Roles of troponin isoforms in pH dependence of contraction in rabbit fast and slow skeletal and cardiac muscles.

Skinned fibers prepared from rabbit fast and slow skeletal and cardiac muscles showed acidotic depression of the Ca2+ sensitivity of force generation, in which the magnitude depends on muscle type in the order of cardiac>fast skeletal>slow skeletal. Using a method that displaces whole troponin-complex in myofibrils with excess troponin T, the roles of Tn subunits in the differential pH dependence of the Ca2+ sensitivity of striated muscle were investigated by exchanging endogenous troponin I and troponin C in rabbit skinned cardiac muscle fibres with all possible combinations of the corresponding isoforms expressed in rabbit fast and slow skeletal and cardiac muscles. In fibers exchanged with fast skeletal or cardiac troponin I, cardiac troponin C confers a higher sensitivity to acidic pH on the Ca2+ sensitive force generation than fast skeletal troponin C independently of the isoform of troponin I present. On the other hand, fibres exchanged with slow skeletal troponin I exhibit the highest resistance to acidic pH in combination with either isoform of troponin C. These results indicate that troponin C is a determinant of the differential pH sensitivity of fast skeletal and cardiac muscles, while troponin I is a determinant of the pH sensitivity of slow skeletal muscle.     

Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins

1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C. 2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity. 3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations. 4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase. 5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.     

Ca2+ binding to skeletal muscle troponin C in skeletal and cardiac myofibrils

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.     

Elucidation of isoform-dependent pH sensitivity of troponin i by NMR spectroscopy

Myocardial ischemia is characterized by reduced blood flow to cardiomyocytes, which can lead to acidosis. Acidosis decreases the calcium sensitivity and contractile efficiency of cardiac muscle. By contrast, skeletal and neonatal muscles are much less sensitive to changes in pH. The pH sensitivity of cardiac muscle can be reduced by replacing cardiac troponin I with its skeletal or neonatal counterparts. The isoform-specific response of troponin I is dictated by a single histidine, which is replaced by an alanine in cardiac troponin I. The decreased pH sensitivity may stem from the protonation of this histidine at low pH, which would promote the formation of electrostatic interactions with negatively charged residues on troponin C. In this study, we measured acid dissociation constants of glutamate residues on troponin C and of histidine on skeletal troponin I (His-130). The results indicate that Glu-19 comes in close contact with an ionizable group that has a pK(a) of ～6.7 when it is in complex with skeletal troponin I but not when it is bound to cardiac troponin I. The pK(a) of Glu-19 is decreased when troponin C is bound to skeletal troponin I and the pK(a) of His-130 is shifted upward. These results strongly suggest that these residues form an electrostatic interaction. Furthermore, we found that skeletal troponin I bound to troponin C tighter at pH 6.1 than at pH 7.5. The data presented here provide insights into the molecular mechanism for the pH sensitivity of different muscle types.     

Site-directed mutation of the trigger calcium-binding sites in cardiac troponin C

The trigger Ca2+-binding sites in troponin C, those which initiate muscle contraction, are thought to be the first two of four potential sites (sites I-IV). In cardiac troponin C, the first Ca2+-binding site is inactive, and initiation of contraction in cardiac muscle appears to involve only the second site. To study this phenomenon and associated Ca2+-dependent protein conformational changes in cardiac troponin C, the cDNA for the chicken protein was incorporated into a bacterial expression plasmid to allow site-specific mutagenesis. Ca2+-binding site I was activated by deletion of Val-28 and conversion of amino acids 29-32 to those found at the first four positions in the active site I of fast skeletal troponin C. In a series of proteins, Ca2+-binding site II was inactivated by mutation of amino acids Asp-65, Asp-67, and Gly-70. All mutated proteins exhibited the predicted calcium-binding characteristics. The single mutation of converting Asp-65 to Ala was sufficient to inactivate site II. Ca2+-dependent conformational changes in the normal and mutated proteins were monitored by labeling with a sulfhydryl-specific fluorescent dye. Activation of Ca2+-binding site I or inactivation of site II, eliminated the large Ca2+-dependent increase in fluorescence seen in the wild type protein and there was, instead, a Ca2+-dependent decrease in fluorescence. All mutant proteins could associate with troponin I and troponin T to form a troponin complex. Activation of Ca2+-binding site I changed the characteristics of contraction in skinned slow skeletal muscle fibers such that the response to Ca2+ was more cooperative. Inactivation of Ca2+-binding site II abolished Ca2+-dependent contraction in skinned muscle fibers. The data provide a direct demonstration that Ca2+-binding site II in cardiac troponin C is essential for triggering muscle contraction and support the hypothesis that site I functions to modify the characteristics of contraction.     

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.     

Effect of substitution of troponin C in cardiac myofibrils with skeletal troponin C or calmodulin on the Ca2+- and Sr2+-sensitive ATPase activity

Troponin C was removed almost completely from the porcine cardiac myofibrils by the same extraction procedure using CDTA as that previously reported for the rabbit skeletal myofibrils (Morimoto, S. & Ohtsuki, I. (1987) J. Biochem. 101, 291-301), and the effects of substitution of troponin C in cardiac myofibrils with rabbit skeletal troponin C or bovine brain calmodulin were examined. While the ATPase activity of intact cardiac myofibrils or cardiac troponin C-reconstituted cardiac myofibrils was activated at only a little higher concentration of Sr2+ than Ca2+, the skeletal troponin C-substituted cardiac myofibrils, as well as intact rabbit skeletal myofibrils, required more than 10 times higher concentration of Sr2+ than Ca2+ for activation of the myofibrillar ATPase activity. However, the concentrations of Ca2+ and Sr2+ required for the activation of the ATPase activity of the skeletal troponin C-substituted cardiac myofibrils were both about 5 times higher than those of intact skeletal myofibrils. The skeletal troponin C-substituted cardiac myofibrils, as well as intact skeletal myofibrils, also showed higher cooperativity in the Ca2+-activation of the ATPase activity than intact or cardiac troponin C-reconstituted cardiac myofibrils. The ATPase activity of calmodulin-substituted cardiac myofibrils was activated at a several times lower concentration of Ca2+ or Sr2+ than that of calmodulin-substituted skeletal myofibrils, while the ratios of the concentration of Sr2+ to Ca2+ required for activation were almost the same in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of troponin I isoform switching in determining the pH sensitivity of Ca(2+) regulation in developing rabbit cardiac muscle

Skinned muscle fibers prepared from fetal rabbit heart (28 days of gestation) showed a marked resistance to acidic pH in the Ca(2+) regulation of force generation, compared to the fibers prepared from adult heart. SDS-PAGE and immunoblot analysis showed that the slow skeletal troponin I was predominantly expressed in the fetal cardiac muscle, while the cardiac isoform was predominantly expressed in the adult cardiac muscle. Direct exchange of purified slow skeletal and cardiac troponin I isoforms into these skinned muscle fibers revealed that cardiac troponin I made the Ca(2+) regulation of contraction sensitive to acidic pH just as in the adult fibers, whereas slow skeletal troponin I made the Ca(2+) regulation of contraction resistant to acidic pH just as in the fetal fibers. These results demonstrate that the troponin I isoform switching accounts fully for the change in the pH dependence of Ca(2+) regulation of contraction in developmental cardiac muscle.     

The effect of heart and skeletal muscle troponin complexes and calmodulin on the Ca2+-dependent reactions of phosphorylase kinase isoenzymes

The dephosphorylated form of phosphorylase kinase was purified 700-fold from rabbit heart extract. The purified enzyme had a pH 6.8/pH 8.2 activity ratio of 0.04-0.08 and was completely dependent on Ca2+ with an apparent Ka value for Ca2+ of 2.59 microM at pH 6.8. At free Ca2+ concentrations between 0.057 microM and 400 microM, 1.5 microM rabbit heart troponin complex had no significant effect on the reaction. However, 1.5 microM rabbit skeletal muscle troponin complex stimulated the reaction 1.5-2-fold with a concomitant decrease in the Ka value for Ca2+ to 1.40 microM. No differences in the effects of these troponin complexes were observed when heart-type and skeletal muscle-type phosphorylase b isoenzymes from either rabbit or pig were used as substrate. Similar effects of heart and skeletal muscle troponin complexes were observed on the Ca2+-dependent reaction of the dephosphorylated form of phosphorylase kinase partially purified from rabbit skeletal muscle. A saturating concentration (1.36 microM) of bovine brain calmodulin stimulated 2-5-fold the Ca2+-dependent reaction of skeletal muscle phosphorylase kinase, but not the reaction of heart phosphorylase kinase. Heart troponin complex (12 microM) suppressed 80-100% the stimulatory effect of skeletal muscle troponin complex on the reactions of phosphorylase kinase isoenzymes, but had no significant effect on the stimulation by calmodulin of skeletal muscle phosphorylase kinase reaction.     

Troponin C-like protein in chicken gizzard muscle.

1. A troponin C-like protein was prepared from frozen chicken gizzard by preparative polyacrylamide gel electrophoresis and its apparent molecular weight was estimated to be about 15,500 daltons. 2. In urea gel electrophoresis, the mobility of the troponin C-like protein increased slightly in the presence of Ca2+, like that of skeletal muscle troponin C. On the other hand, the mobility of the the troponin C-like protein in glycerol gel electrophoresis, unlike that of skeletal muscle troponin C, was significantly decreased by Ca2+. 3. In alkaline gel electrophoresis, the troponin C-like protein formed a Ca2+-dependent complex with troponin I or troponin T from skeletal muscle. 4. The troponin C-like protein could neutralize the inhibitory effect of skeletal muscle troponin I on the Mg2+-activated ATPase of actomyosin from rabbit skeletal muscle, but could not confer Ca2+-sensitivity on the actomyosin in the presence of troponin I and troponin T from skeletal muscle.     

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.     

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.     

Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.

Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction.     

Troponin from the myocardium and skeletal muscles: structure and properties

The literary and experimental data on the structure and properties of cardiac and skeletal muscle troponin are reviewed. The cation--binding sites of cardiac and skeletal muscle troponin C are distinguished by specificity; the sites localized in the C-terminal part of the protein molecule can bind both Ca2+ and Mg2+, whereas the sites localized at the N-end specifically bind Ca2+. The use of bifunctional reagents revealed a number of helical sites within the structure of cardiac troponin C (residues 84-92 and 150-158) and of skeletal muscle troponin C (residues 90-98 and 125-136). A comparison of experimental data with the results of an X-ray analysis testifies to the presence in the central part of the troponin C molecule of a long alpha-helical sequence responsible for troponin C interaction with the inhibiting peptide of troponin I. The efficiency of interaction of troponin components depends on Ca2+ concentration; the integrity of the overall troponin complex is mainly provided for by troponin C interaction with troponin I and by troponin I interaction with troponin T. The interaction between troponins T and C is relatively weak, especially in the case of cardiac troponin components. Both skeletal and cardiac muscles synthesize several troponin T isoforms differing in length and amino acid composition of N-terminal 40-60 member peptides. Troponin T isoforms can undergo phosphorylation by several protein kinases. The single site of troponin T which exists in a phosphorylated state in vivo (residue Ser-1) undergoes phosphorylation by specific protein kinase (troponin T kinase) related to casein kinases II. It was assumed that the phosphorylation of Ser-1 residue of troponin T as well as the synthesis of troponin T isoforms differing in the structure of the N-terminal peptide, provides for the regulation of interaction between two neighbouring tropomyosin molecules.     

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.     

Effect of phosphorylation of porcine cardiac troponin I by 3':5'-cyclic AMP-dependent protein kinase on the actomyosin ATPase activity

1. Porcine cardiac native tropomyosin was phosphorylated by bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Most of the phosphate incorporation was observed in troponin I, the maximum of which was 0.7 mol of Pi per mol of troponin I. 2. In the presence of phosphorylated native tropomyosin, actomyosin ATPase activity was 15-40% lower than that in the presence of the unphosphorylated preparation at all calcium ion concentrations (1.5 x 10(-8) M-2.4 x 10(-5) M). Half-maximum activation of ATPase was obtained with a concentration of 7 x 10(-7) M Ca2+ (unphosphorylated) and 1.3 x 10(-6) M Ca2+ (phosphorylated), respectively. Maximum ATPase activity was reached with 3 x 10(-6) M Ca2+ (unphosphorylated) and 1.0 x 10(-5) M Ca2+ (phosphorylated). 3. Porcine cardiac troponin I isolated by affinity chromatography inhibited ATPase activity of desensitized actomyosin in the presence of tropomyosin. There was little difference between phosphorylated troponin I and a control preparation with regard to the inhibitory effect of ATPase activity. 4. Troponin C from rabbit skeletal muscle neutralized the inhibitory effect of troponin I. The minimum amount of troponin C required for complete neutralization was approximately equimolar to troponin I. The inhibitory effect of phosphorylated troponin I was neutralized by troponin C less effectively than that of unphosphorylated preparation.     

Differential contribution of troponin I phosphorylation sites to the endothelin-modulated contractile response

Cardiac troponin I is a phosphorylation target for endothelin-activated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca2+ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca2+ transient could not explain the divergent relaxation response to endothelin in myocytes expressing cardiac versus slow skeletal troponin I nor could activation of Na+/H+ exchange. Three separate clusters within cardiac troponin I are phosphorylated by protein kinase C, and we set out to determine the contribution of the Thr144 and Ser23/Ser24 clusters to the endothelin-mediated contractile response. Myocyte replacement with a cardiac troponin I containing a Thr144 substituted with the Pro residue found in slow skeletal troponin I resulted in prolonged relaxation in response to acute endothelin compared with control myocytes. Ser23/Ser24 also is a target for protein kinase C phosphorylation of purified cardiac troponin I, and although this cluster was not acutely phosphorylated in intact myocytes, significant phosphorylation developed within 1 h after adding endothelin. Replacement of Ser23/Ser24 with Ala indicated that this cluster contributes significantly to relaxation during more prolonged endothelin stimulation. Overall, results with these mutants provide evidence that Thr144 plays an important role in the acute acceleration of relaxation, whereas Ser23/Ser24 contributes to relaxation during more prolonged activation of protein kinase C by endothelin.     

The highly conserved COOH terminus of troponin I forms a Ca2+-modulated allosteric domain in the troponin complex

The primary structure of the COOH-terminal region of troponin I (TnI) is highly conserved among the cardiac, slow, and fast skeletal muscle TnI isoforms and across species. Although no binding site for the other thin filament proteins is found at the COOH terminus of TnI, truncations of the last 19-23 amino acid residues reduce the activity of TnI in the inhibition of actomyosin ATPase and result in cardiac muscle malfunction. We have developed a specific monoclonal antibody (mAb), TnI-1, against the conserved COOH terminus of TnI. Using this mAb, isolation of the troponin complex by immunoaffinity chromatography from muscle homogenate and immunofluorescence microscopic staining of myofibrils indicate that the COOH terminus of TnI forms an exposed structure in the muscle thin filament. Binding of this mAb to the COOH terminus of cardiac TnI induced extensive conformational changes in the protein, suggesting an allosteric role of this region in the functional integrity of troponin. In the absence of Ca2+, the binding of troponin C and troponin T to TnI had very little effect on the conformation of the COOH terminus of TnI as indicated by the unaffected mAb affinity for the TnI-1 epitope. However, Ca2+ significantly increased the accessibility of the TnI-1 epitope on TnI in the presence of troponin C and troponin T. The results provide evidence that the COOH terminus is an essential structure in TnI and participates in the allosteric switch during Ca2+ activation of contraction.     

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.     

Replacement of three troponin components with cardiac troponin components within single glycerinated skeletal muscle fibers.

The tension of single glycerinated rabbit skeletal muscle fiber was desensitized to a Ca(2+)-concentration after treatment with an excessive amount of bovine cardiac troponin T and reached a level of about 70% of the maximum tension of the untreated fiber. A SDS-gel electrophoretic examination indicated that troponin C.I.T complex in the fiber was replaced with the added cardiac troponin T. The Ca(2+)-sensitivity of the tension of the troponin T-treated fiber was then recovered by the addition of bovine cardiac troponins I and C. The rabbit skeletal muscle fiber thus hybridized with bovine cardiac troponin C.I.T showed the same cooperativity of Ca(2+)-activation as the cardiac muscle.