The Active State of Mammalian Skeletal Muscle

A new technique is proposed for computing the active state of striated muscle, based on the three component model of Fenn and Marsh (8) and of Hill (7). The method permits calculation of the time course of the active state from its peak to the time at which maximum isometric twitch tension is reached. The intormation required for the calculation can be obtained from a single muscle without moving it from its mount in the lever system. The time course of the active state proved to be a function of the length of the muscle. This length dependency led to the predictions that (a) the length at which maximum force is developed during tetanic stimulation is different from that at which it is developed during a twitch, and (b) the tetanus-twitch tension ratio is a function of length. Both predictions were verified in a series of experiments on the rat gracilis anticus muscle at 17.5°C.     

Effect of stretch and release on equatorial X-ray diffraction during a twitch contraction of frog skeletal muscle.

Time-resolved intensity measurements of the x-ray equatorial reflections were made during twitch contractions of frog skeletal muscles, to which stretches or releases were applied at various times. A ramp stretch applied at the onset of a twitch (duration, 15 ms; amplitude, approximately 3% of muscle length) caused a faster and larger development of contractile force than in an isometric twitch. The stretch accelerated the decrease of the 1.0 reflection intensity (I1,0). The magnitude of increase of the 1,1 reflection intensity (I1,1) was reduced by the stretch, but its time course was also accelerated. A release applied at the peak of a twitch or later (duration, 5 ms; amplitude, approximately 1.5%) caused only a partial redevelopment of tension. The release produced clear reciprocal changes of reflections toward their relaxed levels, i.e., the I1,0 increased and the I1,1 decreased. A release applied earlier than the twitch peak had smaller effects on the reflection intensities. The results suggest that a strength applied at the onset of a twitch causes a faster radial movement of the myosin heads toward actin, whereas a release applied at or later than the peak of a twitch accelerates their return to the thick filament backbone. The results are discussed in the context of the regulation of the myosin head attachment by calcium.     

Isometric contractile properties and instantaneous stiffness of amphibian skeletal muscle in the temperature range from 0 to 20 degrees C

The isometric contractile properties of frog (Rana pipiens) and toad (Bufo bufo) sartorii have been studied over the temperature range from 0 to 20 degrees C. The isometric twitch tension was found to vary considerably between these two species and between muscles in the same species. Between 0 and 4 degrees C there was very little change in maximum isometric twitch tension. Between 4 and 12 degrees C several muscles from frog or toad showed a potentiation of twitch tension whereas others showed a decline. Over this temperature range the toad sartorii consistently demonstrated a greater potentiation. By 12 degrees C a steady decline in twitch tension in both muscles was seen as the temperature range the toad sartorii consistently demonstrated a greater potentiation. By 12 degrees C a steady decline in twitch tension in both muscles was seen as the temperature approached 20 degrees C. The maximum isometric tetanic tension recorded between 18 and 20 degrees C increased fractionally to an average of 1.504 +/- 0.029 (n = 4) for frog sartorii and to 1.377 +/- 0.008 (n = 5) for toad sartorii. The time to peak twitch tension and the half-relaxation time decreased markedly with an increase in temperature. Moreover, the half-relaxation time was reduced by a greater proportion than the time to peak twitch tension. Measurements of instantaneous stiffness by controlled velocity releases from the plateau of isometric tetani revealed that the large increase in isometric tetanus tension as the muscle was warmed was not accompanied by a corresponding increase in the total number of active cross-bridges. The possibility that a decreased availability of intracellular Ca2+ ions at the contractile sites contributing to the fall of isometric twitch tension at elevated temperatures is discussed. The possibility exists that at elevated temperatures a change inthe intrinsic contractile ability of the muscle occurs which produces an increased tension per cross-bridge.     

Force-frequency relationship and potentiation in mammalian skeletal muscle.

Repetitive activation of a skeletal muscle results in potentiation of the twitch contractile response. Incompletely fused tetanic contractions similar to those evoked by voluntary activation may also be potentiated by prior activity. We aimed to investigate the role of stimulation frequency on the enhancement of unfused isometric contractions in rat medial gastrocnemius muscles in situ. Muscles set at optimal length were stimulated via the sciatic nerve with 50-micros duration supramaximal pulses. Trials consisted of 8 s of repetitive trains [5 pulses (quintuplets) 2 times per second or 2 pulses (doublets) 5 times per second] at 20, 40, 50, 60, 70, and 80 Hz. These stimulation frequencies represent a range over which voluntary activation would be expected to occur. When the frequency of stimulation was 20, 50, or 70 Hz, the peak active force (highest tension during a contraction - rest tension) of doublet contractions increased from 2.2 +/- 0.2, 4.1 +/- 0.4, and 4.3 +/- 0.5 to 3.1 +/- 0.3, 5.6 +/- 0.4, and 6.1 +/- 0.7 N, respectively. Corresponding measurements for quintuplet contractions increased from 2.2 +/- 0.2, 6.1 +/- 0.5, and 8.7 +/- 0.7 to 3.2 +/- 0.3, 7.3 +/- 0.6, and 9.0 +/- 0.7 N, respectively. Initial peak active force values were 27 +/- 1 and 61.5 +/- 5% of the maximal (tetanic) force for doublet and quintuplet contractions, respectively, at 80 Hz. With doublets, peak active force increased at all stimulation frequencies. With quintuplets, peak active force increased significantly for frequencies up to 60 Hz. Twitch enhancement at the end of the 8 s of repetitive stimulation was the same regardless of the pattern of stimulation during the 8 s, and twitch peak active force returned to prestimulation values by 5 min. These experiments confirm that activity-dependent potentiation is evident during repeated, incompletely fused tetanic contractions over a broad range of frequencies. This observation suggests that, during voluntary motor unit recruitment, derecruitment or decreased firing frequency would be necessary to achieve a fixed (submaximal) target force during repeated isometric contractions over this time period.     

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.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

The Pattern of Activation in the Sartorius Muscle of the Frog

The development of isometric twitch tension has been compared with the redevelopment of isometric tension in the fully active frog sartorius muscle following release. At 0°C the rate of rise of isometric twitch tension is the same as that for the muscle in the fully active state at the same tension but not until about 40 msec. after the stimulus and then only for a few milliseconds. The rates of rise of tension in the twitch and in the redevelopment of tension in the fully active muscle appear to be nearly the same at low tensions. Substitution of nitrate for chloride in the Ringer's solution bathing the muscle retards the development of tension during the early part of the contraction phase of the twitch and the effect reaches a maximum within 3 minutes after changing the solutions. These observations have been discussed in connection with some possible patterns of activation and the hypothesis has been advanced that the rate of activation of a sarcomere is determined mainly by the rate at which the transverse component of the link between excitation and contraction is propagated inwards from the periphery to the center of the fiber. This hypothesis has been discussed in relation to others concerning the nature of excitation-contraction coupling.     

The effect of muscle fatigue on the isometric contractile characteristics of skeletal muscle in the cat

The rise time of an isometric twitch, the tetanic tension, the twitch tetanus ratio, the frequency-tension relationship, and the height of the MUAP (motor unit action potential) were measured in fast twitch (medial gastrocnemius) and slow twitch (soleus) muscles of the cat immediately before, in the middle, and immediately after fatiguing isometric contractions at tensions of 30, 50 and 80% of each muscle's initial strength (tetanic tension recorded from the unfatigued muscle). Although the twitch-tetanus ratio was always less for the soleus than for the medial gastrocnemius muscles, the twitch-tetanus ratio for any one muscle was constant throughout the duration of fatiguing isometric contractions at any of the tensions examined. In contrast, the twitch tension and tetanic tension of the muscles were both less after the contractions, the largest reduction occurring for both muscles during contractions sustained at the lowest isometric tensions. The time to peak tension of an isometric twitch was prolonged for both muscles following the contractions. This was associated with a corresponding shift in the frequency tension relationship such that at the point of muscular fatigue, the muscles tetanized at lower frequencies of stimulation than did the unfatigued muscle. In contrast, the amplitude of the MUAP showed only a modest reduction throughout the duration of the fatiguing contractions.     

Estimation of muscle active state

A method is presented for the estimation of the complete time course of muscle active state. The method is based on the selection of a proper model for the muscle, consisting of linear and non-linear components, and on the estimation of its parameters from a simple experiment. The model's parameters are estimated, using the least square method, from measurements of a tetanized muscle's response to a change of its length. The time course of the active state is calculated from an isometric twitch tension response of the same muscle. The twitch tension response is taken as the system's output, and the active state as its input. The latter can be estimated since the system parameters have already been estimated from the tetanized muscle experiment. Experiments were performed on the gastrocnemius muscle of frogs and cats. Results are given for the whole active state time course of these muscles. The results show that the peak active state force does not reach tetanic value, and a negative force is generated during the relaxation period. Additional experiments were carried out with the purpose of verifying the existence of this force; however, no conclusive results were obtained.This research was supported by the Julius Silver Institute of Bio-Medical Engineering Sciences, Grant 050-304     

Skeletal muscle fatigue in vitro is temperature dependent

Our purpose was to determine the effect of temperature on the fatigability of isolated soleus and extensor digitorum longus (EDL) muscles from rats during repeated isometric contractions. Muscles (70-90 mg) were studied at 20-40 degrees C in vitro. Fatigability was defined with respect to both the time and number of stimuli required to reach 50% of the force (P) developed at the onset of the fatigue test. Fatigue was studied during stimulation protocols of variable [force approximately 70% of maximum force (Po)] and constant frequency (28 Hz). Results for soleus and EDL muscles were qualitatively similar, but fatigue times were longer for soleus than for EDL muscles. During the variable-frequency protocol, development of approximately 70% of Po required an increase in stimulation frequency as temperature increased. During stimulation at these frequencies, fatigue time shortened as temperature increased. For both fatigue protocols, the relationship between temperature and the number of stimuli required to reach fatigue followed a bell-shaped curve, with maximum values at 25-30 degrees C. The temperature optimum for maximizing the number of isometric contractions to reach fatigue reflects direct effects of temperature on muscle function.     

Time-course of force production by fast isometric contraction of the knee extensor in young and elderly subjects

Rapid force production by isometric contraction of the knee extensor was examined in a wide range of force output for 12 healthy elderly (65-86 years) and 12 young (20-35 years) subjects. Time-course of tension development and duration of first burst EMG activities (AG-1) of the vastus medialis muscle were compared between both groups. Significant increase in the elderly as compared with the young was found in duration from the onset of the EMG to the rise of tension (TLT), but not in time from the rise to peak of force (FTmax), although FTmax tended to be longer in the elderly than the young at three different levels of force output. AG-1 duration was also prolonged in the elderly but the difference was not significant. The prolongation of TLT in the elderly suggests that spatio-temporal recruitments of the motor units and/or the percentage of fast twitch fibers decreases with aging.     

Effect of exercise conditioning on excitation-contraction coupling in aged rats

We studied aged (24-26 mo) Fischer 344 rats after they underwent 8 wk of moderate exercise conditioning. Right ventricular papillary muscles were loaded with the calcium indicator aequorin. Electrophysiological recordings were also performed. Time to peak isometric tension in muscles from exercised aged rats (EAR) was shorter than in those from unexercised aged rats (UAR) (126 +/- 7 vs. 167 +/- 7 ms; P less than 0.01). Time to 50% relaxation from peak isometric tension was also shorter in EAR than in UAR (88 +/- 3 vs. 119 +/- 12 ms; P less than 0.05). There was a trend toward decrease in time to peak light and a significant decrease in time to 50% decline from peak light (33 +/- 4 ms in EAR vs. 59 +/- 17 ms in UAR; P = 0.001). Action potential amplitude was smaller in EAR than in UAR (67 +/- 4 vs. 82 +/- 3 mV; P = 0.003); however, action potential duration was longer (137 +/- 6 ms in EAR vs. 100 +/- 10 ms in UAR; P = 0.005). Right ventricular-to-body weight ratios revealed no evidence of hypertrophy in EAR compared with UAR. Cardiac tissue norepinephrine content was significantly greater in EAR than in UAR (1,212 +/- 25 vs. 630 +/- 105 ng/tissue; P = 0.02). In summary, exercise reversed the age-related prolongation of isometric contraction and associated intracellular calcium transient in the aged rat while it prolonged the transmembrane action potential. In addition, exercise in aged rats resulted in an increase in cardiac norepinephrine content.     

Ankle flexor muscles in the cat: Length-active tension and muscle unit properties as related to locomotion

The mechanical properties of the whole muscle and fast-twitch muscle units of the cat hindlimb pretibial flexors have been explored and related to normal locomotion. Tibialis anterior (TA) is parallel-fibered and functionally crosses a single joint, the ankle, whereas extensor digitorum longus (EDL) is pinnate and spans the ankle, knee, metatarsophalangeal and interphalangeal joints. The active tetanic tension of TA remains near its peak value over a range of muscle lengths associated with normal ankle movement. In contrast, the length-tension curve of EDL is sharply peaked. However, normal corollary action of the knee, ankle and metatarsophalangeal joints during stepping minimizes EDL's excursion and maintains it at or near a length optimal for peak tension development. EDL is capable of producing synchronous but sterotyped digit and ankle movements while TA provides for independent ankle flexion at all relevant joint angles. The mechanical properties of 84 TA and 98 EDL fast-twitch muscle units were studied by measuring twitch contraction time (≤45 msec), peak tetanic tension, response to repetitive stimulation, and contractile fatigue resistance during electrical stimulation of single alpha axons, functionally isolated from ventral root filaments. These mechanical properties were essentially similar for both muscles with the exception of mean peak tetanic tension which was 30% lower for TA units (14 gm-wt) than for EDL units (20 gm-wt). A high proportion of units in both muscles demonstrated fatigue resistance which is reflective of the repetitive, phasic demand upon these muscles during locomotion.     

Force-velocity constants in smooth muscle: afterloaded isotonic and quick-release methods

In using pharmacologic stimuli, force-velocity (FV) curves are usually obtained by the method of quick release (QR) and redevelopment of shortening at peak tetanic tension; the advantage of the method being that the active state is at maximum. However, the QR may itself reduce the intensity of the active state and result in reduced values of FV constants. We tested this by delineating FV curves in canine tracheal smooth muscle using both conventional afterloaded isotonic contractions (ALI), and redevelopment of shortening after QR methods. For both these studies a supramaximal tetanizing electrical stimulus was used. The analysis of 11 experiments revealed that the latter method resulted in statistically significant reductions of all FV constants except for Po (maximum isometric tetanic tension). The means and standard errors for the sets of constants for the ALI and QR, respectively, are as follows: Vmax (maximum velocity of shortening) = 0.275 lo (optimal muscle length)/s +/- 0.024 (SE), and 0.216 lo/s + 0.023; a (hyperbolic constant with units of force) = 294 g/cm2 +/- 35 and 236 g/cm2 +/- 32; b (hyperbolic constant with units of velocity) = 0.059 lo +/- 0.004 and 0.039 lo/s +/- 0.005; a/Po = 0.214 +/- 0.028 and 0.182 +/- 0.026; and Po = 1.362 kg/cm2 +/- 0.106 and 1.294 kg/cm2 +/- 0.097. These data clearly show that the quick-release method for measuring force-velocity relationships in canine smooth muscle results in significant underestimates of muscle shortening properties.     

Time-resolved changes in equatorial x-ray diffraction and stiffness during rise of tetanic tension in intact length-clamped single muscle fibers.

We report the first time-resolved x-ray diffraction studies on tetanized intact single muscle fibers of the frog. The 10, 11, 20, 21, 30, and Z equatorial reflections were clearly resolved in the relaxed fiber. The preparation readily withstood 100 1-s duration (0.4-s beam exposure) tetani at 4 degrees C (less than 4% decline of force and no deterioration in the 10, 11 equatorial intensity ratio at rest or during activation). Equatorial intensity changes (10 and 11) and fiber stiffness led tension (t1/2 lead 20 ms at 4 degrees C) during the tetanus rise and lagged during the isometric phase of relaxation. These findings support the existence of a low force cross-bridge state during the rise of tetanic tension and isometric relaxation that is not evident at the tetanus plateau. In "fixed end" tetani lattice expansion occurred with a time course similar to stiffness during the tetanus rise. During relaxation, lattice spacing increased slightly, while the sarcomere length remained isometric, but underwent large changes after the "shoulder" of tension. Under length clamp control, lattice expansion during the tetanus rise was reduced or abolished, and compression (2%) of the lattice was observed. A lattice compression is predicted by certain cross-bridge models of force generation (Schoenberg, M. 1980. Biophys. J. 30:51-68; Schoenberg, M. 1980. Biophys. J. 30:69-78).     

The Effects of Temperature upon Contraction and Ionic Exchange in Rabbit Ventricular Myocardium : Relation to control of active state

The mechanical responses (active and resting tension, dP/dt, TPT) and ionic exchange characteristics (Ca⁺⁺, K⁺, Na⁺) which follow upon a variation in temperature, rate, and [K⁺]₀ were studied in the rabbit papillary muscle and arterially perfused rabbit interventricular setpum. Abrupt changes in temperature provided a means of separating the contributions of rate of development (intensity) of active state and duration of active state to total active tension development (approximated by isometric tension). Threefold changes in duration of active state with proportional changes in active tension can be induced without evidence for alteration of Ca⁺⁺, K⁺, or Na⁺ exchange. Abrupt cooling produced a moderate (~15%) increase of dP/dt which suggests an augmentation of active state intensity. Evidence is presented to suggest that this increase of dP/dt is based upon an increase in membrane Ca⁺⁺ concentration which occurs secondary to inhibition of active Na⁺ transport. The alterations in ionic exchange and active state produced by variation of temperature are discussed in terms of a five-component control system.     

Length-Tension Relations in Cardiac Muscle

The distinguishing mechanical characteristics of cardiac muscleare(1) the presence of a significant and sometimes labile restingtension at functional lengths. Certain invertebrate skeletalmuscles also possess this property, but the structural basisseems to be different.(2)Force-velocity characteristics of heartmuscle are labile and constitute a mechanism for regulatingcardiac performance.(3)Data on quick-stretch and release showthat the active state in heart muscle is slow in its onset,probably preceding the development of isometric tension by onlya short time.The onset of the active state is labile also, andprobably forms the basis of the regulatory function of the force-velocityrelation. Measurements of heat show a slow rate of increaseof heat during a twitch consistent with the onset of contractility.     

Structural changes of cross-bridges on transition from isometric to shortening state in frog skeletal muscle

Structural changes in the myosin cross-bridges were studied by small-angle x-ray diffraction at a time resolution of 0.53 ms. A frog sartorius muscle, which was electrically stimulated to induce isometric contraction, was released by approximately 1% in 1 ms, and then its length was decreased to allow steady shortening with tension of approximately 30% of the isometric level. Intensity of all reflections reached a constant level in 5-8 ms. Intensity of the 7.2-nm meridional reflection and the (1,0) sampling spot of the 14.5-nm layer line increased after the initial release but returned to the isometric level during steady shortening. The 21.5-nm meridional reflection showed fast and slow components of intensity increase. The intensity of the 10.3-nm layer line, which arises from myosin heads attached to actin, decreased to a steady level in 2 ms, whereas other reflections took longer, 5-20 ms. The results show that myosin heads adapt quickly to an altered level of tension, and that there is a distinct structural state just after a quick release.     

Effects of taurine depletion on intrinsic contractility of rat ventricular papillary muscles

Contractile parameters in Krebs-Henseleit media containing various calcium concentrations were compared in left ventricular papillary muscles of two groups of rats: control and taurine depleted. All tests were carried out with the muscles at initial length, Lmax, the length that produced maximal active tension. From measurements of after- and un-loaded contractions, the velocity-tension curves and the derived maximum velocity of shortening were not different between the groups. Time to peak shortening and extent of shortening were not altered, while relaxation times and contraction duration were significantly prolonged for taurine-depleted muscles. Peak isometric tension and its rate of development were significantly reduced in taurine-depleted muscles compared with controls. Postrest (3 min) stimuli and paired stimuli (200-ms interval) evoked similar potentiated contractile responses in both groups, such that the ratio of their peak tensions remained unchanged. For taurine-depleted muscles the force-frequency relationship (a negative staircase) was parallel to, but lower than, control. These experiments suggest that taurine deficiency leads to reductions in action potential triggered calcium release from internal stores, and deficits in calcium sequestration. This may result from disfacilitation of calcium binding to the sarcoplasmic reticulum and other storage sites during taurine deficiency.     

Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines

The purpose of this study was to compare the excitability and contractility of three-dimensional skeletal muscle constructs, termed myooids, engineered from C2C12 myoblast and 10T1/2 fibroblast cell lines, primary muscle cultures from adult C3H mice, and neonatal and adult Sprague-Dawley rats. Myooids were 12 mm long, with diameters of 0.1-1 mm, were excitable by transverse electrical stimulation, and contracted to produce force. After approximately 30 days in culture, myooid cross-sectional area, rheobase, chronaxie, resting baseline force, twitch force, time to peak tension, one-half relaxation time, and peak isometric force were measured. Specific force was calculated by dividing peak isometric force by cross-sectional area. The specific force generated by the myooids was 2-8% of that generated by skeletal muscles of control adult rodents. Myooids engineered from C2C12-10T1/2 cells exhibited greater rheobase, time to peak tension, and one-half relaxation time than myooids engineered from adult rodent cultures, and myooids from C2C12-10T1/2 and neonatal rat cells had greater resting baseline forces than myooids from adult rodent cultures.