Strongly Binding Myosin Crossbridges Regulate Loaded Shortening and Power Output in Cardiac Myocytes
Abstract—This study investigated the possible roles of strongly binding myosin crossbridges in determining loaded shortening and power output in cardiac myocytes. Single skinned cardiac myocytes were attached between a force transducer and position motor, and shortening velocities were measured over a range of loads during varying levels of Ca2+ activation. Lowering the [Ca2+] slowed shortening velocities, decreased relative power output, and increased the curvature of length traces. We tested the hypothesis that Ca2+ activation dependence of loaded shortening is determined primarily by strongly binding crossbridges or by [Ca2+] per se, which was done by measuring loaded shortening before and after addition of N-ethylmaleimide–conjugated myosin subfragment-1 (NEM-S1), a strongly binding myosin analogue that cooperatively enhances thin filament activation. At fixed [Ca2+], NEM-S1 reduced the curvature of length traces and sped loaded shortening velocities. Even when [Ca2+] was adjusted so that force was equal with and without NEM-S1, myocyte shortening was faster and exhibited less curvature with NEM-S1. In the presence of NEM-S1, peak relative power output was also significantly greater during activations either at the same [Ca2+] or when [Ca2+] was adjusted to achieve the same force. Consequently, NEM-S1 eliminated any Ca2+ dependence of relative power output that is normally observed in cardiac myocytes. These results indicate that strongly binding crossbridges play a significant role in determining loaded shortening and power output and suggest that previously observed Ca2+ dependence of power output is mediated by alterations in numbers of crossbridges bound to the thin filament.
Myocardium shortens against an afterload as blood is expelled into the circulation during systole. The amount of blood expelled is ultimately determined by the rate of myocardial shortening, which depends on several factors and varies inversely with afterload. Considerable information regarding the relationship between stroke volume and afterload has been obtained by characterizing force-velocity relationships in isolated, multicellular preparations of myocardium. Although these experiments have enriched our understanding of myocardial contraction, the presence of extracellular elasticity has confounded the interpretation of these relationships and made it difficult to assess how afterload influences the contractile machinery. The recent development of single skinned myocyte preparations for dynamic mechanical measurements circumvents several of the complexities associated with intercellular elasticity in multicellular preparations and has added to our understanding of myofibrillar behavior during loaded shortening. In single skinned myocyte preparations, as with multicellular preparations, force-velocity relationships are hyperbolic and are highly sensitive to changes in myoplasmic [Ca2+].1 2 Also, the time course of loaded shortening changes when Ca2+ activation levels are reduced in skinned myocyte preparations. Lowering the [Ca2+] not only yields slower shortening velocities but also increases the curvature in length traces during load clamps.1 3 Velocity slows progressively throughout isotonic shortening, which may have an important physiological role in determining hemodynamics during the latter part of systole. The purpose of this study was to gain a better understanding of the basis for this Ca2+-dependent slowing of myocyte velocity during shortening and, thus, decreased power output both at the onset of loaded shortening and throughout the isotonic contraction.
Myocardial activation and contraction involve complex interactions among Ca2+, thin filament regulatory proteins, and myosin crossbridges.4 Some current models of regulation propose that thin filaments exist in 3 distinct states, which depend on the relative position of tropomyosin.5 6 In the absence of Ca2+, thin filaments are thought to be in a “blocked” state in which tropomyosin sterically hinders myosin from interacting with actin. When Ca2+ binds to troponin C, the thin filament undergoes a transition to a “closed” state as tropomyosin changes position, which allows increased numbers of weakly bound actomyosin crossbridges to interact with actin. It has recently been proposed that when thin filaments are in the closed state a population of crossbridges undergoes the transition to a strongly bound, non–force-generating state,7 which promotes further movement of tropomyosin into an “open” state. It is only in the open state that strongly bound force-generating crossbridges can form between myosin and actin. Thus, in this model, full activation of thin filaments occurs only in the presence of both Ca2+ and strongly bound myosin crossbridges, wherein Ca2+ first allows crossbridges to bind and these in turn promote additional binding.
One well-established consequence of muscle shortening is a reduction in the number of strongly bound crossbridges,8 9 which, according to the above model, would tend to shift thin filaments from the open state to the “closed” state in a cooperative manner.10 11 In the context of loaded shortening, such a reduction in strongly bound force-generating crossbridges would result in slower crossbridge cycling rates for the muscle to sustain a given load. The resulting cooperative inactivation of the thin filament could mediate the characteristic slowing of muscle shortening under load in submaximally Ca2+-activated myocardium.12 If such a mechanism is involved, we predict that experimental manipulations designed to maintain the open state or activated thin filaments during loaded shortening would attenuate curvilinear shortening. We undertook such experiments by examining the characteristics of loaded shortening in the presence of a strongly bound crossbridge derivative, N-ethylmaleimide–conjugated myosin subfragment-1 (NEM-S1), which is thought to cooperatively enhance thin filament activation. NEM-S1 activates acto-S1 ATPase activity in solution13 and both force and kinetics of force development in skinned muscle preparations.14 15 Thus, our experiments tested the hypothesis that curvilinear shortening in submaximally activated myocardium is modulated primarily by strongly binding crossbridges.
A second aim of this study was to determine whether strongly binding myosin crossbridges influence loaded shortening velocities and relative power output of cardiac myocytes. We investigated the hypothesis that strongly binding myosin crossbridges, as opposed to [Ca2+] per se, play the dominant role in determining power output in skinned cardiac myocytes. This idea was tested by characterizing power-load curves in single skinned cardiac myocyte preparations activated at fixed [Ca2+] before and after addition of NEM-S1. Finally, we investigated whether fixed force levels induced by Ca2+ alone or Ca2+ plus NEM-S1 produced differences in loading shortening speeds and power output in skinned cardiac myocyte preparations. These experiments addressed the possibility that strongly binding crossbridges influence loaded shortening velocities independent of force levels.
Materials and Methods
Cardiac Myocyte Preparation
Cardiac myocyte preparations were obtained by mechanical disruption of hearts from Sprague-Dawley rats as described previously.1 The experimental apparatus for physiological measurements on myocyte preparations was similar to one previously described in detail.1 16
Compositions of relaxing and activating solutions were as follows (in mmol/L): EGTA 7, free Mg2+ 1, imidazole 20, MgATP 4, creatine phosphate 14.5 (pH 7.0), various Ca2+ concentrations between 10–9 (relaxing solution) and 10–4.5 mol/L (maximal Ca2+-activating solution), and sufficient KCl to adjust ionic strength to 180 mmol/L.1
Force-Velocity and Power-Load Measurements
All mechanical measurements were made at 12°C. For measurements of loaded shortening, a servosystem incorporating integrative feedback was used to control the load on the myocyte preparations as previously described.1 3
To test the effects of NEM-S1 on loaded shortening, the myocyte was first maximally Ca2+ activated in pCa 4.5 solution to obtain an initial maximal force (ie, P4.5), which was followed by a series of isotonic contractions (≈15). The myocyte was then transferred to a Ca2+-containing solution that yielded ≈40% P4.5 (pCa solutions ranged between pCa 5.6 and 5.7), and a second series of isotonic contractions was performed. The myocyte was then bathed for 20 minutes in pCa 9.0 solution containing 6 μmol/L NEM-S1; ie, NEM-S1 was only added to pCa 9.0 solution. After NEM-S1 incubation, the myocyte was again transferred to an activating solution that yielded ≈40% P4.5, and another series of isotonic contractions was performed. The pCa solutions yielding ≈40% P4.5 after NEM-S1 treatment varied between preparations, ranging from pCa 9.0 to 5.7. A final series of isotonic contractions was obtained during Ca2+ activation in the identical pCa solution that yielded ≈40% P4.5 before NEM-S1. Last, the myocyte preparation was transferred to a solution of pCa 4.5, and a final maximum force (P4.5) was obtained.
Myocyte length traces during isotonic contractions were fit with a single decaying exponential equation of the following form: where L is cell length at time t, A and C are constants with dimensions of length, and k is the apparent rate constant of shortening (kshortening). Velocity of shortening at any given time t was determined as the slope of a tangent to the fitted curve at that time point. For this study, velocities of shortening were calculated at t=0 ms.
Hyperbolic force-velocity curves were fit to force-velocity data using the Hill equation, and power-load curves were obtained by multiplying force × velocity as previously described.1
One-way repeated-measures ANOVA was performed to determine whether there were significant effects of NEM-S1 on kshortening or peak relative power output. Student-Newman-Keuls tests were used as post hoc tests to assess differences among means. For comparisons of kshortening, values were used if myocytes shortened 5% to 15% of their original length. P<0.05 was chosen as indicating significance. All values are expressed as mean±SD.
Myocyte (n=7) dimensions and force characteristics were as follows: length, 149±52 μm; width, 22±3 μm; and maximum isometric force (P4.5), 12±6 μN. The resting sarcomere length (SL) of these preparations was set to yield passive forces just slightly above 0 (SL≈2.25 μm). During maximal Ca2+ activation (ie, at pCa 4.5), mean SL shortened to ≈2.20 μm during fixed-end, isometric contractions, which is indicative of low end compliance of the myocytes at the points of attachment.
Effects of NEM-S1 on Loaded Shortening in Cardiac Myocytes
Loaded shortening varies as a function of activator [Ca2+] in skinned cardiac myocyte preparations, in that lower [Ca2+] yields slower shortening velocities and greater curvature in length traces during lightly loaded contractions1 2 (Figure 1⇓). To investigate possible mechanisms by which Ca2+ regulates loaded shortening, isotonic contractions were recorded during constant [Ca2+] activations both before and after the addition of 6 μmol/L NEM-S1. This concentration of NEM-S1 was chosen on the basis of previous experiments, which showed that 6 μmol/L NEM-S1 does not alter peak Ca2+-activated force but does increase force at submaximal [Ca2+].14 15 At a given [Ca2+], NEM-S1 potentiated force and altered the time course of loaded shortening by reducing the curvature of length traces (Figure 2⇓). To quantify curvature of length traces, apparent rate constants of shortening (ie, kshortening) were calculated from exponentials fit to length traces before and after addition of NEM-S1. kshortening values during submaximal activations decreased from 4.4±2.9 in controls (n=57) to 2.1±1.0 (n=62) in the presence of NEM-S1 (see inset, Figure 2⇓). These results indicate that strongly bound crossbridges have a marked effect on the time course of loaded shortening even at the same [Ca2+], perhaps by maintaining thin filaments in the open or activated state.
The effects of NEM-S1 on curvature at constant [Ca2+] may result simply from NEM-S1–induced increases in force and presumably greater thin filament activation levels. To examine this idea and further investigate regulation of loaded shortening, isotonic contractions were next assessed during constant force levels before and after addition of NEM-S1. To obtain the same force (≈40% of P4.5) in the presence and absence of NEM-S1, [Ca2+] had to be reduced during activations with NEM-S1. Specifically, Ca2+ concentrations in activating solutions were adjusted between 5.7 and as low as pCa 9.0 to obtain the same force as before NEM-S1 incubation. Even when force levels were equalized, NEM-S1 significantly reduced the curvature of length traces in submaximally activated myocytes (Figure 3⇓). kshortening values were 4.4±2.9 (n=62) in the absence of NEM-S1 compared with 2.4±1.2 (n=65) with NEM-S1 (Figure 3⇓). Interestingly, the curvature of length traces obtained in the presence of NEM-S1 was similar to the curvature during isotonic contractions when the same myocytes were maximally Ca2+ activated (ie, pCa 4.5); that is, NEM-S1 eliminated the activation dependence of curvature in shortening records. During maximal Ca2+ activations (ie, pCa 4.5 solution), kshortening was 1.8±1.1 (n=54). Given that thin filament activation levels are thought to be related to the amount of Ca2+ bound to troponin C and the number of strongly bound crossbridges to actin,4 high [Ca2+] would be predicted to maintain high levels of thin filament activation even during loaded shortening. NEM-S1 is also thought to maintain thin filaments at high activation levels during mechanical perturbations,14 although only a fraction of the length of each thin filament would presumably remain activated (ie, in the open state) in the presence of NEM-S1 at low [Ca2+], given that submaximal isometric forces are produced. The finding that curvature of shortening is similar during maximal Ca2+ activations and during submaximal activations in the presence of NEM-S1 is consistent with the idea that inactivation of the thin filament due to shortening-induced reductions in crossbridge number contributes to curvilinear shortening observed during submaximal isotonic contractions.
Effects of NEM-S1 on Force-Velocity and Power-Load Curves
A second aim of this study was to assess the role of [Ca2+] per se versus strongly binding crossbridges in determining loaded shortening velocities (at the onset of shortening) and relative power output (ie, power output normalized for isometric force) in skinned cardiac myocyte preparations. At a given [Ca2+], increasing the number of strongly binding crossbridges with NEM-S1 elevated isometric force, sped loaded shortening velocities, and thus increased relative power output at nearly all loads (Figure 4⇓). Peak relative power output increased from 0.074±0.016 (n=7) before NEM-S1 to 0.107±0.027 (n=7) in the presence of NEM-S1 during activation in solutions of identical [Ca2+]. Force-velocity and relative power-load curves were also shifted upward by NEM-S1 even when [Ca2+] was adjusted to yield steady forces equal to values before NEM-S1 (Figure 5⇓). Peak relative power output was 0.100±0.025 with NEM-S1 (n=7) compared with 0.074±0.016 (n=7) before NEM-S1. In fact, NEM-S1 eliminated all Ca2+ dependence of relative power output; ie, peak relative power output was the same during maximal Ca2+ activations (0.099±0.026) as during submaximal Ca2+ activations with NEM-S1 (inset Figure 5⇓).
This study examined potential mechanisms by which Ca2+ regulates loaded shortening velocity and relative power output in cardiac myocytes. To do this, we used a strongly bound crossbridge derivative, NEM-S1, which is thought to maintain thin filaments in the open state, allowing endogenous crossbridges to cycle through power-generating transitions. We found that both loaded shortening velocities and relative power output varied predominantly by addition of strongly binding crossbridges, as opposed to [Ca2+] per se. At a fixed [Ca2+], NEM-S1 increased force levels and markedly reduced the curvature of loaded shortening traces, sped loaded shortening, and increased relative power output at nearly all loads in skinned cardiac myocytes. Even when steady isometric force was kept constant by adjusting [Ca2+], NEM-S1 still reduced the curvature of length traces, sped loaded shortening, and increased relative power output. These findings indicate that strongly binding crossbridges modulate loaded shortening and power output and imply that reduced levels of thin filament activation due to decreased numbers of strongly binding crossbridges could mediate the slower shortening velocities reported previously in myocardial preparations activated at low [Ca2+].17 18 19 20 Because we were not able to directly measure thin filament activation levels during these experiments, our conclusion assumes that NEM-S1 maintains the thin filament in an open or activated state, which is based on previous reports that NEM-S1 activates acto-S1 ATPase activity in solution13 and force and kinetics of force development in skinned fibers.14 15
The regulation of muscle contraction is thought to involve complex molecular processes involving both thin and thick filament proteins.21 Current models propose that thin filaments occupy 3 distinct states (ie, blocked, closed, and open) depending on the relative position of tropomyosin.5 6 7 In the Ca2+-bound closed state, there is increased probability that weakly bound crossbridges will make the transition to strongly bound, non–force-generating states. Such strongly bound, non–force-generating crossbridges are thought to move tropomyosin farther into the groove between actin monomers yielding the transition to the open state in which strongly bound, force-generating crossbridges accumulate. In the present study, we used NEM-S1 to potentially increase the probability of thin filaments occupying the open state even at low levels of myoplasmic Ca2+, to study the relative contributions of Ca2+ per se and strongly binding crossbridges in determining loaded shortening velocity and power output in cardiac myocytes. Previous work has shown that loaded shortening and power output increase with the levels of Ca2+ in skinned cardiac myocytes.2 Ca2+ regulation could arise from direct effects of Ca2+ binding to alter crossbridge cycling kinetics and loaded shortening velocity, or Ca2+ might regulate velocity indirectly by affecting the level of thin filament activation by strongly binding crossbridges. Using NEM-S1 to alter the number of strongly bound crossbridges, our results show that Ca2+ likely regulates power output primarily by the second mechanism.
Several earlier studies found that both absolute and relative power output increased when the level of activation (as determined by force) was increased in intact cardiac muscle preparations.18 22 23 In those experiments, activation was varied in one of several ways, including inotropic agents such as β-agonists, increased [Ca2+]o, or measuring loaded shortening at various times during twitch contractions. Despite the consistent finding of Ca2+-induced alterations in power output, the subcellular factors that limit loaded shortening and relative power output in cardiac muscle remained uncertain. One possibility is that crossbridges must work against internal loads during shortening of cardiac muscle. The source of internal load is uncertain but may be the cytoskeleton,12 titin/connectin,19 24 or crossbridges themselves.25 A consequence of an internal load is that the proportion of cycling crossbridges required to overcome these loads is greater during submaximal than maximal Ca2+ activations. Consequently, proportionately fewer crossbridges would be available to work against an external load, so that the rate of crossbridge and muscle shortening will be slowed. A second factor that may also reduce the number of cycling crossbridges is cooperative inactivation of the thin filament as muscle shortens, as previously suggested.2 11 Because muscle shortening reduces the number of crossbridges in strongly bound states, there will be greater likelihood of thin filament transition from open to closed states based on the previously mentioned model of regulation. Cooperative inactivation of the thin filament will further reduce the proportion of cycling crossbridges bearing a relative external load, yielding even slower loaded shortening and decreased power output. Consistent with this idea, loaded shortening in the present study was faster and relative power output greater in the presence of strongly bound crossbridges (ie, NEM-S1) both when [Ca2+] was kept constant and when force-generating capability was kept constant by varying [Ca2+].
Our results also address the mechanisms by which the velocity of loaded shortening progressively slows during submaximal Ca2+ activations. We found that such curvilinear shortening was markedly reduced in the presence of NEM-S1 either at constant [Ca2+] or constant force-generating capabilities, a finding that is consistent with the idea that cooperative inactivation of the thin filament contributes to curvilinear shortening. However, other mechanisms might also contribute to curvilinear shortening. For example, a subpopulation of slowly detaching crossbridges has been proposed to arise in zones of the thin filaments between regions that are in closed and open states.25 Experimental evidence for this idea came from the finding that unloaded shortening was biphasic during submaximal Ca2+ activation of skinned fast-twitch fibers; ie, shortening up to 40 to 80 nm per half-sarcomere was markedly faster than shortening beyond 80 nm per half-sarcomere in fast-twitch skeletal fibers. The idea here is that after shortening of ≈80 nm per half-sarcomere, the slowly detaching crossbridges in these transition zones become compressed and produce a load opposing shortening. The activation effects of NEM-S1 would be predicted to reduce the number of transition zones and thus the number of slowly detaching crossbridges. However, such a crossbridge-dependent internal load is unlikely to be the sole or even the main contributor to curvilinear shortening in cardiac muscle, because considerable curvature was observed in our length traces before sarcomeres had shortened 40 to 80 nm per half-sarcomere.
The ability of the heart to pump blood through the circulation depends on a number of factors, including preload, afterload, and inotropic state. The inotropic state of the heart, and thus its pumping capacity, is determined in large part by variations in myoplasmic [Ca2+]. Changes in [Ca2+] have marked effects on the rate of work produced by individual myocytes, which ultimately determines stroke volume. Results here suggest that [Ca2+]-induced changes in loaded shortening and power output are largely mediated by crossbridge number–induced changes in the level of thin filament activation. Thus, cooperative activation of the thin filament triggered by Ca2+ during isovolumetric contraction seems to be a major contributor to power produced by the myocardium during ejection and to stroke volume.
Another important functional aspect of the cardiac cycle is that peak ventricular and aortic pressures are reached before the end of ejection.26 This arises because the rate that blood exits the aorta exceeds the rate of ventricular ejection, which is determined by myocardial power output. Therefore, the fall in ventricular pressure during the latter part of systole may be due, at least in part, to a slowing of loaded shortening velocity of cardiac myocytes, which is consistent with the progressive slowing of shortening in skinned cardiac myocyte preparations observed during submaximal Ca2+ activations. Evidence from the current study suggests that such slowing of shortening arises at least in part from progressive cooperative inactivation of the thin filaments throughout shortening.
This study was supported by NIH Grant HL-57852 (to K.S.M.) and HL-54581 (to R.L.M). We thank Dr Daniel P. Fitzsimons and Chad Warren for assistance in preparation of NEM-S1.
- Received May 16, 2000.
- Revision received August 17, 2000.
- Accepted August 28, 2000.
- © 2000 American Heart Association, Inc.
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