SUMMARY1. The influence of Ca2+ concentration and ionic strength on the maximum velocity of shortening (Vmax) and the tension generating capability of frog skinned muscle fibres has been studied at temperatures between 1 and 101C.2. Fibre segments were mounted between a force transducer and servo motor, where they could be viewed and photographed through a microscope. Segments in which the striations became non-uniform during activation were discarded.3. Velocity was obtained as a function of load by stepping the tension to values less than the steady isometric tension. Vmax was then determined by an extrapolation technique. Vmax was also obtained using a second, independent method by measuring the times required to take up various amounts of slack imposed on the segments. 4. Vmax was significantly influenced by the Ca2+ concentration, decreasing by about one half when the Ca2+ concentration was reduced to give steady tensions less than half-maximal. 5. Vma. was not influenced by changes in ionic strength, in the range 0-09-018 M.Steady tension was found to increase as ionic strength was decreased in the same range.6. These results indicate that the effect of changes in ionic strength is to alter the numbers or stiffness of attached cross-bridges, while there is no apparent influence of ionic strength on the steady-state kinetics of the actin-myosin interaction during unloaded shortening. The mechanism responsible for the influence of Ca2+ on Vmax is unknown, though possible sites of action for Ca2+ are discussed.

In this study maximum shortening velocity (Vmax) and isometric tension (P0) in skinned single fibers from rat slow soleus (SOL) and fast superficial vastus lateralis (SVL) muscles were examined after varying degrees of filament lattice compression with dextran. In both fiber types Vmax was greatest in the absence of dextran and decreased as the concentration of dextran was increased between 2.5 and 10 g/100 ml. At 10% dextran, which compressed fiber width by 31-38%, Vmax relative to the initial 0% dextran value was 0.28 +/- 0.03 (mean +/- SE) and 0.26 +/- 0.02 in SVL and SOL fibers, respectively. The effect of compression to depress Vmax was reversed completely by returning the fiber to 0% dextran. The force-generating capability of skinned fibers was not as sensitive to variations in cell width. In both the SOL and SVL fibers P0 increased by 3-7% when the concentration of dextran was increased from 0 to 5%. Further compression of lattice volume with 10% dextran resulted in a 8-13% decline in P0 relative to the initial value. While the precise mechanism by which filament lattice spacing modulates contractile function is not known, our results suggest that the major effect is upon the rate constant for cross-bridge detachment.

Considerable interest has been focussed on the role of myosin light chain LC2 in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R. L., G. G. Giulian, and M. L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC2 removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, Vmax, were measured in fiber segments prior to LC2 removal. The segments were then bathed at 30°C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60% of the LC2. Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC2 to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber.Vmax was found to decrease as the LC2 content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC2 were also observed. Reversal of the order of recombination of LC2 and TnC indicated that the reduction in V~x following EDTA treatment was a specific effect of LC2 removal. These results strongly suggest that LC2 may have roles in determining the kinetics and extent of interaction between myosin and actin.The role(s) of the low molecular weight subunits, or light chains, of myosin in the contraction of vertebrate skeletal muscles have not been clearly resolved. The great majority of the work that has been done to investigate this problem has involved in vitro biochemical studies of the isolated contractile proteins, actin and myosin. Results obtained using this approach have generally been unable to indicate specific functions for the light chains. Removal of the so-called alkali light chains with NI-LC1 has been found to result in the loss of actomyosin ATPase activity (11, 15); however, these extraction conditions may denature the remainder of the myosin molecule, rendering it inactive (13). More recently, Wagner and Giniger (26) and Sivaramakrishnan and Burke (22) have shown that significant myosin ATPase activity remains even after the total removal of all light chains. Removal of up to 50% of the LC2 light chain with dithionitrobenzene (DTNB) has been found to have little effect upon actomyosin ATPase activity (16), though recent evidence (21) indicates that LC2 may play a role in modulating the ATPase activity of myosin and regulated actin during Ca 2+ activation.Examination of the mechanical properties of single muscle cells in which the light chain (LC) composition could be manipulated and quantitated would seem to be a useful approach to the study of the physiological function(s) of these subunits. Work in this laboratory has shown that when LC2 was partially extracted from single skinned...

FOR THE purposes of this review, it will be helpful to consider the simple block diagram of a striated muscle system shown in Figure 1. The system has been generalized, in order to be applicable to heart muscle, by including an inotropic state mechanism. The parallel elastic component is necessary to account for resting tension. However, neither of these elements will be discussed further. According to the classical model for muscle contraction proposed by Hill, 1 a contracting muscle can be represented by a contractile component (CC) in series with a series elastic component (SEC). In the resting state, the CC is freely extensible, whereas in the active state it is supposed to resist strongly any attempt to suddenly change its length. The properties of the active CC are such that its force-velocity behavior can be described by the following equation:where Pis the force, V is the velocity of shortening and a and b are constants. The use of P 0J , where / is time, can be explained as follows. During a steady state isometric contraction, as in a tetanus, successive stimuli serve to keep the active state at its maximum level, indicated by the tetanic P o -In this case there is no need for the subscript t in Equation 1, because P o is time-invariant. However, in a twitch the peak developed isometric force does not attain the tetanic level. Hill 2 knew from his thermal measurements that heat production starts off at its maximum rate very soon after a stimulus and before any mechanical response is detectable. He therefore reasoned that the transition in the CC from rest to a fully "active state" following a single stimulus was very rapid, so that the active state was completely developed. Relaxation was produced by a slower decline of the active state to its resting level. Hill 2 defined the intensity of the active state at any instant in time to be equal to the magnitude of the developed force when the velocity of shortening of the CC was zero; that is, in Equation 1, P o> , = P when V = 0. Using this definition, and expressing the force of the CC, P, relative to the tetanic P o , it can be seen that the active state level varies between 0 and 1.In view of the fact that Levin and Wyman 3 showed that

SUMMARY. Heart cells from adult rats were induced to beat in suspension by electric field stimulation. We have gained evidence that all the rod-shaped cells in suspension were indeed beating, and that the beat had dynamic characteristics similar to those of intact heart muscle contracting under zero load. The cells were undamaged in the process, as judged by maintenance of ATP levels, morphology, and ability to beat. In gaining such evidence, we also measured the metabolic cost to the cells of beating under zero load. In cells with oxidative phosphorylation inhibited by rotenone plus oligomycin (termed anaerobic), the rate of beat-dependent lactate production suggested an equivalent rate of ATP utilization of 0.126 ± 0.013 nmol ATP/beat per mg protein (plus isoproterenol), and 0.058 ± 0.005 nmol ATP/beat per mg protein (minus isoproterenol). In respiring cells, the rate of beat-dependent oligomycin-sensitive oxygen consumption gave an equivalent rate of ATP utilization of 0.198 ± 0.009 nmol ATP/beat per mg protein (plus isoproterenol), and 0.126 ± 0.013 nmol ATP/beat per mg protein (minus isoproterenol). The cells beat with the same approximate maximum velocity whether isoproterenol was present or not. We calculate that-in the case of anaerobic cells without isoproterenol-this rate of ATP utilization can account for only about a 15% degree of activation of the contractile proteins. In addition, we have found an oligomycin-insensitive beat-dependent mitochondrial respiration of 0.023 ± 0.006 nanoatom O/beat per mg. The cause of this respiration is not known. The total rate of oxygen consumption of cells and also the rate per beat was comparable to that measured in nonworking whole hearts. (Circ Res 52:342-351,1963)