Cardiac length dependence of force and force redevelopment kinetics with altered cross-bridge cycling

We examined the influence of cross-bridge cycling kinetics on the length dependence of steady-state force and the rate of force redevelopment (k(tr)) during Ca(2+)-activation at sarcomere lengths (SL) of 2.0 and 2.3 microm in skinned rat cardiac trabeculae. Cross-bridge kinetics were altered by either replacing ATP with 2-deoxy-ATP (dATP) or by reducing [ATP]. At each SL dATP increased maximal force (F(max)) and Ca(2+)-sensitivity of force (pCa(50)) and reduced the cooperativity (n(H)) of force-pCa relations, whereas reducing [ATP] to 0.5 mM (low ATP) increased pCa(50) and n(H) without changing F(max). The difference in pCa(50) between SL 2.0 and 2.3 microm (Delta pCa(50)) was comparable between ATP and dATP, but reduced with low ATP. Maximal k(tr) was elevated by dATP and reduced by low ATP. Ca(2+)-sensitivity of k(tr) increased with both dATP and low ATP and was unaffected by altered SL under all conditions. Significantly, at equivalent levels of submaximal force k(tr) was faster at short SL or increased lattice spacing. These data demonstrate that the SL dependence of force depends on cross-bridge kinetics and that the increase of force upon SL extension occurs without increasing the rate of transitions between nonforce and force-generating cross-bridge states, suggesting SL or lattice spacing may modulate preforce cross-bridge transitions.     

Cross-bridge versus thin filament contributions to the level and rate of force development in cardiac muscle

In striated muscle thin filament activation is initiated by Ca(2+) binding to troponin C and augmented by strong myosin binding to actin (cross-bridge formation). Several lines of evidence have led us to hypothesize that thin filament properties may limit the level and rate of force development in cardiac muscle at all levels of Ca(2+) activation. As a test of this hypothesis we varied the cross-bridge contribution to thin filament activation by substituting 2 deoxy-ATP (dATP; a strong cross-bridge augmenter) for ATP as the contractile substrate and compared steady-state force and stiffness, and the rate of force redevelopment (k(tr)) in demembranated rat cardiac trabeculae as [Ca(2+)] was varied. We also tested whether thin filament dynamics limits force development kinetics during maximal Ca(2+) activation by comparing the rate of force development (k(Ca)) after a step increase in [Ca(2+)] with photorelease of Ca(2+) from NP-EGTA to maximal k(tr), where Ca(2+) binding to thin filaments should be in (near) equilibrium during force redevelopment. dATP enhanced steady-state force and stiffness at all levels of Ca(2+) activation. At similar submaximal levels of steady-state force there was no increase in k(tr) with dATP, but k(tr) was enhanced at higher Ca(2+) concentrations, resulting in an extension (not elevation) of the k(tr)-force relationship. Interestingly, we found that maximal k(tr) was faster than k(Ca), and that dATP increased both by a similar amount. Our data suggest the dynamics of Ca(2+)-mediated thin filament activation limits the rate that force develops in rat cardiac muscle, even at saturating levels of Ca(2+).     

Modulation of the rate of cardiac muscle contraction by troponin C constructs with various calcium binding affinities

We investigated whether changing thin filament Ca(2+) sensitivity alters the rate of contraction, either during normal cross-bridge cycling or when cross-bridge cycling is increased by inorganic phosphate (P(i)). We increased or decreased Ca(2+) sensitivity of force production by incorporating into rat skinned cardiac trabeculae the troponin C (TnC) mutants V44QTnC(F27W) and F20QTnC(F27W). The rate of isometric contraction was assessed as the rate of force redevelopment (k(tr)) after a rapid release and restretch to the original length of the muscle. Both in the absence of added P(i) and in the presence of 2.5 mM added P(i) 1) Ca(2+) sensitivity of k(tr) was increased by V44QTnC(F27W) and decreased by F20QTnC(F27W) compared with control TnC(F27W); 2) k(tr) at submaximal Ca(2+) activation was significantly faster for V44QTnC(F27W) and slower for F20QTnC(F27W) compared with control TnC(F27W); 3) at maximum Ca(2+) activation, k(tr) values were similar for control TnC(F27W), V44QTnC(F27W), and F20QTnC(F27W); and 4) k(tr) exhibited a linear dependence on force that was indistinguishable for all TnCs. In the presence of 2.5 mM P(i), k(tr) was faster at all pCa values compared with the values for no added P(i) for TnC(F27W), V44QTnC(F27W), and F20QTnC(F27W). This study suggests that TnC Ca(2+) binding properties modulate the rate of cardiac muscle contraction at submaximal levels of Ca(2+) activation. This result has physiological relevance considering that, on a beat-to-beat basis, the heart contracts at submaximal Ca(2+) activation.     

Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.     

The effects of force inhibition by sodium vanadate on cross-bridge binding, force redevelopment, and Ca2+ activation in cardiac muscle

Strongly bound, force-generating myosin cross-bridges play an important role as allosteric activators of cardiac thin filaments. Sodium vanadate (Vi) is a phosphate analog that inhibits force by preventing cross-bridge transition into force-producing states. This study characterizes the mechanical state of cross-bridges with bound Vi as a tool to examine the contribution of cross-bridges to cardiac contractile activation. The K(i) of force inhibition by Vi was approximately 40 microM. Sinusoidal stiffness was inhibited with Vi, although to a lesser extent than force. We used chord stiffness measurements to monitor Vi-induced changes in cross-bridge attachment/detachment kinetics at saturating [Ca(2+)]. Vi decreased chord stiffness at the fastest rates of stretch, whereas at slow rates chord stiffness actually increased. This suggests a shift in cross-bridge population toward low force states with very slow attachment/detachment kinetics. Low angle x-ray diffraction measurements indicate that with Vi cross-bridge mass shifted away from thin filaments, implying decreased cross-bridge/thin filament interaction. The combined x-ray and mechanical data suggest at least two cross-bridge populations with Vi; one characteristic of normal cycling cross-bridges, and a population of weak-binding cross-bridges with bound Vi and slow attachment/detachment kinetics. The Ca(2+) sensitivity of force (pCa(50)) and force redevelopment kinetics (k(TR)) were measured to study the effects of Vi on contractile activation. When maximal force was inhibited by 40% with Vi pCa(50) decreased, but greater force inhibition at higher [Vi] did not further alter pCa(50). In contrast, the Ca(2+) sensitivity of k(TR) was unaffected by Vi. Interestingly, when force was inhibited by Vi k(TR) increased at submaximal levels of Ca(2+)-activated force. Additionally, k(TR) is faster at saturating Ca(2+) at [Vi] that inhibit force by > approximately 70%. The effects of Vi on k(TR) imply that k(TR) is determined not only by the intrinsic properties of the cross-bridge cycle, but also by cross-bridge contribution to thin filament activation.     

Magnitude of length-dependent changes in contractile properties varies with titin isoform in rat ventricles

The effects of differential expression of titin isoforms on sarcomere length (SL)-dependent changes in passive force, maximum Ca(2+)-activated force, apparent cooperativity in activation of force (n(H)), Ca(2+) sensitivity of force (pCa(50)), and rate of force redevelopment (k(tr)) were investigated in rat cardiac muscle. Skinned right ventricular trabeculae were isolated from wild-type (WT) and mutant homozygote (Ho) hearts expressing predominantly a smaller N2B isoform (2,970 kDa) and a giant N2BA-G isoform (3,830 kDa), respectively. Stretching WT and Ho trabeculae from SL 2.0 to 2.35 μm increased passive force, maximum Ca(2+)-activated force, and pCa(50), and it decreased n(H) and k(tr). Compared with WT trabeculae, the magnitude of SL-dependent changes in passive force, maximum Ca(2+)-activated force, pCa(50), and n(H) was significantly smaller in Ho trabeculae. These results suggests that, at least in rat ventricle, the magnitude of SL-dependent changes in passive force, maximum Ca(2+)-activated force, pCa(50), n(H), and k(tr) is defined by the titin isoform.     

Cross-bridge kinetics in rat myocardium: effect of sarcomere length and calcium activation

We tested the hypotheses that Ca(2+) concentration ([Ca(2+)]) and sarcomere length (SL) modulate force development via graded effects on cross-bridge kinetics in chemically permeabilized rat cardiac trabeculae. Using sinusoidal length perturbations, we derived the transfer functions of stiffness over a range of [Ca(2+)] at a constant SL of 2.1 micrometer (n = 8) and at SL of 2.0, 2.1, and 2.2 micrometer (n = 4). We found that changes in SL affected only the magnitude of stiffness, whereas [Ca(2+)] affected the magnitude and phase-frequency relations. The data were fit to complex functions of two exponential processes. The characteristic frequencies (b and c) of these processes are indexes of cross-bridge kinetics, with b relating to cross-bridge attachment to and c to detachment from certain non-force-generating states. Both were significantly affected by [Ca(2+)], with an increase in b and c of 140 and 44%, respectively, over the range of [Ca(2+)] studied (P < 0.01). In contrast, SL had no effect on the characteristic frequencies (P > 0.6). We conclude that Ca(2+) activation modulates force development in rat myocardium, at least in part, via a graded effect on cross-bridge kinetics, whereas SL effects are mediated mainly by recruitment of cross bridges.     

Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium

Transgenic mice expressing an allele of cardiac troponin T (cTnT) with a COOH-terminal truncation (cTnT(trunc)) exhibit severe diastolic and mild systolic dysfunction. We tested the hypothesis that contractile dysfunction in myocardium expressing low levels of cTnT(trunc) (i.e., <5%) is due to slowed cross-bridge kinetics and reduced thin filament activation as a consequence of reduced cross-bridge binding. We measured the Ca(2+) sensitivity of force development [pCa for half-maximal tension generation (pCa(50))] and the rate constant of force redevelopment (k(tr)) in cTnT(trunc) and wild-type (WT) skinned myocardium both in the absence and in the presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1). Compared with WT mice, cTnT(trunc) mice exhibited greater pCa(50), reduced steepness of the force-pCa relationship [Hill coefficient (n(H))], and faster k(tr) at submaximal Ca(2+) concentration ([Ca(2+)]), i.e., reduced activation dependence of k(tr). Treatment with NEM-S1 elicited similar increases in pCa(50) and similar reductions in n(H) in WT and cTnT(trunc) myocardium but elicited greater increases in k(tr) at submaximal activation in cTnT(trunc) myocardium. Contrary to our initial hypothesis, cTnT(trunc) appears to enhance thin filament activation in myocardium, which is manifested as significant increases in Ca(2+)-activated force and the rate of cross-bridge attachment at submaximal [Ca(2+)]. Although these mechanisms would not be expected to depress systolic function per se in cTnT(trunc) hearts, they would account for slowed rates of myocardial relaxation during early diastole.     

Filament compliance effects can explain tension overshoots during force development

Spatially explicit stochastic simulations of myosin S1 heads attaching to a single actin filament were used to investigate the process of force development in contracting muscle. Filament compliance effects were incorporated by adjusting the spacing between adjacent actin binding sites and adjacent myosin heads in response to cross-bridge attachment/detachment events. Appropriate model parameters were determined by multi-dimensional optimization and used to simulate force development records corresponding to different levels of Ca(2+) activation. Simulations in which the spacing between both adjacent actin binding sites and adjacent myosin S1 heads changed by approximately 0.06 nm after cross-bridge attachment/detachment events 1), exhibited tension overshoots with a Ca(2+) dependence similar to that measured experimentally and 2), mimicked the observed k(tr)-relative tension relationship without invoking a Ca(2+)-dependent increase in the rate of cross-bridge state transitions. Tension did not overshoot its steady-state value in control simulations modeling rigid thick and thin filaments with otherwise identical parameters. These results underline the importance of filament geometry and actin binding site availability in quantitative theories of muscle contraction.     

Regulation of contraction kinetics in skinned skeletal muscle fibers by calcium and troponin C

The influences of [Ca(2+)] and Ca(2+) dissociation rate from troponin C (TnC) on the kinetics of contraction (k(Ca)) activated by photolysis of a caged Ca(2+) compound in skinned fast-twitch psoas and slow-twitch soleus fibers from rabbits were investigated at 15 degrees C. Increasing the amount of Ca(2+) released increased the amount of force in psoas and soleus fibers and increased k(Ca) in a curvilinear manner in psoas fibers approximately 5-fold but did not alter k(Ca) in soleus fibers. Reconstituting psoas fibers with mutants of TnC that in solution exhibited increased Ca(2+) affinity and approximately 2- to 5-fold decreased Ca(2+) dissociation rate (M82Q TnC) or decreased Ca(2+) affinity and approximately 2-fold increased Ca(2+) dissociation rate (NHdel TnC) did not affect maximal k(Ca). Thus the influence of [Ca(2+)] on k(Ca) is fiber type dependent and the maximum k(Ca) in psoas fibers is dominated by kinetics of cross-bridge cycling over kinetics of Ca(2+) exchange with TnC.     

Phosphate release and force generation in cardiac myocytes investigated with caged phosphate and caged calcium.

The phosphate (P(i)) dissociation step of the cross-bridge cycle was investigated in skinned rat ventricular myocytes to examine its role in force generation and Ca(2+) regulation in cardiac muscle. Pulse photolysis of caged P(i) (alpha-carboxyl-2-nitrobenzyl phosphate) produced up to 3 mM P(i) within the filament lattice, resulting in an approximately exponential decline in steady-state tension. The apparent rate constant, k (rho i), increased linearly with total P(i) concentration (initial plus photoreleased), giving an apparent second-order rate constant for P(i) binding of 3100 M(-1) s(-1), which is intermediate in value between fast and slow skeletal muscles. A decrease in the level of Ca(2+) activation to 20% of maximum tension reduced k (rho i) by twofold and increased the relative amplitude by threefold, consistent with modulation of P(i) release by Ca2+. A three-state model, with separate but coupled transitions for force generation and P(i) dissociation, and a Ca(2+)-sensitive forward rate constant for force generation, was compatible with the data. There was no evidence for a slow phase of tension decline observed previously in fast skeletal fibers at low Ca(2+), suggesting differences in cooperative mechanisms in cardiac and skeletal muscle. In separate experiments, tension development was initiated from a relaxed state by photolysis of caged Ca(2+). The apparent rate constant, k(Ca), was accelerated in the presence of high P(i) consistent with close coupling between force generation and P(i) dissociation, even when force development was initiated from a relaxed state. k(Ca) was also dependent on the level of Ca(2+) activation. However, significant quantitative differences between k (rho i) and k(Ca), including different sensitivities to Ca(2+) and P(i) indicate that caged Ca(2+) tension transients are influenced by additional Ca(2+)-dependent but P i-independent steps that occur before P(i) release. Data from both types of measurements suggest that kinetic transitions associated with P(i) dissociation are modulated by the Ca(2+) regulatory system and partially limit the physiological rate of tension development in cardiac muscle.     

Force kinetics and individual sarcomere dynamics in cardiac myofibrils after rapid ca(2+) changes

Kinetics of force development and relaxation after rapid application and removal of Ca(2+) were measured by atomic force cantilevers on subcellular bundles of myofibrils prepared from guinea pig left ventricles. Changes in the structure of individual sarcomeres were simultaneously recorded by video microscopy. Upon Ca(2+) application, force developed with an exponential rate constant k(ACT) almost identical to k(TR), the rate constant of force redevelopment measured during steady-state Ca(2+) activation; this indicates that k(ACT) reflects isometric cross-bridge turnover kinetics. The kinetics of force relaxation after sudden Ca(2+) removal were markedly biphasic. An initial slow linear decline (rate constant k(LIN)) lasting for a time t(LIN) was abruptly followed by an ~20 times faster exponential decay (rate constant k(REL)). k(LIN) is similar to k(TR) measured at low activating [Ca(2+)], indicating that k(LIN) reflects isometric cross-bridge turnover kinetics under relaxed-like conditions (see also. Biophys. J. 83:2142-2151). Video microscopy revealed the following: invariably at t(LIN) a single sarcomere suddenly lengthened and returned to a relaxed-type structure. Originating from this sarcomere, structural relaxation propagated from one sarcomere to the next. Propagated sarcomeric relaxation, along with effects of stretch and P(i) on relaxation kinetics, supports an intersarcomeric chemomechanical coupling mechanism for rapid striated muscle relaxation in which cross-bridges conserve chemical energy by strain-induced rebinding of P(i).     

Effects of low-level &alpha;-myosin heavy chain expression on contractile kinetics in porcine myocardium

Myosin heavy chain (MHC) isoforms are principal determinants of work capacity in mammalian ventricular myocardium. The ventricles of large mammals including humans normally express ～10% α-MHC on a predominantly β-MHC background, while in failing human ventricles α-MHC is virtually eliminated, suggesting that low-level α-MHC expression in normal myocardium can accelerate the kinetics of contraction and augment systolic function. To test this hypothesis in a model similar to human myocardium we determined composite rate constants of cross-bridge attachment (f(app)) and detachment (g(app)) in porcine myocardium expressing either 100% α-MHC or 100% β-MHC in order to predict the MHC isoform-specific effect on twitch kinetics. Right atrial (～100% α-MHC) and left ventricular (～100% β-MHC) tissue was used to measure myosin ATPase activity, isometric force, and the rate constant of force redevelopment (k(tr)) in solutions of varying Ca(2+) concentration. The rate of ATP utilization and k(tr) were approximately ninefold higher in atrial compared with ventricular myocardium, while tension cost was approximately eightfold greater in atrial myocardium. From these values, we calculated f(app) to be ～10-fold higher in α- compared with β-MHC, while g(app) was 8-fold higher in α-MHC. Mathematical modeling of an isometric twitch using these rate constants predicts that the expression of 10% α-MHC increases the maximal rate of rise of force (dF/dt(max)) by 92% compared with 0% α-MHC. These results suggest that low-level expression of α-MHC significantly accelerates myocardial twitch kinetics, thereby enhancing systolic function in large mammalian myocardium.     

Protein kinase A-induced myofilament desensitization to Ca(2+) as a result of phosphorylation of cardiac myosin-binding protein C

In skinned myocardium, cyclic AMP-dependent protein kinase A (PKA)-catalyzed phosphorylation of cardiac myosin-binding protein C (cMyBP-C) and cardiac troponin I (cTnI) is associated with a reduction in the Ca(2+) responsiveness of myofilaments and an acceleration in the kinetics of cross-bridge cycling, although the respective contribution of these two proteins remains controversial. To further examine the relative roles that cTnI and cMyBP-C phosphorylation play in altering myocardial function, we determined the Ca(2+) sensitivity of force (pCa(50)) and the activation dependence of the rate of force redevelopment (k(tr)) in control and PKA-treated mouse myocardium (isolated in the presence of 2,3-butanedione monoxime) expressing: (a) phosphorylatable cTnI and cMyBP-C (wild type [WT]), (b) phosphorylatable cTnI on a cMyBP-C-null background (cMyBP-C(-/-)), (c) nonphosphorylatable cTnI with serines(23/24/43/45) and threonine(144) mutated to alanines (cTnI(Ala5)), and (d) nonphosphorylatable cTnI on a cMyBP-C-null background (cTnI(Ala5)/cMyBP-C(-/-)). Here, PKA treatment decreased pCa(50) in WT, cTnI(Ala5), and cMyBP-C(-/-) myocardium by 0.13, 0.08, and 0.09 pCa units, respectively, but had no effect in cTnI(Ala5)/cMyBP-C(-/-) myocardium. In WT and cTnI(Ala5) myocardium, PKA treatment also increased k(tr) at submaximal levels of activation; however, PKA treatment did not have an effect on k(tr) in cMyBP-C(-/-) or cTnI(Ala5)/cMyBP-C(-/-) myocardium. In addition, reconstitution of cTnI(Ala5)/cMyBP-C(-/-) myocardium with recombinant cMyBP-C restored the effects of PKA treatment on pCa(50) and k(tr) reported in cTnI(Ala5) myocardium. Collectively, these results indicate that the attenuation in myofilament force response to PKA occurs as a result of both cTnI and cMyBP-C phosphorylation, and that the reduction in pCa(50) mediated by cMyBP-C phosphorylation most likely arises from an accelerated cross-bridge cycling kinetics partly as a result of an increased rate constant of cross-bridge detachment.     

Calcium regulation of tension redevelopment kinetics with 2-deoxy-ATP or low [ATP] in rabbit skeletal muscle.

The correlation of acto-myosin ATPase rate with tension redevelopment kinetics (k(tr)) was determined during Ca(+2)-activated contractions of demembranated rabbit psoas muscle fibers; the ATPase rate was either increased or decreased relative to control by substitution of ATP (5.0 mM) with 2-deoxy-ATP (dATP) (5.0 mM) or by lowering [ATP] to 0.5 mM, respectively. The activation dependence of k(tr) and unloaded shortening velocity (Vu) was measured with each substrate. With 5.0 mM ATP, Vu depended linearly on tension (P), whereas k(tr) exhibited a nonlinear dependence on P, being relatively independent of P at submaximum levels and rising steeply at P > 0.6-0.7 of maximum tension (Po). With dATP, Vu was 25% greater than control at Po and was elevated at all P > 0.15Po, whereas Po was unchanged. Furthermore, the Ca(+2) sensitivity of both k(tr) and P increased, such that the dependence of k(tr) on P was not significantly different from control, despite an elevation of Vu and maximal k(tr). In contrast, lowering [ATP] caused a slight (8%) elevation of Po, no change in the Ca(+2) sensitivity of P, and a decrease in Vu at all P. Moreover, k(tr) was decreased relative to control at P > 0.75Po, but was elevated at P < 0.75Po. These data demonstrate that the cross-bridge cycling rate dominates k(tr) at maximum but not submaximum levels of Ca(2+) activation.     

Activation of striated muscle: nearest-neighbor regulatory-unit and cross-bridge influence on myofilament kinetics

We have formulated a three-compartment model of muscle activation that includes both strong cross-bridge (XB) and Ca(2+)-activated regulatory-unit (RU) mediated nearest-neighbor cooperative influences. The model is based on the tight coupling premise--that XB retain activating Ca(2+) on the thin filament. Using global non-linear least-squares, the model produced excellent fits to experimental steady-state force-pCa and ATPase-pCa data from skinned rat soleus fibers. In terms of the model, nearest-neighbor influences over the range of Ca(2+) required for activation cause the Ca(2+) dissociation rate from regulatory-units (k(off)) to decrease and the cross-bridge association rate (f) to increase each more than ten-fold. Moreover, the rate variations occur in separate Ca(2+) regimes. The energy of activation governing f is strongly influenced by both neighboring RU and XB. In contrast, the energy of activation governing k(off) is less affected by neighboring XB than by neighboring RU. Nearest-neighbor cooperative influences provide both an overall sensitization to Ca(2+) and the well-known steep response of force to free Ca(2+). The apparent sensitivity for Ca(2+)-activation of force and ATPase is a function of cross-bridge kinetic rates. The model and derived parameter set produce simulated behavior in qualitative agreement with steady-state experiments reported in the literature for partial TnC replacement, increased [P(i)], increased [ADP], and MalNEt-S1 addition. The model is an initial attempt to construct a general theory of striated muscle activation-one that can be consistently used to interpret data from various types of muscle manipulation experiments.     

Ablation of myosin-binding protein-C accelerates force development in mouse myocardium

Myosin-binding protein-C (MyBP-C) is a thick filament-associated protein that binds tightly to myosin. Given that cMyBP-C may act to modulate cooperative activation of the thin filament by constraining the availability of myosin cross-bridges for binding to actin, we investigated the role of MyBP-C in the regulation of cardiac muscle contraction. We assessed the Ca(2+) sensitivity of force (pCa(50)) and the activation dependence of the rate of force redevelopment (k(tr)) in skinned myocardium isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) mice. Mechanical measurements were performed at 22 degrees C in the absence and presence of a strong-binding, nonforce-generating analog of myosin subfragment-1 (NEM-S1). In the absence of NEM-S1, maximal force and k(tr) and the pCa(50) of isometric force did not differ between WT and cMyBP-C(-/-) myocardium; however, ablation of cMyBP-C-accelerated k(tr) at each submaximal force. Treatment of WT and cMyBP-C(-/-) myocardium with 3 muM NEM-S1 elicited similar increases in pCa(50,) but the effects of NEM-S1 to increase k(tr) at submaximal forces and thereby markedly reduce the activation dependence of k(tr) occurred to a greater degree in cMyBP-C(-/-) myocardium. Together, these results support the idea that cMyBP-C normally acts to constrain the interaction between myosin and actin, which in turn limits steady-state force development and the kinetics of cross-bridge interaction.     

Troponin T isoforms modulate calcium dependence of the kinetics of the cross-bridge cycle: studies using a transgenic mouse line

Alternative splicing of troponin T (TnT) in striated muscle during development results in expression of different isoforms, with the splicing of a 5(') exon of TnT resulting in the expression of low-molecular-weight basic adult TnT isoforms and high-molecular-weight acidic embryonic TnT isoforms. Although other differences exist, the main differences between cardiac TnT (cTnT) and fast skeletal muscle TnT (fTnT) are in the NH(2) terminus, with fTnT being less acidic than cTnT. A transgenic mouse line expressing chicken fTnT in the heart was used to investigate the functional significance of TnT NH(2)-terminal charge differences on cardiac muscle contractility. The rates of force redevelopment (k(tr)) at four levels of Ca(2+) activation were recorded for skinned left ventricular trabeculae from control and transgenic mice. The k(tr) vs Ca(2+) relationship was different in control mice and transgenic mice, suggesting that the structure of TnT, and possibly the NH(2)-terminal region, is involved in determining the kinetics of cross-bridge cycle. These results suggest that isoform shifts in TnT may be an important molecular mechanism for determining the Ca(2+) dependence of cardiac muscle contractility.     

Ca(2+) activation of myofilaments from transgenic mouse hearts expressing R92Q mutant cardiac troponin T

The functional consequences of the R92Q mutation in cardiac troponin T (cTnT), linked to familial hypertrophic cardiomyopathy in humans, are not well understood. We have studied steady- and pre-steady-state mechanical activity of detergent-skinned fiber bundles from a transgenic (TG) mouse model in which 67% of the total cTnT in the heart was replaced by the R92Q mutant cTnT. TG fibers were more sensitive to Ca(2+) than nontransgenic (NTG) fibers [negative logarithm of half maximally activating molar Ca(2+) (pCa(50)) = 5.84 +/- 0.01 and 6.12 +/- 0.01 for NTG and TG fibers, respectively]. The shift in pCa(50) caused by increasing the sarcomere length from 1.9 to 2.3 microm was significantly higher for TG than for NTG fibers (DeltapCa(50) = 0.13 +/- 0.01 and 0.29 +/- 0.02 for NTG and TG fibers, respectively). The relationships between rate of ATP consumption and steady-state isometric tension were linear, and the slopes were the same in NTG and TG fibers. Rate of tension redevelopment was more sensitive to Ca(2+) in TG than in NTG fibers (pCa(50) = 5.71 +/- 0.02 and 6.07 +/- 0.02 for NTG and TG fibers, respectively). We concluded that overall cross-bridge cycling kinetics are not altered by the R92Q mutation but that altered troponin-tropomyosin interactions could be responsible for the increase in myofilament Ca(2+) sensitivity in TG myofilaments.     

Functional consequences of the human cardiac troponin I hypertrophic cardiomyopathy mutation R145G in transgenic mice

In this study, we addressed the functional consequences of the human cardiac troponin I (hcTnI) hypertrophic cardiomyopathy R145G mutation in transgenic mice. Simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca(2+)-activated force without changes in the maximal ATPase activity and an increase in the Ca(2+) sensitivity of both ATPase and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state), showing that changes in Ca(2+) sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared with Tg-WT fibers. The addition of 3 mm 2,3-butanedione monoxime at pCa 9.0 showed that there was approximately 2-4% of force generating cross-bridges attached in Tg-R145G fibers compared with less than 1.0% in Tg-WT fibers, suggesting that the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca(2+)] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared with Tg-WT. These results suggest that the phenotype of hypertrophic cardiomyopathy is most likely caused by the compensatory mechanisms in the cardiovascular system that are activated by 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge, 2) slowed relaxation (diastolic dysfunction) caused by prolonged [Ca(2+)] and force transients, and 3) an inability of the cardiac TnI to completely inhibit activation in the absence of Ca(2+) in Tg-R145G mice.