Mechanisms of Ageing and Development 130 (2009) 742–747

Age-related decline in stress responses of human myocardium may not beexplained by changes in mtDNA

Francis Miller a,b,*, Phillip Nagley b, Justin A. Mariani a, Ruchong Ou a, Vincent W.S. Liu b, Chunfang Zhang b,Anthony W. Linnane c, Salvatore Pepe a, Franklin Rosenfeldt a

a Cardiac Surgical Research Unit, Alfred Hospital, Dept of Surgery, Monash University, and Baker Heart Research Institute, Commercial Road Prahran, Victoria 3181, Australiab Department of Biochemistry and Molecular Biology, Monash University, PO Box 13D, Clayton, Victoria 3800, Australiac Centre for Molecular Biology and Medicine, Epworth Medical Centre, 185-187 Hoddle Street, Richmond, Victoria 3121, Australia

A R T I C L E I N F O

Article history:

Received 31 August 2008

Received in revised form 3 September 2009

Accepted 20 September 2009

Available online 9 October 2009

Keywords:

Mitochondrial DNA

mtDNA

Ageing

Myocardium

A B S T R A C T

Elderly patients undergoing cardiac surgery are more likely to suffer postoperative heart failure than

younger patients. This phenomenon is mirrored by an age-related loss of mitochondrial function and by

an in vitro loss of myocardial contractile force following a stress. To examine the possibility that loss of

mtDNA integrity may be responsible, we quantified representative age-associated mtDNA mutations

(mtDNA4977 and mtDNAA3243G) and mtDNA copy number using quantitative polymerase chain reaction

in atrial samples obtained during cardiac surgery. The myocardium underwent organ bath contractility

testing before and after either an ischaemic or hypoxic stress. We found that with age, recovery of

developed force after either stressor significantly declined (p < 0.0001). The abundance of mtDNA4977

correlated weakly with loss of contractility (R2 = 0.09, p = 0.047). However, the abundance level was low

(average 0.0075% of total mtDNA) and the correlation disappeared when age was included in a

multivariate analysis. Neither the abundance of mtDNAA3243G nor mtDNA copy number correlated with

reduced recovery of developed force after stress. We conclude that, although mtDNA mutations (as

exemplified by mtDNA4977) accumulate in the ageing heart, they are unlikely to make a major

contribution to loss of contractile function.

� 2009 Elsevier Ireland Ltd. All rights reserved.

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1. Introduction

Ageing patients undergoing cardiac surgery manifest increasedmortality and a higher incidence of postoperative complicationssuch as cardiac failure and atrial fibrillation than youngercounterparts (Horneffer et al., 1987; Mullany et al., 1990;Naunheim et al., 1990; Glower et al., 1992). We previouslydemonstrated that human atrial myocardium obtained at the timeof cardiac surgery and tested in vitro demonstrates an age-relateddecline in ability to recover contractile force following anischaemic or hypoxic stress (Mariani et al., 2000). However themechanism of these age-related impairments in cardiac functionhas not been elucidated.

At a subcellular level, myocardial mitochondrial respiratorychain function decreases with age (Papa, 1996). It is alsorecognised that ageing human post-mitotic cells accumulate anincreased burden of mtDNA mutations. The mtDNA mutations thatoccur in tissues of ageing humans comprise genomic deletions,

* Corresponding author at: National Trauma Research Institute, Alfred Hospital,

PO Box 315, Prahran, Victoria 3181, Australia.

E-mail address: francismiller1066@hotmail.com (F. Miller).

0047-6374/$ – see front matter � 2009 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.mad.2009.09.003

point mutations and duplications. These mutations correspond tothose found in overt mitochondrial diseases but occur at a muchlower abundance (Ozawa, 1997; Papa, 1996; Nagley and Wei,1998). Almost two decades ago, it was suggested that mtDNAmutations in normally ageing tissues may be a major cause ofcellular dysfunction (Linnane et al., 1989). Studies examining theimpact that alterations of mtDNA integrity have on cellularfunction have yielded conflicting results. For example, a study insensory nerves in rats found that an accumulation of mtDNAdeletions paralleled age-associated loss of function (Nagley et al.,2001), yet the actual abundance of measured deletion (less than1%) was well below the mutation abundances of between 40 and80% that breach the ‘‘phenotypic threshold’’ (Rossignol et al., 2003)to cause mitochondrial disorders such as Kearn-Sayre syndrome.Nonetheless, recent studies have shown that distribution ofmutated mtDNA molecules can be focused in particular tissueregions, leading to highly localized deficits in function withinparticular tissues (Bua et al., 2006). In this study, we hypothesizedthat the age-associated decline in function in human atrialmyocardium and the consequential cardiac failure after heartsurgery might be best explained by an age-associated increase inmtDNA deletions. Assessment of deletion abundance mightrepresent a measure of biological age and predict the likely

Fig. 1. Recovery of developed force after ischaemia/reperfusion versus age. There

was a highly significant decline in contractile force after stress with increasing age

(n = 52, R2 = 0.167, p = 0.0014).

F. Miller et al. / Mechanisms of Ageing and Development 130 (2009) 742–747 743

response to surgery. With this in mind, we measured contractilefunction in small strips of right atrial myocardium derived frompatients of a wide range of ages undergoing cardiac surgery andcorrelated this with the level of the mtDNA4977 and A3243G pointmutations in the same strips, representative examples of a mtDNAdeletion and point mutation, respectively. Due to the small size ofthe samples, biochemical testing of the atrial strips was notfeasible. To further characterise mtDNA status in these tissues,mtDNA copy number was also measured.

2. Methods

2.1. Human tissues

Myocardium was obtained from right atrial tissue discarded during cannulation

for cardiopulmonary bypass from 45 consenting patients (aged between 3 weeks

and 87 years, designated M1–45) undergoing cardiac surgery at the Alfred Hospital,

Melbourne and the Royal Children’s Hospital, Melbourne. None of the patients had

overt mitochondrial disease. The study protocol was approved by the Monash

University Standing Committee on Ethics in Research on Humans.

2.2. Isolated human atrial strip preparation

Immediately following removal during surgery, the right atrial sample was

immersed in oxygenated ice-cold modified Ringer’s solution (2,3-butanedione

monoxime 30 mM ((BDM)), NaCl 125.8 mM, KCl 3.6 mM, MgSO4 0.6 mM, NaH2PO4

1.3 mM, NaHCO3 25.0 mM, glucose 11.2 mM, CaCl2 2.5 mM). After immediate

transport to the laboratory, 10 mg strips approximately 1 mm � 3 mm in size were

dissected from the lumenal aspect of the sample. Samples considered too small for

contractility testing were immediately frozen for later DNA analysis.

Each strip was connected to a Grass FT03 force transducer and held between two

platinum field-stimulation electrodes. The muscle preparation was lowered into

BDM-free Ringer’s solution in a water-jacketed tissue bath kept at 37 8C and pO2

was maintained above 600 mmHg. The bathing solution was replaced prior to

commencing the experiment in order to remove residual BDM. During the initial

stabilisation period, tissues were maintained unstretched and field stimulated at

1 Hz with a 10 ms square wave pulse via an isolated stimulator (Grass ST48).

Transduced muscle contraction was recorded via a Neomedix 8-channel recorder.

2.3. Contractile function assessment protocol

After 30 min of stabilisation, each muscle strip was stretched incrementally over

20 min to a length that developed maximum force. A further 30 min stabilisation

ensued at the end of which steady-state contractile developed force (DF) in mg was

assessed. The muscle strips were then subjected to either an ischaemic (ischaemic

protocol) or hypoxic (hypoxic protocol) stress as previously described (Mariani

et al., 2000). Both protocols were considered to simulate the stressful conditions

that occur at a myocardial level during cardiac surgery.

Ischaemia was simulated by draining fluid from the chamber which was then

filled with humidified 95% N2/5% CO2. Hypoxia was induced by exchanging oxygen

flow to the organ bath with 95% N2/5% CO2. Tissues in both protocols were

electrically stimulated at 2 Hz. After 30 min the tissues were reperfused and

reoxygenated (ischaemic protocol) or reoxygenated (hypoxic protocol). Both sets of

tissues were then paced at 1 Hz for a further 30 min and post-stress function was

measured.

Weight and muscle strip length under tension were determined at the end of

each experiment in order to calculate cross-sectional area (CSA). CSA was calculated

by dividing muscle mass by the product of length and density, assuming a

cylindrical shape and a density of 1.06 mg/mm3 (Keon et al., 1991). Tissues with a

CSA of greater than 1.2 mm2 were excluded from the study since larger tissues are

more likely to develop an anoxic core (Keon et al., 1991). DF was normalised for CSA

so that tension was not affected by variations in muscle size. The tissues were then

snap frozen in liquid nitrogen pending DNA analysis.

2.4. DNA extraction

Total cellular DNA was extracted from muscle samples (average wet wt 10 mg)

as described previously (Zhang et al., 1992). The DNA was stored in TE buffer

(10 mM Tris–HCl, 1 mM EDTA, pH 8.0). Absorbance readings (260 nm) of DNA

extracts indicated the DNA concentration to be in the range 50–300 ng/ml.

2.5. Quantification of mtDNA4977, mtDNAA3243G, and mtDNA copy number

Quantitative PCR, using external plasmid standards as we have detailed

elsewhere (Liu et al., 1997, 1998; Zhang et al., 1998), was used to measure the

abundance of mutations mtDNA4977 and mtDNAA3243G. mtDNA copy number per

diploid nuclear genome was precisely quantified by a PCR-based technique we

devised using dual insert plasmid standards (Miller et al., 2003). In total, tissues

from 49 patients were analysed. Of these, right atrial samples from 28 patients were

subjected to functional testing and then correlated to mtDNA4977 abundance and

mtDNA copy number. These tissues yielded a total of 52 atrial strips that were

suitable for testing for recovery of developed force and also tabulation against age.

All of these strips were analysed for mtDNA4977 abundance while only 39 were

analysed for mtDNA copy number. A further 7 atrial samples that were not

employed in functional testing due to their small size were analysed for mtDNA4977

abundance or mtDNA copy number, or both, depending on the amount of DNA

extract available after the extraction process. Right atrial samples from a further 14

patients were subjected to functional testing and mtDNAA3243G abundance testing

only. At no stage of the quantification process were any tissues selected or rejected

on the basis of functional performance, save in the case of tissues that were judged

to small to be used at all.

2.6. Statistical methods

Comparison between groups was performed by one-way analysis of variance.

The coefficient of variation (COV, calculated by dividing the standard deviation of a

set of values by the mean) was used to estimate the variability of sets of values.

Linear regression analysis was used to relate two variables. Multiple regression

analysis was used to compare the effects of multiple factors on functional recovery.

Statistical significance was set at p < 0.05.

3. Results

3.1. Post-ischaemic recovery

3.1.1. Recovery of developed force as a function of age

Multiple strips were dissected from right atrial appendagesamples from 28 patients (tissues M1–28) such that a total of 52were obtained. Most atrial samples generated at least two stripsbut the small size of some samples precluded dissection of morethan one strip. The age of the patients from whom these tissueswere obtained ranged from 4 to 80 years old. These tissues weresubjected to an ischaemic protocol. The intra-appendage stripvariability for recovery of function was acceptably low (COV 16%).

In keeping with previous work in our laboratory on an earliergroup of patient right atrial samples (Mariani et al., 2000), wefound that recovery of pre-stress developed force decreased withincreasing age (Fig. 1). Linear regression though, highly significant(p = 0.0014), explained only 17% (R2 = 0.167) of the variability inrecovery of developed force. Of note was that of the 11 stripsrecovering less than 50% of developed force, 10 were from patientsaged over 66 years.

3.1.2. Recovery of developed force following stress as a function of

percentage mtDNA4977 abundance

Each of the strips undergoing ischaemic stress was individuallytested for abundance of the 4977 bp deletion. The abundance of

Fig. 2. mtDNA4977 abundance in myocardium versus age. mtDNA abundance (%

total mtDNA, expressed as the natural log (ln)) showed an increase in deletion

abundance with age (n = 35, R2 = 0.78, p < 0.001).

Table 1Predictors of recovery of developed force after ischaemia/reperfusion by multiple

regression. Of the three variables (mtDNA4977, mtDNA copy number, age) that were

correlated with recovery of developed force after stress, only age was significant

(p = 0.0001).

mtDNA4977 Copy no. Age

Slope �0.36 �1.04 �0.76

p value 0.86 0.73 0.0001

F. Miller et al. / Mechanisms of Ageing and Development 130 (2009) 742–747744

mtDNA4977 in this group as a whole was very low (0.0075% totalmtDNA), and varied widely from 2.4 � 10�6% of total mtDNA in a4-year-old tissue to 0.06% in a 63-year-old tissue but this did showa highly significant increase with age (R2 = 0.78, p < 0.001, Fig. 2).These data correlate well with earlier findings on another set ofcardiac tissues (Liu et al., 1998).

Values of recovery of developed force following ischaemia/reperfusion versus abundance of mtDNA4977 were subjected tolinear regression analysis. A statistically significant inverserelationship, although weakly so, was apparent between increas-ing levels of deletion and recovery of contractile force (R2 = 0.09,p = 0.047, Fig. 3). In order to determine the relative influences ofmtDNA4977 abundance and age (significant univariate predictors)on post-ischaemic recovery of developed force, we performed amultiple regression analysis of recovery of developed force againstrecovery of developed force against mtDNA4977 and age. When theeffect of age (p = 0.0001) and copy number (p = 0.73) wereaccounted for, the mtDNA4977 abundance ceased to be significant(p = 0.86, Table 1).

Fig. 3. Recovery developed force after ischaemia/reperfusion versus mtDNA4977

abundance. Increased abundance of the 4977 deletion correlated weakly with loss

of function (n = 52, R2 = 0.08, p = 0.047).

3.1.3. Recovery of developed force as a function of mtDNA copy

number

Thirty-nine atrial strips from 25 patients (among patients M1–28) that were tested according to the ischaemia/reperfusionprotocol were also analysed for mtDNA copy number per diploidnuclear complement. The mtDNA copy number range in this groupwas between 1667 and 33,333. We have previously shown norelationship between mtDNA copy and age in such cardiac muscle(Miller et al., 2003). Linear regression was performed betweenrecovery of developed force and mtDNA copy number. However,no relationship was identified (R2 = 0.01, p = 0.46, Fig. 4). Also, theregression was performed without the outlying value of mtDNAcopy number 33,333 but, once again, significance was not reached(p = 0.41).

3.2. Post-hypoxic recovery

3.2.1. Recovery of developed force as a function of abundance of

mtDNAA3243G

A further set of tissues (M29–38) were subjected to stresstesting according to the hypoxia protocol. In total 14 strips weredissected and underwent analysis for abundance of the pointmutation A3243G. Mutation abundance was correlated withrecovery of developed force following stress according to thehypoxia protocol. In this small group of tissues, there was noassociation between mutation level and recovery of function(R2 = 0.04, p = 0.51, Fig. 5).

3.3. Correlation of mtDNA4977 and mtDNA copy number

To test the possibility of a compensatory increase in mtDNAcopy number due to increasing abundance of the commondeletion, a correlation between mtDNA4977 and mtDNA copynumber was sought. All thirty-nine strips that underwent testingfor mtDNA copy number and recovery from ischaemia/reperfusion

Fig. 4. Recovery developed force after ischaemia/reperfusion versus mtDNA copy

number (n = 39, R2 = 0.01, p = 0.46).

Fig. 5. Recovery developed force after hypoxia versus mtDNAA3243G abundance. No

relationship was found between the A3243G deletion and function (n = 14,

R2 = 0.04, p = 0.51).

Fig. 6. mtDNA copy number per diploid nuclear genome versus mtDNA4977

abundance. No correlation between the two variables was found (n = 45,

R2 = 0.000016, p = 0.98).

F. Miller et al. / Mechanisms of Ageing and Development 130 (2009) 742–747 745

stress (from patients M1–28) also had quantification of mtDNA4977

performed. In addition, a further 7 atrial samples (M39–45) thathad been analysed for mtDNA4977 and mtDNA copy number buthad not been tested for functional recovery were included. Thislarger group of 45 samples underwent comparison betweenabundance of the 4977 deletion and mtDNA copy number perdiploid nuclear genome complement. However, linear regressionfailed to reveal any correlation between the two variables(R2 = 0.000016, p = 0.98, Fig. 6).

4. Discussion

In this study of cardiac tissue obtained from patients ranging inage from 3 weeks to 87 years, we found that with increasing age,there was a decline in the ability of the myocardium to recovercontractile function after stress. The abundance of mtDNA4977

increased with age and when the abundance of the commondeletion was correlated with loss of function after an ischaemia/

reperfusion stress, a weakly positive inverse relationship wasfound (p = 0.047). However, this was lost as an independentpredictor of recovery when age and mtDNA copy number wereincluded in multiple regression analysis. Following hypoxia, theabundance of mtDNAA3243G (p = 0.51) did not correlate withreduced recovery of developed force. No relationship was foundbetween mtDNA copy number per diploid nucleus and recovery offunction following ischaemic stress (p = 0.46). Also, mtDNA copynumber did not correlate with the abundance of mtDNA4977

(p = 0.98). We felt it was appropriate to test each strip as anindividual unit rather than take an average of strips from the samepatient, in light of the heterogeneity for both physiologicalperformance and genetic makeup between atrial strips derivedfrom the same heart. In this way, we maintained a focus on thevariables we were testing rather than other unknown factors thatmay differ in the myocardium in one person’s heart compared toanother. We chose to use linear lines of best fit in our graphs, asopposed to curvilinear approaches to data analysis, since the latterdo not lend themselves well to statistical treatment. The paucity ofpatients in the mid-teen to forty year-old group (due to patientrecruitment constraints) emphasized the need to be cautious inthis respect.

We found a significant (p < 0.0001) association betweenimpaired recovery of developed force following an ischaemia/reperfusion stress and increased age. This mirrors the clinicalfinding that the elderly suffer more readily from heart failure afterthe stress of cardiac interventions than younger patients. However,regression analysis suggested that only 21% of the variability ofrecovery of developed force was due to age. In other words, factorsother than age contributed to the observed decrease in recovery ofdeveloped force in vitro. This conclusion was supported by thevariability in performance between different strips taken from asingle sample of atrial myocardium (COV = 16.3%); this value,whilst relatively low, nevertheless indicates functional hetero-geneity. Technical factors, such as differences in atrial strip widthor length, may partly have accounted for some of the variation infunctional performance between strips. Nonetheless, it appearsthat intrinsic myocardial factors (which in themselves may or maynot be associated with ageing) accounted for such variability, notonly between the strips dissected from a single atrial sample butalso between patients.

In post-ischaemic myocardium, diminished contractile perfor-mance is related to mitochondrial adenine nucleotide depletion(Sandhu and Asimakis, 1991), augmentation of cytosolic (Steen-bergen et al., 1987; Marban et al., 1990) and mitochondrial calcium(Miyata et al., 1992), free radical production (Zweier, 1988) andmembrane disruption (Jennings and Reimer, 1991). In the agedheart, compared to the mature adult myocardium, accumulation ofintracellular calcium induced by ischaemia/reperfusion injury andreduced recovery of pre-ischaemic contractile function have bothbeen shown to be more marked (Ataka et al., 1992; Hano et al.,1995). The ischaemia-induced, reduced availability of ATP maylead to the failed maintenance of ATP-dependent ionic gradients byCa++-ATPase, and Na+-K+-ATPase with resulting inability to re-establish calcium homeostasis (Ataka et al., 1992; Jennings et al.,1983). The majority of cellular ATP is produced by the mitochon-drial respiratory chain. Furthermore, essential subunits of com-plexes I, III, IV and V, mitochondrial ribosomal and transfer RNA arecoded by the mitochondrial DNA. A substantial alteration inmtDNA genome integrity would, therefore, be expected to result inreduced mitochondrial function, a concept first proposed byLinnane in the late 1980s (Linnane et al., 1989).

The average abundance of mtDNA4977 in the myocardialsamples we tested was very low (0.0075%), and several ordersof magnitude below the deletion abundance (often quoted as>60%) associated with mitochondrial diseases such as Kearn-Sayre

F. Miller et al. / Mechanisms of Ageing and Development 130 (2009) 742–747746

syndrome. We found that reduced recovery of developed forceafter ischaemia/reperfusion (p = 0.047) correlated significantlywith the abundance of mtDNA4977. However, the associationwas weak (R2 = 0.08). When the effects on function of mtDNA4977

abundance and age were subjected to multiple regression analysis,the deletion abundance became non-significant. Furthermore, theabundance of mtDNAA3243G did not correlate at all with reducedcontractile function (p = 0.51). A study that correlated mtDNA4834

abundance (the rat counterpart of mtDNA4977) with loss of sensorynerve function in ageing rats found a somewhat strongerassociation between deletion abundance and neural dysfunctionalthough the mutation levels were low and very similar to thelevels we measured (Nagley et al., 2001). It is well established thatageing humans accrue a variety of mtDNA mutations. We choosethe mtDNA4977 and mtDNAA3243G mutations as they are recognizedas mutations that do increase with age. While we did not attemptto measure either the total mutational load of all mtDNAmutations, it is unlikely that other mtDNA mutations would havesignificantly higher levels than the ones measured in this study(Miller et al., 2003) and so mtDNA4977 and mtDNAA3243G wereconsidered to be representatives of the mutational burden in thetissues studied. Also, by necessity, only right atrial myocardiumwas available for this study. Other chambers of the human hearthave been found to harbour mtDNA mutations, higher by a factorof 2 magnitudes in the case of the left ventricle (Corral-Debrinskiet al., 1991, 1992). At present, it is not possible to directlyextrapolate right atrial functional correlations with mtDNAparameters with other chambers of the human heart.

Previous work in our laboratory has found that the number ofmtDNA genomes per nucleus does not change in the ageing humanheart (Miller et al., 2003). The reliability of using quantitative PCRto measure mtDNA copy number has recently been attested byseveral other groups (Chabi et al., 2003, 2005; Bai et al., 2004; Baiand Wong, 2005; Frahm et al., 2005). The possibility that the age-associated loss of myocardial function was due to a low cellularcomplement of mtDNA was examined by correlating recovery ofdeveloped force after stress with mtDNA copy number in twenty-nine atrial strips. The average copy number in this group was 7000mtDNA genomes per diploid nuclear complement. The range ofvalues was 1667–33,333. We have previously documented markedvariations in copy number between adjacent strips dissected fromone sample of right atrium suggesting a tissue mosaicism (Milleret al., 2003). Even so, in the present study when copy number wascompared to function on a strip by strip basis, no relationshipbetween the two variables was found suggesting that depletion ofmtDNA was not a cause of myocardial dysfunction. In contrast, incertain rare infantile cases of mitochondrial disease (Moraes et al.,1991) and other mitochondriopathies (Bai et al., 2004; Bai andWong, 2005), severe mtDNA depletion, in some cases less than 20%of normal, is found in affected tissues.

Thirty-nine atrial strips were concomitantly tested for bothmtDNA4977 abundance and mtDNA copy number. However, therewas no relationship between the two quantified characteristics(R2 = 0.00002, p = 0.98). One would expect that if the cumulativemutation load in a cell was high enough to cause dysfunction inenergy production there might be a regulated increase in mtDNAcopy number to compensate for that mutation load. It is certainlywell documented that muscle can respond to increased metabolicneed such as exercise (Williams et al., 1986, 1987; Menshikovaet al., 2006) or increased afterload on the heart (Rajamanickamet al., 1979) with higher levels of mitochondrial genomes. Also,recent work has indicated that some patients with mitochondrialdisease show a proliferation of mitochondrial genomes in effectedorgans (Bai et al., 2004; Bai and Wong, 2005). This increase washypothesized to represent a cellular compensation to the reducednumbers of intact mitochondrial genomes. Some of these patients

also exhibited ‘‘ragged red fibres’’ in the region of mtDNAproliferation (Bai et al., 2004). While the absence of a compensa-tory rise in mtDNA copy number in the current set of tissues doesnot prove a low cumulative abundance of mutations, it doessuggest that preoperatively at least, the myocardium containedenough functional mtDNA to avoid the need for proliferation inorder to maintain functional output of normally ageing myocar-dium.

The low abundances of mtDNA mutation that we measuredcontrast to much higher levels found in a variety of ageing tissuesby some other authors. Although very small samples of rightatrium (10 mg) were used to determine mtDNA mutationabundance, the resultant DNA extract would be derived fromscores if not hundreds of individual cardiomyocytes. In keepingwith previous studies from our laboratory (Liu et al., 1997; Zhanget al., 1998), the measured abundance of mtDNA4977 had anaverage value of 0.0075%. However, when other authors havemeasured mtDNA mutation levels in successively smaller tissuesamples including individual cells, either the mutation level tendsto approach zero or increases dramatically (Schwarze et al., 1995).One study for example, found that some individual humancardiomyocytes obtained from centenarians contained as muchas 64% abundance of a specific mtDNA deletion while most of theexamined cells had no deletions at all (Khrapko et al., 1999). In ourown work on right atrial appendages (Zhang et al., 1999), wereported a pronounced mosaic distribution in the abundance ofmtDNA4977 in a study of multiple samples from a number ofindividual patients.

While the mosaic pattern of mtDNA mutation may explain thevariability in mutation measurements between relatively largehomogenized samples versus individual cells or even regions ofcells, it does not account for loss of function on the macroscopiclevel. We found that there was an age-associated loss of recovery ofdeveloped force after stress in the atrial muscle strips. While therewas an associated or at least parallel increase in mtDNA4977 it wassmall, and in light of the mosaic distribution outlined above, itwould presumably represent sparsely scattered deletion enrichedcardiomyocytes among fibres that would predominantly containthe wild type genome. There is evidence that increasing abundanceof mutated mtDNA in ageing tissues does have a deleterious effecton function. For example, high levels (up to 99%) of mutatedmtDNA have been found in cytochrome oxidase-negative regionsof individual skeletal muscle fibres (Bua et al., 2006). Also, knock-inmice that have a defective mtDNA proofreading capacity have beenfound to demonstrate premature ageing and cardiac dysfunction(Trifunovic et al., 2004). Whether or not widely scattereddysfunctional cardiomyocytes could adversely affect the functionof the whole heart is a difficult problem to test and remainsunknown at this time. The lack of a strong association betweenheart function and mutation abundance as was identified in thisstudy would suggest that they would not.

In conclusion, we found that elderly tissues regain lesscontractile force after stress than do younger ones. While theabundance of mtDNA4977 and mtDNAA3243G increased with age,neither correlated strongly with loss of function. It remains to bedetermined however what role they have in the loss of contractileperformance seen in the ageing human heart. The evidencepresented in this study of myocardial function failed to findconclusive or even supportive evidence of a major contributionfrom mtDNA mutation.

Acknowledgements

The authors would like to thank Dr Michael Bailey for statisticaladvice and Ms Heling Ng for her assistance with the preparation ofthe figures.

F. Miller et al. / Mechanisms of Ageing and Development 130 (2009) 742–747 747

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