end of the thermopile had thermocouples that were not connected to the recording
region to ensure that heat loss from the preparation was uniform along its length and
thus prevented the development of thermal gradients along the muscles length due to
varied heat loss through the thermocouples. Muscles were stimulated via two platinum
wire electrodes placed on either side of the preparation.
5.2.1 Heat measurements
The thermopile was enclosed in a glass chamber containing Krebs-Henseleit solution.
The chamber was submerged in a 40 L temperature-controlled water bath (F-10-HC,
Julabo Labortechnik, Germany) maintained at 27°C. The thermopile output was
proportional to the difference in temperature between the muscle and the frame of the
thermopile. The frame was kept at a constant temperature by contact with the
temperature-controlled reservoir. The rate of muscle heat output was calculated from the
difference in muscle temperature between that measured when the chamber was full of
solution (heat produced by the muscle dissipates into the solution so muscle temperature
equals the chamber temperature) and that measured when the solution was drained from
the chamber (air is a poor conductor of heat so muscle temperature increases until rate
of heat loss through the thermocouples to the frame equals the rate of heat production).
50
The chamber was continuously aerated with 95% O2–5% CO2 that had been thermally
equilibrated and saturated with water vapour.
5.2.2 Calculation of rate of resting heat production
Resting metabolic rate was defined as the rate measured when the muscle was
mechanically quiescent and had been so long enough for metabolic activity associated
with any preceding contractile activity to have ceased. In preliminary experiments, it
was found that after a series of contractions active metabolic rate decreased with a time
constant of ∼15 s at 27°C. Therefore, contractile activity must have ended at least 5 × 15
= 75 s prior to measurement of resting metabolism. In the current study, muscles
performed a brief series of twitches, to measure contractile force, after each
measurement of resting metabolism and the muscle was then quiescent for 10 min
before the next measurement.
The rate of heat production of the resting muscles (AR) was calculated from the change
in thermopile output (∆V, corrected for amplifier gain) that occurred upon draining the
solution from the muscle chamber using the following formula (Hill, 1928).
R
VkCA
nα
∆= (25)
where n is the number of thermocouples, α the Seebeck coefficient (µV °C-1
couple-1
), k
the rate of heat loss from the muscle (s-1
) and C the heat capacity of the muscle and any
adhering saline (mJ °C-1
). C and k were determined from the time course of cooling
following heating of the muscle using the Peltier effect (Kretzschmar & Wilkie, 1972).
To check the resolution of the method, a series of measurements was made using a dead
papillary muscle (stored in ethanol for 24 h prior to measurements and then rehydrated
in saline). The “resting heat rate” was -0.1 ± 0.4 mW g-1
(mean ± standard deviation; 21
measurements). For comparison, the minimum measured resting heat rate for live
preparations was 8.4 ± 1.6 mW g-1
.
5.2.3 Measurement of myocyte sarcomere length by diffraction of laser light
The performance of the heart depends on the length of the sarcomeres (the basis of
Starling’s Law of the heart). The sarcomere is the functional contractile unit of the
muscle cell and consists of two opposing sets of thin filaments (composed of actin)
extending towards the middle of the sarcomere where they overlap with the thick
51
filament (myosin) (see Fig. 5.1). The varying optical density of regions of sarcomeres
allows muscles to act as a diffraction grating when monochromatic light is shone
through the fibres. The sarcomere length sets the width of the diffracting slits.
The equation for determining sarcomere length, SL, is:
2 LSL
X
λ= (26)
where λ is the wavelength of light (in this case 632.8 nm), L the distance from the
muscle fibre to the screen for measuring the diffraction pattern and X the distance
between the two first order diffraction bands.
In a series of preliminary experiments, muscle length was set to that giving a passive
force of 5 mN mm-2
. It has been shown that with this pre-load sarcomere length is ~2.1
µm (see Fig. 4 in ref. Stuyvers et al., 2002). The muscles were then fixed in ethanol
(Josephson & Stokes, 1994). After 24 h, the muscles were transferred to glycerol, small
bundles of myocytes were teased free and their sarcomere length measured using the
diffraction pattern formed when laser light was shone through the myocytes. The mean
sarcomere length was 2.12 ± 0.03 µm (mean ± SEM; n = 5 preparations). Subsequently,
at the start of each experiment, muscles were stretched until the passive force was ~5
mN mm-2
.
Fig. 5.1. Example of myocyte sarcomere. The thin filaments are composed of actin and the associated proteins troponin
and tropomyosin. Actin is attached to the Z-lines at either end of the sarcomere
and the thin filaments extend in towards the middle of the sarcomere where they
overlap with thick filaments. The latter are mainly composed of the contractile
protein myosin. The area consisting of thin filaments is called the I-band and the
region where the thin filaments overlap with the thick filaments is referred to as
the A-band. Finally, the remaining area with only thick filaments is named the
H-band. Illustration from Huxley (1957).
thick
filaments
thin
filaments
52
5.2.4 Experimental protocols
The thermopile system required 15 min for thermal stabilisation before the first
measurement of resting metabolism could be made. Measurements were then made at
10 min intervals for 40 min. In experiments in which the effect of metabolic substrate
(glucose or pyruvate) on resting metabolism was determined, the protocol was
performed as described above using one substrate and, 40 min after the first
measurement, the solution was changed to one containing the other substrate and the
measurement protocol was repeated. The order of presentation of the substrates was
alternated in successive experiments.
Sucrose was used to study the effect of hyperosmolarity on resting metabolism. For
these experiments, three measurements of resting heat rate were made in the standard
Krebs solution (∼150 mOsM) and then that solution was replaced with Krebs containing
300 mOsM sucrose in addition to the normal Krebs for the following three
measurements. Thus, the solution osmolarity went from 150 mOsM to 450 mOsM. The
solution was then changed back to the standard Krebs for three more measurements (see
Fig. 5.5).
5.2.5 Analysis of diffusive O2 supply
The radius to which O2 can diffuse (the critical radius, RC) was calculated using the
equation derived by Hill (1928; 1965). For consideration of steady-state O2
consumption, Eq (23) simplifies to the following:
04C
R
KYR
A= (27)
where K is the diffusivity of O2 in muscle (cm2 atm
-1 min
-1), Yo is the partial pressure
of O2 (PO2) at the muscle surface (atm) and AR is the rate of O2 consumption of the
resting muscle (cm3 min
-1 g
-1). In this model it is assumed that the rate of O2
consumption is constant. K is 2.37 × 10-5
cm2 atm
-1 min
-1 at 22.8°C (Mahler et al.,
1985) and was adjusted to 27°C using a Q10 of 1.06 (Mahler et al., 1985), giving a value
of 2.43 × 10-5
cm2 atm
-1 min
-1. The PO2 in the chamber solution was measured using an
O2-sensitive microelectrode (OX500, Unisense, Aarhus, Denmark) and was 0.95 atm.
Rates of resting metabolism were converted to rates of O2 consumption using an
energetic equivalent of 19 mJ µL-1
(see Section 2.1.2).
53
5.2.6 Statistical analysis
The statistical significance of variations in metabolic rate with muscle mass was
analysed using a one-way analysis of variance. Comparisons of substrates and the effect
of hyperosmolarity were made using a paired Student’s t-test. Decisions concerning
statistical significance were made at the 95% level of confidence.
5.3 Results
5.3.1 Resting metabolic rate depended on time and muscle mass
The rate of heat output from resting papillary muscles decreased with time during an
experiment (Fig. 5.2). The records in Fig. 5.2 show thermopile records from one muscle
made at 10 min intervals. The amplitude of the record is proportional to resting heat
rate. The rate of heat production declined with time and the difference between
successive 10 min intervals decreased as the experiment progressed.
The time course of the decline could be well described by a single exponential function
with an asymptote approximating the eventual steady value (AR(∞), Equation (28)).
-( ) [ (0) - ( )] ( )tR R R RA t A A e Aτ= ∞ + ∞ (28)
where AR(0) was the first value measured in an experiment (i.e. 15 min after placing the
muscle in the chamber) and τ was the time constant. The mean time constant was 18 ± 2
min (n = 13) so that ~5 × 18 = 90 min were required before AR attained a steady value.
Fig. 5.3 illustrates that AR did eventually stop declining and become fairly steady.
Despite the changes in AR, there was no significant effect of time on isometric force
production during the time course of the experiments.
54
The absolute values of AR(t), for a given value of t, depended on muscle mass, being
greater for small muscles than large muscles. To illustrate this, muscles were sorted
according to mass and mean values calculated for each group (Table 5.1 and Fig. 5.4).
The mean resting metabolic rate of muscles with mass <1 mg (mean mass, 0.74 ± 0.03
mg; n = 3) was 47 ± 11 mW g-1
compared to a rate of 14 ± 2 mW g-1
for muscles >2 mg
(mean mass, 2.6 ± 0.2 mg; n = 4). There was no significant difference in the time
constant for decline in rate among the groups and the slope of a line through a plot of
the individual data as a function of muscle radius did not differ significantly from zero
(r2 = 0.04, n = 13).
On eight occasions, two papillary muscles from the same heart were tested. In those
cases, the second muscle was left attached to the ventricular wall of the isolated heart
for ~2 h, exposed to oxygenated Krebs, before it was transferred to the experimental
chamber. The initial values of AR for the second muscles were appropriate for their
mass, as judged by data from muscles that were tested shortly after excision of the heart,
and AR declined with the same time course as for the other muscles.
0
10
20
30
40R
ate
of
he
at
ou
tpu
t (µ
J s
-1)
25
15
55
7565
45
35
1 s
A.
0 20 40 60 80
Time (min)
0
6
12
18 Re
stin
g m
eta
bo
lism
(mW
g-1)
B.
Fig. 5.2. Example of decline in resting heat rate with time during an
experiment. A. Thermopile records from one papillary muscle (mass, 2.06 mg; length, 4.6
mm) at different times during an experiment. The time (in min) at which each
recording was made is given beside the record. The sections of records on the
left are the baseline measurements made while the muscle was immersed in
solution. The sections on the right were made after the solution had been
drained. B. Resting metabolic rate, normalised by muscle mass () taken from
the records in A. An exponential curve (solid line) was fitted through the points
by least squares regression. Time constant for this example was 28 min and the
estimated steady value was 3.4 mW g-1
.
55
Table 5.1. Characteristics of mouse papillary muscles.
<1 mg 1–2 mg >2 mg
Number of muscles 3 6 4
Wet mass (mg) 0.74 ± 0.03 1.5 ± 0.1 2.6 ± 0.2
Length (mm) 2.8 ± 0.3 3.8 ± 0.2 4.2 ± 0.4
Cross-sectional area (mm2) 0.26 ± 0.02 0.39 ± 0.04 0.61 ± 0.09
Radius (mm) 0.28 ± 0.01 0.35 ± 0.02 0.44 ± 0.03
Fig. 5.3. Exponential decline in resting
metabolism. Resting metabolic rate of a papillary muscle
(mass, 1.78 mg; length, 3.9 mm) measured at
10 min intervals. The fitted exponential curve
is shown by the solid line. The time constant
was 20 min and the steady value was 3.6 mW
g-1
.
0 50 100 150 200
Time (min)
0
5
10
15
20
Re
stin
g m
eta
bolis
m (
mW
g-1
)
Fig. 5.4. Effect of muscle mass on resting
metabolic rate. Muscles were divided according to wet mass
into three groups (<1mg, 1–2 mg, >2 mg) and
for each group, the mean resting metabolic rate
is plotted as a function of mean mass. The
number of muscles in each group is shown in
parentheses. Data are shown for the first
measurement made in an experiment (; solid
line) and for the estimated steady values (;
dashed line). The steady values were estimated
from the exponential curves fitted to the data
for each muscle (Fig. 5.2 and Fig. 5.3). The
symbols represent the mean values and the
error bars represent the SEM. The effect of
muscle mass on resting metabolic rate was
statistically significant.
0 1 2 3
Mass (mg)
0
10
20
30
40
50
60
Re
stin
g m
eta
bo
lism
(m
W g
-1)
AR(0)
AR(∞)
(3)
(6)
(4)
56
5.3.2 The effect of BDM on resting metabolic rate
To determine whether changes in either cross-bridge activity or Ca2+
cycling may have
contributed to the decline in rate with time, the resting metabolism of four muscles was
measured with 30 mM BDM in the Krebs solution. BDM at this concentration inhibits
both cross-bridge cycling and Ca2+
cycling (Backx et al., 1994). AR still declined
exponentially with a time constant of 17 ± 4 min (n = 4). Although the experiment was
not specifically designed to test whether the magnitude of AR at the start of an
experiment was affected by BDM, the mean value in the first measurement (9 ± 2 mW
g-1
; mean mass, 2.2 ± 0.6 mg) was comparable to that for muscles of similar mass
measured without BDM. After measuring AR in the presence of BDM for ~90 min, the
solution was changed to Krebs without BDM and, after a 15 min interval (sufficient for
mechanical activity to recover), AR was measured again. At that time, AR measured
without BDM present did not differ significantly from that measured previously with
BDM.
5.3.3 The effect of hyperosmolarity on resting metabolic rate
A characteristic of resting metabolism in striated muscles from other species is that it
increases with increasing osmolarity of the bathing solution (e.g. Loiselle et al., 1996).
To see whether this also applied to mouse papillary muscles, resting metabolism
measured in normal Krebs solution was compared to that measured in the presence of
the impermeant solute sucrose (300 mM ≡ 300 mOsmol L-1
, a 3-fold increase in
osmolarity). In the hyperosmotic solution, AR increased greatly (Fig. 5.5A) and active
force output (Fig. 5.5B) was almost abolished. The mean resting heat rate with sucrose
was 247 ± 20% (n = 4) of that in normal Krebs solution. Upon returning the muscle to
normal solution, both AR and force output returned to previous values within 15 min.
57
5.3.4 The effect of metabolic substrate on resting metabolism and force output
Two exogenous substrates are commonly used in experiments with isolated papillary
muscles: glucose and pyruvate. In all preparations studied (n = 10), resting metabolic
rate was higher with pyruvate than with glucose (Fig. 5.6). The mean difference (AR
with pyruvate–AR with glucose) was significant and was 2.8 ± 0.8 mW g-1
, which
corresponded to the value in pyruvate being 210 ± 50% of that in glucose. There was no
significant difference in the maximum isometric force output between the two substrates
(mean values: pyruvate, 20 ± 3 mN mm-2
; glucose, 19 ± 2 mN mm-2
(n = 9)).
Fig. 5.5. Example of the effect of
hyperosmolarity on resting heat
output and force production. A. The protocol used to determine the
effect of hyperosmolarity on resting
metabolism. The first three measurements
of resting metabolism () were made in
the presence of isosmotic saline (I). The
decline in rate was fitted with an
exponential curve (dashed line). Three
measurements were then made in the
hyperosmotic solution (time of exposure is
indicated by H and solid horizontal line;
resting heat rate, ), followed by another 4
in isosmotic solution. The magnitude of
the hyperosmotic stimulation of resting
metabolism (indicated by the arrow) was
determined by comparing the resting
metabolism to that predicted by
extrapolation of the exponential fitted
through the first three points (dashed line).
Hyperosmolarity caused a three-fold
increase in resting heat rate in this
example. B. Records of the time course of
isometric force production made before
(left), during (middle) and after (right)
exposure to the hyperosmolar solution.
Hyperosmolarity reversibly decreased
isometric force production.
0 20 40 60 80 100 120 140 160
Time (min)
0
2
4
6
8
Re
stin
g m
eta
bo
lism
(m
W g
-1)
A
H II
0
6
12
18
Fo
rce
ou
tpu
t (m
N m
m-2
)
500 ms
B
58
5.4 Discussion
The data described in this chapter are the first measurements of the metabolism of
mouse papillary muscles. The primary purpose of this study was to obtain data that
could be used to assess the adequacy of diffusive O2 supply. In addition, the
characterisation of resting metabolism of mouse papillary muscles provides some
further insight into the metabolism of in vitro preparations of cardiac muscle and these
will be discussed first.
5.4.1 Comparison with other studies
A prominent characteristic of the resting metabolism of papillary muscles is that it
declines with time (Figs. 5.2 and 5.3). This has been observed in papillary muscles from
cats and rats (Loiselle & Gibbs, 1979) but not those from guinea-pigs (Loiselle &
Gibbs, 1979; Daut & Elzinga, 1988). For papillary muscles from rats, the time constant
for the decline in resting heat rate is greater (~60 min at 27°C; Loiselle & Gibbs, 1979;
Loiselle, 1985b) than that for mouse papillary muscles (~20 min at 27°C). The cause of
the decline in resting metabolism with time remains obscure. It is not a reflection of a
dying muscle, because the mechanical performance does not alter with time, nor does it
represent an irreversible or only slowly reversible depression of metabolic capacity,
because the resting metabolism can be stimulated several-fold by changing substrate
from glucose to pyruvate and increasing the osmolarity of the bathing solution (Fig.
5.5). The decline is related to isolation of the muscles, since it probably does not occur
Fig. 5.6. The effect of glucose and
pyruvate on the resting metabolic
rate of mouse papillary muscle. Resting metabolic rate measured in the
presence of either 10 mM glucose or 10
mM pyruvate. Values in glucose are
indicated by symbols on the left and those
in pyruvate by symbols on the right. The
lines join values from the same muscle.
The measurements were made after 1 hour
in vitro and the order of presentation of
substrates was alternated to negate time-
dependent effects.
0
5
10
15
Re
stin
g m
eta
bo
lism
(m
W g
-1)
Glucose Pyruvate
59
in isolated hearts (for a detailed discussion, see Gibbs & Loiselle, 2001). Gibbs &
Loiselle (2001) have suggested that the decline in resting metabolism may arise from
accumulation of an inhibitory endothelial factor that accumulates in the absence of
microvascular circulation. Our data suggest that the rate does not decline from the time
the heart is excised, as previously suggested (Loiselle & Gibbs, 1979; Loiselle, 1985b;
Gibbs & Loiselle, 2001) because in our experiments the resting metabolism was the
same whether muscles were tested within 20 min of excision of the heart or had
remained in the open, superfused ventricle for two hours before dissection. This
suggests that the decline in metabolic rate is initiated by dissection of the papillary
muscle from the ventricle. This raises the possibility that the resting metabolic heat rate
is high initially due to dissection damage, as has been described for skeletal muscle
(Biron et al., 1979). The only part of the papillary muscles that necessarily incurred
damage during dissection was the end that attached to the ventricular wall (one of the
clips holding the preparation was crimped onto a piece of ventricular wall muscle) but
heat output from that section of the preparation was not included in our recordings. It
has also been suggested that the first values measured (i.e. AR(0)) may be high due to
waves of contractile activity (Denis Loiselle, personal communication). However, there
was no visual evidence of writhing in these experiments and treatment with BDM did
not affect metabolic rate. Thus, it is unlikely that the high metabolic rate recorded at the
start of an experiment was related to dissection damage. However, the cause of the
change in resting metabolic rate with time in isolated papillary muscles remains unclear.
It is of interest to know how resting metabolic rates measured in papillary muscles
compare to those of intact mouse hearts. The resting metabolism of mouse hearts can be
estimated from published data for energy cost as a function of pressure−volume area
(PVA). The y-intercept of these relationships (i.e. the energy cost corresponding to 0
PVA is the sum of resting heat rate and metabolism associated with excitation-
contraction coupling (or activation metabolism). There are two reports that provide the
data required to estimate resting metabolic rate in mouse hearts.
60
It was assumed that ∼19% (see Section 6.3.6) of the force-dependent metabolism
corresponds to activation metabolism and that the remainder is associated with resting
metabolism. Using data from How et al. (2005), the resting metabolism was predicted to
be ∼20 mW g-1
when supplied with medium containing a low fatty acid concentration
(Fig. 5.7A). Kameyama et al. (1998), on the other hand, showed with their perfused
mouse hearts a very high y-intercept on the PVA graphs, corresponding to a resting
metabolism of ∼60 mW g-1
(Fig. 5.7B). These hearts were perfused with a buffer
containing pyruvate. In the current study, it was shown that pyruvate doubled the resting
metabolic rate of papillary muscles. If this is also the case with mouse hearts, this may
account for some of the difference in resting heart rate between these two studies. In
summary, these mouse heart studies suggest that resting metabolic rate of hearts is
higher than the steady values for papillary muscles but lower than the highest values
recorded for papillary muscles. However, it should be noted that neither of the heart
studies appeared to be aware of the potential for trans-epicardial O2 exchange in isolated
hearts (Loiselle, 1989; Gibbs & Loiselle, 2001). This has potential to cause substantial
0.000 0.005 0.010 0.015 0.020
Pressure-volume area (J beat-1 g dry wt-1)
0.0
0.5
1.0
1.5
2.0O
2 c
on
su
mp
tio
n (
ml O
2 b
ea
t-1 g
-1)
10-3A.
0 5 10 15 20
Pressure-volume area (mmHg ml g-1)
0.0
0.5
1.0
1.5
2.0
Activation metabolism
Resting metabolism
Pressure-dependent
Pressure-independent
10-3B.
Fig. 5.7. Prediction of in vivo resting metabolism. Schematic diagrams of published relationships between O2 consumption and
pressure−volume area in the mouse heart. The O2 consumption is equivalent to
the total energy cost. The energy cost of the heart is made up of two components,
pressure-dependent and pressure-independent, or activation, metabolism. The y-
intercept indicates the in vivo energetic requirements of pressure-independent
metabolism. This is in turn made up of two components, resting and activation
metabolism. From the relationships shown the contribution of resting
metabolism was predicted to be ∼20 mW g-1
(A) by How et al. (2005) and ∼60
mW g-1
(B) by Kameyama et al. (1998).
61
errors in calculated rates of O2 consumption. Therefore, it is difficult to be sure exactly
how mouse papillary muscle data compares with that for isolated mouse hearts.
The inverse relationship between muscle size and resting metabolism has been
described previously for rabbit papillary muscles (Loiselle & Gibbs, 1983), although in
the current study the magnitude (both absolute and relative) of this effect was greater
than that for rabbit muscles. An obvious explanation for such an effect is that larger
muscles are unable to get enough O2 by diffusion to support their metabolism, so an
anoxic core develops. However, this explanation has been shown to be incorrect, by
both modelling and experiment (Loiselle & Gibbs, 1983; Loiselle, 1985a; Loiselle,
1987). In fact, as will be demonstrated in the following section, it was the small
preparations used in the current study that were most likely to be O2 diffusion limited.
5.4.2 Adequacy of diffusive oxygen supply
The main purpose of these experiments was to obtain data to enable an assessment of
the adequacy of diffusive O2 supply to meet the metabolic requirements of resting
mouse papillary muscles. If the metabolic rate of an isolated muscle is high compared to
the rate at which O2 diffuses into the muscle, an anoxic region may develop in the core
of the muscle. Furthermore, if an anoxic region subsequently becomes oxygenated, for
example as metabolic rate declines with time, then the cellular characteristics associated
with ischaemia-reperfusion damage (for a review, see Bolli & Marban, 1999) may
become apparent.
The results of the analysis of diffusive O2 supply are illustrated in Fig. 5.8 in which the
resting metabolic rates of the muscles used in this study are plotted as a function of
muscle radius. Data are shown both for the first values measured (Fig. 5.8A) and for the
estimated steady values (Fig. 5.8B). The curved lines indicate the calculated critical
radius (RC; Equation (27)) for each combination of radius and metabolic rate. For
muscles lying to the right of these lines, O2 diffusion would have been inadequate to
meet the O2 demand. For the metabolic rates measured early in an experiment, the
calculations indicate that six muscles may have had inadequate O2 supply (Fig. 5.8A).
Three of these were muscles with masses below 0.8 mg. Once resting metabolic rate had
declined to its steady value, all the muscles used were below the critical radius for their
metabolic rate.
62
This analysis illustrates that despite the small size of the mouse papillary muscles, at
early times in an experiment their metabolic rate can be sufficiently high that formation
of an anoxic core is likely and, in an apparent paradox, this is particularly likely to occur
for the smallest muscles because they have high metabolic rates. Furthermore, because
the resting metabolic rate is doubled if pyruvate is used as a substrate, achieving
adequate oxygenation would be more difficult using that substrate; doubling metabolic
rate reduces RC by a factor of ≈ 0.7.
Experimental temperature also has an important effect on diffusive O2 supply with
lower temperatures favouring O2 supply because resting metabolism is more
temperature sensitive (Q10 ~1.3; Loiselle & Gibbs, 1983; Loiselle, 1985c) than O2
diffusivity (Q10 = 1.06; Mahler et al., 1985). Some mouse papillary muscle experiments
have been performed at 37°C (He et al., 1997; Meyer et al., 1999; Bluhm et al., 2000).
At that temperature, metabolic rate would be 1.3-times greater than the values in Fig.
5.8 and the critical radius would be decreased by a factor of 1.06 1.3 = 0.9. The
metabolic rate of contracting mouse papillary muscles has not yet been measured but
contractile activity at physiological frequencies might be expected to double metabolic
0.0 0.2 0.4 0.6
Radius (mm)
0
20
40
60
80
100R
estin
g m
eta
bo
lism
(m
W g
-1)
A.
0.0 0.2 0.4 0.6
Radius (mm)
0
20
40
60
80
100
B.
Fig. 5.8. Adequacy of diffusive O2 supply to resting mouse papillary
muscle. The resting metabolic rates measured 15 min after placing the muscles in the
chamber (A) and the estimated steady value (B) plotted as a function of muscle
radius. Muscle radii were calculated from the average cross-sectional area for
each muscle. Data are shown for muscles with glucose () and pyruvate () as
substrate. The curved lines are the critical radii for diffusive O2 supply at 27°C;
to the left of the curves, muscles are adequately oxygenated and to the right, O2
supply would have been inadequate to maintain PO2>0 throughout the muscle
cross-section. Critical radii were calculated using Equation (27).
63
rate (Gibbs & Loiselle, 2001), decreasing the critical radius by an additional factor of
0.7. If we consider a mid-range papillary muscle (mass ~1.5 mg; radius 0.35 mm), at
27°C it has an eventual steady AR of ~4.5 mW g-1
and RC would be 0.8 mm (RC>actual
radius implies adequate O2 supply). Two hours after dissection, at 37°C, contracting and
with pyruvate as substrate, total metabolic rate could be ~18 mW g-1
and RC would be
reduced to 0.4 mm but still greater than the critical radius. However, under those
conditions RC would be <0.3 mm, favouring anoxic core formation, throughout the first
30 min in vitro. These calculations emphasise that the adequacy of diffusive O2 supply
cannot be taken for granted simply because the preparation is small.
5.5 Recommendations for performing experiments with isolated papillary muscles
In summary, even for quiescent mouse papillary muscles it is possible that diffusive O2
supply is inadequate, especially shortly after removal of the muscle from the heart. On
the basis of the data obtained in this study we would recommend the following
procedures to minimise the chances of O2 supply becoming inadequate: (1) avoid using
very small papillary muscles (<1 mg); (2) use glucose rather than pyruvate as substrate;
(3) use a temperature lower than 37°C; (4) avoid or minimise contractile activity during
the equilibration period after dissecting the muscle. Implementing recommendations
(2)–(4) just during the time that AR is high (the first 40–60 min after dissection) would
also minimise the chances of developing anoxia early in an experiment.
64
65
CChhaapptteerr 66:: CChhaarraacctteerriissaattiioonn ooff aaccttiivvee
mmeettaabboolliissmm
This Chapter characterises the active metabolism of the mouse papillary muscle. The
energy requirements of a contracting muscle were examined using different frequencies
and in combination with various metabolic substrates and at different temperatures.
Muscle efficiency was studied using a realistic range of shortening velocities. Finally,
the number of cross-bridges that are activated in a cardiac twitch was calculated.
6.1 Introduction
The basic contractile event in striated muscle is the twitch, which is the response of a
muscle or fibre to a single neural or electrical stimulus. Given its fundamental nature, it
is of interest to characterise the molecular events that underlie the twitch. Under both
physiological and typical laboratory conditions peak twitch force is less than the
maximum force that a muscle or fibre can produce. Support for this idea comes from the
observations that skeletal muscle twitch force is less than tetanic force and that cardiac
twitch force can be increased substantially by inotropic agents and changes in
experimental conditions (e.g. cooling) that increase intracellular [Ca2+
] (for a review,
see Endoh, 2004). Because twitch force is sub-maximal, it is unlikely that the force
output reflects the simultaneous action of all the available myosin cross-bridges.
66
One purpose of this study was to quantify the fraction of cross-bridges that cycle during
a cardiac twitch. This has not been determined before. Under most conditions, each
force-generating interaction between a myosin cross-bridge and an adjacent actin
filament is associated with the hydrolysis of one ATP molecule. Therefore, one way of
counting the number of cross-bridge cycles that occur during a contraction is to measure
the number of ATP molecules used; that is, to measure the energy cost of the
contraction. In the current study, the fraction of cycling cross-bridges was estimated
from measurements of the energy cost of contraction using isolated papillary muscles
taken from the left ventricle of the mouse heart. Energy cost was determined using the
myothermic technique, which has the chemical and temporal resolution to accurately
monitor the biochemical changes occurring within a brief twitch (Woledge, 1998). In
addition, with this technique it is possible to separate energy used by the cross-bridges
from energy used by other cellular ATPases (i.e. those associated with pumping ions
across membranes) (Gibbs et al., 1988; Alpert et al., 1989). Mouse cardiac muscle was
used because the advent of heart-focussed genetic manipulations (e.g. Headrick et al.,
1998; Bluhm et al., 2000) has made it important to develop experimental techniques and
protocols that can be used to probe the basic physiology of cardiac muscle in this
species. For example, although in a number of studies the intracellular free Ca2+
and
Ca2+
content of the sarcoplasmic reticulum (SR) of mouse cardiac muscle have been
measured (Gao et al., 1998; Georgakopoulos & Kass, 2001; Stull et al., 2002), in no
cases has the amount of Ca2+
released been quantified.
This study is the first in which the energetics of contracting, isolated mouse cardiac
muscle have been measured. It is important to ensure that diffusive O2 supply is
adequate to meet the needs of isolated muscles so the metabolic data obtained in the
current study was combined with a theoretical model of diffusive O2 supply (Barclay,
2005) to confirm that the muscles used were small enough to maintain a favourable
balance between O2 supply and consumption.
6.2 Methods
The materials and methods used for the experiments reported in this chapter have been
described in Chapter 3. Only those methods specific to these experiments will be
described here. Muscle characteristics are listed in Table 6.1.
67
6.2.1 Mechanical output
The time course of a twitch was followed and the time taken to reach peak force
calculated. The time to peak force output reflects how quickly cross-bridges attach and
generate force, which allows comparison between muscles that produce different peak
forces under different conditions.
6.2.2 Contribution of recovery heat to initial heat measurements
As described in the Methods (Section 3.4), initial enthalpy output was calculated by
measuring the enthalpy produced over the first three contraction cycles. As for the rat
papillary muscle (Section 4.2.1), an estimate was made of how much recovery heat
might have been included in the measured enthalpy over the first three cycles. The R/I
for mouse papillary muscles contracting at 2 Hz was 1.2 (see Section 6.3.5) and was
independent of muscle mass. The steady state recovery heat output was estimated using
that R/I and assuming that in an energetic steady state this amount of recovery heat
would be produced within each contraction cycle (i.e. in 0.5 s when contracting at 2
Hz).
At a contraction frequency of 2 Hz, the initial enthalpy output was ~2.3 mJ g-1
twitch-1
so R,SSQg
= (1.2 × 2.3) / 0.5 = 5.5 mW g-1
. Substituting this value into Equation (18), the
amounts of recovery heat produced between the start of recording and the ends of the
first, second and third contraction cycles were 0.028, 0.11, and 0.25 mJ g-1
, respectively,
which was 1.2, 2.4 and 3.6% of the total heat produced by the end of those cycles (Fig.
3.6B).
6.2.3 Experimental protocols
Once in the experimental chamber, muscle length was adjusted to give a passive force
of 5 mN mm-2
; this length was designated L0. It has previously been shown that this
passive force corresponds to a sarcomere length of ~2.1 µm in these muscles (see
Table 6.1. Characteristics of mouse papillary muscles.
Number of muscles 24
Wet mass (mg) 1.7 ± 0.1
Length (mm) 3.4 ± 0.1
Cross-sectional area (mm2) 0.47 ± 0.03
Radius (mm) 0.38 ± 0.01
68
Section 5.2.3). Muscles were then allowed to equilibrate for 60 min. When first
dissected, the resting metabolism of isolated papillary muscles is high and central
anoxia can develop (see Fig. 5.8). To minimise the possibility of this occurring, muscles
were not stimulated during the equilibration period. After 60 min, the muscles were
stimulated at 0.2 Hz for 10 min. For 5 min prior to making heat measurements, muscles
were not stimulated to allow metabolic rate to decrease to its resting value. This
provided the thermal baseline for heat measurements.
To investigate the effects of contraction frequency on number of cross-bridge cycles in
a twitch, an isometric contraction protocol was used; the protocol consisted of 20 s of
contractions at frequencies of 1, 2, 3 and 4 contractions s-1
.
The effect of metabolic substrate (glucose or pyruvate) on isometric force production
and enthalpy output was first measured using one substrate. After the last measurement
in that substrate the solution was changed to one containing the other substrate and
thirty min were allowed for stabilisation in the substrate before the first measurement
took place and the measurement protocol repeated. Muscles were allowed to equilibrate
in each substrate and then force and enthalpy output were measured using the
abovementioned frequencies. The order of presentation of the substrates was alternated
in successive experiments.
The effect of temperature (22, 27 and 37°C) was also investigated using 40 twitches at a
contraction frequency of 2 Hz.
To investigate the effects of shortening on energetics, a protocol that incorporated a
cyclic strain pattern in each twitch was used (Mellors & Barclay, 2001). The strain
pattern was designed to mimic strains experienced by papillary muscles in vivo
(Semafuko & Bowie, 1975). The protocol consisted of a 60 ms isometric phase after
delivery of the stimulus pulse, isovelocity shortening with an amplitude of 0.15L0 s-1
,
followed by isovelocity lengthening back to L0 (see Fig. 4.1). The velocity of the
shortening phase ranged between 1 and 5 mm s-1
(~0.4 to 1.7 L0 s-1
). These velocities
were calculated by dividing the strain amplitude by the reported duration of the ejection
phase in isolated, working mouse hearts (Larsen et al., 1999). To accommodate the sub-
physiological temperature used in this study, it was assumed that the Q10 for shortening
velocity (i.e. change in velocity for a 10°C temperature change) was 2. This particular
strain pattern was used because it was found in preliminary experiments to maximise
work output at 2 Hz.
69
6.2.4 Calculation of number of cross-bridge cycles
The number of cross-bridge cycles that occurred in a twitch was estimated from the
amount of ATP used and assuming that each cross-bridge cycle was associated with
hydrolysis of one ATP molecule. It was further assumed that under the conditions used
in this study ATP splitting was fully buffered by the CK reaction so that the enthalpy
output arises from the net breakdown of PCr. The number of ATP molecules hydrolysed
(NATP; ATP g-1
twitch-1
) was calculated as follows.
(1 )I CB A
ATP
PCr
H f NN
H
∆ −=
∆ (29)
∆HI is the initial heat per twitch (mJ g-1
), (1−fCB) is the force-independent enthalpy
output expressed as a fraction of the initial enthalpy output, ∆HPCr is the molar enthalpy
change for PCr hydrolysis and NA is Avogadro’s constant. ∆HPCr was calculated from
the R/I ratio (Section 6.3.5), as described by Woledge and colleagues (p.219, Woledge
et al., 1985; Woledge & Reilly, 1988), and was 34 kJ mol-1
. To compare this value to
the number of cross-bridges present in mouse cardiac muscle, we scaled NATP to a
volume of muscle containing a precisely known number of cross-bridges, the unit
sarcomere rhombus. This volume contains a total of 600 cross-bridges (for calculation
see Appendix I). Then the number of ATP used can be related to the number of cross-
bridge cycles that occurred in this volume per twitch (NCB) as follows.
sCB ATP
M
VN N
V
ρ= (30)
where Vs is the volume of a sarcomere rhombus, ρ is the density of muscle (1.06 g cm-
3) and VM is the fraction of muscle volume occupied by myofibrils. The volume of a
sarcomere unit cell was calculated assuming that sarcomere length was 2.1 µm (see
Section 5.2.3) and that the spacing between the thick filaments was 41 nm (Yagi et al.,
2004), giving a sarcomere cell volume of 3.5 × 10-15
cm3 (see Appendix I). VM takes
account of the volume density of myofibrils in myocytes (0.52 in mouse myocytes;
Barth et al., 1992) and the fraction of muscle volume that is occupied by myocytes (0.79
in rat myocardium; Dobson & Cieslar, 1997); therefore, VM is 0.52 × 0.79 = 0.41 ×
muscle volume.
70
6.2.5 Analysis of diffusive O2 supply
The adequacy of diffusive O2 supply to papillary muscles was assessed as described in
Section 3.7. The value of PO2 at the muscle centre was calculated for 20 s of contractile
activity (as used in this study) and for contraction frequencies of 1, 2, 3 and 4 Hz (Fig.
6.1). PO2 at the muscle surface, measured using an O2-sensitive microelectrode (OX500,
Unisense, Aarhus, Denmark), was 0.84 atm (85 kPa). At rest, PO2 at the muscle centre
was calculated to be ~0.7 atm. Central PO2 decreased upon stimulation (Fig. 6.1).
However, even in the worst case (contraction frequency 4 Hz), at the end of the
contraction protocol central PO2 (minimum value ~4 kPa) would have remained greater
than the values at which mitochondrial respiration becomes compromised (~1.3 kPa;
Schenkman, 2001).
6.2.6 Statistical analysis
The statistical significance of variations in initial and net enthalpy use with contraction
frequency and shortening velocity was assessed using one-way analysis of variance
(ANOVA); for the contraction frequency data, a repeated measures one-way ANOVA
was used. The significance of variations in force production, measured at different times
during the contraction series, with contraction frequency were assessed using a 2-way
ANOVA. Where appropriate, post-hoc analyses were performed using Dunnett’s test for
comparisons with a control group (Hancock & Klockars, 1996). Decisions concerning
statistical significance were made at the 95% level of confidence.
71
6.3 Results
6.3.1 Force output of mouse papillary muscle
Metabolic substrate, pyruvate or glucose, had no effect on force or enthalpy output in
isometric contractions. Isometric force output decreased significantly with contraction
frequency. This analysis was performed using the average forces in each of the four 5 s
intervals in the contraction protocol. The effect of contraction frequency was
independent of the time interval over which force was averaged (Fig. 6.2). There was no
difference in peak force output between muscles in a bathing solution containing
glucose; 16 ± 2 mN mm2, and pyruvate; 15 ± 2 mN mm
2 (n = 11).
0 5 10 15 20
Time after start of contraction series (s)
0.0
0.2
0.4
0.6
0.8
PO
2 a
t m
uscle
ce
ntr
e (
atm
)
1 Hz
2 Hz
3 Hz
4 Hz
Fig. 6.1. Simulations of time course of PO2 at muscle centre during
contraction series. Simulations of the PO2 profile through muscle were made using Equation (23).
The diffusivity of O2 through muscle was taken to be 2.43 × 10-5
ml cm-1
min-1
atm-1
at 27°C (adjusted from value at 22.8°C using a Q10 of 1.06; Mahler et al.,
1985). Prior to the start of the contraction series, metabolic rate was assumed to
be constant and equal to the resting metabolic rate (∼5 mW g-1
). It was further
assumed that during the series of contractions the rate of O2 consumption
increased exponentially towards a steady value of 7 mJ g-1
twitch-1
with a time
constant equal to that for the decline in rate of heat output when contractile
activity ended (i.e. 12.2 ± 0.8 s, n = 11). Metabolic rates were converted to rates
of O2 consumption using an energetic equivalent of ∼19 mJ µL-1
, which was
calculated on the basis that the primary substrate for mitochondrial oxidation
was glucose for which the molar enthalpy is 2820 kJ mol-1
. Note, however, that
the calculated PO2 values are only ~2.5% smaller if it were assumed that fat
oxidation, yielding 17.3 mJ µL-1
, fuelled the contractions. The PO2 in the
solution surrounding the muscle was 0.84 atm. Muscle radius was taken to be
0.38 mm. Simulations were made for 20 s of contractile activity (as used in this
study) and for contraction frequencies of 1, 2, 3, and 4 Hz.
72
Fig. 6.2. Contraction frequency
dependence of normalised
isometric force output. Isometric force output is shown as a
function of contraction frequency. For
each muscle, the average force output
was calculated over 5 s intervals and
normalised by the average force over the
corresponding interval at 1 Hz. At all
frequencies the full protocol was of 20 s
duration. The symbols represent the
mean values of data from 11 muscles.
The data for different 5 s intervals are
distinguished as indicated by the key in
the Figure. Only at 4 Hz did the mean
values for all groups differ significantly
from those at 1 Hz (indicated by ∗).
0 1 2 3 4
Frequency (Hz)
0
20
40
60
80
100
Active
fo
rce
ou
tpu
t (%
)
0-5 s5-10 s10-15 s
15-20 s
6.3.2 Energy cost of a twitch and effects of contraction frequency, substrate and temperature
The energy cost of papillary muscle contraction was first assessed using a series of
isometric contractions at frequencies between 1 and 4 Hz. At a frequency of 1 Hz, the
initial enthalpy output, averaged over the first three contractions (Fig. 3.6B), was 3.3 ±
0.6 mJ g-1
twitch-1
and the net enthalpy output, from all the twitches, was 6.8 ± 1.1 mJ
g-1
twitch-1
(n = 11). Both the initial and net enthalpy outputs declined significantly with
increasing contraction frequency (Fig. 6.3). At a frequency of 4 Hz the mean initial
enthalpy output was 1.2 ± 0.2 mJ g-1
twitch-1
and the mean net enthalpy output was 3.9
± 0.6 mJ g-1
twitch-1
(n = 11). There was no significant difference in heat output
between the two substrates as the mean absolute heat output was 6.8 ± 1.1 mJ g-1
twitch-1
in glucose and 5.9 ± 1.2 mJ g-1
twitch-1
in pyruvate when stimulated at 1 Hz
(Table 6.2).
73
Increasing temperature from 22 to 27°C caused a reduction in peak twitch force and
enthalpy output and shortened the time taken for force to develop (Table 6.3). Two
muscles were also studied at 37°C and data from these preparations support the
conclusions from muscles studied at 22 and 27°C. At 37°C, twitch enthalpy output was
further reduced from 6.5 mJ g-1
twitch-1
(22°C) to 3.7 mJ g-1
twitch-1
. However, there
were no significant effects of temperature on rate of heat output or the time for recovery
heat rate to return to baseline (Table 6.3). The resulting Q10 values (where Q10 is the
magnitude of change in rate for 10°C change in temperature) of active rate of enthalpy
output were 0.78 between 22 and 27°C and 0.64 between 27 and 37°C (Table 6.3).
Fig. 6.3. Enthalpy output per twitch
in isometric contractions. The initial (grey bar) and net (black bar)
enthalpy output per twitch was measured
in isometric contractions. The heat output
of the papillary muscles was measured at
four different frequencies (1−4 Hz) using
glucose as substrate. The enthalpy output
per twitch decreased as twitch rate
increased. Asterisks (*) indicate statistical
significant difference between enthalpy
outputs of higher frequencies compared to
1 Hz.
0 1 2 3 4
Frequency (Hz)
0
2
4
6
8
En
tha
lpy o
utp
ut
(mJ g
-1 t
witch
-1)
Net enthalpy
Initial enthalpy
Table 6.2 Effect of glucose and pyruvate on enthalpy output (n = 11).
Contraction frequency
(Hz)
Glucose
(mJ g-1
twitch-1
)
Pyruvate
(mJ g-1
twitch-1
)
1 6.8 ± 1.1 5.9 ± 1.2
2 5.8 ± 0.8 6.3 ± 1.3
3 4.8 ± 0.7 4.6 ± 0.9
4 4.0 ± 0.6 5.1 ± 1.1
74
6.3.3 Effects of shortening on twitch energy cost
The effects of shortening velocity on energy cost were investigated using a contraction
frequency of 2 Hz with a period of shortening in each contraction at one of a range of
velocities. The shortening velocities used ranged from 0 (isometric) to 1.7 L0 s-1
and
enthalpy data were sorted into velocity bins, with bin width determined using Sturge’s
rule (no. bins = 1 + 3.322 × log(no. data points)). By plotting force output as a function
of change in muscle length a work-loop is formed. The work-loop has its three-
dimensional analogue in the pressure−volume diagram for the heart. Although the shape
of the work-loop changed with shortening velocity, there was only a marginal effect on
the work output (Fig. 6.4). Shortening velocity, in the range tested, had no significant
effect on either initial or net enthalpy output (Fig. 6.5). The net enthalpy output,
averaged across all velocities, was 5.6 ± 0.4 mJ g-1
twitch-1
and the corresponding initial
enthalpy output was 2.1 ± 0.2 mJ g-1
twitch-1
(n = 9).
Table 6.3. Characteristics of mechanical and energetic properties of
mouse papillary muscles performing isometric contractions (2 Hz) at
22, 27 and 37°C using glucose as a substrate.
22°C 27°C 37°C
Number of preparations 5 11 2
Active force (mN mm-2
) 20 ± 2 15 ± 2 14 ± 4
Twitch time (ms) 474 ± 5 381 ± 9 195 ± 27
Time to peak tension (ms) 253 ± 10 163 ± 2 95 ± 9
Enthalpy output (mJ g-1
twitch-1
) 6.5 ± 0.9 5.8 ± 0.8 3.70 ± 0.04
Time constant for recovery heat rate (s) 13 ± 2 13 ± 1 11 ± 2
75
Length (mm)
Fo
rce
(m
N)
0.53 L0 s-1
1.14 mJ g-1
0.79 L0 s-1
1.38 mJ g-1
1.05 L0 s-1
1.41 mJ g-1
1.32 L0 s-1
1.28 mJ g-1
1.58 L0 s-1
1.18 mJ g-1
Fig. 6.4. Effect of shortening velocity on work-loop. Work-loops produced by a mouse papillary muscle contracting at 2 Hz. In each
set of records the first stimulus pulse resulted in a large twitch force and the
second in much reduced force output. In the subsequent contractions there was a
gradual build-up of the force back towards the initial level. In this example the
shortening velocity varied from 0.5 to 1.6 L0 s-1
. Muscle mass: 1.57 mg; muscle
length: 3.8 mm.
0.0 0.5 1.0 1.5 2.0
Shortening velocity (L0 s-1)
0
2
4
6
8
En
tha
lpy o
utp
ut
(mJ g
-1 t
witch
-1)
Net enthalpy
Initial enthalpy
Fig. 6.5. Enthalpy output per twitch in shortening contractions. The initial (grey bar) and net (black bar) enthalpy output per twitch was
measured in contractions using a realistic contraction protocol. Enthalpy output
of a muscle shortening using a realistic contraction protocol. Heat and work were
expressed as a function of shortening velocity normalised to muscle length at a
realistic range of velocities.
76
6.3.4 Mechanical efficiency
There was no significant effect of shortening velocity on initial mechanical efficiency
(Fig. 6.6A). Peak initial efficiency was 31.1 ± 1.3%. Net mechanical efficiency includes
both the initial and recovery processes and there was significant effect of shortening
velocity on net mechanical efficiency (Fig. 6.6A). Peak net mechanical efficiency was
16.9 ± 1.5%. There was no significant effect of shortening velocity on the ratio between
net efficiency and initial efficiency (Fig. 6.6B). The mean ratio was 2.20 ± 0.09.
6.3.5 Ratio of recovery to initial enthalpy output
The ratio of recovery to initial enthalpy output (R/I) quantifies the coupling between
energy supply and energy demand and its value depends on the cost of recovery
metabolism relative to the cost associated with contractile activity. R/I was derived from
the initial and net mechanical efficiency (Equation (22)) and was 1.20 ± 0.09. This ratio
indicates the relative enthalpy change of values associated with PCr hydrolysis and its
0.0 0.5 1.0 1.5 2.0
Shortening velocity (L0 s-1)
0
5
10
15
20
25
30
35
Me
ch
an
ica
l e
ffic
ien
cy (
%)
ε initial
ε net
A.
0.0 0.5 1.0 1.5 2.0
Shortening velocity (L0 s-1)
0
1
2
3
4ε in
itia
l/εn
et
B.
Fig. 6.6. Initial and net mechanical efficiency. (A) Initial () and net () mechanical efficiency were expressed as functions of
shortening velocity normalised to muscle length. Peak efficiency was ∼31% and
net mechanical efficiency was ∼17%. There was a small but statistically
significant effect (∗) of shortening velocity on net mechanical efficiency. (B)
Variation in the ratio of initial to net mechanical efficiency with shortening
velocity. The mean ratio across all velocities was 2.2. There was no statistically
significant effect of shortening velocity on the ratio of initial and net mechanical
efficiency.
77
oxidative resynthesis. If ∆H for PCr hydrolysis is 34 kJ mol-1
(Woledge & Reilly,
1988), then ∆H for PCr resynthesis must be 1.2 × 34 = 41 kJ mol-1
.
6.3.6 Partitioning energy cost between force-dependent and force-independent components
Initial enthalpy output, measured over the first three contraction cycles, was partitioned
into force-dependent and force-independent components by selectively inhibiting force
output using BDM (Fig. 6.7). Force-independent enthalpy output accounted for 18.6 ±
1.9% (n = 7) of the initial enthalpy output.
0 20 40 60 80 100
FTI (%)
0
20
40
60
80
100
Initia
l h
ea
t (%
)
Fig. 6.7. Example of determining force-independent enthalpy output. Force-independent enthalpy output was determined from the relationship between initial
enthalpy output and force-time integral (FTI). FTI was varied by bathing muscles in Krebs
solution containing increasing concentrations of BDM. Initial enthalpy output is expressed
as a percentage of that measured in the absence of BDM. The lowest force-time integral was
obtained using a combination of 5 mM BDM and 150 mM sucrose. A line was fitted
through the data using the method of least squares; its equation is Relative initial heat = 0.82
× FTI + 13.6 (r2 = 0.97). The value of the y-intercept was used to quantify the force-
independent fraction of initial enthalpy output.
6.4 Discussion
The overall purpose of this part of the study was to characterise the active metabolism
of the mouse papillary muscle. The first point to note is that it was possible to make
reliable measurements of these preparations for the 90 min required to do these
experiments (i.e. 60 min equilibration and 30 min for measurements).
The peak isometric forces produced compared well with other reports of experiments
using left ventricular mouse papillary muscles for mechanical experiments (see Fig 5,
ref. Bluhm et al., 2000; Redel et al., 2002) but appears to be slightly lower than that
78
from papillary muscles from other species. For example, the peak isometric force for rat
muscles is typically between 30 and 50 mN mm-2
(Loiselle & Gibbs, 1979; Baxi et al.,
2000; Peterson et al., 2001). It is interesting to note, however, that there are several
published accounts of experiments with mouse papillary muscles which report very low
forces (eg ∼2−5 mN mm-2
) (He et al., 1997; Meyer et al., 1999; Bluhm et al., 2000;
Golenhofen et al., 2006) under similar conditions to those used in the current study. It
seems likely that the preparations in those studies were either not in good condition or
were incompletely stimulated.
6.4.1 Number of cross-bridge cycles per twitch
Using Equations (29) and (30), the number of ATP molecules split was calculated and
compared to the number of cross-bridges in the same volume of muscle. At 2 Hz and
contracting isometrically, it was calculated that 290 ATP molecules were used in the
unit sarcomere. There are 600 cross-bridges in that volume and if one cross-bridge cycle
involves splitting of one ATP, then the amount of ATP used is consistent with 48% of
the cross-bridges completing one ATP-splitting cycle in each twitch. These values can
also be expressed in µmol per g of muscle mass (i.e. taking into account non-
myofibrillar intracellular volume and extracellular volume). In that case, the amount of
ATP split is:
-1
23 -15
290 1ATP split = × 0.41 = 53 nmol g
6.023 × 10 3.5 × 10 × 1.06×
The cross-bridge concentration, worked out similarly, is 110 nmol g-1
.
The initial enthalpy measurements encompassed just the first three twitches so it is
possible that these calculations are not representative of energetics during more
prolonged activity. To see whether energy use averaged over 20 s can be accounted by a
similar number of ATP splitting cycles per twitch, the calculations were repeated using
the net enthalpy output produced in response to the complete 20 s protocol (i.e. 40
twitches when contraction frequency was 2 Hz). In that case, the denominator was the
molar enthalpy output associated with substrate oxidation, expressed per mol of ATP
formed. The enthalpy of glucose (the exogenous substrate provided in this study)
oxidation is 2820 kJ mol-1
(Crabtree & Nicholson, 1988). If this generates 38 ATP (i.e.
P/O2 ratio of 6.3) then glucose oxidation produces 2820/38 = 74 kJ per mole of ATP.
Substituting 5.6 mJ g-1
twitch-1
for ∆HI and 74 kJ mol-1
for ∆HPCr in Equation (29) gives
the number of ATP used per twitch per unit sarcomere cell of 336, which is equal to
79
56% of the number of cross-bridges. If the energy came from oxidation of endogenous
fat (which gives 76 kJ per mole of ATP) rather than glucose, then the number of ATP
used per twitch per unit sarcomere cell would be 328, which is equal to 55% of the
number of cross-bridges.
The simplest interpretation of this observation is that half the cross-bridges completed
one ATP-splitting cycle in each twitch. That only a fraction of cross-bridges complete
one ATP-splitting cycle has been suggested previously for rabbit papillary muscle (Mast
& Elzinga, 1990; see Discussion following Gibbs & Barclay, 1995) and could be
inferred from estimates of cross-bridge cycling rate for rat papillary muscle (e.g. Hoh et
al., 1988) but the current study is the first to attempt to quantify the fraction. It is
possible that fewer than half the cross-bridges completed more than one cycle, in which
case it might be expected that the number of cross-bridge cycles could be modulated by
events, such as shortening, occurring during the twitch. However, the lack of influence
of shortening velocity on energy cost is consistent with the idea that the amount of
energy to be used is determined early in a twitch (Gibbs & Barclay, 1995) and is not
greatly influenced by events that occur during the contraction. Two factors that strongly
influence twitch force and that are established at the start of a contraction are pre-load
and the amount of Ca2+
entering the cell, each of which, via different mechanisms (e.g.
Yagi et al., 2004), influences the number of cross-bridges that can bind. Contraction
frequency also influences the status of muscles at the start of the twitch by, for instance,
determining how much Ca2+
is available for release from the SR (e.g. Stull et al., 2002).
The enthalpy output observed at 4 Hz was ~60% of the value for 2 Hz, so the number of
cross-bridge ATP splitting cycles would have been reduced similarly. Consistent with
this the mean force output at 4 Hz was reduced by the same fraction as the enthalpy
output relative to that at 2 Hz.
The observation that, across a realistic range of shortening velocities, energy cost was
independent of shortening velocity is equivalent to stating that in a beating heart at
constant pre-load, varying stroke work does not alter energy cost. In terms of Suga’s
time-varying elastance model (for reviews, see Suga, 1990; Gibbs, 1995; Suga, 2003a),
the current papillary muscle protocol was equivalent to varying work output by altering
stroke volume (i.e. shortening) while maintaining a constant pressure−volume area
(PVA); rate of O2 consumption depends on PVA rather than upon stroke work (e.g.
Kameyama et al., 1998; Suga, 2003a; How et al., 2005). Equating energy cost to
80
number of cross-bridge cycles provides a mechanistic insight into this observation: the
number of cross-bridges cycles is not much altered by what the muscle does during the
twitch. Thus, sliding of the contractile filaments, at least when the sliding starts once
force has begun to develop (as in the current study), does not promote additional or
accelerated cross-bridge cycling, as occurs in skeletal muscle. Rather, the number of
cross-bridge cycles that will occur is set early in a twitch, presumably by the pre-load
and by the amount of Ca2+
released.
The twitch energy cost of mouse left ventricular papillary muscles was lower than that
for rat left papillary muscles (∼6 mJ g-1
twitch-1
in mouse (Table 6.2) versus ∼11 mJ g-1
twitch-1
in rat (Table 4.2)) stimulated at a contraction frequency of 2 Hz. In terms of the
analysis presented in this chapter this suggests that a greater fraction of cross-bridges
cycle in each twitch of rat papillary muscle compared to mouse. The number of cross-
bridge cycles in the rat preparations was about 70% of the total number of cross-bridges
in the muscle. ∆HPCr was calculated from the R/I ratio (1.1) and was 35 kJ mol-1
(Woledge & Reilly, 1988) and VM 0.61 in rat myocytes (Barth et al., 1992). It is
possible that the greater number of cross-bridge cycles estimated to occur in twitches of
rat muscles underlies the higher forces reported for these muscles as mentioned earlier
(Section 6.4).
6.4.2 Amount of Ca2+ released from the SR in each twitch
The approach taken to calculating NCB can also be applied to calculate the amount of
Ca2+
cycled through the SR of a mouse cardiac cell in each twitch. This was done by
modifying Equation (29) to take account of Ca2+
pump energetics and combining the
result with Equation (30).
2
2 sI A
CaPCr M
Vkf H NN
H V
ρ+
∆= ⋅
∆ (31)
where k is the fraction of the force-independent enthalpy output associated with Ca2+
pumping (assumed to be the same as in rabbit myocytes, 0.77; Delbridge et al., 1996)
and the factor of 2 reflects the stoichiometry of the SR Ca2+
pump (2 Ca2+
pumped for
each ATP hydrolysed). It should be noted that in mouse cardiac cells >90% of Ca2+
cycling is via the SR Ca2+
pump (Georgakopoulos & Kass, 2001). Substituting
appropriate values, again for the case for a muscle contracting at 2 Hz, gives a value of
98 Ca2+
released per twitch in the sarcomere cylinder volume, which, taking account of
the non-myofibrillar volume of muscle, is equivalent to 18 nmol g-1
twitch-1
. The SR
81
Ca2+
content in mouse myocytes contracting steadily at 0.5 Hz at 22°C has been
estimated to be 43 nmol (g muscle mass)-1
(Terracciano et al., 1998). Thus, the amount
estimated to be released in the current study is probably about half the SR Ca2+
content,
consistent with other estimates (e.g. Delbridge et al., 1996).
If there are 290 ATP-splitting cross-bridge cycles in the sarcomere unit volume in each
twitch and 98 Ca2+
are released into the same volume, then about three cross-bridge
cycles were completed for each Ca2+
released. Most Ca2+
released into muscle cells is
bound: peak free Ca2+
concentration (~0.5−1 nmol g-1
; Gao et al., 1998; Stull et al.,
2002) is <5% of the total Ca2+
released into the cell in each twitch (e.g. 18 nmol g-1
).
The concentration of troponin-C-tropomyosin regulatory units (each of which binds 1
Ca2+
in cardiac muscle) is 25% of the cross-bridge concentration (for a review, see
Gordon et al., 2000), which would be 0.25 × 110 = 28 nmol g-1
. If it were assumed that
all the 18 nmol g-1
Ca2+
released in a twitch were bound to troponin-C, then for mouse
muscle contracting at 2 Hz and 27°C about two-thirds of the regulatory units would
have been occupied by Ca2+
.
The dependence of the number of Ca2+
released per twitch on the assumed magnitude of
the force-independent enthalpy output is shown in Fig. 6.8. The greater the fraction of
enthalpy output that is independent of force generation, the greater the number of Ca2+
ions released. If it were assumed that under physiological conditions, the maximum
amount of Ca2+
that was released into the cell was just sufficient to saturate the
troponin-C Ca2+
binding sites, then this would correspond to 28 nmol Ca2+
g-1
twitch-1
or 150 Ca2+
per sarcomere cylinder. From Fig. 6.8, it can be seen that this would be
consistent with a relative force-independent enthalpy output of ~25%. This would then
be the maximum relative force-independent enthalpy output, which supports the idea
that estimates that were substantially higher than this value may have been in error.
Also shown in Fig. 6.8 is the variation in number of ATP molecules split with
magnitude of force-independent enthalpy output. This number decreases as force-
independent enthalpy output increases but, over the likely range of force-independent
enthalpy output, it is always greater than the number of Ca2+
ions released.
82
Fig. 6.8. Dependence of Ca2+
released and ATP used on
magnitude of force-independent
enthalpy output. The number of Ca
2+ released into a
sarcomere cylinder or the number of ATP
molecules hydrolysed in the sarcomere
cylinder volume per twitch is shown as a
function of the magnitude of the force-
independent enthalpy output (expressed
relative to the total initial enthalpy
output). The horizontal dashed line
corresponds to the number of troponin-C
Ca2+
binding sites in a sarcomere cylinder
in cardiac muscle (i.e. 150 or one-quarter
of the number of cross-bridges; Gordon et
al., 2000). The vertical dotted line is the
relative force-independent enthalpy output
measured in the current study.
0 10 20 30 40 50
Force-independent enthalpy output (%)
0
100
200
300
400
Nu
mb
er
of
mo
lecu
les
ATP
Ca2+
6.4.3 Partitioning of energy between force-dependent and force-independent components
The calculation of the number of ATP molecules split depends on the partitioning of
energy between cross-bridge and non-cross-bridge processes. In this study, it was
assumed that BDM inhibited cross-bridge cycling without affecting Ca2+
cycling (Alpert
et al., 1989; Higashiyama et al., 1994). Alpert et al. (1989) described a comprehensive
set of experiments, using rabbit papillary muscles, that were designed to establish
whether BDM selectively inhibited cross-bridge cycling and those experiments
supported the notion that this was correct. In contrast, the effects of BDM on the
relationship between rate of O2 consumption and pressure−volume area (PVA) for
blood-perfused dog hearts have been interpreted as indicating that BDM acted primarily
by reducing Ca2+
release (Takasago et al., 1997). If that also applied to the papillary
muscles used in this study, then force-time integral and enthalpy output would decline
in proportion so the enthalpy-FTI relation would pass through the origin; this was not so
(Fig. 6.7). It remains possible that the linear relationship between enthalpy output and
FTI reflects a partial inhibitory effect on Ca2+
cycling as well as a direct effect on cross-
bridge cycling; in that case, our estimate of force-independent enthalpy output
represents a lower limit.
Schramm et al. (1994) also used BDM (1−10 mM) to measure force-independent
enthalpy output of guinea pig trabeculae at 37°C. They found that force-independent
83
enthalpy output was 23% of the total energy cost. Gibbs and colleagues (Gibbs et al.,
1988; Kiriazis & Gibbs, 2001) used a different technique for measuring force-
independent enthalpy output: force output was reduced by rapidly shortening muscles
during the latent period between the delivery of the stimulus and the start of the
mechanical response. Using this technique, force-independent enthalpy output was
calculated to account for 25 to 30% of energy cost in papillary muscles from the rabbit
(Gibbs et al., 1988) and rat (Kiriazis & Gibbs, 2001). The cause of the discrepancy in
values from the latency release method and the BDM method is unclear, although it has
been suggested that in the former residual cross-bridge cycling may occur, increasing
estimates of force-independent enthalpy output (Alpert et al., 1989). Further insight into
the force-independent enthalpy output can be gained from the estimated amount of Ca2+
released from the SR in each twitch.
6.4.4 Mitochondrial efficiency
The efficiency of mitochondrial oxidative phosphorylation was estimated as described
previously (Equation (21)). This equation uses the ratio εNet/εI which had a mean value
of 0.45 ± 0.02 (the inverse of the ratio of εI/εNet shown in Fig. 6.6B). Assuming ∆GATP is
59 kJ mol-1
(Table 2.1), ∆HPCr is 34 kJ mol-1
(Woledge & Reilly, 1988) and that the sole
source of uncertainty is the ratio of the efficiencies (a relative error of 0.02/0.45 = 0.04),
then ηR for mouse papillary muscle would be 0.81 ± 0.03. That is, the free energy in
ATP produced in the mitochondria was 81% of the free energy available in the
metabolic substrate. Thus, if exogenous glucose were the sole substrate, with a molar
free energy change of 2878 kJ mol-1
, then the amount of free energy in ATP from one
mole of glucose is (81/100) × 2878 = 2331 kJ mol-1
. If ∆GATP = 59 kJ mol-1
, then each
mole of substrate oxidised produced 2331/59 = 39 moles of ATP. If the O2:substrate
ratio for glucose oxidation is 6, then the P:O2 ratio is 39/6 = 6.5 (± 0.3; i.e. a 4%
uncertainty). This compares favourably with the expected stoichiometry of 6.3 that
arises from production of 38 ATP from each mole of substrate oxidised.
6.5 Conclusion
In conclusion, this study has demonstrated that the energetics of isolated preparations of
mouse cardiac muscle can be satisfactorily determined using the myothermic technique.
By combining a number of approaches used in previous studies, we have calculated that
the energy required for a twitch under the conditions used in this study can be accounted
84
for by ATP-splitting cycles by half the available cross-bridges and cycling of about one
Ca2+
for every three cross-bridge cycles.
85
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In Chapter 4 alterations in the cellular energy balance in isolated rat papillary muscles
following a period of simulated ischaemia were studied. Before the start of the
ischaemia protocol, the muscles were allowed to equilibrate for 60 min to allow resting
metabolism to decrease to a level that would not compromise the balance between O2
use and supply. The ischaemia protocol consisted of an equilibration period lasting 15
min, followed by 60 min exposure to conditions mimicking ischaemia, before the
preparation was allowed to recover for 30 min. Thus, the minimum duration of an
experiment was 2 hours 45 min. Therefore, the first step for replicating a protocol of
this type with mouse papillary muscles was to demonstrate that muscles would remain
viable (i.e. keep producing force) and, preferably, maintain a relatively stable
mechanical and energetic performance for almost 3 h in vitro. This proved extremely
difficult to achieve. This chapter describes some of the attempts made to improve the
stability of the performance of mouse papillary muscles and concludes with an
assessment of the suitability of mouse papillary muscles studying various aspects of
cardiac function.
86
7.1 The observations
In many trials, using a protocol like that used for the control studies in the study of
ischaemia in rat papillary muscles, it was found to be difficult to maintain a consistent
mechanical performance for 2 or 3 h. Instead, muscle performance often abruptly
decreased at some time after 90 min in vitro. An example of this behaviour is shown in
Fig. 7.1A, in which records made using the same stimulus pattern after 90 min and 155
min in vitro are compared. In the later recording active force output was much reduced
and passive force was elevated. However, this was not always the case, as shown by the
example in Fig. 7.1B. In that example, there was a small increase in passive force output
but little change in active force output over the same time interval. This inconsistent and
unpredictable behaviour would make it very difficult to identify changes due to an
experimental intervention such as ischaemia. A series of experiments was performed to
try to overcome the time-dependent changes in function and these are briefly described
in the following sections.
0
2
4
6
8
Fo
rce
ou
tpu
t (m
N)
10 s
155 min90 min
A.
0
5
10
15
20
10 s
105 min 175 min
B.
Fig. 7.1. Changes in mechanical performance with time. The stability of the mechanical performance of mouse papillary muscle is
illustrated above. A. The muscle was stimulated at a contraction frequency of 2
Hz and first measurement was made 90 min after the muscle was mounted in the
set-up and the force output remained >5 mN throughout the series of
contractions. The next measurement was made 65 min later and at that stage the
force output was reduced by about 80%, or was <1 mN. B. Another preparation
was stimulated at a contraction frequency of 1 Hz. The passive force increased
slightly over time but there was little change in active force output.
87
7.2 Solution
To ensure that the bathing solution was optimal, Krebs-Henseleit solution was made up
fresh on the day of the experiment. Solutions were usually made using deionised water
and a comparison was made with solutions made using more pure, osmotically filtered
water (18.2 Ω, Milli-RO 3 plus and Milli-Q plus 185, Millipore, Australia). However,
this had no discernable effect.
In initial experiments, the Krebs solution contained 1.5 mM Ca2+
. However, with this
solution muscle performance, from the start of experiments, was variable and many
muscles produced very little force. By increasing the concentration to 2.5 mM muscles
consistently produced more force and exhibited prolonged viability (see Section 3.1).
To further enhance the contractility of the muscle much effort was spent comparing
muscle performance using either glucose or pyruvate as the exogenous metabolic
substrate. It is well-accepted that the provision of pyruvate as a substrate has a positive
inotropic effect on cardiac muscle of many species. For instance it has been reported
that pyruvate, when substituted for glucose, increased the isometric force output in
isolated rabbit papillary muscles (Chapman, 1972; Chapman & Gibbs, 1974; Chapman
et al., 1976) and guinea pig trabeculae (Daut & Elzinga, 1989). The enhanced
contractile performance with pyruvate as a metabolic substrate has also been shown in
isolated perfused hearts from guinea pigs (Zweier & Jacobus, 1987; Mallet & Sun,
1999) and rats (Dos Santos et al., 2000). In contrast, the energetics of mouse papillary
muscles in this study was unaltered by substitution of pyruvate for glucose (both at 10
mM) (Table 6.2). This may be a chacteristic of mouse cardiac muscle because a similar
result has been observed in isolated, perfused mouse hearts (Flood et al., 2003). In fact,
in that study isovolumic pressure development was slightly lower in the presence of
pyruvate than glucose.
In a final modification to the bathing solution with the aim of enhancing the success rate
of prolonged experiments, the perfusion medium was supplemented with fetal bovine
serum. The inspiration for this came from a study by Lännergren & Westerblad (1987)
in which skeletal muscle fibre survival and force production improved when serum was
included in the solution. Unfortunately there were no beneficial effects of this on the
mouse papillary muscle.
88
7.3 Experimental set-up
Great care was taken to clean the apparatus thoroughly after each experiment and the
muscle bath was rinsed with both water and ethanol. The apparatus was also taken apart
for extra cleaning on a regular basis. All the tubes supplying the chamber with solution
and gas were also regularly replaced to prevent bacterial growth.
A thin layer of silicone heat transfer compound (Unick, Unick Chemical Corp.) was
always applied between the jaws of the thermopile frame and the thermopile and a
check was made to see whether leaching of this substance into the bathing solution may
have affected muscles. The silicone compound (detailed composition unknown) was
replaced with silicone vacuum grease, which has been used previously on the
thermopile used in this study. However, this change did not alter muscle performance.
The stimulating system was the part of the experimental set-up that was thought to be
the most likely to cause problems with consistent muscle performance. The stimulating
electrode arrangement was modified and replaced multiple times! The original
arrangement consisted of relatively thick platinum electrodes that were positioned to
touch each sides of the preparation. It was thought that this may damage the myocytes.
The first modification was to carefully tie very thin platinum wires (25 µm) in a knot
around each end of the muscle (Yin, 1990). This proved difficult and may also have
exposed the muscles to damage. Finally, the arrangement was altered so that muscles
were stimulated via the platinum clips that were used to connect the muscle to the rods
attached to the force transducer and motor. The stimulating wire was wound in fine
coils around the connecting rod and the hook connecting the muscle to the recording
unit. This proved the most reliable technique although it still proved necessary to
change the wires at regular intervals.
The treatment of the muscles during the stabilisation phase was also investigated. Two
approaches were taken: (1) stimulate muscles at 0.2 Hz for 60 min (as typically used for
rat papillary muscles) or (2) leave preparations unstimulated for the same amount of
time. However, there was no beneficial effect of either of these approaches on the
prolonged function of muscles.
89
7.4 Adequate oxygenation
The depressed active force output and raised passive force observed in some muscles
(Fig. 7.1A) is reminiscent of the effects of ischaemia and reperfusion (e.g. Fig. 4.3)
which lead to the idea that maybe, despite the model predictions, diffusive O2 supply to
the muscles was sometimes inadequate. The PO2 of the solution in the thermopile
chamber used for the resting heat experiments (Chapter 5) was higher than that in the
horizontal thermopile used for the other experiments (0.95 atm and 0.84 atm,
respectively), because the former could be bubbled more vigorously. So a trial was
performed in which some muscles were placed in the vertical chamber bubbling with
95% O2. However, muscles subjected to this protocol performed no better than those
placed in the horizontal thermopile chamber.
7.5 Method of euthanasia
The animals were rendered unconscious by inhalation of 80% CO2–20% O2 gas mixture
(Kohler et al., 1999) and killed by cervical dislocation. The inhalation of CO2 can
potentially lead to myocardial acidosis. However, the animals were only briefly exposed
to the gas before being killed and the hearts were still beating when removed from the
animal and were bright red in colour. There were no signs of consistent increase in force
output during the equilibration period, as might be expected if accumulated H+ were
being gradually removed from the cells (cardiac muscle force output is quite sensitive to
intracellular pH; Vaughan-Jones et al., 1987).
Furthermore, when mice were killed by cervical dislocation alone, in vitro performance
was not improved, supporting the idea that the method of euthanasia was not
compromising the mouse papillary muscles’ performance.
7.6 Assessment of the suitability of isolated mouse papillary muscles for investigating cardiac muscle physiology
The experiments described in this Thesis demonstrate that the isolated mouse papillary
muscle can be used to study cardiac muscle physiology. The preparations produce good
forces and the measured rates of energy use compare well with data from other species.
Furthermore, as long as experiments are of less than ∼2 h duration the inter-preparation
variability was acceptable. It has been demonstrated that the preparation can be used to
explore the integrated functioning of the activation and contraction processes. However,
90
the proviso is that the duration of the in vitro investigation should be at most 2 h. For
longer protocols, such as typically required for studies of ischaemia and reperfusion the
inability to obtain consistently stabile performance limits the usefulness of the
preparation.
It is interesting to note that in a recent study (Golenhofen et al., 2006) papillary muscles
from wild-type and transgenic mice were used to study changes in mechanical
performance during simulated ischaemia. Studying the paper in detail reveals a number
of limitations. (1) The peak active force output of these preparations was 2.5 ± 0.5 and
2.1 ± 0.5 mN mm-2
in wild-type and transgenic mice, respectively. This can be
compared to typical values of 20 mN mm-2
in the current study (see Section 5.3.4 and
6.3.1). A small fraction of this difference can be accounted for by the use of a higher
[Ca2+
] in the current study (2.5 mM versus 1.5 mM) as the force output doubles at the
higher concentration (Redel et al., 2002; Stuyvers et al., 2002) but the comparison
suggests that the muscles in the study by Golenhofen et al. (2006) were either in poor
condition or were inadequately stimulated. (2) Ischaemia was simulated by withdrawal
of glucose and by replacing O2 with 95% N2−5% CO2, but no data has been supplied
explaining the partial pressure of O2 in the chamber. In the current study, it was found
that considerable ingenuity and care was required to get chamber PO2 to very low
values. (3) The study lacked a suitable control group from which the effects of time in
vitro could be distinguished from those due to ischaemia. Instead muscles from wild-
type mice, also subjected to ischaemia, were used as a control for muscles from
transgenic mice. Ideally, these data would have been complemented with measurements
of the mechanical stability of muscles from the wild-type mice under non-ischaemic
conditions.
91
CChhaapptteerr 88:: CCoonncclluuddiinngg ccoommmmeennttss
In this Thesis the first measurements of the metabolism of mouse papillary muscles
have been described. These measurements encompassed both resting and active
metabolism and the partitioning of active metabolism between initial and recovery
reactions and between force-dependent and force-independent processes. In addition to
the work on mouse papillary muscles, there is also a section describing alterations in the
energetics of rat papillary muscles during and after a period of simulated ischaemia. In
this concluding chapter, the highlights of the study are briefly recounted and
experiments that may further address some of the issues raised in each chapter are
suggested.
8.1 Resting metabolism
The most striking outcome of experiments on resting metabolism was the exceptionally
high initial resting metabolic rate, particularly in small muscles (<1 mg). However, in
most respects, the characteristics of the resting metabolic rate of mouse papillary
muscles were similar to those of papillary muscles of other species. For example, it
declined exponentially with time (although with a much shorter time constant than that
for papillary muscles from larger species), was increased by changing substrate from
glucose to pyruvate and by exposure of the muscles to hyperosmotic solutions. The
difference in time course of the decline in resting metabolism between muscles from rat
92
and mouse may provide a clue to the mechanism underlying the decline: is it related, in
some way, to the size of the preparation? Is it diffusion related (in which case the
temperature dependence of the time constant would be quite small)?
Another interesting question from the resting metabolism study was how the resting
metabolism of the heart compares to that of the isolated papillary muscle. The available
data (see Section 5.4.1), obtained indirectly from the relationship between myocardial
O2 consumption and PVA, suggests that resting metabolism of isolated mouse hearts is
higher than the eventual steady values in the isolated papillary muscles. An interesting
project would be to measure resting metabolism of both the isolated heart and the
papillary muscle. Resting metabolism could first be measured in the isolated heart,
taking the transepicardial O2 exchange into account (Loiselle, 1989; Gibbs & Loiselle,
2001), followed by measurement of the resting metabolism of a papillary muscle
dissected from the same heart.
8.2 Active metabolism
Active metabolism of mouse papillary muscles was studied by using both isometric and
realistic contraction protocols. The active metabolic cost per twitch was lower in the
mouse papillary muscle compared to that of the rat papillary muscle but total
metabolic rate at realistic contraction frequencies would be higher in the mouse muscle
due to the higher heart rate of the mouse. Interestingly, there was no effect of pyruvate
on either force output or energy cost and, through reference to published data for
isolated mouse hearts, it was suggested that this may be a characteristic peculiar to
mouse cardiac muscle. The mechanical efficiency of mouse cardiac muscle was found
to be similar to that of cardiac muscle from other species, indicating that the need for
more rapid force dynamics and more rapid energy use and supply in the smaller muscles
does not compromise efficiency.
The enthalpy output of the mouse papillary muscle was used to calculate the number of
cross-bridge cycles that occur in a single twitch. It was concluded that the energy used
in a twitch of mouse papillary muscle can be accounted by cycling of about half the
cross-bridges and the uptake into the SR of about one Ca2+
for every three cross-bridge
cycles. Furthermore, the estimated number of Ca2+
released in a twitch was fewer than
the number of Ca2+
binding sites on troponin-C. Thus, it can be seen that twitch force
can be modulated (both increased and decreased) by varying the amount of Ca2+
93
released or, even in the absence of changes in Ca2+
release, by altering the kinetics of
cross-bridge attachment or detachment.
Overall, the experiments described in Chapter 6 demonstrated that energetic
measurements could be made using mouse papillary muscles and that it is possible to
use these to quantify the molecular and ionic changes that underlie contraction.
Most of the characterisation of mouse papillary muscles was carried out at a sub-
physiological temperature (27°C). This was regarded as a good balance between
preserving physiological properties evident at 37°C while ensuring that diffusive O2
supply is able to match metabolic O2 demands. However, recent modelling of diffusive
O2 supply to isolated papillary muscles (Barclay, 2005) suggested that these
experiments actually are favoured by the physiological temperature. This arises from the
negative temperature dependence of energy cost per twitch (Table 6.3), which
overcomes the small positive temperature dependence of resting metabolism. Therefore,
in future studies, it may be worth attempting to use physiological temperatures.
8.3 Ischaemia and reperfusion
Isolated rat papillary muscles were exposed to 60 min of simulated ischaemia, which
resulted in depressed work and enthalpy output but with no change in efficiency or
activation metabolism. These results suggested that the likely mechanism underlying the
depressed work output in post-ischaemic rat papillary muscle was that fewer cross-
bridges cycled in each contraction. This may come about because of reduced sensitivity
of the myofibrils to the Ca2+
release. Although this argument is supported by other
studies that investigated Ca2+
handling, these conclusions could be supported by further
work. For example, by measurements of Ca2+
transients or by comparing maximum
Ca2+
-activated force output pre- and post-ischaemia. It would also be of interest to
perform a test of the idea that reactive oxygen species might damage the myofibrils,
reducing their Ca2+
sensitivity (Bolli & Marban, 1999) by introducing antioxidants into
the superfusate.
One aim of this study was to investigate the effect of simulated ischaemia using the
mouse papillary muscle. This cannot be recommended under the conditions described
using the rat papillary muscle, as the prolonged stability of the mouse papillary muscle
is in doubt. However, one possibility could be to work out a compromise between the
94
long equilibration period (due to the high resting metabolic rate) and the start of the
ischaemic protocol.
8.4 Conclusion
It has been demonstrated that the mouse papillary muscle is a viable model for studying
energetic aspects of cardiac muscle contraction. This is important because, as a result of
the use of mice for studies involving genetic modifications, the mouse has become the
favoured animal model for work in the field of cardiovascular physiology. This
preparation would be ideal to study the physiological and functional consequences of
heart-focussed genetic manipulations. However, improvements in maintaining
preparation viability in vitro for periods greater than 2 h are required before extending
the scope of this model to ischaemia and reperfusion experiments.
95
AAppppeennddiixx II
The unit sarcomere cylinder is a region bounded transversely by four neighbouring thick
filaments and longitudinally by successive Z-lines and encloses four thin filaments (Fig.
AI. 1). The volume of a sarcomere unit cell was calculated assuming that sarcomere
length was 2.1 µm (see Section 5.2.3) and that the spacing between the thick filaments
was 41 nm (Yagi et al., 2004).
-6 ° -9 2 -15 3Volume sarcomere unit = 2.1 × 10 × sin 60 × (41 × 10 ) = 3.1 × 10 cm
This volume can be used to calculate the concentration of cross-bridges in mouse
papillary muscle. The length of the myosin filament containing cross-bridges is ∼700
nm. There are three myosin molecules (i.e. six cross-bridges) every 14.3 nm along the
filament (Cooke, 1986), giving a total of (700/14.3) × 6 = 294 cross-bridges per myosin
filament. From the geometrical arrangement of the filaments, each myosin filament can
interact with six neighbouring actin filaments so, 294/6 = 49 cross-bridges. As each thin
filament has three adjacent thick filaments, there are 3 × 49 = 147 cross-bridges that can
interact with each thin filament, so this volume contains a total of 588 cross-bridges. In
this case, the cross-bridge concentration in the sarcomere cell is 588/3.1×10-15
= 1.9 ×
1017
cm-3
= 0.32 mM.
Correcting for the volume density of myofibrils in myocytes (0.52 in mouse myocytes;
Barth et al., 1992) and the fraction of muscle volume that is occupied by myocytes (0.79
96
in rat myocardium; Dobson & Cieslar, 1997) the muscle cross-bridge concentration is
0.32 × 0.52 × 0.79 = 0.13 mM.
AI. 1. Unit sarcomere rhombus. Arrangement of thick () and thin (•) filaments in striated muscles (reproduced
from Squire et al., 1990, courtesy L. de Beus). Each myosin filament in a half
sarcomere is surrounded by six equally-spaced thin filaments. The unit
sarcomere rhombus is bounded transversely by four neighbouring thick filaments
and lengthwise by successive Z-lines and encloses four thin filaments, two in
each half of the sarcomere unit. The area enclosed by the four thick filaments is
given by the formula for the area of a rhombus: Area = sin(θ) × l2 where θ is the
internal angle of the sides and l the length of the sides. For mammalian striated
muscle, θ = 60° or 120° and l = 41 nm.
97
AAppppeennddiixx IIII
The energetic and mechanical properties of four right ventricular rat papillary muscles
were measured (Table AII. 1 and AII. 3 and Fig. AII. 2). The muscles were dissected
∼7−11 h after cardiectomy, mounted in the set-up and stabilised for 15 min before the
first measurement. During the stabilisation period, muscle length was adjusted to give
maximal force output (Lmax). The experiments were performed at 27°C and the muscles
stimulated to contract at a frequency of 2 Hz. The contraction protocol lasted for 20 s
and both isometric and realistic contractions were used. The realistic protocol was
similar to that used for measurements with the left ventricular muscle of the rat, that is,
shortening velocity was normalised to muscle length and corresponded to 0.54 Lmax s-1
.
AII. 1. Right rat ventricular papillary muscle characteristics.
Number of preparations 4
Wet muscle mass (mg) 1.2 ± 0.1
Muscle length (mm) 3.1 ± 0.2
Cross-sectional area (mm2) 0.38 ± 0.05
Muscle radius (mm) 0.35 ± 0.02
Peak active force (mN mm-2
) 32 ± 2
Twitch time 409 ± 11
Time to peak tension (ms) 184 ± 8
98
Isometric Realistic
Net heat output (mJ g-1
twitch-1
) 7.6 ± 0.6 5.8 ± 0.6
Net work output (mJ g-1
twitch-1
) - 0.8 ± 0.1
Net enthalpy output (mJ g-1
twitch-1
) 7.6 ± 0.6 6.6 ± 0.7
Initial heat output (mJ g-1
twitch-1
) 4.2 ± 0.6 3.0 ± 0.4
Initial work output (mJ g-1
twitch-1
) - 1.00 ± 0.06
Net efficiency (%) - 12.7 ± 0.4
Initial efficiency (%) - 26.3 ± 4.1
R/I - 1.1 ± 0.3
Time constant for recovery heat rate, τ (s) 11 ± 2 11 ± 2
AII. 2. Examples of changes in
recordings of right rat ventricular
papillary muscle with time. Examples of recordings of the change in
muscle length, force output, temperature and
cumulative heat production in rat right
ventricular papillary muscles. This muscle
was mounted in the set-up ∼11 h after
cardiectomy and the first measurement was
made after an equilibration period of 15 min.
The first stimulus pulse resulted in a large
twitch force and the second a much reduced
force output. In the subsequent contractions
there was a gradual build-up of the force back
towards the initial level. A realistic
contraction protocol was used, indicated by
the change in muscle length. The protocol
consisted of a 110 ms long phase where the
muscle ends were held fixed, followed by a
shortening phase lasting 185 ms (amplitude
10% of muscle length), before being
relengthened at constant velocity. The
enthalpy output was recorded both in the form
of heat produced by the muscle and the work
generated. The peak force output was ∼26 mN
mm-2
and force output reached a steady-state
producing ∼24 mN mm-2
. The total amount of
heat and work produced was ∼300 mJ g-1
.
Muscle mass: 1.02 mg; muscle length: 2.9
mm.
-0.3
-0.2
-0.1
0.0
∆ L
en
gth
(m
m)
0
2
4
6
8
10
Fo
rce
(m
N)
0
2
4
6
8
10
∆ T
em
pe
ratu
re (
m°C
)
0 20 40 60 80Time (s)
0
100
200
300
400
En
tha
lpy o
utp
ut
(mJ g
-1)
Heat
Enthalpy
Work
AII. 3. Contractile properties of right papillary muscle.
99
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