Cardiac Troponin C Research Articles

Effects of troponin C isoforms on pH sensitivity of contraction in mammalian fast and slow skeletal muscle fibres.

1. The effects of troponin C (TnC) isoforms on the acidic pH-induced rightward shift in the tension-pCa (-log[Ca2+]) relationship were examined in slow soleus and fast psoas skeletal muscle fibers. Endogenous TnC was partially extracted from skinned single fibres and the extracted fibres were subsequently reconstituted with purified TnC. The pCa producing one-half maximal tension (pCa50) was determined at pH 7.00 and 6.20 in each fibre and then the pH-induced shift in pCa50 (delta pCa50) was calculated. 2. In control fast fibres which express fast skeletal TnC (sTnC), the delta pCa50 was 0.64 +/- 0.02 pCa units (n = 10), and this increased significantly to 0.78 +/- 0.04 pCa units (n = 8) following extraction and reconstitution with cardiac TnC (cTnC). In each fibre, the reconstituted delta pCa50 was subtracted from the control delta pCa50 which yielded a significant shift of -0.13 +/- 0.05 pCa units (n = 8; P < 0.05). Thus, the pH sensitivity of contraction was increased in the cTnC-reconstituted psoas fibres. 3. In extracted psoas fibres that were reconstituted with fast sTnC the pH sensitivity of contraction was unchanged, indicating that the above effects were related to the TnC isoform and not a non-specific effect of the extraction procedure. 4. In a second series of experiments cTnC was specifically extracted from slow soleus fibres which were subsequently reconstituted with purified fast sTnC. Skeletal TnC reconstituted soleus fibres demonstrated a significant decrease in pH sensitivity. In each fibre, the reconstituted delta pCa50 (mean, 0.58 +/- 0.02 pCa units) was subtracted from the control delta pCa50 (mean, 0.63 +/- 0.02 pCa units) which yielded a significant shift of 0.05 +/- 0.01 pCa units (n = 4; P < 0.05). The pH sensitivity was not altered in cTnC-reconstituted soleus fibres (-0.01 +/- 0.01 pCa units, n = 4). 5. These findings indicate that TnC isoforms alter the pH sensitivities of contraction in slow and fast skeletal muscle fibres. However, the magnitude of the change in pH sensitivity is muscle lineage dependent, indicating that differential expression of other myofilament protein isoforms, together with TnC, is necessary to confer full pH sensitivity of contraction in striated muscles.

Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.

Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin-I mutants and troponin-C

Troponin C (TnC) could be extracted from skinned porcine cardiac muscle fibres and their Ca 2+ sensitivity restored by reconstitution with recombinant human cardiac TnC. After extraction of troponin I (TnI) and TnC using the vanadate treatment method of Strauss et al. [Strauss, J. D., Zeugner, C., Van Eyk, J.E., Bletz, C., Troschka, M. and Rüegg, J.C. (1992) FEBS Lett. 310, 229–234], skinned porcine cardiac muscle fibres were reconstituted with wild-type recombinant human cardiac TnC and either wild-type cardiac TnI or several mutant isoforms of human TnI. Reconstitution with wild-type proteins restored the Ca 2+ sensitivity of the tissue and phosphorylation of the TnI with the catalytic subunit of protein kinase A reduced the Ca 2+ sensitivity (i.e. -log[Ca 2+] for 50% of maximal force) as has been shown by others. However, reconstitution with the TnI mutant Ser-23Asp/Ser-24Asp mimicking the phosphorylated form of cardiac TnI, led to a reduced Ca 2+ sensitivity compared with reconstitution with wild-type TnI, whereas the mutant Ser-23Ala/Ser-24Ala behaved as the dephosphorylated form of TnI. These data confirm the importance of negative charge in this region of the TnI molecule in altering the Ca 2+ responsiveness in this system.

Phosphorylation of Both Serine Residues in Cardiac Troponin I Is Required to Decrease the Ca2+ Affinity of Cardiac Troponin C

The phosphorylation of cardiac muscle troponin I (CTnI) at two adjacent N-terminal serine residues by cAMP-dependent protein kinase (PKA) has been implicated in the inotropic response of the heart to beta-agonists. Phosphorylation of these residues has been shown to reduce the Ca2+ affinity of the single Ca(2+)-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca2+ dissociation from this site (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Recent studies (Zhang, R., Zhao, J., and Potter, J. D. (1995) Circ. Res. 76, 1028-1035) have correlated this increase in Ca2+ dissociation with a reduced Ca2+ sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation. To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine. The wild type and the mutated CTnIs were expressed in Escherichia coli and purified. Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca2+ sensitivity of force development seen in a skinned cardiac muscle preparation. The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca2+ sensitivity. The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.

The Effects of Phosphorylation of Cardiac Troponin-I on Its Interactions with Actin and Cardiac Troponin-C

It is known that the phosphorylation of two serine residues on the NH2-terminal extension specific to cardiac troponin-I (Tn-I) modulates the calcium-dependent activation of the myofilaments. The process by which this occurs remains an unsolved puzzle. We have applied a dissective approach to study the effect of this phosphorylation on the interactions between Tn-I and its partner proteins, actin and troponin-C (Tn-C). Using N-[14C]ethylmaleimide-labelled Tn-I in sedimentation assays with F-actin, we found that the dephosphorylated Tn-I binds to F-actin with pronounced positive cooperativity, both in the absence and the presence of tropomyosin. Phosphorylation of the protein slightly weakens the interaction in the absence of tropomyosin, but the cooperativity remained. In the presence of tropomyosin, phosphorylation of the Tn-I also appeared to slightly weaken the interaction as well but, more significantly, the cooperativity was eliminated. These data can only be explained simply by a cooperative interaction between the monomer units in the actin filament. The interactions between cardiac Tn-I and Tn-C were studied by labelling the Tn-C with the fluorescent probe dansyl aziridine. As expected, the binding of the dephosphorylated Tn-I to Tn-C was strengthened by over 20-fold upon the addition of calcium to the assay. Phosphorylation of the protein, however, had a dramatic effect on the interaction in that it appeared to desensitise the complex to the effect of calcium: the Ka values obtained for both interactions (+/- Ca2+) were almost identical. These results clearly indicate that the phosphorylation of Tn-I in the cardiac system has dramatic effects on the isolated inter-protein interactions. We also discuss the possible significance of such an effect on the interactions of the isolated proteins in their roles within the intact cardiac regulatory complex.

The Effects of Phosphorylation of Cardiac Troponin-I on Its Interactions with Actin and Cardiac Troponin-C

The Effects of Phosphorylation of Cardiac Troponin-I on Its Interactions with Actin and Cardiac Troponin-C

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)

Kinetics of tension development in skinned cardiac myocytes measured by photorelease of Ca2+

The rate of activation of tension development by free Ca2+ was examined in skinned rat ventricular myocytes. Pulse photolysis of the photosensitive Ca2+ chelator, Nitr-7, was used to rapidly elevate Ca2+ in the vicinity of the myofilaments. Tension increased exponentially with a first-order rate constant (kCa) that depended on the level of Ca2+ activation. kCa increased approximately linearly from 0.9 +/- 0.2 s-1 at 20% maximal Ca(2+)-activated tension (Po) to 4.0 +/- 0.9 s-1 at 85% Po, representing a fourfold increase in kCa with activation. Reducing free Mg2+ from 1 to 0.1 mM accelerated kCa by about twofold at all levels of Ca2+. Tension development kinetics were significantly different in skinned rabbit psoas fibers: kCa increased nonlinearly from 1.2 +/- 0.2 s-1 at 15% Po to a maximum of 17.5 +/- 1.3 s-1 at 85% Po, representing a 15-fold increase in kCa with activation. Moreover, lowering free Mg2+ increased kCa only at submaximal Ca2+ in psoas fibers. Extraction of troponin C (TNC) from psoas fibers and recombination with bovine cardiac TNC did not significantly alter any of these characteristics of tension development kinetics. We conclude that significant differences exist between cardiac and fast-twitch skeletal muscles in terms of the effects of Ca2+ and Mg2+ on contraction kinetics and that these differences cannot be attributed solely to differences in TNC isoforms.

The role of troponin C in modulating the Ca2+ sensitivity of mammalian skinned cardiac and skeletal muscle fibres.

1. We investigated the effects of acidosis, inorganic phosphate (Pi) and caffeine on the Ca2+ affinity of isolated fast-twitch skeletal and cardiac troponin C (TnC), labelled with fluorescent probes to report Ca2+ binding to the regulatory sites. We also measured the effects of these interventions on the maximum force development and the Ca2+ sensitivity of skinned fibres from fast-twitch skeletal muscle and cardiac muscle, as has been done previously. The two types of experiment were carried out under similar solution conditions, so that we could assess the contribution of any direct actions on TnC to the modulation of Ca2+ sensitivity in the skinned muscle fibres. 2. In skinned fibres, acidosis (decreasing pH from 7.0 to 6.2) and Pi (20 mM) suppressed maximum force to the same extent within a given muscle type, but had greater effects on cardiac fibres compared with skeletal fibres. Caffeine (20 mM) depressed maximum force equally in cardiac and skeletal muscle. Thus, the fall of force induced by acidosis or Pi may involve a different mechanism from that induced by caffeine. 3. Skinned skeletal fibres were more Ca2+ sensitive than cardiac fibres by 0.29 pCa units (pCa = -log10[Ca2+]). Isolated skeletal TnC also had a greater Ca2+ affinity than cardiac TnC, by 0.20 pCa units. These results suggest that the Ca2+ sensitivity of skinned fibres is at least partly determined by the type of TnC present. 4. Acidosis reduced the Ca2+ sensitivity of force in skinned fibres profoundly and had a 2-fold greater effect in cardiac muscle than skeletal muscle (falls in pCa for 50% activation, pCa50, were 1.09 and 0.55, respectively). Acidosis also reduced the Ca2+ affinity of TnC, again having double the effect on the pCa50 for cardiac TnC (0.58) as on that for skeletal TnC (0.28). The greater effect of acidosis on cardiac skinned fibres, compared with skeletal, may be partly explained, therefore, by the type of TnC present, and one-half of the effect on fibres may be attributed to the direct effect of H+ on TnC. 5. Pi reduced the Ca2+ sensitivity of force in skeletal and cardiac skinned fibres by 0.30 and 0.19 pCa units, respectively. However, the Ca2+ affinity of isolated cardiac and skeletal TnC was unaffected by Pi, indicating that the decrease in muscle Ca2+ sensitivity is not mediated by a direct action of Pi on TnC. 6. Caffeine increased the Ca2+ sensitivity of cardiac skinned fibres by 0.31 pCa units, which was 3 times greater than for the skeletal fibres (0.09 pCa units).(ABSTRACT TRUNCATED AT 400 WORDS)

Formation of Fetal Rat Cardiac Cell Clones by Retroviral Transformation: Retention of Select Myocyte Characteristics

Ventricular myocyte (cardiomyocyte) growth is exquisitely regulated such that embryonic and fetal development are the primary periods of active cellular division. This report describes formation of three separate cardiomyocyte cell clones obtained by replication-defective retroviral (v/ myc and v-H- ras) transformation of primary cultures of day-16 fetal rat cardiomyocytes. The cell clones do not spontaneously contract, yet they express several cardiac-specific (cardiac troponin-C, alpha-cardiac actin) and associated genes (Gonnexin 43, Early growth response gene-1) with stable expression of several genes determined through the 28th passage. None of these cell clones express skeletal muscle actin or the skeletal muscle regulatory gene. MyoD1; yet all display ultrastructural and biochemical evidence of their cardiac muscle lineage. Molecular and biochemical studies of cardiac-specific gene regulation can be anticipated from the cell clones as it pertains to nuclear transcription factors and transient CAT-based reporter gene constructs. The formation of these cell clones will enable further studies of growth and development of this unique muscle cell population of the cardiovascular system to be performed at the cellular and molecular level.

Interaction of troponin I and troponin C: use of the two-dimensional transferred nuclear Overhauser effect to determine the structure of a Gly-110 inhibitory troponin I peptide analog when bound to cardiac troponin C

The structure of a peptide analog of the inhibitory region of cardiac troponin-1 ( N-acetyl-G110-TnI(104–115)amide) when bound to cardiac troponin-C has been determined by 2-dimensional 1H-NMR techniques. The bound structure determined for this peptide is similar to that determined previously for the skeletal peptide (which has a proline at position 110) bound to skeletal troponin-C (Campbell and Sykes (1991) J. Mol. Biol. 222, 405–412). This structure shows a helical like peptide backbone ”bent“ around P109—G110 to bring the hydrophobic residues F1106, L111 and V114 closer together. The other ”side“ of this structure is surrounded by the basic residues extending outwards towards the protein or solution. While the bound structures of the cardiac and skeletal peptides are shown to be quite similar, the cardiac peptide appears more flexible near the central glycine residue.

Time-resolved tryptophan emission study of cardiac troponin I.

We have carried out a time-resolved fluorescence study of the single tryptophanyl residue (Trp-192) of bovine cardiac Tnl (CTnl). With excitation at 300 nm, the intensity decay was resolved into three components by a nonlinear least-squares analysis with lifetimes of 0.60, 2.22, and 4.75 ns. The corresponding fractional amplitudes were 0.27, 0.50, and 0.23, respectively. These decay parameters were not sensitive to complexation of CTnl with cardiac troponin C (CTnC), and magnesium and calcium had no significant effect on the decay parameters. After incubation with 3':5'-cyclic AMP-dependent protein kinase, the intensity decay of CTnl required a fourth exponential term for satisfactory fitting with lifetimes of 0.11, 0.81, 1.95, and 6.63 ns and fractional amplitudes of 0.06, 0.37, 0.27, and 0.29, respectively. When bound to CTnC, the intensity decay of phosphorylated CTnl (p-CTnl) also required four exponential terms for satisfactory fitting, but the longest lifetime increased by a factor of 1.7. The decay parameters obtained from the complex formed between p-CTnl and CTnC were not sensitive to either magnesium or calcium. The anisotropy decay was resolved into two components with rotational correlation times of 0.90 and 23.48 ns. Phosphorylation resulted in a decrease of the long correlation time to 14.61 ns. The anisotropy values recovered at zero time suggest that the side chain of the Trp-192 had considerable subnanosecond motional freedom not resolved in these experiments. Within the CTnl.CTnC complex, the unresolved fast motions appeared sensitive to calcium binding to the calcium-specific site of CTnC. The observed emission heterogeneity is discussed in terms of possible excited-state interactions in conjunction with the predicted secondary structure of CTnl. The loss of molecular asymmetry of cardiac troponin I induced by phosphorylation as demonstrated in this work may be related to the known physiological effect of beta-agonists on cardiac contractility.

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.

The role of troponin C in the length dependence of Ca(2+)‐sensitive force of mammalian skeletal and cardiac muscles.

1. Skinned fibre preparations of right ventricular trabeculae, psoas and soleus muscles from hamster and rabbit were activated by Ca2+ and the length dependencies of their pCa (-log [Ca2+])-force relationships were compared. 2. Ca2+ sensitivity of the myocardium was higher at 2.2-2.4 microns than that at 1.7-1.9 microns. The length dependence was at least twofold greater in cardiac muscle than in fast skeletal fibres at identical temperatures and salt concentrations. Slow-twitch fibres gave a response similar to that in the myocardium. 3. The effect of the troponin C (TnC) phenotype on the length dependence of Ca2+ sensitivity was measured on both fast skeletal fibres and cardiac muscle with TnC exchange in situ. The length-induced increase in Ca2+ sensitivity was found to be greater in the presence of cardiac TnC than with fast skeletal TnC. Thus the results indicate that a certain domain of TnC is specialized in this length function, and that this domain is different in the two phenotypes. 4. The possibility that the enhanced length dependence of Ca2+ sensitivity after cardiac TnC reconstitution was attributable to reduced TnC binding was excluded when the length dependence of partially extracted fast fibres was reduced to one-half the normal value after a 50% deletion of the native TnC. 5. Two recombinant forms of cardiac TnC (kindly provided by Dr John Putkey, Houston, TX, USA) were used next, to investigate the roles of two specific domains in TnC in the control of length dependence of Ca2+ sensitivity and in the contraction-relaxation switching of cardiac muscle: 6. Using mutant CBM1 [corrected], in which site 1 was modified such as to bind the 4th Ca2+ ion, as in skeletal TnC, the length-induced Ca2+ sensitivity in cardiac muscle was suppressed. The effect was intermediate between cardiac and skeletal TnCs under the same conditions. The pSr (-log [Sr2+])-force relationship of cardiac muscle was also measured. In the presence of the mutant, skinned trabeculae manifest pSr-activation curves identical to those of fast fibres. This indicates that the metal ion binding properties of site 1 in TnC modulate the regulatory action of site 2. 7. Using mutant CBM2A, in which site 2 was inactivated, the activation of cardiac muscle by both Ca2+ and Sr2+ ions was completely blocked. This is the expected result, since both regulatory sites were now inactive, regulatory site 1 being normally inactive in cardiac muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

The low-affinity Ca2(+)-binding sites in cardiac/slow skeletal muscle troponin C perform distinct functions: site I alone cannot trigger contraction.

Both troponin C (TnC) and calmodulin share a remarkably similar tertiary motif that may be common to other Ca2(+)-binding proteins with activator activity. TnC plays a critical role in regulating muscle contraction and is particularly well-suited for structural analysis by site-directed mutation. Fast-twitch skeletal muscle TnC has two low-affinity Ca2(+)-binding sites (sites I and II), while in cardiac and slow-twitch skeletal muscle TnC site I is inactive. Recently, using protein engineering, we directly demonstrated that binding of Ca2+ to the low-affinity site(s) initiates muscle contraction. In the present study, we use mutagenesis to determine whether either of the low-affinity sites in cardiac TnC can trigger contraction in slow-twitch skeletal muscle fibers. In one Ca2(+)-binding mutant, Ca2(+)-binding to the dormant low-affinity site I was restored (CBM+I). In a second mutant, site I was activated while site II was inactivated (CBM+I-IIA). Both proteins had the predicted CA2(+)-binding characteristics, and both were able to associate with troponin I and troponin T to form a troponin complex and integrate into permeabilized slow-twitch skeletal muscle fibers. A comparison of NMR spectra shows the aromatic regions in the two proteins to be qualitatively similar without divalent cations but markedly different with Ca2+. Mutant CBM+I supported force generation in skinned slow skeletal muscle fibers but had Sr2+ and Ca2+ sensitivities similar to fast skeletal TnC. Mutant CBM+I-IIA was unable to restore Ca2(+)-dependent contraction to TnC-depleted skinned slow muscle fibers. The data directly demonstrate that low-affinity sites I and II have distinct functions and that only site II in cardiac TnC can trigger muscle contraction in slow-twitch skeletal muscle fibers. This principle of distinct, modular activities for Ca2(+)-binding sites in the same protein may apply to other members of the TnC/calmodulin family.

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.

Modification of temperature dependence of myofilament Ca sensitivity by troponin C replacement

The Ca sensitivity of chemically skinned right ventricular trabeculae from the rat heart was determined at 22 and 8 degrees C. Endogenous troponin C (TnC) was then extracted with EDTA and replaced with either bovine cardiac TnC or rabbit fast-twitch skeletal TnC. The temperature dependence of myofilament Ca sensitivity was then reevaluated. Cooling native cardiac tissue from 22 to 8 degrees C reduced the pCa (-log10 [Ca2+]), generating half-maximal tension (K1/2) from 5.20 +/- 0.07 to 4.89 +/- 0.08 (SD, n = 14), and also reduced maximum Ca-activated force to 33 +/- 6% of its value at 22 degrees C. After extraction of endogenous TnC and reconstitution with cardiac TnC, cooling from 22 to 8 degrees C caused a similar shift in mean K1/2 from 4.93 +/- 0.08 to 4.69 +/- 0.06 (n = 7). When skeletal TnC was reconstituted into TnC-extracted ventricular fibers, cooling from 22 to 8 degrees C led to a much smaller mean shift in K1/2 from 4.88 +/- 0.07 to 4.78 +/- 0.04 (n = 7). The results show that the magnitude of the cooling-induced shift in myofilament Ca sensitivity observed in the native state (or after reconstitution with cardiac TnC) is significantly reduced if the fiber is reconstituted with skeletal TnC (P less than 0.001). This indicates that the temperature dependence of myofilament Ca sensitivity of cardiac muscle can be modified by incorporation of skeletal TnC. Thus Ca binding to TnC plays an important role in determining the temperature dependence of myofilament Ca sensitivity.

Molecular cloning and expression of chicken cardiac troponin C.

We have isolated a full length complementary DNA clone (pCTnC1) from a 19-day embryonic chicken heart library corresponding to cardiac troponin C (TnC). Sequence analysis demonstrated varying homologies with TnC complementary DNA clones isolated from developing chick skeletal muscle. Using pCTnC1 as a hybridization probe, we have determined that cardiac TnC is constitutively expressed in both atria and ventricles of the developing and adult heart. These data along with previous immunochemical studies of TnC expression demonstrate that the slow skeletal and cardiac muscle isoform is the only TnC expressed in the heart. In contrast, expression of slow skeletal and cardiac muscle TnC is developmentally regulated in skeletal muscles of the chicken. The tightly controlled expression of slow skeletal and cardiac muscle TnC in the varying myocyte types of the heart suggests a physiologically significant role of this regulatory protein.

Ca2+, pH and the regulation of cardiac myofilament force and ATPase activity

When the pH surrounding myofilaments of striated muscle is reduced there is an inhibition of both the actin-myosin reaction as well as the Ca2+-sensitivity of the myofilaments. Although the mechanism for the effect of acidic pH on Ca2+-sensitivity has been controversial, we have evidence for the hypothesis that acidic pH reduces the affinity of troponin C (TNC) for Ca2+. This effect of acidic pH depends not only on a direct effect of protons on Ca2+-binding to TNC, but also upon neighboring thin filament proteins, especially TNI, the inhibitory component of the TN complex. Using fluorescent probes that report Ca2+-binding to the regulatory sites of skeletal and cardiac TNC, we have shown, for example, that acidic pH directly decreases the Ca2+-affinity of TNC, but only by a relatively small amount. However, with TNC in whole TN or in the TNI-TNC complex, there is about a 2-fold enhancement of the effects of acidic pH on Ca2+-binding to TNC. Acidic pH decreases the affinity of skeletal TNI for skeletal TNC, and also influences the micro-environment of a probe positioned at Cys-133 of TNI, a region of interaction with TNC. Other evidence that the effects of acidic pH on Ca2+-TNC activation of myofilaments are influenced by TNI comes from studies with developing hearts. In contrast, to the case with the adult preparations, Ca2+-activation of detergent extracted fibers prepared from dog or rat hearts in the peri-natal period are weakly affected by a drop in pH from 7.0 to 6.5. This difference in the effect of acidic pH appears to be due to a difference in the isoform population of TNI, and not to differences in isotype population or amount of TNC.

Down-regulation of fast-twitch skeletal muscle fiber with cardiac troponin-C and recombinant mutants Structure/function studies with site-directed mutagenesis

Structure/function relationships in troponin C are studied with vertebrate fast-twitch fibers by exchanging the skeletal troponin C with two bacterially synthesized recombinant proteins designed by site-directed mutagenesis of cardiac troponin C. One mutant (CBM1) contained an additional active site, by deleting Val-28 and converting Leu-29, Gly-30, Ala-31 and Glu-32 to Asp, Ala, Asp and Gly, respectively, in the normally inactive trigger site 1 in the N-terminus. In another mutant (CBM2A), the normally active site 2 was inactivated by conversion of Asp-65 to Ala. The fibers were found to be down-regulated with recombinant cardiac troponin C (CTnC3), as with tissue-cardiac-troponin-C. With mutants, in one case (CBM1) the regulation was unmodified despite Ca2+ coordination by all sites. In contrast, regulation was found to be completely blocked with the mutant (CBM2A) where both trigger sites were inactive. The results provide the first indication that structural specification of the entire EF-hand motif of site 1, and not just Ca2+ coordination, is needed to operate fully the Ca2+ switch in fast-twitch fibers.