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.     

Myosin binding-induced cooperative activation of the thin filament in cardiac myocytes and skeletal muscle fibers.

Myosin binding-induced activation of the thin filament was examined in isolated cardiac myocytes and single slow and fast skeletal muscle fibers. The number of cross-bridge attachments was increased by stepwise lowering of the [MgATP] in the Ca(2+)-free solution bathing the preparations. The extent of thin filament activation was determined by monitoring steadystate isometric tension at each MgATP concentration. As pMgATP (where pMgATP is -log [MgATP]) was increased from 3.0 to 8.0, isometric tension increased to a peak value in the pMgATP range of 5.0-5.4. The steepness of the tension-pMgATP relationship, between the region of the curve where tension was zero and the peak tension, is hypothesized to be due to myosin-induced cooperative activation of the thin filament. Results showed that the steepness of the tension-pMgATP relationship was markedly greater in cardiac as compared with either slow or fast skeletal muscle fibers. The steeper slope in cardiac myocytes provides evidence of greater myosin binding-induced cooperative activation of the thin filament in cardiac as compared with skeletal muscle, at least under these experimental conditions of nominal free Ca2+. Cooperative activation is also evident in the tension-pCa relation, and is dependent upon thin filament molecular interactions, which require the presence of troponin C. Thus, it was determined whether myosin-based cooperative activation of the thin filament also requires troponin C. Partial extraction of troponin C reduced the steepness of the tension-pMgATP relationship, with the effect being significantly greater in cardiac than in skeletal muscle. After partial extraction of troponin C, muscle type differences in the steepness of the tension-pMgATP relationship were no longer apparent, and reconstitution with purified troponin C restored the muscle lineage differences. These results suggest that, in the absence of Ca2+, myosin-mediated activation of the thin filament is greater in cardiac than in skeletal muscle, and this apparent cooperativity requires the presence of troponin C on thin filament regulatory strands.     

A pH-sensitive interaction of troponin I with troponin C coupled with strongly binding cross-bridges in cardiac myofilament activation

Slow skeletal muscle troponin I (ssTnI) expressed predominantly in perinatal heart confers a marked resistance to acidic pH on Ca(2+) regulation of cardiac muscle contraction. To explore the molecular mechanism underlying this phenomenon, we investigated the roles of TnI isoforms (ssTnI and cardiac TnI (cTnI)) in the thin filament activation by strongly binding cross-bridges, by exchanging troponin subunits in cardiac permeabilized muscle fibers. Fetal cardiac muscle showed a marked resistance to acidic pH in activation of the thin filament by strongly binding cross-bridges compared to adult muscle. Exchanging ssTnI into adult fibers altered the pH sensitivity from adult to fetal type, indicating that ssTnI also confers a marked resistance to acidic pH on the cross-bridge-induced thin filament activation. However, the adult fibers containing ssTnI or cTnI but lacking TnC showed no pH sensitivity. These findings provide the first evidence for the coupling between strongly binding cross-bridges and a pH-sensitive interaction of TnI with TnC in cardiac muscle contraction, as a molecular basis of the mechanism conferring the differential pH sensitivity on Ca(2+) regulation.     

The effects of partial extraction of TnC upon the tension-pCa relationship in rabbit skinned skeletal muscle fibers

The activation of contraction in vertebrate skeletal muscle involves the binding of Ca2+ to low-affinity binding sites on the troponin C (TnC) subunit of the regulatory protein troponin. The present study is an investigation of possible cooperative interactions between adjacent functional groups, composed of seven actin monomers, one tropomyosin, and one troponin, along the same thin filament. Single skinned fibers were obtained from rabbit psoas muscles and were then placed in an experimental chamber containing relaxing solution maintained at 15 degrees C. Isometric tension was measured in solutions containing maximally and submaximally activating levels of free Ca2+ (a) in control fiber segments, (b) in the same segments after partial extraction of TnC, and finally (c) after recombination of TnC into the segments. The extraction was done at 11-13 degrees C in 20 mM Tris, 5 mM EDTA, pH 7.85 or 8.3, a procedure derived from that of Cox et al. (1981. Biochem. J. 195:205). Extraction of TnC was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the control and experimental samples. Partial extraction of TnC resulted in reductions in tension during maximal Ca activation and in a shift of the relative tension-pCa (i.e., -log[Ca2+]) relationship to lower pCa's. The readdition of TnC to the extracted fiber segments resulted in a recovery of tension to near-control levels and in the return of the tension-pCa relation to its original position. On the basis of these findings, we conclude that the sensitivity to Ca2+ of a functional group within the thin filament may vary depending upon the state of activation of immediately adjacent groups.     

Kinetics of a Ca(2+)-sensitive cross-bridge state transition in skeletal muscle fibers. Effects due to variations in thin filament activation by extraction of troponin C

The rate constant of tension redevelopment (ktr; 1986. Proc. Natl. Acad. Sci. USA. 83:3542-3546) was determined at various levels of thin filament activation in skinned single fibers from mammalian fast twitch muscles. Activation was altered by (a) varying the concentration of free Ca2+ in the activating solution, or (b) extracting various amounts of troponin C (TnC) from whole troponin complexes while keeping the concentration of Ca2+ constant. TnC was extracted by bathing the fiber in a solution containing 5 mM EDTA, 10 mM HEPES, and 0.5 mM trifluoperazine dihydrochloride. Partial extraction of TnC resulted in a decrease in the Ca2+ sensitivity of isometric tension, presumably due to disruption of near-neighbor molecular cooperativity between functional groups (i.e., seven actin monomers plus associated troponin and tropomyosin) within the thin filament. Altering the level of thin filament activation by partial extraction of TnC while keeping Ca2+ concentration constant tested whether the Ca2+ sensitivity of ktr results from a direct effect of Ca2+ on cross-bridge state transitions or, alternatively, an indirect effect of Ca2+ on these transitions due to varying extents of thin filament activation. Results showed that the ktr-pCa relation was unaffected by partial extraction of TnC, while steady-state isometric tension exhibited the expected reduction in Ca2+ sensitivity. This finding provides evidence for a direct effect of Ca2+ on an apparent rate constant that limits the formation of force-bearing cross-bridge states in muscle fibers. Further, the kinetics of this transition are unaffected by disruption of near-neighbor thin filament cooperativity subsequent to extraction of TnC. Finally, the results support the idea that the steepness of the steady-state isometric tension-calcium relationship is at least in part due to mechanisms involving molecular cooperativity among thin filament regulatory proteins.     

Alterations in Ca2+ sensitive tension due to partial extraction of C- protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers

C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca(2+)-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23 degrees C; a similar amount was extracted from psoas fibers during a 3-h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40-60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension-pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 microns which, because of the distribution of C-protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction.     

The control of myocardial contraction with skeletal fast muscle troponin C

The present study describes experiments on the myocardial trabeculae from the right ventricle of Syrian hamsters whose troponin C (TnC) moiety was exchanged with heterologous TnC from fast skeletal muscle of the rabbit. These experiments were designed to help define the role of the various classes of Ca2+-binding sites on TnC in setting the characteristic sensitivities for activations of cardiac and skeletal muscles. Thin trabeculae were skinned and about 75% of their troponin C extracted by chemical treatment. Tension development on activations by Ca2+ and Sr2+ was found to be nearly fully blocked in such TnC extracted preparations. Troponin C contents and the ability to develop tension on activations by Ca2+ and Sr2+ was permanently restored after incubation with 2-6 mg/ml purified TnC from either rabbit fast-twitch skeletal muscle (STnC) or the heart (CTnC, cardiac troponin C). The native (skinned) cardiac muscle is characteristically about 5 times more sensitive to activation by Sr2+ than fast muscle, but the STnC-loaded trabeculae gave response like fast muscle. Attempts were also made to exchange the TnC in psoas (fast-twitch muscle) fibers, but unlike cardiac muscle tension response of the maximally extracted psoas fibers could be restored only with homologous STnC. CTnC was effective in partially extracted fibers, even though the uptake of CTnC was complete in the maximally extracted fibers. The results in this study establish that troponin C subunit is the key in setting the characteristic sensitivity for tension control in the myocardium above that in the skeletal muscle. Since a major difference between skeletal and cardiac TnCs is that one of the trigger sites (site I, residues 28-40 from the N terminus) is modified in CTnC and has reduced affinity for Ca2+ binding, the possibility is raised that this site has a modulatory effect on activation in different tissues and limits the effectiveness of CTnC in skeletal fibers.     

Influence of length on force and activation-dependent changes in troponin c structure in skinned cardiac and fast skeletal muscle

Linear dichroism of 5' tetramethyl-rhodamine (5'ATR) was measured to monitor the effect of sarcomere length (SL) on troponin C (TnC) structure during Ca2+ activation in single glycerinated rabbit psoas fibers and skinned right ventricular trabeculae from rats. Endogenous TnC was extracted, and the preparations were reconstituted with TnC fluorescently labeled with 5'ATR. In skinned psoas fibers reconstituted with sTnC labeled at Cys 98 with 5'ATR, dichroism was maximal during relaxation (pCa 9.2) and was minimal at pCa 4.0. In skinned cardiac trabeculae reconstituted with a mono-cysteine mutant cTnC (cTnC(C84)), dichroism of the 5'ATR probe attached to Cys 84 increased during Ca2+ activation of force. Force and dichroism-[Ca2+] relations were fit with the Hill equation to determine the pCa50 and slope (n). Increasing SL increased the Ca2+ sensitivity of force in both skinned psoas fibers and trabeculae. However, in skinned psoas fibers, neither SL changes or force inhibition had an effect on the Ca2+ sensitivity of dichroism. In contrast, increasing SL increased the Ca2+ sensitivity of both force and dichroism in skinned trabeculae. Furthermore, inhibition of force caused decreased Ca2+ sensitivity of dichroism, decreased dichroism at saturating [Ca2+], and loss of the influence of SL in cardiac muscle. The data indicate that in skeletal fibers SL-dependent shifts in the Ca2+ sensitivity of force are not caused by corresponding changes in Ca2+ binding to TnC and that strong cross-bridge binding has little effect on TnC structure at any SL or level of activation. On the other hand, in cardiac muscle, both force and activation-dependent changes in cTnC structure were influenced by SL. Additionally, the effect of SL on cardiac muscle activation was itself dependent on active, cycling cross-bridges.     

Evidence that the Sr2+ activation properties of cardiac troponin C are altered when substituted into skinned skeletal muscle fibers

Troponin C (TnC) was extracted from skinned skeletal muscle fibers by a method similar to that used previously on myofibrils (Zot, H.G., and Potter, J.D. (1982) J. Biol. Chem. 257, 7678-7683) and replaced with either skeletal (fast-twitch) or cardiac TnC. The relationship between isometric tension and Sr2+ concentration remained essentially the same before removal and after replacement with skeletal or cardiac TnC. Therefore, the origin of the TnC made no difference in the Sr2+ activation properties of the skinned fiber. In contrast, the activation of skinned cardiac fibers is approximately an order of magnitude more sensitive to Sr2+ than skinned skeletal fibers. These results show that the affinity of cardiac TnC for Sr2+ is altered when substituted into skinned skeletal muscle fibers through protein-protein interactions.     

Altered Ca2+ dependence of tension development in skinned skeletal muscle fibers following modification of troponin by partial substitution with cardiac troponin C

Binding of Ca2+ to the troponin C (TnC) subunit of troponin is necessary for tension development in skeletal and cardiac muscles. Tension was measured in skinned fibers from rabbit skeletal muscle at various [Ca2+] before and after partial substitution of skeletal TnC with cardiac TnC. Following substitution, the tension-pCa relationship was altered in a manner consistent with the differences in the number of low-affinity Ca2+-binding sites on the two types of TnC and their affinities for Ca2+. The alterations in the tension-pCa relationship were for the most part reversed by reextraction of cardiac TnC and readdition of skeletal TnC into the fiber segments. These findings indicate that the type of TnC present plays an important role in determining the Ca2+ dependence of tension development in striated muscle.     

Effects of partial extraction of troponin complex upon the tension-pCa relation in rabbit skeletal muscle. Further evidence that tension development involves cooperative effects within the thin filament

Partial extraction of troponin C (TnC) decreases the Ca2+ sensitivity of tension development in mammalian skinned muscle fibers (Moss, R. L., G. G. Giulian, and M. L. Greaser. 1985. Journal of General Physiology. 86:585), which suggests that Ca2+-activated tension development involves molecular cooperativity within the thin filament. This idea has been investigated further in the present study, in which Ca2+-insensitive activation of skinned fibers from rabbit psoas muscles was achieved by removing a small proportion of total troponin (Tn) complexes. Ca2+-activated isometric tension was measured at pCa values (i.e., -log[Ca2+]) between 6.7 and 4.5: (a) in control fiber segments, (b) in the same fibers after partial removal of Tn, and (c) after recombination of Tn. Tn removal was accomplished using contaminant protease activity found in preparations of LC2 from rabbit soleus muscle, and was quantitated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning densitometry. Partial Tn removal resulted in the development of a Ca2+-insensitive active tension, which varied in amount depending on the duration of the extraction, and concomitant decreases in maximal Ca2+-activated tensions. In addition, the tension-pCa relation was shifted to higher pCa values by as much as 0.3 pCa unit after Tn extraction. Readdition of Tn to the fiber segments resulted in the reduction of tension in the relaxing solution to control values and in the return of the tension-pCa relation to its original position. Thus, continuous Ca2+-insensitive activation of randomly spaced functional groups increased the Ca2+ sensitivity of tension development in the remaining functional groups along the thin filament. In addition, the variation in Ca2+-insensitive active tension as a function of Tn content after extraction suggests that only one-third to one-half of the functional groups within a thin filament need to be activated for complete disinhibition of that filament to be achieved.     

Interplay of Troponin- and Myosin-Based Pathways of Calcium Activation in Skeletal and Cardiac Muscle: The Use of W7 as an Inhibitor of Thin Filament Activation

To investigate the interplay between the thin and thick filaments during calcium activation in striated muscle, we employed n-(6-aminohexyl) 5-chloro-1-napthalenesulfonamide (W7) as an inhibitor of troponin C and compared its effects with that of the myosin-specific inhibitor, 2,3-butanedione 2-monoxime (BDM). In both skeletal and cardiac fibers, W7 reversibly inhibited ATPase and tension over the full range of calcium activation between pCa 8.0 and 4.5, resulting in reduced calcium sensitivity and cooperativity of ATPase and tension activations. At maximal activation in skeletal fibers, the W7 concentrations for half-maximal inhibition (K_I) were 70–80 μM for ATPase and 20–30 μM for tension, nearly >200-fold lower than BDM (20 mM and 5–8 mM, respectively). When W7 (50 μM) and BDM (20 mM) were combined in skeletal fibers, the ATPase and tension-pCa curves exhibited lower apparent cooperativity and maxima and higher calcium sensitivity than expected from two independent activation pathways, suggesting that the interplay between the thin and thick filaments varies with the level of activation. Significantly, the inhibition of W7 increased the ATPase/tension ratio during activation in both muscle types. W7 holds much promise as a potent and reversible inhibitor of thin filament-mediated calcium activation of skeletal and cardiac muscle contraction.     

Modulation of contractile activation in skeletal muscle by a calcium-insensitive troponin C mutant

Calcium controls the level of muscle activation via interactions with the troponin complex. Replacement of the native, skeletal calcium-binding subunit of troponin, troponin C, with mixtures of functional cardiac and mutant cardiac troponin C insensitive to calcium and permanently inactive provides a novel method to alter the number of myosin cross-bridges capable of binding to the actin filament. Extraction of skeletal troponin C and replacement with functional and mutant cardiac troponin C were used to evaluate the relationship between the extent of thin filament activation (fractional calcium binding), isometric force, and the rate of force generation in muscle fibers independent of the calcium concentration. The experiments showed a direct, linear relationship between force and the number of cross-bridges attaching to the thin filament. Further, above 35% maximal isometric activation, following partial replacement with mixtures of cardiac and mutant troponin C, the rate of force generation was independent of the number of actin sites available for cross-bridge interaction at saturating calcium concentrations. This contrasts with the marked decrease in the rate of force generation when force was reduced by decreasing the calcium concentration. The results are consistent with hypotheses proposing that calcium controls the transition between weakly and strongly bound cross-bridge states.     

Thin filament cooperativity as a major determinant of shortening velocity in skeletal muscle fibers.

The mechanism underlying the calcium sensitivity of the velocity of shortening of skeletal muscle fibers was investigated using a multiple shortening protocol: within a single contraction, skinned rabbit psoas fibers were made to shorten repetitively under a light load by briefly stretching back to their initial length at regular intervals. At saturating [Ca2+], the initial fast shortening pattern was repeated reproducibly. At submaximal [Ca2+], the first shortening consisted of fast and slow phases, but only the slow phase was observed in later shortenings. When the fibers were held isometric after the first shortening, the velocity of the second shortening recovered with time. The recovery paralleled tension redevelopment, implying a close relationship between the velocity and the number of the preexisting force-producing cross-bridges. However, this parallelism was lost as [Ca2+] was increased. Thus, the velocity was modified in a manner consistent with the cooperative thin filament activation by strong binding cross-bridges and its modulation by calcium. The present results therefore provide evidence that the thin filament cooperativity is primarily responsible for the calcium sensitivity of velocity. The effect of inorganic phosphate to accelerate the slow phase of shortening is also explained in terms of the cooperative activation.     

Fast skeletal muscle skinned fibers and myofibrils reconstituted with N-terminal fluorescent analogues of troponin C

Glycerinated rabbit fast skeletal muscle fibers were chemically skinned with 1% Brij 35 and partially depleted of endogenous troponin C subunit (TnC) by exposure of the fibers to EDTA (Zot, H. G., and Potter, J. D. (1982) J. Biol. Chem. 257, 7678-7683). The TnC-depleted fibers exhibited a decrease in maximal tension that was mostly restored by readdition of TnC or by the addition of the fluorescent 5-dimethylaminonaphthalene-1-sulfonyl aziridine analogue, TnCDanz. TnCDanz is known to undergo an increase in fluorescence intensity when Ca2+ binds to the two low affinity Ca2+-specific regulatory sites of TnC. Steady-state fractional fluorescence and tension changes were measured simultaneously as a function of Ca2+. The Ca2+ sensitivity of the fluorescence curve was about 0.6 log unit greater than the tension curve. This difference in sensitivity could be explained if separate conformational states of TnC, brought about by Ca2+ binding to the Ca2+-specific sites, produce the fluorescence and tension changes. TnC-depleted fibers were also reconstituted with the fluorescent 2-[(4'-iodoacetamido)analino]naphthalene-6-sulfonic acid analogue, cardiac TnCIaans, which undergoes an increase in fluorescence intensity when Ca2+ binds to the single Ca2+- specific regulatory site. The steady-state fractional fluorescence and tension curves for fibers reconstituted with cardiac TnCIaans had nearly the same Ca2+ sensitivity. The steady-state fractional fluorescence of myofibrils reconstituted with TnCDanz was found to have a greater sensitivity to Ca2+ than the simultaneously measured ATPase. In all cases paired fractional fluorescence and activity curves tended to have parallel dependence on Ca2+. These procedures make it possible to study the Ca2+ binding properties of the Ca2+- specific sites in intact myofibrils and skinned fibers; the results presented suggest that the Ca2+ affinity of the Ca2+-specific sites of troponin are reduced in the thin filament compared to that of troponin in solution.     

Co-operative interactions between troponin-tropomyosin units extend the length of the thin filament in skeletal muscle

Ca2+ binding to troponin C (TnC), a subunit of the thin filament regulatory strand, activates vertebrate skeletal muscle contraction. Tension, however, increases with Ca2+ too abruptly to be the result of binding to sites on individual TnCs. Because extraction of one TnC on average per regulatory strand dramatically reduces the slope of the tension/Ca2+ relationship, we proposed that all 26 troponin-tropomyosin complexes of the regulatory strand form a co-operative system. This study of permeabilized (chemically skinned) rabbit psoas fibers analyzes the extraction time-course, the distribution of extraction sites on regulatory strands and the effects of extraction on the co-operativity of the tension/Ca2+ relationship. Two components of TnC are resolved in the time-course of extraction: a "rapidly extracting" component that can be selectively removed without affecting tension or co-operativity, and a "slow extracting" component whose loss reduces tension and co-operativity. Extraction of [14C]TnC shows that the slowly extracting component is lost randomly, so that, after removal of 5% of the TnC, most extracted strands have lost one TnC. Extraction interrupts the transmission of co-operativity by dividing the regulatory strand into smaller, independent co-operative systems; it reduces tension by preventing Ca2+ activation of TnC-depleted regulatory units. Co-operativity of the tension/Ca2+ relationship is modeled with the concerted-transition formalism for intact systems of 26 regulatory units, and for the smaller systems in extracted fibers.     

Effect of troponin I phosphorylation by protein kinase A on length-dependence of tension activation in skinned cardiac muscle fibers

We examined the effect of troponin I (TnI) phosphorylation by cAMP-dependent protein kinase (PKA) on the length-dependent tension activation in skinned rat cardiac trabeculae. Increasing sarcomere length shifted the pCa (-log[Ca2+])-tension relation to the left. Treatment with PKA decreased the Ca2+ sensitivity of the myofilament and also decreased the length-dependent shift of the pCa-tension relation. Replacement of endogenous TnI with phosphorylated TnI directly demonstrated that TnI phosphorylation is responsible for the decreased length-dependence. When MgATP concentration was lowered in the absence of Ca2+, tension was elicited through rigorous cross-bridge-induced thin filament activation. Increasing sarcomere length shifted the pMgATP (-log[MgATP])-tension relation to the right, and either TnI phosphorylation or partial extraction of troponin C (TnC) abolished this length-dependent shift. We conclude that TnI phosphorylation by PKA attenuates the length-dependence of tension activation in cardiac muscle by decreasing the cross-bridge-dependent thin filament activation through a reduction of the interaction between TnI and TnC.     

Filament compliance influences cooperative activation of thin filaments and the dynamics of force production in skeletal muscle

Striated muscle contraction is a highly cooperative process initiated by Ca²⁺ binding to the troponin complex, which leads to tropomyosin movement and myosin cross-bridge (XB) formation along thin filaments. Experimental and computational studies suggest skeletal muscle fiber activation is greatly augmented by cooperative interactions between neighboring thin filament regulatory units (RU-RU cooperativity; 1 RU = 7 actin monomers+1 troponin complex+1 tropomyosin molecule). XB binding can also amplify thin filament activation through interactions with RUs (XB-RU cooperativity). Because these interactions occur with a temporal order, they can be considered kinetic forms of cooperativity. Our previous spatially-explicit models illustrated that mechanical forms of cooperativity also exist, arising from XB-induced XB binding (XB-XB cooperativity). These mechanical and kinetic forms of cooperativity are likely coordinated during muscle contraction, but the relative contribution from each of these mechanisms is difficult to separate experimentally. To investigate these contributions we built a multi-filament model of the half sarcomere, allowing RU activation kinetics to vary with the state of neighboring RUs or XBs. Simulations suggest Ca²⁺ binding to troponin activates a thin filament distance spanning 9 to 11 actins and coupled RU-RU interactions dominate the cooperative force response in skeletal muscle, consistent with measurements from rabbit psoas fibers. XB binding was critical for stabilizing thin filament activation, particularly at submaximal Ca²⁺ levels, even though XB-RU cooperativity amplified force less than RU-RU cooperativity. Similar to previous studies, XB-XB cooperativity scaled inversely with lattice stiffness, leading to slower rates of force development as stiffness decreased. Including RU-RU and XB-RU cooperativity in this model resulted in the novel prediction that the force-[Ca²⁺] relationship can vary due to filament and XB compliance. Simulations also suggest kinetic forms of cooperativity occur rapidly and dominate early to get activation, while mechanical forms of cooperativity act more slowly, augmenting XB binding as force continues to develop.     

Evidence that both Ca(2+)-specific sites of skeletal muscle TnC are required for full activity

To investigate the role of the Ca2(+)-specific (I and II) sites of fast skeletal muscle troponin C (TnC) in the regulation of contraction, we have produced two TnC mutants which have lost the ability to bind Ca2+ at either site I (VG1) or at site II (VG2). Both mutants were able to partially restore force to TnC-depleted skinned muscle fibers (approximately 25% for VG1 and approximately 50% for VG2). In contrast, bovine cardiac TnC (BCTnC), which like VG1 binds Ca2+ only at site II, could fully reactivate the contraction of TnC-depleted fibers. Higher concentrations of both mutants were required to restore force to the TnC-depleted fibers than with wild type TnC (WTnC) or BCTnC. VG1 and VG2 substituted fibers could not bind additional WTnC, indicating that all of the TnC-binding sites were saturated with the mutant TnC's. The Ca2+ concentration required for force activation was much higher for VG1 and VG2 substituted fibers than for WTnC or BCTnC substituted fibers. Also, the steepness of force activation was much less in VG1 and VG2 versus WTnC and BCTnC substituted fibers. These results suggest cooperative interactions between sites I and II in WTnC. In contrast, BCTnC has essentially the same apparent Ca2+ affinity and steepness of force activation as does WTnC. Thus, cardiac TnC must have structural differences from WTnC which compensate for the lack of site I, while in WTnC, both Ca2(+)-specific sites are probably crucial for full functional activity.     

Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers

Intrinsic troponin C (TnC) was extracted from small bundles of rabbit psoas fibers and replaced with TnC labeled with dansylaziridine (5-dimethylaminonaphthalene-1-sulfonyl). The flourescence of incorporated dansylaziridine-labeled TnC was enhanced by the binding of Ca2+ to the Ca2+-specific (regulatory) sites of TnC and was measured simultaneously with force (Zot, H.G., Güth, K., and Potter, J.D. (1986) J. Biol. Chem. 261, 15883-15890). Various myosin cross-bridge states also altered the fluorescence of dansylaziridine-labeled TnC in the filament, with cycling cross-bridges having a greater effect than rigor cross-bridges; and in both cases, there was an additional effect of Ca2+. The paired fluorescence and tension data were used to calculate the apparent Ca2+ affinity of the regulatory sites in the thin filament and were shown to increase at least 10-fold during muscle activation presumably due to the interaction of cycling cross-bridges with the thin filament. The cross-bridge state responsible for this enhanced Ca2+ affinity was shown to be the myosin-ADP state present only when cross-bridges are cycling. The steepness of the pCa force curves (where pCa represents the -log of the free Ca2+ concentration) obtained in the presence of ATP at short and long sarcomere lengths was the same, suggesting that cooperative interactions between adjacent troponin-tropomyosin units may spread along much of the actin filament when cross-bridges are attached to it. In contrast to the cycling cross-bridges, rigor bridges only increased the Ca2+ affinity of the regulatory sites 2-fold. Taken together, the results presented here indicate a strong coupling between the Ca2+ regulatory sites and cross-bridge interactions with the thin filament.