Journal of Muscle Research and Cell Motility 17, 425--438 (1996).

Limits of titin extension in single cardiac myofibrils

W O L F G A N G A. L I N K E l*, M A R C L. B A R T O O 2, M A R C I V E M E Y E R 1 a n d G E R A L D H . P O L L A C K 2

1Institute of Physiology II, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany 2Center for Bioengineering WD-12, University of Washington, Seattle, WA 98195, USA

Received 24 May 1995; revised 15 February 1996; accepted 20 March 1996

Summary

Passive force and dynamic stiffness were measured in relaxed, single myofibrils from rabbit ventricle over a wide range of sarcomere lengths, from -2-5 prn. Myofibril stretch up to sarcomere lengths of - 3 pan resulted in a steady increase in both force and stiffness. The shape of the length-force and the length-stiffness curves remained fully reproducible for repeated extensions to a sarcomere length of -2.7 pin. Above this length, myofibrillar viscoelastic properties were apparently changed irreversibly, likely due to structural alterations within the titin (connectin) filaments. Stretch beyond ~3 ~rn sarcomere length resulted in a markedly reduced slope of the passive force curve, while the stiffness curve became flat. Thus, cardiac sarcomeres apparently reach a strain limit near a length of 3 pan. Above the strain limit, both curve types frequently showed a series of inflections, which we assumed to result from the disruption of titin-thick filament bonds and consequent addition of previously bound A-band titin segments to the elastic I-band titin portion. Indeed, we confirmed in immunofluorescence microscopic studies, using a monoclonal antibody against titin near the A/I junction, that upon sarcomere stretch beyond the strain limit length, the previously stationary antibody epitopes suddenly moved into the I-band, indicating A-band titin release. Altogether, the passive force/stiffness-length relation of cardiac myofibrils was qualitatively similar to, but quantitatively different from, that reported for skeletal myofibrils. From these results, we inferred that cardiac myofibrils have an approximately two times greater relative I-band titin extensibility than skeletal myofibrils. This could hint at differences in the maximum passive force-bearing capacity of titin filaments in the two muscle types.

Introduction

Titin (connectin), an extremely high molecular weight polypepfide, is the protein responsible for sarcomere elasticity in relaxed striated muscle (for recent re- views, see Maruyama, 1994; Trinick, 1994; Keller, 1995). It is now established that the elastic por t ion of the titin filament lies within the I-band segment, whereas the A-band segment is bound along the thick filament and therefore stiff unde r physiological conditions (e.g., Itoh et al., 1988; Trombitas et al., 1991, 1995; Wang et al., 1991). To s tudy titin's elastic properties, most work to date has focused on skeletal muscle preparations, and has employed immunoelec- t ron microscopic and mechanical techniques. Com- parable studies on cardiac muscle specimens, on the other hand, have only been sparse (e.g., Hill & Weber, 1986; Funatsu et al., 1993; Granzier & Irving, 1995). This is chiefly a consequence of the relatively high stiffness of conventional cardiac preparations, such as papil lary muscles or trabeculae, which cannot

*To whom correspondence should be addresse&

0142-4319/96 © 1996 Chapman & Hall

be stretched beyond sarcomere lengths (SLs) of approximately 2.4 pan, where stiff, non-sarcomeric structures preclude further extension. Hence, the mechanical propert ies of cardiac titin filaments still await detailed exploration, a task now made easier with the recently repor ted molecular structure of titin in hand (Labeit & Kolmerer, 1995).

We have previously shown that single, isolated myofibrils can be used to s tudy cardiac muscle mechanics (Linke et al., 1994). In that study, we focused on tension measurements within the phy- siological SL range, a l though we were able to extend the cardiac myofibril preparat ion to SLs well beyond 3 pan. In the present study, we focused on passive mechanical propert ies at longer SLs. Although under physiological conditions, mammal ian cardiac muscle does not reach SLs above approximately 2.4 pan (Rodriguez et al., 1992), it is useful to s tudy myofibril elasticity over a wide SL range, in order to gather information about the mechanical charac- teristics of titin filaments. Our main goal was to

426 LINKE et al.

investigate the extensibility limits of titin filaments in cardiac myofibrils, and to compare the results with those reported for skeletal muscle titin.

Passive force and stiffness of relaxed myofibrils were measured over a wide range of SLs, from --2 to 5 I~n. We found that with small to moderate stretch, both parameters increased steadily. With extreme stretch, however, the slopes of the passive length-tension curve and the stiffness-length curve became greatly reduced. The two curves were similarly shaped, indicating that a single element is responsible for both passive tension and stiffness, presumably the titin filament. Al though the passive force/stiffness-length relation of cardiac myofibrils appeared to be qualitatively similar to that of skeletal myofibrils, we found large quantitative differences between the two muscle types. Possible reasons for these differences are discussed.

Materials and methods

Myofibril preparation and solutions

Isolated myofibrils were prepared from rabbit right ventricular wall tissue as described previously (Linke et al., 1993). Briefly, thin strips of muscle tissue were dissected for storage in rigor/glycerol (50/50 by volume) solution for a minimum of 5 days at -20 ° C. To obtain single myofibrils, the glycerinated strips were minced, and the pieces were further skinned in 4°C rigor solution containing 0.5% Triton X-100 for 0.5h. After washing with fresh rigor solution, the tissue pieces were homogenized in a blender (Sorvall Omni Mixer) at low speed for 5-6 s in the same buffer. A drop of this suspension was placed in the specimen chamber, and myofibrils were allowed to settle. From myofibrils that adhered lightly to the chamber bottom, one specimen (a single myofibril or a doublet) was picked up by two microneedles, which could be controlled by hydraulic micromanipulators (Narishige, model 102, Tokyo, Japan).

All experiments were performed at room temperature (20-22°C). Normal relaxing solution had a total ionic strength of 200 mM (adjusted with KOH) in a MOPS buffer (pH 7.1), and contained 3 mM magnesium methanesulfo- nate, 5 mM dipotassium methanesulfonate, 4 mM Na2ATP, and 15 mM EGTA. In some experiments, the active-force suppressing drug 2,3-butanedione monoxime (BDM) at a concentration of 50ram was applied to the relaxing sMution. Finally, to all solutions used in this study, we always added 20 ~gm1-1 of the protease inhibitor leupep- tin.

Experimental apparatus

An experimental setup to measure SL and tension of single myofibrils has been developed, and is described in detail elsewhere (Bartoo et aL, 1993; Fearn et al., 1993; Linke et al., 1993). Briefly, the apparatus is centered around a Zeiss Axiovert 35 inverted microscope, equipped with phase- contrast optics. The myofibril image can be projected onto either a high resolution CCD video camera (Sony XC-

77RR), or a 256-element linear photodiode array (Reticon Electronics). Tension and SL data are collected with a Macintosh II computer and MacAdios analog/digital board. Length and appearance of each sarcomere within the specimen can be clearly observed, and by calculating the centre of mass of individual A-bands (Bartoo et al., 1989), we could follow SL changes during stretch protocols. Average SL could be measured to a precision of -10 nm, individual SLs to -100 nm (Linke et al., 1993). During fast sinusoidal oscillations, peaks in the striation pattern appeared broadened because the digitization rate of the image (10.6 ms per line scan) was slow, compared with the applied oscillation frequency (500 or 1300 Hz).

Force transducer and motor movement

Each end of a myofibril was glued to the tip of a glass needle (glue, 3145 RTV, Dow Corning), one of which was attached to a piezoelectric motor, the other to a force transducer. The force transducer operates on the basis of a stiff, displaceable optical fibre (beam diameter, 70 pro), which, at its distal end, emits a cone of white light. When force is exerted onto the fibre by an attached myofibril, the beam is deflected according to the amount of force applied. Beam deflection is monitored differentially by a pair of receiving optical fibres, and is used to generate a voltage proportional to the applied force. We used two different transducer beams in this study. The longer beam had a resonant frequency of 750 Hz and a sensitivity of -10 nN, and was used for most experiments. A shorter beam, with a resonant frequency of 1500 Hz and lower resolution, was used for stiffness measurements at 1300 Hz.

We performed control experiments (with no myofibril mounted) to study the force transducer response to imposed motor movement. For this, the glass needle connected to the motor was positioned such that it could touch the force-transducer needle lightly, and was able to move the transducer beam. We then generated both 1 Hz and 500 Hz oscillations of varying amplitude. As shown in Fig. 1A, a linear increase in peak-to-peak motor-input voltage resulted in a proportional increase in the ampli- tude of motor movement. Likewise, the force transducer output increased in a linear manner, and was not different for 1 Hz or 500 Hz oscillations. Figure 1B shows that an increased static deflection of the force-transducer beam did not affect the magnitude of transducer response to small° amplitude, 500 Hz motor oscillations. For a given oscilla- tion amplitude, we found no significant differences in force output, even when the transducer beam deflection was larger than that usually observed during stretch of a mounted myofibril. The standard deviations calculated from an average of 30 oscillations ranged between 3 and 6% of the total amplitude of transducer response. In sum, under the experimental conditions selected, the force transducer responded accurately to imposed motor move- ment.

SDSogel electrophoresis

The protein composition of cardiac myofibrils in suspension was analysed by SDS-polyacrylamide-gel electrophoresis, which was carried out according to the method of Laemmli (1970), using 12% polyacrylamide gels. For the detection of

Cardiac titin extension 427

the high-molecular weight polypeptide, titin, we used agarose-strengthened 2% polyacrylamide slab gels with a Laemmli buffer system (Tatsumi & Hattori, 1995). Glyceri- nated and homogenized tissue was dissolved in a buffer containing 1% SDS, 4M urea, 15% glycerol, 50 mM DTT, 50mM Tris-HC1 (pH 6.8). Protease inhibitors leupeptin, aprotinin, and pepstatin A were added at concentrations of 10 ~g m1-1. Protein bands were visualised with Coomassie Brillant Blue R. For comparison, we also analysed SDS gels from myofibril suspensions prepared from fresh cardiac muscle tissue (12% gels) and from glycerinated psoas muscle (2% gels).

Immunofluorescence microscopy Myofibrils suspended between two needles under the phase-contrast microscope were labelled with either of two monoclonal antibodies: (1) T12 (Boehringer, No. 1248-

634; cf Ffirst et al., 1988), which stains the Nl-line titin region -100nm from the Z-line within the I-band (Trombitas et al., 1995); and (2) BD6 (a kind gift from Dr J. Trinick, Bristol, UK), which labels titin near the A/I junction, -40 nm from the end of the thick filament within the A-band (Whiting et al., 1989). To visualize the antibody epitopes by fluorescence microscopy, we used rhodamine- conjugated anti-mouse IgG (whole molecule; SIGMA, No. T-5393). The primary and secondary antibodies were used in dilutions of 1:50 and 1:80 (in relaxing solution), respectively; exposure time to myofibrils was 10-30 min, which was sufficient to result in strong labelling. Antibody position within the sarcomere was measured by using the CCD camera, video recorder, frame grabber board, and image processing software (Global Lab Image, Data Trans- lation). For stretch experiments with BD6-1abeUed myofi- brils, two alternative protocols were used. In the first, a

A 16

Force transducer output (V)

12

8 ¸

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1 H z ~ ~ - - - - 500 Hz

Force ~ ~ ~ t r ansduceZ ._~Moto r

I 0 I I I 1 0 200 300

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0 400 50(

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movement (~m)

Measured force (nN)

90

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0.0 0.2

80 nm

t Peak-to-peak amplitude of motor oscillation

I I I I I I

0.4 0.6 0.8 1.0 1.2 1.4

Force transducer displacement (~tm)

Fig. 1. Control of force transducer performance during sinusoidal analysis. No myofibril was mounted in the apparatus. (A) Force transducer response (filled circles) to varying amplitudes of imposed motor oscillations (open squares); sinusoids of 1 Hz (dashed line) and 500 Hz (solid line), respectively, were generated. (B) Effect of static force-transducer beam deflection (displacement of the transmit fibre tip) on the magnitude of transducer response; 500 Hz sinusoids of three different amplitudes were compared.

428 LINKE et al.

relaxed myofibril was set to slack SL, was labelled with the primary antibody and, after washout, with the secondary antibody. Then, the myoflbril was stretched slowly (about 10% of its initial length per minute) to a series of SLs, and the translational movement of the antibody was recorded. In a second experimental protocol, the myofibril was stretched first to a desired SL above slack and was then labelled with the antibody and fluorophore. Both measure- ment protocols gave similar results. As a control, we labelled myofibrils with the secondary antibody only, and found no fluorescence.

with less accuracy (error, approximately 6% RMS), which was a result of the analog-to-digital conversion at the higher frequency. Furthermore, with shortening of the force-transducer beam to raise its resonant frequency (to reliably measure the response to 1300 Hz oscillations), the transducer's sensitivity to low force levels dropped significantly. This drop in sensitivity interfered with our ability to measure stiffness at short SLs, because of the low signal-to-noise ratio. Thus, we preferred to use the longer transducer beam for most experiments.

Experimental protocols Passive force was measured by two different protocols. In the first, a myofibril was held at slack length and was then stretched directly to the desired length. Stretch duration was 3-10 s. At the stretched length, we generally observed a stress-relaxation-based force decay. When force ap- proached the steady state level (after - 3 min; cf., Fig. 4), the specimen was returned to slack length, before being stretched to the next experimental length. In the second protocol, a myofibril was stretched in stages from its slack length to the desired length, without return to slack length. After a series of stretches, interrupted by 3-minute-long hold periods, a desired maximum SL was reached. Then, a progressive release protocol was performed, so that finally the specimen returned to slack length. Both measurement protocols gave similar results. Force was measured every 12s, for a period of 0.5s (at intervals of 160 ~s). We expressed force in units of ~N, and did not scale it to myofibril cross-sectional area, since our ability to precisely measure myofibril diameter under the phase-contrast microscope was limited (error, -+0.2 ~rn; cf., Linke et al., 1994).

Stiffness was estimated from the amplitude of force response to small-magnitude, usually 6-8 nm per half- sarcomere peak-to-peak, sinusoidal oscillations (500 and 1300 Hz, respectively), imposed by the motor (cf., Granzier & Wang, 1993). Figure 2 shows motor output waveforms at both oscillation frequencies (A) and the resulting force oscillations, which we took as a measure of myofibril stiffness (B). The phase difference between motor and force transducer oscillations was minimal and comparable at both frequencies. Motor oscillations were imposed at 12 s intervals, for a period of 0.5 s, and the force response during that period was measured every 160 ~s. Stiffness was calculated by averaging the peak-to-peak amplitude of 30 (500Hz) or 80 (1300Hz) force oscillations and was expressed in units of nNnm -1 (per half-sarcomere). The sinusoidal length amplitude was determined by measuring myofibril end displacement divided by the nu~mber of haif- sarcomeres per specimen. To investigate stiffness at differ- ent SLs, sinusoids were superimposed onto the above- mentioned stretch-release ramps used to measure passive tension. At short SLs (where the signal-to-noise ratio was relatively low), we usually averaged data from several measurements at the same SL.

We chose to perform most stiffness experiments at an oscillation frequency of 500 Hz, and only a few at 1300 Hz. This was done because 500Hz sinusoids could be generated accurately (error of peak-to-peak amplitude, <0.5% RMS), whereas 1300 Hz sinusoids were generated

Results

Myofibrillar protein composition From cardiac muscle strips glycerinated for one week, we p repa red myofibri ls as described in Materials and Methods and analysed the protein composi t ion by both 12% and 2% SDS-PAGE. Typical 12% gels, such as that shown in Fig. 3A (columns a-e), revealed the characteristic protein bands previous ly repor ted by others for skinned cardiac tissue (e.g. Pfitzer & Rfiegg, 1984). Particularly distinct bands corre- sponded to myos in heavy chain, R-actinin, actin, the myos in light chains 1 and 2, and the regulatory proteins (Fig. 3A, co lumns b and c). For comparison, we also invest igated the protein composi t ion of cardiac myofibri ls p repared f rom fresh muscle (Fig. 3A, co lumn d), and found no obvious differences in protein preservat ion be tween the two preparat ions. Two percent po lyacry lamide slab gels, p repa red according to the me thod of Tatsumi and Hattor i (1995), revealed the characteristic two bands for the titin polypept ide , wi th a s trong u p p e r band (titin 1) and a weak lower band (titin 2; Fig. 3A, columns f and g). Thus, the appearance of the fitin bands was not different f rom that repor ted by others (e.g., Maruyama , 1994; Granzier & Irving, 1995). Also, cardiac titin 1 migra ted farther than psoas muscle titin 1 (columns h and i), as expected f rom the lower molecular weight of the cardiac isoform (cf., Labeit & Kolmerer, 1995). Finally, in gels p repared f rom psoas myofibrils , we found an addit ional s trong band for nebulin, which is absent in cardiac muscle.

Titin preservat ion was also confirmed on indivi- dual myofibri ls by immunof luorescence microscopy. We used f luorophore-marked monoclonal antibodies to label titin at the Nl- l ine (with the T12 ant ibody) and near the A / I junction (with the BD6 antibody), respectively, i.e., at the two sites flanking the elastic I -band titin portion. Within minutes of exposure, we found avid labelling with both antibodies (Fig. 3B). No obvious difference in immunof luorescence inten- sity was observed in myofibri ls p repared f rom either fresh or 1-week-long glycerinated cardiac muscle (data not shown). Further, the BD6 epi topes m o v e d a w a y f rom the Z-disc wi th small to modera t e myofibri l stretch (see also Fig. 6B) and returned to

Cardiac titin extension 429

0.2

0.0

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500 Hz oscillations

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60 301 A force 0

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Fig. 2. Example of stiffness measurements on a myofibril doublet (20 sarcomeres, SL = 2.9 gin). (A) Motor oscillations (peak-to-peak amplitude, 8 nm per half-sarcomere) and (B) force transducer response. At the higher oscillation frequency (1300 Hz - bottom panels in each figure), the force response was larger than at the lower frequency (500 Hz - top panels).

their initial sa rcomere posit ion u p o n release, as expected for prepara t ions wi th intact titin. Alto- gether, we concluded that prote in preservat ion of isolated cardiac myofibri ls was not noticeably differ- ent than that of larger cardiac preparat ions .

Viscoelastic response to stretch

Single cardiac myofibri ls were remarkab ly extensible and could general ly be stretched to at least 5 gm SL wi thout breakage. Even wi th ex t reme stretch, SLs remained relatively homogeneous (average SL varia-

430 LINKE et al.

Fig. 3. (A) SDS-PAGE patterns of myofibril suspensions prepared according to Materials and Methods. (a-e) 12% gel; (f-i) 2% gel. (a) standard for b-e; (b) glycerinated cardiac myofibrils, high loading (30 ~g); (c) the same, low loading (15 ~g); (d) myofibrils from fresh cardiac muscle (loading, 20 ~tg); (e) loading of a cardiac troponin complex only, as a standard to identify TnT, TnI, and TnC. The band for TnC stained only lightly with Coomassie Blue, but could be more clearly visualized after silver staining (not shown). (f) 2% gel of glycerinated cardiac myofibrils, low loading (15 ~tg); (g) the same, high loading (30 ~g); (h) 2% gel of glycerinated psoas myofibrils, low loading (15 ~g); (i) the same, high loading (30 ~tg). (B) Phase-contrast (top) and fluorescence (bottom) images of single myofibrils,,labelled with the T12 (left) and the BD6 (right) titin antibody, and a fluorophore-marked secondary antibody. T12 binds to titin close to the Z-disc (-100 nm from its center) in each half-sarcomere, so that the antibody epitopes usually appeared as one single, broad, stripe in the Z-disc region (Fi~rst et al., 1988). BD6 labels titin near the A/I junction, 40 nm from the end of the thick filament within the A- band (Whiting et al., 1989). Scale bar = 5 ~m.

tion at 4.5 ~m: +6.7%; n = 11 myofibrils; 143 sarco- meres). More distinct SL inhomogeneit ies were often seen near the myofibril ends. Following a release, the contrast between I and A bands was still good under the phase-contrast microscope, indicating that fila- ment arrangement was still orderly. However , slack SL (average before stretch: 1.84 + SD 0.12 ~rn; n = 18 myofibrils) was generally increased (cf Figs 5-7).

Passive force increased steadily with stretch, but dur ing the hold period, it decayed exponentially towards a steady-state plateau value (Fig. 4). The time course of this decay appeared to be multi- phasic, with a very rapid phase followed by one or two slower phases. Such decay, or 'stress relaxation', is typical for viscoelastic material. During this stress relaxation, SL remained almost constant, the slight

increase measured being entirely attributable to a small deflection of the force-transducer beam atten- dant with the force change. Stress relaxation was observed at SLs both with and without overlap of thick and thin filaments, and is thus likely to be an intrinsic proper ty of the passive viscoelastic struc- tures within a myofibril, probably the titin filaments. The phenomenon possibly results from rearrange- ment within, a n d / o r between, the individual do- mains (such as tandem immunoglobul in repeats), which make up the I-band titin segment (Labeit & Kolmerer, 1995). Finally, we also observed another remarkable proper ty of the myofibrils: a large hysteresis. At comparable SLs, force following stretch was much higher than that following release (Fig. 4).

Cardiac titin extension

Motor ramp

Passive force (pN)

0.18.

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3.65

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Fig. 4. Stress relaxation of a stretched cardiac myofibril. Top: relative motor needle displacement and SLs (pan) during the hold period (values given above the tracing). Bottom: passive force level, measured every 12s for a period of 0.5 s, at 160 ~ intervals (filled circles indicate average force during that period). Stretch duration was 4 s, the hold period lasted - 3 min. Care was taken to measure force precisely at the end of a stretch, in order to estimate the full magnitude of stress relaxation. Upon release, force decreased (large hysteresis), but recovered slightly during the first few seconds of the hold period.

Strain limit of cardiac myofibrils

Plots of plateau force (i.e., the force after 3 min of stress relaxation) versus SL revealed a characteristic multiphasic shape (Fig. 5). Passive force became apparent at a round 2 ~ and increased steadily up to an SL of approximate ly 3 ~rn. A first decrease in the slope was observed at 2.6-2.7 ~rn (open arrow- head). This slope change was more clearly detectable

0.4

Passive force (IIN)

0 . 2

0 . 1

0 . 0 , . . . . . , - , . , . , . , . , • ,

1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 5.0 Sarcomere length (pm)

Fig. 5. Passive force versus SL, for progressive stretch and release of a myofibril. Notable curve segments are indicated by the open and closed arrowheads, and the arrow (for further details, see text). Inset: log force versus SL curve, from the same experimental data.

431

in plots of log force versus SL (inset of Fig. 5). At a round 3 ~rn, the curve flattened, indicating that a strain limit was reached at this SL. Interestingly, the curve never became completely flat. A small majority of specimens (6 out of 11) exhibited inflections, i.e., a quasi-plateau phase followed by a small force increase. We observed most ly two to three such inflections, spaced at intervals of 0.3-0.5 ~m (closed arrowheads). The magni tude of the inflections was clearly above the resolution limit of the force t ransducer (--10nN; Linke et al., 1993). In the remainder of specimens, no inflections at SLs beyond 3 ~rn were detectable; instead, a s teady increase in force was seen. This increase was smaller, compared with that at SLs below the strain limit. Finally, in most specimens, the slope of the force-SL curve became steeper at extreme SLs above 4.0--4.5 ~rn (arrow). Release of a myofibril resulted in a steep decrease in force, indicating a large hysteresis. In the example of Fig. 5, force decreased to zero at - 2 . 8 pan SL. Although we were unable to detect any passive force below 2.8 ~n , the myofibril still shortened further to a slack SL of -2 .1 ~n .

Passive force was usually measured in normal relaxing solution, but we also performed a few experiments after adding 50 mM BDM to the relaxing solution. At this concentration, BDM is known to suppress active myosin-act in interactions (e.g., Brotto et al., 1995). By applying this d rug to the solution, we could therefore investigate whether such interactions contribute to passive tension. Up to 3 ~ SL, we repor ted elsewhere (Linke et al., 1994) that passive force was not affected by BDM. In the current experiments, no effect was found beyond 3 ~ as well (data not shown). Thus, residual interactions do not appear to underl ie passive force. The characteristic multiphasic shape of the myofi- brillar passive force curve is therefore likely to be based solely on passive viscoelastic elements.

Test of segmental titin extension hypothesis

In skeletal muscle, several studies have shown that A-band titin is ordinari ly bound to the thick filament, and therefore functionally stiff under physiological conditions (e.g., Itoh et al., 1988). Intrinsically, how- ever, the bound segment has viscoelastic properties similar to those of I-band titin (Trombitas & Pollack, 1993; Wang et al., 1993). Evidence has been presented (e.g., Trombitas et al., 1991; Wang et al., 1991) that dur ing extreme sarcomere stretch, A-band titin segments become free and add to the elastic I-band port ion of titin (segmental titin extension hypothesis). Such length increase in the extensible I-band segment affects passive force of the sarcomere. Therefore, a good means of probing this hypothesis is to subject a muscle preparat ion to several cycles of stretch and release, and progressively increase the max imum SL

432

in each cycle. Such cycles were applied to cardiac myofibrils to test whether the segmental extension hypothesis may also be valid for cardiac titin.

Figure 6A shows the results of a representative experiment. Passive force was measured while per- forming four stretch-release cycles on a myofibril (doublet). In the first cycle, force increased steadily with stretch to -2 .5 pan SL, and decreased upon release, with some hysteresis. Slack SL was the same as before the stretch. In the second cycle, stretch resulted in a force increase similar to that of the first cycle, indicating that the structures responsible for this increase (presumably titin) had not been altered. Only between approximately 2.7 and 2.9 pan SL, a small change in slope became apparent. Upon release, force dropped with a large hysteresis. Slack SL was now slightly increased. In the third cycle, force initially increased less steeply than in the two previous cycles, but beyond -2 .9 pxn, rose above the maximum force value reached during cycle 2. Above 3 ~rn SL, the curve flattened. Upon release from 3.3 ~un (large hysteresis), force became undetectable at -2 .7 pan, but the myofibril still shortened further to a slack SL

Fig. 6. (A) Passive force recordings during four stretch- release cycles, performed on a myofibril doublet. Max- imum SL in each cycle was progressively increased. Cycle 1: open circles; cycle 2: filled triangles; cycle 3: open squares; cycle 4: filled circles; solid lines: stretch; dotted lines: release. For further details, see text. (B) Stretch of BD6 antibody-labelled myofibril. Shown are five fluores- cence images of the same myofibril, stretched to progres- sively increasing SLs. At -3.4 pm SL, the previously constant epitope-epitope distance across the M-line in- creased, and A-band titin segments were released into the I-band (arrows). Left: SLs values (prn); I = I-band titin segment; A = A-band titin region; scale bar = 5 pan.

LINKE et al.

slightly above 2 prn. In the fourth cycle, the slope was markedly reduced at shorter SLs and decreased even further at longer SLs, but no sudden slope change was evident. Remarkably, the force level at 3.8 pan SL was about the same as that of the third cycle at 3.3 prn. Following release, passive force approached zero at - 3 prn, while slack SL was 2.2 ~rn.

The results of such stretch-release protocols imply that in cardiac myofibrils, the elements responsible for passive tension undergo irreversible alterations at SLs above -2 .7 prn. Those alterations become parti- cularly clear at around 3 pm SL. Since we assume that the elements responsible for passive force in the sarcomere are the titin filaments, it follows that at SLs around 3 pan, titin filaments may reach their strain limit. Further, after extreme stretch and release, myofibrillar slack SL is increased, depending on the maximum SL reached during that stretch. Such increase in slack length can be explained by 'recruitment' of previously bound A-band titin segments, which are added to the elastic I-band portion of titin (Wang et al., 1993). Also, even after stretch to extreme SLs, the maximum force-bearing capacity of titin filaments appears to remain intact: al though the slope of the SL-passive force curve is reduced after the onset of structural changes, subsequent stretches to very long lengths can still increase the force level to values equal to, or greater than, those measured at the strain limit. Taken together, these data suggest that extreme stretch results mainly not in breakage of titin filaments, but rather in addition of previously bound A-band titin segments to I-band titin.

To further test this hypothesis, we fluorescently labelled cardiac titin near the A / I junction, using the monoclonal antibody BD6 (cf Fig. 3B), and measured the position of the antibody epitopes upon myofibril stretch (Fig. 6B). With small to moderate degrees of stretch, the epitopes in each half-sarcomere moved away from the Z-line but remained stationary relative to the center of the A-band. However, in myofibrils stretched beyond strain limit SL (usually, at SLs between 3.3 and 3.4 pro), the epitope-epitope distance across the M-line could change dramati- cally: beginning in one or two sarcomeres in the center or near the ends of the myofibril, the epitopes were suddenly 'pulled away' from their A-band location, so that the previously constant A-band titin length was now markedly increased (arrows in Fig. 6B). With further stretch, such 'yielding' of A-band titin arbitrarily progressed along the specimen. Even at extremely stretched SLs of -4 .5-5 pan, however, we could sometimes still find sarcomeres with no A- band titin yield. Interestingly, when we imposed rapid sinusoidal oscillations (such as those applied during stiffness experiments) onto a myofibril, we could induce the A-band titin release to occur at

Cardiac titin extension

somewhat shorter SLs than in specimens s imply stretched wi thout the oscillations. To be sure that yielded was not a side-effect of the glycerination procedure, we also labelled titin in myofibrils p repared from fresh muscle; upon extreme stretch, we found the same yielding phenomenon and thus, considered it to be genuine. Finally, when tension after an extreme stretch was released, the BD6 epitopes moved back toward the Z-disc and the M-line, respectively; however , slack SL was now markedly increased.

These results indicate that principal predictions of the segmental titin extension hypothesis appear to be valid also for cardiac muscle. However , large quantitative differences in titin extension behavior exist in cardiac and skeletal myofibrils and will be considered below (see Discussion).

Dynamic stiffness of titin

In an additional set of experiments, we measured the force response of relaxed cardiac myofibrils to fast sinusoidal oscillations of small ampli tude, in order to de termine dynamic stiffness. Under the experimental conditions selected, stiffness is likely to result f rom the viscoelastic behavior of titin filaments rather than from actively cycling weakly binding crossbridges (as p roposed for skeletal muscle), since: (1) at normal ionic strength and room tempera ture used here, those weak bridges should exist only in small numbers (Brenner et al., 1986), if at all (Bagni et al., 1995); and (2) in cardiac myofibrils, titin stiffness is - 1 0 times higher than in skeletal myofibrils at comparable SLs (Linke et al., 1994), so that weak bridge stiffness, if present, is negligibly small relative to titin-based stiffness.

Figure 7 shows stiffness (frequency, 500Hz) dur ing stretch-release cycles, measured on the same myofibril preparat ion as in Fig. 6A. At the short SLs, signal-to-noise ratio was low and stiffness measure- ments were unreliable; thus, the measured shape of the curves may differ somewhat f rom the true shape at those lengths. On the whole, however , it is evident that the shape of the SL-stiffness curves is similar to that of the SL-passive force curves. At - 2 . 7 ~ SL (cycle 2), we observed that the s teady stiffness increase became less steep than that at shorter SLs. Near 3 ~ml SL, where titin filaments reach their strain limit, the curve became flat (cycle 3). In the fourth cycle, stiffness still rose to the max imum value of cycle 3, a l though at a longer SL of -3 .5 ~rn. Hysteresis was present in all four cycles, and was dependent on the max imum SL reached dur ing the stretch. We found one particular differ- ence between the stiffness and the passive force curves: while force at SLs beyond the strain limit generally cont inued to rise, stiffness usual ly re- mained on a plateau (or sometimes even decreased slightly, cf., Fig. 9). Leaving aside this difference, the

433

12 c y c l ~ ~ 7

(St ~lffners ~ cycle 1 / , / /

4 .n ..- ; . , - ' ' " .dl"

0 "'e'~ .... 1.8 2.2 2.6 3.0 3.4 3.8 Sarcomere length (pm)

Fig. 7. Stiffness during four cycles of stretch-release, measured on the same myofibril preparation as in Fig. 6A (stiffness and force were recorded simultaneously). In cycles 3 and 4, some stiffness values could not be determined even well above slack SL, since large hysteresis had resulted in a low signal-to-noise ratio at these lengths. Oscillation frequency, 500 Hz. Coding was used as in Fig. 6A. See text for further details.

similarity of the curves suggests that both static force and dynamic stiffness originate f rom a common structure. Evidence from this and a related s tudy (Bartoo et al., submitted) implies that the responsible structure is likely to be the titin filaments.

Multiphasic stiffness curves

With progressive stretch of myofibrils from short to very long SLs, we found characteristic multiphasic stiffness curves, similar in shape to the passive force curves described above (Fig. 5). An example is shown in Fig. 8. Stiffness increased exponentially with stretch to - 2 . 7 ~m. At this SL, a slope change became apparent and was usually more pronounced than that of the passive force curves (open arrowhead). In two specimens, we found the stiffness curve at - 2 . 7 ~ to become almost flat. In most myofibrils, however, the

12

8

Stiffness (nN/nm)

4

~ e

| | i | i .9 2.4 2.9 3.4 3.9 4.4 4.9

Sarcomere length (~m)

Fig. 8. Stiffness recordings during progressive myofibril stretch. Oscillation frequency, 500 Hz. Error bars indicate SDS. Emphasized by the arrow, as well as the open and closed arrowheads, are curve segments of specific interest (see text).

434 LINKE et al.

15

S,.nes; °

0 1.8 2.2 2.6 3.0 3.4 3.8

Sarcomere length (~m)

Fig. 9. Stiffness measurements on the same myofibril preparation, at two different oscillation frequencies. A short force-transducer beam with a resonant frequency of 1500Hz was used. Stiffness was generally higher at 1300 Hz than at 500 Hz. Only at the shortest SL was the signal-to-noise ratio too low to allow reliable stiffness measurements at 500 Hz.

curve flattened at a round 3 ~rn SL. The average SLs, at which we observed a first s lope change and a strain limit, are s u m m a r i z e d in Table 1 (left two columns).

We also found that the inflections seen in the SL- force curves (Fig. 5) could frequent ly be detected in the SL-stiffness curves (Fig. 8, closed ar rowheads; Table 1). The s tandard deviat ions d r a w n in Fig. 8 show that the inflections, a l though of relatively small magn i tude (range, 0 .8--1 .2nNnm-l) , could still be resolved by the m e a s u r e m e n t system. Table 1 lists the average spatial per iodici ty of the inflections, which was - 0 . 4 ~rn (for all exper iments at 500 Hz oscillation frequency).

Finally, beyond SLs of 4-4.5 ~ n , stiffness usual ly increased more smooth ly and steeply (arrow in Fig. 8). This slope increase at extremely stretched SLs can potent ial ly be explained b y a stiffness contr ibution resulting f rom increased strain of endosarcomeric in termediate f i laments (Wang et al., 1993; Granzier & Irving, 1995).

In sum, as was seen in the SL-passive force curves, the SL-stLffness curves exhibited a character-

istic shape that is likely to result f rom the propert ies of titin filaments. H o w the observed curve shapes correlate wi th molecular cardiac titin properties, will be discussed be low (see Discussion).

Frequency dependence of titin stiffness

We assumed that the viscoelastic proper t ies of titin f i laments should result in a clear stiffness dependence on the imposed oscillation frequency. This assump- tion was conf i rmed in experiments, in which stiffness was measu red at two different oscillation frequencies, 500 and 1300 Hz, on the same myofibril . Figure 9 shows a representat ive result. The noise level in these exper iments was relatively high, since we used a short, low-sensit ivity force-transducer b e a m with increased resonant frequency (cf Materials and Methods). We found that stiffness was larger at 1300 Hz, by an average factor of 1.7 (range, 1.4-1.9). Such stiffness differences persisted f rom short (2.0 ~rn) to long (3.65 ~rn) SLs, indicating that over lap of actin and myos in filaments was not a critical factor. Fur thermore, the stiffness curves at both frequencies exhibited the characteristic mult iphasic shape a l ready described: a strain limit appea red at - 3 ~rn SL, fol lowed by a d rop in stiffness, an increase in stiffness at - 3 . 3 ~rn SL, and a pla teau phase. Since it is titin that is ve ry likely to be responsible for this characteristic curve shape, it seems reasonable that titin is also the critical factor for the frequency- dependen t stiffness difference.

Discussion

Use of isolated myofibrils for mechanical studies on titin

In this s tudy, we have invest igated the mechanical proper t ies of relaxed cardiac myofibri ls dur ing ext reme stretch, in order to gather information about the mechanical characteristics of titin filaments. The high stiffness and l imited extensibility of conven- tional, multicellular, cardiac prepara t ions (SL exten- sion range, -1 .7 -2 .4 ~m) have usual ly been obstacles to the direct mechanical investigation of titin proper- ties in cardiac muscle. Recently, cardiac titin elasticity was inferred f rom the results of mechanical measure- ments on isolated cells f rom rat heart (Granzier &

Table 1. Sarcomere lengths, at which a first slope change of the stiffness curves and a strain limit were observed. The spatial periodicity of inflections at SLs beyond the strain limit is listed as well. Because successive stiffness values were usually measured at 0.1-0.2 ~m SL intervals, any SL value considered here was taken as a centroid coordinate, obtained from the SL values of two successive recordings. Average from 13 experiments at an oscillation frequency of 500 Hz

SL at first SL at the Extent of inflections above the strain limit SL slope change strain limit (mean + SD) ([~m) (mean + SD) (mean + SD) (~m) (~m) Inflection 1 Inflection 2 Inflection 3

2.69 + 0.10 2.99 + 0.12 0.39 + 0.09 0.38 + 0.11 0.37 + 0.15 (n = 11) (n = 13) (n = 9) (n = 9) (n = 4)

Cardiac titin extension 435

Irving, 1995). Even in a single cell, however, many structures can support passive tension, and potential contributors must be eliminated by chemical inter- ventions. Since the specificity of such interventions is still unclear, conclusions about titin elasticity can only be drawn indirectly. In a single cardiac myofibril, however, can titin's mechanical properties be mea- sured more directly, for such preparation lacks any extramyofibrillar structures associated with larger cardiac specimens. And such myofibril preparation has been amply shown to exhibit structural and functional properties similar to those of larger, conventional cardiac muscle preparations (Linke et al., 1994).

In the present study, we addressed the issue of specimen quality further. We investigated protein preservation in isolated myofibrils. In 12% SDS gels, we found that the proteins were well preserved, even after 1 week of glycerination (Fig. 3A). In 2% gels, titin migrated as a doublet band, as has generally been reported, with the upper band (titin 1) thought to relate to the native form of titin and the lower band (titin 2) to a proteolytically degraded form that in SDS-gels coexists with the intact titin (e.g., Maruyama, 1994; Granzier & Irving, 1995). Although, a priori, the presence of a weak titin 2 band in this study does not rule out some titin degradation (Fig. 3A, f-g), the strong intensity of the upper band argues for a good preservation of titin in myofibrils from glycerinated and homogenized mus- cle. The notion of a well-preserved titin was also supported by the results of immunofluorescence microscopic studies, which showed that monoclonal titin antibodies T12 and BD6 avidly labelled single, isolated myofibrils (Fig. 3B). Presumably, the pre- sence of relatively high amounts of leupeptin - a thiol protease inhibitor - in all solutions used in this study helps prevent a measurable titin degradation (cf. Maruyama, 1994). Since single cardiac myofibrils also show strong labelling with other I-band titin antibodies we have been using in recent experiments (Linke et al., 1996), we assume that the functions ascribed to titin in the present study relate to the intact polypeptide.

Segmental extension model of passive force and stiffness The organisation of sarcomeric titin into a stiff (but intrinsically elastic) A-band portion and an extensible I-band portion (Itoh et al., 1988; Trombitas et al., 1991) formed the basis for a segmental extension model of passive tension. This model postulates that during sarcomere stretch, the titin filament acts as a dual- stage molecular spring (Wang et al., 1991). Small to moderate stretch results in extension of the I-band segment and exponential tension rise. At high degrees of stretch, I-band titin reaches its maximum extension capacity, or strain limit, as indicated by a

flattening of the passive tension curve. It was thought that at the strain limit SL, a segment of previously bound A-band titin becomes free and adds to the length of I-band titin ('yield point' or 'yield region'). The maximum extensibility of the elastic I-band titin has been suggested to be four to five times the slack length, in both skeletal and insect indirect flight muscle (Wang et al., 1991; 1993; Granzier & Wang, 1993).

The results of this study show that significant quantitative differences in the passive mechanical properties exist between skeletal and cardiac muscle preparations.

First, both passive force and stiffness of cardiac myofibrils begin to increase at much shorter SLs than in skeletal muscle. This is a consequence of the short I-band length of cardiac muscle sarcomeres (0.2-0.3 ~rn), which in turn is due to a small size and short length of cardiac I-band titin (Suzuki et al., 1993; Labeit & Kotmerer, 1995). In the present study, force and stiffness became detectable at an SL around 2 ~ , but due to resolution limitations, we could not exclude small force responses even below this SL (and above 1.84 pro, the average slack SL). In skeletal muscle, on the other hand, slack sarcomeres are generally longer (range, 2.2-2.5 ~lrn), and within a range of 0.3-0.5 ~'n above slack SL, there is no significant passive force (Wang et al., 1993). Our results indicate that such low-force SL range observed in skeletal muscle appears to be much shorter in cardiac muscle.

A second difference between skeletal and cardiac preparations is the much higher stiffness of cardiac myofibrils, compared with that of skeletal myofibrils at the same SLs. This has been discussed in a recent publication from our laboratory (Linke et al., 1994) and may result from differences in I-band titin length between the two muscle types (Labeit & Kolmerer, 1995).

Third, at the strain limit, the SL is shorter in cardiac myofibrils ( - 3 ~m), compared with skeletal myofibrils (3.8-4.5~un; Wang et al., 1991). Using single, isolated, myofibrils from rabbit psoas muscle, we found in preliminary experiments that a strain limit occured at SLs between 3.5-3.8 j~n, in good agreement with the values found by others (Wang et al., 1993; Granzier & Wang, 1993). As for cardiac preparations, a value similar to that of the present study was recently reported for single rat cardiac myocytes (Granzier & Irving, 1995): in those pre- parations, titin-based stiffness reached a maximum at about 2.9 ~ SL, which was interpreted by the authors as the strain limit SL.

Maximum titin extension capacity

As has been done for skeletal muscle, we can calculate in cardiac myofibrils the maximum exten-

436 LINKE et al.

sion capacity of the elastic I-band titin segment at the strain limit SL. For this, we assume, in the first place, a certain slack length of cardiac I-band titin. From A- band length, -1 .6prn and slack SL, 1.84+SD 0.12 ~rn, it follows that the I-band length, including Z-line width, is -0.2-0.3 ~lrn. Similarly, literature values range between 0.2 and 0.35 pro, as inferred from the slack SLs in cardiac cells (Brady, 1991). However, the I-band length of the sarcomere is not the same as the I-band length of the extensible titin segment, because an -100 nm-long portion of I-band titin between the Z-line and the Nl-line is stiff under physiological conditions (Trombitas et al., 1995). Taken together, these data suggest that the extensible I-band titin segment at slack SL, in each half- sarcomere, is at most -0.05 ~rn long. Using this value, we calculate that at the strain limit SL of - 3 pan, the elastic cardiac I-band titin segment is extended by a factor of approximately 10. This value is much higher than that suggested for the maximum extensibility of skeletal I-band titin (four to five times the slack I-band length). It is also higher than the value calculated by Granzier and Irving (1995) for rat cardiac muscle (-fourfold titin extension at the strain limit). That number, however, was corrected in a more recent paper from the same laboratory, in light of new electron microscopic findings, and was assumed to be ~8 (Trombitas et al., 1995), a value closer to that calculated by us. From these calcula- tions, it appears that skeletal and cardiac muscle indeed differ in their maximum I-band titin extensi- bilities. Our results thus do not support the hypoth- esis that different striated muscle types have a similar maximum I-band titin extension capacity (Wang et al., 1993; Granzier & Wang, 1993). Such conclusion is not surprising when the recently reported primary structure of I-band titin is considered: this structure differs greatly in cardiac and skeletal muscle and is also much more heterogeneous than previously assumed (Labeit & Kolmerer, 1995).

To explain the apparently high relative extensi- bility of cardiac titin, the following scenario can be envisioned: I-band extension could be brought about by stretch of two distinctly different I-band titin regions in series, one consisting of tandem immuno- globulin domains, the other comprising the PEVK element, a sequence rich in proline (P), glutamate (E), valine (V), and lysine (K) residues (Labeit & Kolmerer, 1995). At slack SL, both regions might be flaccid, while small stretch could induce straighten- ing of the Ig domain region (Erickson, 1994). At those shorter SLs, the PEVK element might also begin to extend, which may account for the onset of an exponential passive tension rise. Further stretch perhaps induces extension of immunoglobulin do- mains, which unfold (Soteriou et al., 1993a) until they reach their maximum extension capacity,

suggested to be approximately seven times their slack length (Erickson, 1994). Taken together, straigh- tening and extension of the immunoglobulin and the PEVK regions could potentially explain an 8-10-fold extension of the elastic I-band titin segment at the strain limit. However, it must be reminded that in cardiac muscle, SLs above -2.4 ~ do not occur in vivo (Rodriguez et al., 1992), so that under physio- logical conditions, immunoglobulin domains may not unfold appreciably.

The higher relative extension capacity of cardiac titin, compared with that of skeletal titin, suggests that in cardiac muscle, titin domains are perhaps completely folded at the slack SL and fully extended at the strain limit SL. In contrast, skeletal muscle titin domains may be somewhat extended already at slack SL, perhaps due to the presence of nebulin in this muscle type, so that the maximum I-segment extension ratio at the strain limit is lower than in cardiac sarcomeres. Alternatively, cardiac titin could bind stronger than skeletal titin to other sarcomeric proteins, such as thin filament proteins at the N1- line and thick filament proteins at the A/ I junction (Funatsu et al., 1993), so that the titin filament can be stressed to a higher degree than in skeletal muscle. This latter assumption is supported by the fact that the passive force level at the strain limit SL is higher in cardiac myofibrils, relative to that in skeletal myofibrils (Bartoo et al., 1993; Linke et al., 1994). It is thus reasonable to assume that intrinsically, cardiac and skeletal titins have different maximum passive- force bearing capacity.

In conclusion, the segmental titin extension hy- pothesis can account for part, but not all, of this study's findings. While both the SL-passive force and the SL-stiffness curves of cardiac myofibrils flatten at the strain limit SL - just as they do in skeletal myofibrils - the maximum I-band titin extensibility at the strain limit appears to be greater in cardiac than in skeletal myofibrils. This difference could hint at the possibility that cardiac titin is potentially able to bear higher stresses than skeletal titin. It remains to be established, how such differences in the stress-bearing capacity might be brought about.

Differences between passive force and stiffness

Although the overall shape of the SL-force and the SL-stiffness curves was similar, we observed a major difference between both curve types: beyond the strain limit SL, passive force generally continued to increase, albeit less steeply than at shorter SLs (Fig. 6A), whereas stiffness usually remained on a plateau (Fig. 7). Comparable differences between passive force and stiffness have also been observed in rat cardiac cells (Granzier & Irving, 1995). In those cells, stiffness approached a plateau at -2.9 pm SL,

Cardiac titin extension 437

while passive force continued to increase up to an SL of approximately 4 prn. In contrast to the present study, however, the authors did not observe a distinct decrease in the slope of the SL-force curve at SLs near 3 prn. This might be related to the fact that Granzier and Irving (1995) measured passive force during continuous cell stretch, so that a steady- state force level (following stress relaxation) was never obtained. On the whole, however, it appears that in both rabbit (this study) and rat (Granzier & Irving, 1995) cardiac myofibrils above the strain limit SL, the shape of the stiffness curve differs from that of the passive force curve. A possible explanation might be that even in a single myofibril, the population of titin filaments is not entirely homo- geneous. Therefore, during a slow stretch, such as that applied during passive force measurements, some titin filaments may be able to sustain higher forces (stress) than others, so that the SL-force curve never shows a true plateau. In contrast, during rapid sinusoidal oscillations used to estimate stiffness, titin filaments may 'pop off' relatively uniformly in response to the high tensile forces, so that a more average population is measured, and the SL-stiffness curve becomes flat.

Curve inflections above the strain limit SL

Beyond the strain limit, both the passive force and the stiffness curves frequently exhibited a series of inflections, spaced at average intervals of -0.4 pan. Evidence for these inflections was particularly clear in the case of the stiffness curves (Fig. 8, Table 1). At an earlier stage of this study, we assumed that the distinct spatial periodicity of the inflections might hint at the presence of regularly spaced titin-thick filament binding sites, which upon extreme stretch, could become disrupted in a stepwise fashion. This assumption had been supported by the fact that both light meromyosin and C-protein have titin binding capacity (Fiirst et al., 1992; Labeit et al., 1992; Soteriou et al., 1993b) and, like titin in the C-zone of the A- band, exhibit longitudinally repetitive structures, spaced --43 nm apart (F/irst et al., 1989). However, we later considered a common basis for both the 43 nm periodicity of titin-thick filament bonds and the observed spatial periodicity of the inflections unlikely, since from the latter, we would have calculated the distance between two titin-thick fila- ment bonds to be near 20 nm, about half the expected value (as follows from the value of 8-10 for titin's maximum extensibility). Thus, the inflections did not appear to arise from disruption of distinct, regularly spaced, sites of titin binding to the thick filament.

The results of immunofluorescence measurements (Fig. 6B) instead suggested that the inflections occurred due to sudden release of relatively large portions of A-band titin into the I-band region. Such

release was observed in a given sarcomere stretched to shortly above strain limit length, and was subsequently seen in other sarcomeres, once they had apparently reached a critical force level. From such progressing A-band titin release, inflections of both the force and the stiffness curves can be anticipated, since addition of A-band segments, which have been shown to possess intrinsic elastic properties similar to those of I-band titin (Wang et al., 1993), to the elastic titin portion affects the stiffness of the sarcomere. The appearance of A-band titin release in only one or two sarcomeres at a given SL might also explain why the inflections occurred with a certain spatial periodicity.

In summary, this study allows the following principal conclusions: (1) the passive force and stiffness curves of cardiac myofibrils are likely to represent the mechanical properties of titin (con- nectin) filaments; (2) cardiac myofibrils reach a strain limit at shorter SLs than skeletal myofibrils (-3.0 pan versus 3.6-4.5/am); (3) at the strain limit, the elastic I-band segment of cardiac titin is extended by a factor approximately two times larger than that reported for skeletal titin; and (4) above the strain limit SL, previously bound segments of A-band titin are released into the I-band, thereby affecting passive force of the sarcomere.

Acknowledgements

We would like to thank John Myers and Jeff Magula (Seattle) for expert technical assistance. We are also grateful to Dr J. Trinick (Bristol, UK) for kindly supplying the BD6 antibody. The authors also thank Professor J. C. R/iegg (Heidelberg), Professor G. Pfitzer (Berlin), and Dr A. Steusloff (Berlin) for helpful discussions and Drs S. Labeit and B. Kolmerer (EMBL Heidelberg) for communicating unpublished results.

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