The cardiac troponin C isoform and the length dependence of Ca2+ sensitivity of tension in myocardium.

The Ca2+ sensitivity of tension in cardiac muscle is length dependent, such that the sensitivity is diminished with decreasing sarcomere length below 2.4 microm. This length dependence of Ca2+ sensitivity of tension also forms the basis for the Frank-Starling mechanism in the heart. The fast-twitch skeletal muscle has a much lower length dependence of Ca2+ sensitivity. In a recent study of skinned cardiotrabeculae, we indicated that the exchange of endogenous cardiac troponin C (TnC) for skeletal troponin C also resulted in a major reduction in the length dependence to the level of skeletal muscle. These findings suggested that cardiac troponin C has a key role in the length-sensing mechanism. The present investigation supports this conclusion and delineates the specific domain in cardiac TnC responsible for the length effect. Chimeras splicing either 41, 61, or 96 N-terminal cardiac amino acids with the remaining skeletal residues have indicated that while Ca2+ binding in all three constructs is similar to that in wild type cardiac TnC, the functional responsiveness of the 96-cardiac residue construct is improved over the other two. This 96-cardiac residue construct yielded a tension response indistinguishable from that of wild-type cardiac TnC. A tryptophan variant of the chimera indicated fluorescence characteristics indistinguishable from cardiac troponin C. The findings provide further support for the idea that cardiac troponin C in situ is modified in response to sarcomere length change and thereby participates in the Frank-Starling mechanism. Moreover, the study indicates that the tropinin C length-sensing attribute originates within the N-terminal domain constituted by these 96 residues.

Molecular basis of depression of Ca2+ sensitivity of tension by acid pH in cardiac muscles of the mouse and the rat.

BACKGROUND: Acid pH decreases the Ca2+ sensitivity of myocardial tension generation, and recent studies have suggested that regulatory proteins are involved. The current study defines the molecular basis of this effect on troponin C (TnC) and troponin I (TnI) and also addresses previous differences between the rat and mouse. METHODS AND RESULTS: Endogenous cardiac TnC and cardiac TnI in isolated trabeculae from mice and rats were exchanged with their fast-twitch skeletal muscle counterparts. A cardiac-skeletal TnC chimera was used to define the target region for proton action on cardiac TnC. Finally, cardiac TnC and skeletal TnC were genetically modified by insertion of a tryptophan for phenylalanine-26 to probe the pH effects with fluorescence spectroscopy. The pH 6.2 effects on Ca2+ sensitivity of force development in mouse and rat cardiotrabeculae are largely accounted for by the proton influences on TnC (23%) and TnI (53%). In cardiac TnC, residues 1 to 41 provide the target region. Comparison of the Ca(2+)-induced fluorescence in isolated cardiac TnC and skeletal TnC also indicated a greater pH effect in the cardiac isoform. CONCLUSIONS: The studies provide firm evidence that both TnC and TnI moieties are involved in the mechanism of acidosis causing reduction in the Ca sensitivity of force development in the myocardium. The findings rule out the possibility of interspecies variations in the underlying mechanisms. The genetically designed TnCs and a chimera demonstrate that the observed TnC-mediated difference in the pH effects on Ca2+ sensitivity of tension between cardiac and skeletal muscles is preserved in these isolated proteins. The N-terminal amino acid residues 1 to 41 in cardiac TnC are established as the pH sensor of this protein in the mouse as in the rat.

Functional role of arginine-11 in the N-terminal helix of skeletal troponin C: combined mutagenesis and molecular dynamics investigation.

The two main structural differences between calmodulin (CaM) and skeletal troponin C (sTnC) are the absence in CaM of (i) the short N-terminal helix in TnC and (ii) the triplet KGK (residues 91-93; numbering according to chicken sTnC). It was recently shown that deletion of both structural groups from sTnC imparted to the resulting construct the CaM-like ability to activate phosphodiesterase (PDE) and to regulate force development in smooth muscle. To continue probing of the structural basis of the differential behavior of sTnC and CaM, residue Arg-11 in rabbit sTnC was mutated to Ala because the interactions of Arg-11 with distal residues in the N-terminal domain seem to link the N-terminal helix to the rest of the structure. The mutant exhibits CaM-like function in its ability to activate PDE (about 50% of CaM at 5 microM concentration). If, in addition, the KGK triplet is also deleted, PDE activation increases to about 80%. Both constructs retain their TnC function to nearly 100%. To explore the mechanistic basis of this remarkable observation, computational simulations of the molecular dynamics (MD) were carried out for both wild-type 4Ca2+.sTnC and the 4Ca2+.R11A mutant, and the results were compared to those from earlier simulations of 4Ca2+.CaM. Two types of structural changes observed from such simulations of the molecular dynamics of CaM had been considered to have a functional role: (i) a compaction to a more globular form and (ii) a reorientation of the Ca-binding domains around the central tether helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Diminished Ca2+ sensitivity of skinned cardiac muscle contractility coincident with troponin T-band shifts in the diabetic rat.

We have measured the apparent Ca2+ sensitivities of force development in skinned cardiac trabeculae at different sarcome lengths together with shifts in troponin (Tn) T subunits on specimens from the same hearts and drawn insights into the pathogenesis of myocardial dysfunction in the diabetic rat. The Ca(2+)-force relations were measured at a long (2.4-microns) and a short (1.9-microns) sarcomere length. In disease, compared with the control condition, the apparent Ca2+ sensitivity was greatly diminished at a sarcomere length of 1.9 microns but not affected at all at the long length (2.4 microns). We also examined the alterations in contractile regulatory proteins TnT and TnI by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots. The TnI band was largely unperturbed, but major changes were discerned in TnT. The normal rat heart indicated two major bands (TnT1 and TnT2) and a faint third band (TnT3); in the diabetic rat heart, there was a significant shift in intensity from TnT1 to TnT3. Since myosin isozyme shifts also accompany diabetes in the rat, we used a prototypical hypothyroid rat as well to evaluate the myosin influence in the length-induced effects on Ca2+ sensitivity. Myosin shifts during hypothyroidism were unaccompanied by significant changes in TnT, and there were also no length-dependent modifications in Ca2+ sensitivity. The findings raise the possibility that diabetic Ca(2+)-sensitivity changes in the myocardium are coupled with TnT alterations. A plausible explanation is offered whereby these TnT alterations modify the length dependence of Ca2+ sensitivity.

Contributions of troponin I and troponin C to the acidic pH-induced depression of contractile Ca2+ sensitivity in cardiotrabeculae.

Acid pH diminishes the Ca2+ sensitivity for force generation in both cardiac and skeletal muscles, but the mechanisms for these remain undetermined. In permeabilized (skinned) single myofibers of fast-twitch skeletal muscle of the rat, we find that pCa50 of the pCa-force relationship was 5.73 in pH 7 and 5.02 in pH 6.2 (delta pKskeletal = pCa50 in pH 7-pCa50 in pH 6.2 = 0.71 pCa unit); on the other hand, in skinned cardiotrabeculae, the hpCa50 was 5.79 in pH 7 decreasing to 4.14 in pH 6.2 (delta pKcardiac = 1.65 pCa units). We have used this large differential between cardiac/skeletal delta pKs to probe the mechanisms of the pH effects. Since troponin C (TnC) and troponin I (TnI) each have a central role in the Ca2+ switch, we exchanged these proteins in cardiac muscle with their skeletal counterparts and reinvestigated the pH effects. Firstly, with fast-twitch skeletal muscle (sTnC) substituting for 80% of the endogenous cardiac TnC (cTnC), the cardiac pH effect was decreased marginally (modified delta pK = 1.39 pCa units). This TnC-mediated change was further probed with two distinct cardiac-skeletal TnC chimeras, c1/s and CBc1/s (the Ca(2+)-binding c1/s), in which a majority of the N-terminal 41 amino acid residues was made cardiac and the rest skeletal [Gulati, J., & Rao, V. G. (1994) Biochemistry 33, 9052-9056]. The phenotype shift following sTnC/cTnC exchange in the trabeculae was blocked when c1/s was used in lieu of sTnC; on the other hand, interestingly, CBc1/s exactly mimicked sTnC.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mobility of the Ca(2+)-deficient EF-hand of cardiac troponin C as revealed by fluorescence polarization of genetically inserted tryptophan.

To probe attitudinal features of the Ca(2+)-deficient site (site I) in the Ca2+ switch of cardiac troponin C (cTnC), we have examined steady-state fluorescence emission and polarization of a Trp26 inserted in a recombinant cardiac TnC (cTnC3.W) and compared these with the properties of the Ca(2+)-competent site I in skeletal TnC (sTnC4.W). The Ca(2+)-induced fluorescence emission in cTnC3.W was a fraction (25-30%) of that in sTnC4.W, in agreement with previous observations on the Ca(2+)-deficient site incorporated in a cardiac/skeletal chimera c1/s.W [Gulati, J. & Rao, V. G. (1994) Biochemistry 33, 9052-9056]. Thus, the fractional quantum yield reflected intrinsic properties of the cardiac metal ion-deficient site I. Conversely, in sTnC-1.W, where the skeletal site I also was made Ca(2+)-deficient by D27-->A substitution, the Ca(2+)-induced quantum yield was lower than that in cTnC3.W. Nevertheless, similar steady-state fluorescence polarizations for Ca(2+)-saturated sTnC4.W and cTnC3.W indicated indistinguishable final conformations in the two activated TnC isoforms. In EGTA, the polarization parameter (PEGTA) of sTnC4.W is greater than that of cardiac TnC, and the cardiac PEGTA value is closer to the activated PCa. Comparison of the chimera c1/s.W with sTnC-1.W indicated that the differences in conformation of the site I Trp for the EGTA-treated cardiac/skeletal isoforms were due to the structural disparities in this region. This contention was further supported by examination of the chimera CBc1/s.W, where the cardiac EF-hand was altered by 27VLGA30-->DAD substitution. Polarization of the relaxed form was similar to that for sTnC4.W. These findings suggest that the relaxed conformation of the cardiac Ca2+ switch is more favorably predisposed to activation than the skeletal switch.

The role of glycine (residue 89) in the central helix of EF-hand protein troponin-C exposed following amino-terminal alpha-helix deletion.

Because an N-terminal alpha-helical (N-helix) arm and a KGK-triplet (residues 88KGK90) in the central helix of troponin-C (TnC) are missing in calmodulin, several recent studies have attempted to elucidate the structure-function correlations of these units. Presently, with a family of genetically manipulated derivatives especially developed for this study and tested on permeabilized isolated single skeletal muscle fiber segments, we explored the specificities of the amino acid residues within the N-helix and the KGK-triplet in TnC. Noticeably, the amino acid compositions vary between the N-helices of the cardiac and skeletal TnC isoforms. On the other hand, the KGK-triplet is located similarly in both TnC isoforms. We previously indicated that deletion of the N-helix (mutant delta Nt) diminishes the tension obtained on activation with maximal calcium, but the contractile function is revived by the superimposed deletion of the 88KGK90-triplet (mutant delta Nt delta KGK; see Gulati J, Babu A, Su H, Zhang YF, 1993, J Biol Chem 268:11685-11690). Using this functional test, we find that replacement of Gly-89 with a Leu or an Ala could also overcome the contractile defect associated with N-helix deletion. On the other hand, replacement of the skeletal TnC N-helix with cardiac type N-helix was unable to restore contractile function. The findings indicate a destabilizing influence of Gly-89 residue in skeletal TnC and suggest that the N-terminal arm in normal TnC serves to moderate this effect. Moreover, specificity of the N-helix between cardiac and skeletal TnCs raises the possibility that resultant structural disparities are also important for the functional distinctions of the TnC isoforms.

Disparate contributions of Tyr10 and Tyr109 to fluorescence intensity of rabbit skeletal muscle troponin C identified using a genetically engineered mutant.

Intrinsic tyrosines, as monitored by fluorescence spectroscopy, are sensitive reporters of local, Ca(2+)-induced conformational changes in troponin C (TnC). Rabbit skeletal TnC contains two tyrosines (Y10 in the N-helix, and Y109 in site 3 in the C-terminal domain) in distinct microenvironments: their individual contributions to total fluorescence intensity are elucidated here utilizing bacterially synthesized rabbit skeletal TnC (sTnC4) and a genetically engineered variant, termed 109YF, lacking one of the tyrosines (Y109 replaced with F109). The steady-state fluorescence emission spectra following excitation at 280 nm were recorded in EGTA (Ca(2+)-free) and Ca(2+)-saturated (pCa4) solutions. For the wild-type sTnC4, pCa4 causes a significant (46%) increase in the peak fluorescence intensity over the value in EGTA. For the mutant 109YF, the EGTA fluorescence is only marginally affected (74% of the wild-type FEGTA), but interestingly the Ca2+ effect is completely suppressed (delta F = FpCa4-FEGTA = 2% of the wild-type value). These results indicate that the two tyrosines make disparate contributions to the fluorescence spectrum of wild-type sTnC, both in the presence and absence of Ca2+; whereas Y10 in the N-helix is dominant in Ca(2+)-free solution, Y109 is the sole contributor to the Ca2+ effect. Furthermore, to explain the biphasic fluorescence response of Y109 obtained during Ca2+ titrations, the findings yield the most unequivocal evidence that Ca(2+)-induced conformational changes in the trigger sites operating the contractile switch modify properties of the C-terminal sites in TnC pari passu.

Shifts in contractile regulatory protein subunits troponin T and troponin I in cardiac hypertrophy.

To examine the molecular basis of hypertrophied heart failure, we investigated the changes in cardiac contractile regulatory proteins. The guinea pigs were subjected to chronic pressure overload with aortic banding to induce ventricular hypertrophy, and in-situ pressure-volume relations were recorded together with biochemical characterizations to ascertain the contractile modifications. Immunoblots of left and right ventricular samples revealed four distinct troponin T isoforms, which underwent alterations during hypertrophy. The higher molecular weight bands TnT1 and TnT2 shifted towards the lower molecular weight isoforms TnT3 and TnT4. For TnI, a single prominent band was detected, whose intensity also increased with pump failure. The findings provide the first direct evidence of TnT and TnI shifts in an experimentally induced hypertrophied heart failure and has novel mechanistic implications for the future studies.