Hystereses in the force-length relation and regulation of cross-bridge recruitment in tetanized rat trabeculae

Various mechanisms have been suggested to explain cardiac force-length Ca2+ relations. The existence of a cooperativity mechanism, whereby cross-bridge (XB) recruitment is affected by the number of active XBs, suggests that the force response to length oscillations should lag length oscillations. Consequently, the oscillatory force response should be larger during shortening than during lengthening. To test this prediction, force responses to large-sarcomere length (SL) oscillations (36.7 +/- 16.0 nm) at different SLs (n = 6) and frequencies (n = 7) were studied in intact tetanized trabeculae dissected from rat right ventricle (n = 13). Stable tetani were obtained by utilizing 30 microM cyclopiazonic acid in Krebs-Henseleit solution containing 6 mM extracellular Ca(2+) at 25 degrees C. SL was measured by laser diffraction techniques (Dalsa). Force was measured by silicone strain gauge. Instantaneous dynamic stiffness during large oscillations was measured by superimposing additional fast (50 or 200 Hz) and small-amplitude (2.25 +/- 0.25 nm) oscillations. The force responses lagged the SL oscillations at slow frequencies (112 +/- 41 ms at 1 Hz), and counterclockwise hystereses were obtained in the force-length plane: the force was higher during shortening than during lengthening. The delay in the force response decreased as the frequency of the SL oscillation was increased. Clockwise hysteresis, where the force preceded the SL, was obtained at frequencies >4 Hz. Similar hysteresis characteristics were obtained in the force-SL and stiffness-SL planes. Maximal lag was observed at the shortest SL, and the delay decreased with sarcomere elongation: 131.1 +/- 31.7 ms at 1.78 +/- 0.03 microm vs. 14.7 +/- 18.5 ms at 1.99 +/- 0.015 microm. The results establish the ability of cardiac fiber to adapt XB recruitment to changes in prevailing loading conditions. This study supports the stipulated existence of a cooperativity mechanism that regulates XB recruitment and highlights an additional method to characterize regulation of the force-length relation.     

Exercise training alters length dependence of contractile properties in rat myocardium.

Myocardial function is enhanced by endurance exercise training, but the cellular mechanisms underlying this improved function remain unclear. Exercise training increases the sensitivity of rat cardiac myocytes to activation by Ca(2+), and this Ca(2+) sensitivity has been shown to be highly dependent on sarcomere length. We tested the hypothesis that exercise training increases this length dependence in cardiac myocytes. Female Sprague-Dawley rats were divided into sedentary control (C) and exercise-trained (T) groups. The T rats underwent 11 wk of progressive treadmill exercise. Heart weight increased by 14% in T compared with C rats, and plantaris muscle citrate synthase activity showed a 39% increase with training. Steady-state tension was determined in permeabilized myocytes by using solutions of various Ca(2+) concentration (pCa), and tension-pCa curves were generated at two different sarcomere lengths for each myocyte (1.9 and 2.3 microm). We found an increased sarcomere length dependence of both maximal tension and pCa(50) (the Ca(2+) concentration giving 50% of maximal tension) in T compared with C myocytes. The DeltapCa(50) between the long and short sarcomere length was 0.084 +/- 0.023 (mean +/- SD) in myocytes from C hearts compared with 0.132 +/- 0.014 in myocytes from T hearts (n = 50 myocytes per group). The Deltamaximal tension was 5.11 +/- 1.42 kN/m(2) in C myocytes and 9.01 +/- 1.28 in T myocytes. We conclude that exercise training increases the length dependence of maximal and submaximal tension in cardiac myocytes, and this change may underlie, at least in part, training-induced enhancement of myocardial function.     

Structure and function of skeletal muscle in zebrafish early larvae

Zebrafish muscles were examined at an early developmental stage (larvae 5-7 d). Using aluminum clips, preparations (approximately 1.5 mm length, 150 microm diameter) were mounted for force registration and small angle x-ray diffraction. Sarcomeres were oriented mainly in parallel with the preparation long axis. Electrical stimulation elicited fast and reproducible single twitch contractions. Length-force relations showed an optimal sarcomere length of 2.15 microm. X-ray diffraction revealed clear equatorial 1.1/1.0 reflections, showing that myofilaments are predominantly arranged along the preparation long axis. In contrast, reflections from older (2 mo) zebrafish showed two main filament orientations each at an approximately 25 degrees angle relative to the preparation long axis. Electrical stimulation of larvae muscles increased the 1.1/1.0 intensity ratio, reflecting mass transfer to thin filaments during contraction. The apparent lattice volume was 3.42 x 10(-3) microm(3), which is smaller than that of mammalian striated muscle and more similar to that of frog muscles. The relation between force and stimulation frequency showed fusion of responses at a comparatively high frequency (approximately 186 Hz), reflecting a fast muscle phenotype. Inhibition of fast myosin with N-benzyl-p-toluene sulphonamide (BTS) showed that the later phase of the tetanus was less affected than the initial peak. This suggests that, although the main contractile phenotype is fast, slow twitch fibers can contribute to sustained contraction. A fatigue stimulation protocol with repeated 220 ms/186 Hz tetani showed that tetanic force decreased to 50% at a train rate of 0.1 s(-1). In conclusion, zebrafish larvae muscles can be examined in vitro using mechanical and x-ray methods. The muscles and myofilaments are mainly orientated in parallel with the larvae long axis and exhibit a significant fast contractile component. Sustained contractions can also involve a small contribution from slower muscle types.     

Stretch and quick release of rat cardiac trabeculae accelerates Ca2+ waves and triggered propagated contractions

Rapid shortening of active cardiac muscle [quick release (QR)] dissociates Ca2+ from myofilaments. We studied, using muscle stretches and QR, whether Ca2+ dissociation affects triggered propagated contractions (TPCs) and Ca2+ waves. The intracellular Ca2+ concentration was measured by a SIT camera in right ventricular trabeculae dissected from rat hearts loaded with fura 2 salt, force was measured by a silicon strain gauge, and sarcomere length was measured by laser diffraction while a servomotor controlled muscle length. TPCs (n = 27) were induced at 28 degrees C by stimulus trains (7.5 s at 2.65 +/- 0.13 Hz) at an extracellular Ca2+ concentration ([Ca2+]o) = 2.0 mM or with 10 microM Gd3+ at [Ca2+]o = 5.2 +/- 0.73 mM. QR during twitch relaxation after a 10% stretch for 100-200 ms reduced both the time between the last stimulus and the peak TPC (PeakTPC) and the time between the last stimulus and peak Ca2+ wave (PeakCW) and increased PeakTPC and PeakCW (n = 13) as well as the propagation velocity (Vprop; n = 8). Active force during stretch also increased Vprop (r = 0.84, n = 12, P < 0.01), but Gd3+ had no effect (n = 5). These results suggest that Ca2+ dissociation by QR during relaxation accelerates the initiation and propagation of Ca2+ waves.     

Sarcomere shortening in pressure overload hypertrophy

Sarcomere shortening during contraction was measured by using laser diffraction, in thin, rabbit right ventricular (RV) trabeculae from normal hearts (N) (n = 5) and from hearts subjected to RV pressure overload by pulmonary banding (H) (n = 5). Banding resulted in substantial RV hypertrophy after 2 wk. Hypertrophied preparations had the same resting muscle length (H = 3.15 +/- 0.29 mm) and resting sarcomere lengths (H = 2.16 +/- 0.005 micron) as the normal preparations (3.10 +/- 0.37 mm, 2.16 +/- 0.008 micron, respectively). Total tension at the peak of isometric twitches was the same as normal in the hypertrophied muscles (N = 8.06 +/- 1.20, H = 8.51 +/- 1.95 g/mm2). However, the amount of auxotonic sarcomere shortening was much less than normal in the hypertrophied preparations (N = 0.39 +/- 0.028, H = 0.19 +/- 0.034 micron; P less than 0.001). In isotonic contractions in which the ratio of muscle shortening to resting muscle length was the same in both the normal and hypertrophied muscles (ratio of 0.05 in both groups), the extent of sarcomere shortening relative to resting sarcomere length was less in the hypertrophied muscles than in the normal preparations (N = 0.14 +/- 0.01), H = 0.07 +/- 0.01; P less than 0.01). Series elasticity was the same as normal in the hypertrophied muscle P less than 0.05). Less auxotonic sarcomere shortening for a given level of isometric tension development and less isotonic sarcomere shortening per unit muscle shortening indicate that there is less than normal work per sarcomere during contraction in hypertrophied myocardium. These findings may have important implications for intracellular compensatory adaptation in pressure overload cardiac hypertrophy.     

Conduction of hyperpolarization along hamster feed arteries: augmentation by acetylcholine

The conduction of vasodilation along resistance vessels has been presumed to reflect the electrotonic spread of hyperpolarization from cell to cell along the vessel wall through gap junction channels. However, the vasomotor response to acetylcholine (ACh) encompasses greater distances than can be explained by passive decay. To investigate the underlying mechanism for this behavior, we tested the hypothesis that ACh augments the conduction of hyperpolarization. Feed arteries (n = 23; diameter, 58 +/- 4 microm; segment length, 2-8 mm) were isolated from the hamster retractor muscle, cannulated at each end, and pressurized to 75 mmHg (at 37 degrees C). Vessels were impaled with one or two dye-containing microelectrodes simultaneously (separation distance, 50 microm to 3.5 mm). Membrane potential (E(m)) (rest, approximately -30 mV) and electrical responses were similar between endothelium and smooth muscle, as predicted for robust myoendothelial coupling. Current injection (-0.8 nA, 1.5 s) evoked hyperpolarization (-10 +/- 1 mV; membrane time constant, 240 ms) that conducted along the vessel with a length constant (lambda) = 1.2 +/- 0.1 mm; spontaneous E(m) oscillations (approximately 1 Hz) decayed with lambda = 1.2 + 0.1 mm. In contrast, ACh microiontophoresis (500 nA, 500 ms, 1 microm tip) evoked hyperpolarization (-14 +/- 2 mV) that conducted with lambda = 1.9 +/- 0.1 mm, 60% further (P < 0.05) than responses evoked by purely electrical stimuli. These findings indicate that ACh augments the conduction of hyperpolarization along the vessel wall.     

Time-resolved X-ray diffraction by skinned skeletal muscle fibers during activation and shortening

Force, sarcomere length, and equatorial x-ray reflections (using synchrotron radiation) were studied in chemically skinned bundles of fibers from Rana temporaria sartorius muscle, activated by UV flash photolysis of a new photolabile calcium chelator, NP-EGTA. Experiments were performed with or without compression by 3% dextran at 4 degrees C. Isometric tension developed at a similar rate (t(1/2) = 40 +/- 5 ms) to the development of tetanic tension measured in other studies (Cecchi et al., 1991). Changes in intensity of equatorial reflections (I(11) t(1/2), 15-19 ms; I(10) t(1/2), 24-26 ms) led isometric tension development and were faster than for tetanus. During shortening at 0.14P(o), I(10) and I(11) changes were partially reversed (18% and 30%, respectively, compressed lattice), in agreement with intact cell data. In zero dextran, activation caused a compression of A-band lattice spacing by 0.7 nm. In 3% dextran, activation caused an expansion of 1.4 nm, consistent with an equilibrium spacing of 45 nm. But, in both cases, discharge of isometric tension by shortening caused a rapid lattice expansion of 1.0-1.1 nm, suggesting discharge of a compressive cross-bridge force, with or without compression by dextran, and the development of an additional expansive force during activation. In contrast to I(10) and I(11) data, these findings for lattice spacing did not resemble intact fiber data.     

The amplitude and time course of the myoplasmic free [Ca2+] transient in fast-twitch fibers of mouse muscle

Bundles of 10-100 fibers were dissected from the extensor digitorum longus muscle of mouse, mounted in an apparatus for optical recording, and stretched to long sarcomere length (> or = 3.6 microns). One fiber within the bundle was microinjected with furaptra, a fluorescent indicator that responds rapidly to changes in myoplasmic free [Ca2+] (delta [Ca2+]). Twitches and brief tetani were initiated by external stimulation. At myoplasmic furaptra concentrations of approximately 0.1 mM, the indicator's fluorescence signal during fiber activity (delta F/F) was well resolved. delta F/F was converted to delta [Ca2+] under the assumption that furaptra's myoplasmic dissociation constant for Ca2+ is 98 microM at 16 degrees C and 109 microM at 28 degrees C. At 16 degrees C, the peak amplitude of delta [Ca2+] during a twitch was 17.8 +/- 0.4 microM (+/-SEM; n = 8) and the half-width of delta [Ca2+] was 4.6 +/- 0.3 ms. At 28 degrees C, the peak and half-width values were 22.1 +/- 1.8 microM and 2.0 +/- 0.1 ms, respectively (n = 4). During a brief high-frequency tetanus, individual peaks of delta [Ca2+] were also well resolved and reached approximately the same amplitude that resulted from a single shock; the initial decays of delta [Ca2+] from peak slowed substantially during the tetanus. For a single twitch at 16 degrees C, the amplitude of delta [Ca2+] in fast-twitch fibers of mouse is not significantly different from that recently measured in fast- twitch fibers of frog (16.5 +/- 0.9 microM; Zhao, M., S. Hollingworth, and S.M. Baylor. 1996. Biophys. J. 70:896-916); in contrast, the half- width of delta [Ca2+] is surprisingly brief in mouse fibers, only about half that measured in frog (9.6 +/- 0.6 ms). The estimated peak rate at which Ca2+ is released from the sarcoplasmic reticulum in response to an action potential is also similar in mouse and frog, 140-150 microM/ms (16 degrees C).     

Length-tension relationship of the external anal sphincter muscle: implications for the anal canal function

The length at which a muscle operates in vivo (operational length) and the length at which it generates maximal force (optimal length) may be quite different. We studied active and passive length-tension characteristics of external anal sphincter (EAS) in vivo and in vitro to determine the optimal and operational length of rabbit EAS. For the in vitro studies, rings of EAS (n = 4) were prepared and studied in a muscle bath under isometric conditions. For in vivo studies, female rabbits (n = 19) were anesthetized and anal canal pressure was recorded by use of a sleeve sensor placed in the custom-designed catheter holders of 4.5-, 6-, and 9-mm diameters. Measurements were obtained at rest and during EAS electrical stimulation. Sarcomere length of EAS muscle was measured by laser diffraction technique with no probe and three probes in the anal canal. In vitro studies revealed 2,054 mN/cm(2) active tension at optimal length. In vivo studies revealed a probe size-dependent increase in anal canal pressure and tension. Maximal increase in anal canal tension with stimulation was recorded with the 9-mm probe. Increases in anal canal tension with increase in probe size were completely abolished by pancuronium bromide. EAS muscle sarcomere length without and with 9-mm probe in the anal canal were 2.11 +/- 0.08 and 2.99 +/- 0.07 microm, respectively. Optimal sarcomere length, based on the thin filament length measured by thin filament analysis, is 2.44 +/- 0.10 microm. These data show that the operational length of EAS is significantly shorter than its optimal length. Our findings provide insight into EAS function and we propose the possibility of increasing anal canal pressure by surgical manipulation of the EAS sarcomere length.     

The variation of characteristics of twitch and tetanic contractions with sarcomere length in isolated muscle fibres of the frog.

The relation between sarcomere length, tension and time course of tension development in twitch and tetanic contractions at 20 degrees C was determined for isolated fibres from the semitendinosus muscle of the frog (Rana esculenta). In twenty fibres at about 2.15 micron sarcomere length, the peak twitch tension, the maximum tetanic tension and the twitch/tetanus ratio ranged, respectively, from 0.22 to 1.6 kg/cm2, from 2.13 o 3.96 kg/cm2 an from 0.07 to 0.53. The peak twitch tension was found to be: i) directly correlated with the twitch/tetanus ratio and the time to the peak of the first derivative of the twitch tension, ii) inversely correlated with the time to the peak of the first derivative of tetanic tension. No significant correlation was found between the maximal tetanic tension and the peak twitch tension or the twitch/tetanus ratio. Peak twitch tension and twitch/tetanus ratio were not correlated with the fibre cross-sectional area which ranged from 1.052 to 6,283 micron2. Sarcomere length-tension curves for twitch and tetanic isometric contractions at 20 degrees C were determined in twelve fibres. Increases in sarcomere length from about 2.15 to 2.85 micron produced, depending on the peak twitch tension or the twitch/tetanus ratio at about 2.15 micron, either decrease and no change or increase in peak twitch tension, but constantly enhanced the twitch/tetanus ratio and the degree of this potentiation was inversely correlated with the twitch/tetanus ratio at 2.15 micron. Increase in sarcomere length above 2.15 micron did not alter the course of the early development of twitch and tetanic tensions, reduced considerably the variation in peak twitch tension and twitch/tetanus ratio, without altering that of tetanic tension and swamped the correlation between the peak twitch tension and the time to peak of the differentiated twitch tension. However, the peak twitch tension at about 2.85 micron resulted to be directly correlated with the peak twitch tension at about 2.15 micron and in addition the relative length-dependent change in the time of the peak of the first derivative of the twitch tension resulted to be directly correlated with the relative length-dependent change in the peak twitch tension. It is concluded that both the duration of the active state and the rate factors of activation contribute to the determining of the large variation in peak twitch tension at about 2.15 micron, whereas the length-dependent increase in twitch/tetanus ratio appears to be mainly determined by prolongation of the active state duration.     

Effects of passive tension on unloaded shortening speed of frog single muscle fibers.

Experiments were performed to determine the influence of sarcomere length and passive tension on the velocity of unloaded shortening (Vu) as measured by the slack test technique. Slack test results were obtained from intact twitch fibers isolated from the frog (Rana temporaria). Measurements were made both in the absence and presence of passive tension using two different protocols. In one, all releases were initiated from the same sarcomere length and passive tension level; in the other, all releases ended at the same sarcomere length. In the absence of passive tension, no difference was observed between the results from the two slack test protocols. When passive tension was present, performing all releases from the same initial sarcomere length and passive tension level resulted in linear step size-slack time relationships in which the slopes (Vu) were independent of length over a sarcomere length range extending to 3.1 microns, and the intercepts increased with increasing sarcomere length. Performing all releases to the same final sarcomere length in the presence of passive tension produced nonlinear step size-slack time relationships. The results presented here show that, in the presence of significant levels of passive tension, the traditional interpretation of the slope of the slack test plot as the constant unloaded shortening velocity is only correct when all length steps are initiated from the same initial sarcomere length and level of passive tension.     

Time-resolved X-ray diffraction studies of frog skeletal muscle isometrically twitched by two successive stimuli using synchrotron radiation

In order to clarify the delay between muscular structural changes and mechanical responses, the intensity changes of the equatorial and myosin layer-line reflections were studied by a time-resolved X-ray diffraction technique using synchrotron radiation. The muscle was stimulated at 12-13 degrees C by two successive stimuli at an interval (80-100 ms) during which the second twitch started while tension was still being exerted by the muscle. At the first twitch, the intensity changes of the 1.0 and 1.1 equatorial reflections reached 65 and 200% of the resting values, and further changes to 55 and 220% were seen at the second twitch, respectively. Although the second twitch decreased not only the time to peak tension but also that to the maximum intensity changes of the equatorial reflections (in both cases, about 15 ms), the delay (about 20 ms) between the intensity changes and the development of tension at the first twitch were still observed at the second twitch. On the other hand, the intensities of the 42.9 nm off-meridional and the 21.5 nm meridional myosin reflections decreased at the first twitch to the levels found when a muscle was isometrically tetanized, and no further decrease in their intensities was observed at the second twitch. These results indicate that a certain period of time is necessary for myosin heads to contribute to tension development after their arrival in the vicinity of the thin filaments during contraction.     

Influence of temperature on the calcium sensitivity of the myofilaments of skinned ventricular muscle from the rabbit

The steady-state myofilament Ca sensitivity was determined in skinned cardiac trabeculae from the rabbit right ventricle (diameter, 0.13-0.34 mm) at 36, 29, 22, 15, 8, and 1 degree C. Muscles were stimulated to 0.5 Hz and stretched to a length at which maximum twitch tension was generated. The preparation was then skinned with 1% vol/vol Triton X-100 in a relaxing medium (10 mM EGTA, pCa 9.0). Each preparation was exposed to a series of Ca-containing solutions (pCa 6.3-4.0) at two of the six temperatures studied (temperature was regulated to +/- 0.1 degree C). The pCa values (mean +/- SD, n = 6) corresponding to half maximal tension at 36, 29, 22, 15, 8, and 1 degree C were 5.47 +/- 0.07, 5.49 +/- 0.07, 5.34 +/- 0.05, 5.26 +/- 0.09, 4.93 +/- 0.06, and 4.73 +/- 0.04, respectively. Mean (+/- SD) maximum tension (Cmax) developed by the preparation as a percentage of that at 22 degrees C was 118 +/- 10, 108 +/- 5, 74 +/- 6, 57 +/- 7, and 29 +/- 5% at 36, 29, 15, 8, and 1 degree C, respectively. As cooling led to a shift of Ca sensitivity towards higher [Ca2+] and a reduction of Cmax, the Ca sensitivity curves over this range of temperatures do not cross over as has been described for canine Purkinje fibers (Fabiato 1985). Since tension is decreased by cooling at all levels of [Ca2+] it is unlikely that changes in myofilament Ca sensitivity play a role in the large hypothermic inotropy seen in rabbit ventricular muscle. The increase in sensitivity of the myofilaments to Ca on warming from 1 to 29 degrees C might be related to the increase in force seen on rewarming from a rapid cooling contracture in intact rabbit ventricular muscle.     

Spontaneous contractions in rat cardiac trabeculae. Trigger mechanism and propagation velocity

It has previously been observed that spontaneous contractions start in a region of damage of isolated right ventricular trabeculae of rat, propagate along the muscle, and induce triggered arrhythmias (Mulder, B.J.M., P.P. de Tombe, and H.E.D.J. ter Keurs. 1989. J. Gen. Physiol. 93:943-961). The present study was designed to analyze the mechanisms that lead to triggered propagated contractions (TPCs). TPCs were elicited in 29 trabeculae by stimulation with trains (2 Hz; 15-s intervals) at varied number of stimuli (n), lowered temperature (19-21 degrees C), and varied [Ca++]o (1.5-4 mM) in the superfusate. Length (SL) and shortening of sarcomeres in the muscle were measured at two sites using laser diffraction techniques; twitch force (Ft) was measured with a silicon strain gauge. Time between the last stimulus in the train and the onset of sarcomere shortening due to a TPC at a site close to the damaged end region (latency) and propagation velocity of the contraction (Vprop) were correlated with Ft. For 10 trabeculae, TPCs were calculated to start in the end region itself 586 +/- 28 ms (mean +/- 1 SEM) after the last stimulus of a train (n = 15; [Ca++]o: 1.5 mM), i.e., at the end of or after the rapid release of the damaged end during twitch relaxation. When Ft was increased by increasing either SL prior to stimulation or the afterload during twitches, methods that do not affect intracellular calcium levels, latency decreased, but Vprop remained constant. No TPC occurred when Ft was less than 20% of maximal Ft. Both increasing [Ca++]o and n increased Ft to a maximum, increased Vprop progressively (maximum Vprop, 17 mm/s), but decreased latency. These observations suggest that initiation of TPCs depends on the force developed by the preceding twitch, and therefore on the degree of stretch and subsequent rapid release of damaged areas in the myocardium, while Vprop along the trabeculae is determined by intracellular calcium concentration.     

Frequency-dependent contractile response of isolated cardiac trabeculae under hypo-, normo-, and hyperthermic conditions.

The body is from time to time exposed to nonnormothermic conditions; both hypo- and hyperthermia can occur as a result of external (environment) or internal (pathogens, allergens) stressors. To preserve life under hypo- and hyperthermic conditions, adequate perfusion of vital organs is mandated. Although cardiac output regulation under hyperthermic conditions has been studied, the mechanical response of basic contractile function of the myocardium itself is incompletely understood. Accordingly, we set out to test mechanical output of isolated myocardium under hyperthermic conditions and to compare the results with the hypo- and normothermic response in the same tissue. We observed that, in absence of a frequency change, developed force decreased markedly. At a physiological normal stimulation rate of 6 Hz, developed force decreases to 67.2 +/- 2.6% at 42 degrees C compared with 37 degrees C. In addition, twitch timing characteristics also accelerate, allowing for a faster relaxation; time from peak tension to 50% relaxation is approximately 23% faster (from 31.4 +/- 2.6 to 24.4 +/- 1.7 ms). Although this faster relaxation in turn prevents a steep increase in diastolic tension at high frequencies, the very fast calcium kinetics now prevent a more complete activation of the myofilaments, resulting in a lower twitch-force maximum at hyperthermic conditions. Even at maximal beta-adrenergic stimulation, developed force is well below levels reached at physiological temperature.     

Comparison of twitch potentiation in the gastrocnemius of young and elderly men

The capacity for twitch potentiation in the gastrocnemius muscle was determined following maximal voluntary contractions (MVC) in 11 elderly (means +/- SD; 66.9 +/- 5.3 years) and 12 young (25.7 +/- 3.8 years) men. Potentiation was observed by applying selective stimulation to the muscle belly, 2 s after a 5 s MVC. With this procedure, both groups showed significant (P less than 0.05) increases in twitch tension in the gastrocnemius (ratios of potentiated twitch to baseline were means = 1.68 +/- 0.40 for young vs means = 1.40 +/- 0.20 for the elderly, P less than 0.001). Time to peak tension of the twitch decreased from means = 101.5 +/- 17.9 ms to means = 88.0 +/- 15.8 ms in the young men following potentiation; the respective values for the older men were 136.7 +/- 17.9 ms and 133.1 +/- 28.6 ms. These changes resulted in a greater rate of tension development in the potentiated state. The elderly gastrocnemius thus showed qualitatively similar changes in the isometric twitch following potentiation, but reduced and prolonged responses in comparison to young adults. Slowed muscle contraction and reduced capacity for potentiation may be physiological correlates of the reported morphological changes in aged skeletal muscle.     

Tension relaxation after stretch in resting mammalian muscle fibers: stretch activation at physiological temperatures.

Tension responses to ramp stretches of 1-3% Lo (fiber length) in amplitude were examined in resting muscle fibers of the rat at temperatures ranging from 10 degrees C to 36 degrees C. Experiments were done using bundles of approximately 10 intact fibers isolated from the extensor digitorum longus (a fast muscle) and the soleus (a slow muscle). At low temperatures (below approximately 20 degrees C), the tension response consisted of an initial rise to a peak during the ramp followed by a complex tension decay to a plateau level; the tension decay occurred at approximately constant sarcomere length. The tension decay after a standard stretch at approximately 3-4.Lo/s contained a fast, an intermediate, and a (small amplitude) slow component, which at 10 degrees C (sarcomere length approximately 2.5 microns) were approximately 2000.s-1, approximately 150.s-1, and approximately 25.s-1 for fast fibers and approximately 2000.s-1, approximately 70.s-1 and approximately 8.s-1 for slow fibers, respectively. The fast component may represent the decay of interfilamentary viscous resistance, and the intermediate component may be due to viscoelasticity in the gap (titin, connectin) filament. The two- to threefold fast-slow muscle difference in the rate of passive tension relaxation (in the intermediate and the slow components) compares with previously reported differences in the speed of their active contractions; this suggests that "passive viscoelasticity" is appropriately matched to contraction speed in different muscle fiber types. At approximately 35 degrees C, the fast and intermediate components of tension relaxation were followed by a delayed tension rise at approximately 10.s-1 (fast fibers) and 2.5.s-1 (slow fibers); the delayed tension rise was accompanied by sarcomere shortening. BDM (5-10 mM) reduced the active twitch and tetanic tension responses and the delayed tension rise at 35 degrees C; the results indicate stretch sensitive activation in mammalian sarcomeres at physiological temperatures.     

Myofilament-generated tension oscillations during partial calcium activation and activation dependence of the sarcomere length-tension relation of skinned cardiac cells

During partial Ca2+ activation, skinned cardiac cells with sarcoplasmic reticulum destroyed by detergent developed spontaneous tension oscillations consisting of cycles (0.1-1 Hz) of rapid decrease of tension corresponding to the yield of some sarcomeres and slow redevelopment of tension corresponding to the reshortening of these sarcomeres. Such myofilament-generated tension oscillations were never observed during the full activation induced by a saturating [free Ca2+] or during the rigor tension induced by decreasing [MgATP] in the absence of free Ca2+ or when the mean sarcomere length (SL) of the preparation was greater than 3.10 microm during partial Ca2+ activation. A stiff parallel elastic element borne by a structure that could be digested by elastase hindered the study of the SL--active tension diagram in 8-13-microm-wide skinned cells from the rat ventricle, but this study was possible in 2-7-microm-wide myofibril bundles from the frog or dog ventricle. During rigor the tension decreased linearly when SL was increased from 2.35 to 3.80 microm. During full Ca2+ activation the tension decreased by less than 20% when SL was increased from 2.35 to approximately 3.10 microm. During partial Ca2+ activation the tension increased when SL was increased from 2.35 to 3.00 microm. From this observation of an apparent increase in the sensitivity of the myofilaments to Ca2+ induced by increasing SL during partial Ca2+ activation, a model was proposed that describes the tension oscillations and permits the derivation of the maximal velocity of shortening (Vmax). Vmax was increased by increasing [free Ca2+] or decreasing [free Mg2+] but not by increasing SL.     

A non-cross-bridge stiffness in activated frog muscle fibers

Force responses to fast ramp stretches of various amplitude and velocity, applied during tetanic contractions, were measured in single intact fibers from frog tibialis anterior muscle. Experiments were performed at 14 degrees C at approximately 2.1 microm sarcomere length on fibers bathed in Ringer's solution containing various concentrations of 2,3-butanedione monoxime (BDM) to greatly reduce the isometric tension. The fast tension transient produced by the stretch was followed by a period, lasting until relaxation, during which the tension remained constant to a value that greatly exceeded the isometric tension. The excess of tension was termed "static tension," and the ratio between the force and the accompanying sarcomere length change was termed "static stiffness." The static stiffness was independent of the active tension developed by the fiber, and independent of stretch amplitude and stretching velocity in the whole range tested; it increased with sarcomere length in the range 2.1-2.8 microm, to decrease again at longer lengths. Static stiffness increased well ahead of tension during the tetanus rise, and fell ahead of tension during relaxation. These results suggest that activation increased the stiffness of some sarcomeric structure(s) outside the cross-bridges.     

Myofilament sensitivity to Ca2+ in ventricular myocytes from the Goto–Kakizaki diabetic rat

The chronic effects of type 2 diabetes mellitus on myofilament sensitivity to Ca(2+) in ventricular myocytes from the Goto-Kakizaki (GK) rat have been investigated. Experiments were performed in ventricular myocytes isolated from 17-month GK rats and age-matched Wistar controls. Myocytes were loaded with fura-2 (an indicator for intracellular Ca(2+) concentration) and the fura-2 ratio (340/380 nm), and shortening were measured simultaneously in electrically stimulated myocytes. Myofilament sensitivity to Ca(2+) was assessed from phase-plane diagrams of fura-2 versus cell length by measuring the gradient of the fura-2-cell length trajectory during late relaxation of the twitch contraction. Non-fasting and fasting blood glucose were elevated in GK rats compared to controls. Fasting blood glucose was 151.5 +/- 15.3 mg/dl (n = 8) in GK rats compared to 72.1 +/- 3.6 mg/dl (n = 9) in controls. At 120 min after intraperitoneal injection of glucose (2 g/kg body weight), blood glucose was 570.8 +/- 36.8 mg/dl in GK rats compared to 148 +/- 8.6 mg/dl in controls. Amplitude of shortening was significantly increased in myocytes from GK rats (6.56 +/- 0.54%, n = 31) compared to controls (5.05 +/- 0.43%, n = 36), and the amplitude of the Ca(2+) transient was decreased in myocytes from GK rats (0.23 +/- 0.02 RU, n = 31) compared to controls (0.30 +/- 0.02 RU, n = 36). The fura-2-cell length trajectory during the late stages of relaxation of the twitch contraction was steeper in myocytes from GK rats (89.2 +/- 16.6 microm/RU, n = 27) compared to controls (31.9 +/- 5.9 microm/RU, n = 35). Increased amplitude of shortening, accompanied by a decrease in amplitude of the Ca(2+) transient, might be explained by an increased myofilament sensitivity to Ca(2+).