Cardiac Troponin C Research Articles

Challenging Current Paradigms Related to Cardiomyopathies: ARE CHANGES IN THE Ca2+ SENSITIVITY OF MYOFILAMENTS CONTAINING CARDIAC TROPONIN C MUTATIONS (G159D AND L29Q) GOOD PREDICTORS OF THE PHENOTYPIC OUTCOMES?

Two novel mutations (G159D and L29Q) in cardiac troponin C (CTnC) associate their phenotypic outcomes with dilated (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Current paradigms propose that sarcomeric mutations associated with DCM decrease the myofilament Ca2+ sensitivity, whereas those associated with HCM increase it. Therefore, we incorporated the mutant CTnCs into skinned cardiac muscle in order to determine if their effects on the Ca2+ sensitivities of tension and ATPase activity coincide with the current paradigms and phenotypic outcomes. The G159D-CTnC decreases the Ca2+ sensitivity of tension and ATPase activation and reduces the maximal ATPase activity when incorporated into regulated actomyosin filaments. Under the same conditions, the L29Q-CTnC has no effect. Surprisingly, changes in the apparent G159D-CTnC Ca2+ affinity measured by tension in fibers do not occur in the isolated CTnC, and large changes measured in the isolated L29Q-CTnC do not manifest in the fiber. These counterintuitive findings are justified through a transition in Ca2+ affinity occurring at the level of cardiac troponin and higher, implying that the true effects of these mutations become apparent as the hierarchical level of the myofilament increases. Therefore, the contractile apparatus, representing a large cooperative machine, can provide the potential for a change (G159D) or no change (L29Q) in the Ca2+ regulation of contraction. In accordance with the clinical outcomes and current paradigms, the desensitization of myofilaments from G159D-CTnC is expected to weaken the contractile force of the myocardium, whereas the lack of myofilament changes from L29Q-CTnC may preserve diastolic and systolic function.

Treatment of experimental verapamil poisoning with levosimendan utilizing a rodent model of drug toxicity

Background. Levosimendan is an inotropic agent used in the treatment of heart failure. It is a myocardial calcium sensitizer, binding to cardiac troponin-C, and a vascular K+ATP-channel agonist producing peripheral vasodilatation. Aims. To assess the effect of levosimendan on cardiac output (CO), blood pressure (BP), and heart rate (HR) in a rodent model of severe verapamil poisoning. Methods. Male Wistar rats were anesthetized, ventilated, and canulated with jugular and femoral venous catheters and a femoral arterial catheter. CO, systolic BP, MAP, and HR were recorded. Verapamil was infused at 6 mg/kg/h until MAP dropped to 50% of baseline (time-0) and then reduced to 4 mg/kg/h. There were five treatment groups (n = 7 per group): 1) normal saline infusion (control); 2) CaCl2 loading dose and infusion (CaCl2); 3) levosimendan 24 microgram/kg loading dose and 0.6 microgram/kg/min infusion (Levo-24); 4) levosimendan 6 microgram/kg loading dose and 0.4 microgram/kg/min infusion (Levo-6); and 5) levosimendan 0.4 microgram/kg/min infusion with concurrent CaCl2 loading dose and infusion (Levo + CaCl2). Hemodynamic parameters were recorded for 70 minutes. Primary outcome measures were changes observed in CO, BP, and HR with treatments compared to control. Secondary outcome measure was survival. Results were analyzed using one-way ANOVA with Dunnet's post-test comparison with the control group. Results. All groups had similar BP, HR, and CO at base line and peak toxicity. The control group's HR, BP, and CO progressively fell during the verapamil infusion. Levo-24, Levo + CaCl2, and CaCl2 maintained CO compared with control from t = 20 min and Levo6 from t = 30min (p < 0.05). CaCl2 (from t = 10 min) and Levo + CaCl2 (from t = 20 min) produced significant improvements in BP compared to control. However, BP did not return to pre-toxicity levels. Levo-6 and Levo-24 groups did not recover from the hypotension seen at pre-treatment maximal toxicity. HR was maintained in all treatment groups compared to control animals. Twenty-nine percent (2/7) of control, 86% (6/7) levosimendan, 100% (7/7) CaCl2, and Levo + CaCl2 animals survived to the end of the protocol. Conclusions. Levosimendan increased CO in this model of verapamil poisoning to a similar degree as CaCl2 alone, but it did not improve BP from time of maximal toxicity. The addition of CaCl2 to Levosimendan did not appear to result in any further improvement in CO and BP compared to CaCl2 alone. The failure of levosimendan to improve BP may result from vasodilation induced by levosimendan peripheral vascular K+ATP-channel agonism. This may compound the vasodilatory effects of verapamil and offset any hemodynamic improvements produced by increased cardiac output.

Functional and evolutionary relationships of troponin C

Striated muscle contraction is initiated when, following membrane depolarization, Ca(2+) binds to the low-affinity Ca(2+) binding sites of troponin C (TnC). The Ca(2+) activation of this protein results in a rearrangement of the components (troponin I, troponin T, and tropomyosin) of the thin filament, resulting in increased interaction between actin and myosin and the formation of cross bridges. The functional properties of this protein are therefore critical in determining the active properties of striated muscle. To date there are 61 known TnCs that have been cloned from 41 vertebrate and invertebrate species. In vertebrate species there are also distinct fast skeletal muscle and cardiac TnC proteins. While there is relatively high conservation of the amino acid sequence of TnC homologs between species and tissue types, there is wide variation in the functional properties of these proteins. To date there has been extensive study of the structure and function of this protein and how differences in these translate into the functional properties of muscles. The purpose of this work is to integrate these studies of TnC with phylogenetic analysis to investigate how changes in the sequence and function of this protein, integrate with the evolution of striated muscle.

Myosin Binding Protein C in the Heart

See related article, pages 1290–1298 Myocardial force generation is initiated on the release of calcium ions from the sarcoplasmic reticulum and subsequent activation of the contractile apparatus.1 A schematic of the cardiac sarcomere and some of its component proteins is shown in the Figure.2 Contractile activation involves binding calcium to Tn-C, redistribution of the affinity of Tn-I for Tn-C away from Tn-T, and movement of tropomyosin on the thin filament, thereby exposing binding sites on actin. Binding of the myosin S1 domain to actin leads to cross-bridge force development. However, this simple two state model of muscle contraction is not sufficient to explain many features of muscle contraction.2 For example, the rate of force development critically depends on the activation state of the thin filament.2 Also, binding of a modified myosin (NEM-S1 which cannot hydrolyze ATP) has been shown to abolish this phenomenon.3 This, and other structural evidence, has led to the hypothesis that the thin filament must have at least three states: a blocked state (top left), where tropomyosin sterically blocks S1 binding; a closed state, induced by calcium binding to Tn-C, where tropomyosin has shifted away somewhat allowing weak interaction with the S1 domain; and an open state, where tropomyosin has moved away fully allowing for a strong interaction with S1 and force generation (bottom).2 One interesting aspect of this theory is that the binding of myosin to actin by itself induces further activation of the thin filament, thus facilitating S1 domain binding along the filament; an elegant, yet simple molecular model of cooperative activation. Cardiac muscle activation is highly cooperative, even though calcium binding to isolated cardiac troponin-C itself is not cooperative.2,4 However, it is less clear how modulation of cross-bridge cycling rate could be accommodated by such a …

Effects of Phe‐to‐Trp mutation and fluorotryptophan incorporation on the solution structure of cardiac troponin C, and analysis of its suitability as a potential probe for in situ NMR studies

19F NMR spectroscopy is potentially a powerful tool for probing protein properties in situ. However, results obtained using this technique are relevant only if the 19F probe offers minimal perturbation to the surrounding environment. In this paper, we examine the effect of 5-fluorotryptophan (5fW) incorporation on the three-dimensional structure of cardiac troponin-C (cTnC), with the intention of developing a 19F-labeled TnC for use in in situ 19FNMR. We find that, in general, 5fW does not perturb the structure of the protein significantly. Replacement of residue Phe 153 with 5fW produces no noticeable change in protein conformation. However, replacement of residue Phe 104 with 5fW produces a folding behavior that is dependent on the Escherichia coli strain used to express the mutant. The orientations of the indole rings in these mutants are such that the Trp residue adopts a chi2 of approximately 90 degrees in the F104W mutant and approximately -100 degrees in the F153W mutant. Using results from 19F-1H heteronuclear NOE experiment, we show the replacement of L-Trp with 5fW at these positions does not change the orientation of the indole ring and the spread of the 5fW side-chain dihedral angles increases moderately for the F104(5fW) mutant and not at all for the F153(5fW) mutant. Based on these structures, we conclude that the substitution of Phe by 5fW at these two positions has minimal effects on the structure of cTnC and that the 5fW indole rings in both mutants have well defined orientation, making the two mutants viable candidates for use in in situ 19F NMR spectroscopy.

Effects of troponin C isoform on the action of the cardiotonic agent EMD 57033

The effects of the cardiotonic potentiator EMD 57033 on different TnC (troponin C) isoforms were investigated. Endogenous skeletal TnC was extracted from glycerinated, permeabilized rabbit psoas fibres and replaced with either purified native rabbit psoas TnC (fast TnC) or human recombinant cTnC (cardiac TnC) (3 mg/ml in relaxing solution for 30 min). In both conditions, 10 microM EMD 57033 increased maximal calcium-activated force (Pmax) and gave a leftward shift in the pCa-tension curve. With cTnC, the increase in Pmax was much greater (228%) compared with the effect seen for fast TnC (137%), which was the same as that in unextracted control fibres. When the whole troponin was replaced rather than just TnC, the effects of EMD 57033 on fibres replaced with cTn were the same as with the cTnC subunit alone, except that the force at low Ca2+ concentrations was not increased as much. If TnC was only partially extracted, it was found that the degree of extraction did not influence the effect of EMD 57033, except when force was decreased to below 10% of the pre-extraction Pmax. Dynamic stiffness was not altered by EMD 57033 in any of the preparations. The rate of tension recovery following a release-restretch method (ktr) was decreased by EMD 57033. We conclude that EMD 57033 acts by a rate-modulating effect, and that the quantitative response of this effect is dependent on the TnC isoform present.

Site-Directed Spin Labeling Electron Paramagnetic Resonance Study of the Calcium-Induced Structural Transition in the N-Domain of Human Cardiac Troponin C Complexed with Troponin I

Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggers skeletal and cardiac muscle contraction. The salient feature of this structural transition is the movement of the B and C helices, which is termed the "opening" of the N-domain. This movement exposes a hydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seen in the crystal structure of the troponin ternary complex [Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2003) Nature 424, 35-41]. In contrast to skeletal TnC, Ca(2+)-binding site I (an EF-hand motif that consists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising from comparisons with skeletal TnC is how both helices move according to Ca(2+) binding or interact with TnI in cardiac TnC. In this study, we examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directed spin labeling electron paramagnetic resonance (EPR). Doubly spin-labeled TnC mutants were prepared, and the spin-spin distances were estimated by analyzing dipolar interactions with the Fourier deconvolution method. An interspin distance of 18.4 A was estimated for mutants spin labeled at G42C on the B helix and C84 on the D helix in a Mg(2+)-saturated monomer state. The interspin distance between Q58C on the C helix and C84 on the D helix was estimated to be 18.3 A under the same conditions. Distance changes were observed by the addition of Ca(2+) ions and the formation of a complex with TnI. Our data indicated that the C helix moved away from the D helix in a distinct Ca(2+)-dependent manner, while the B helix did not. A movement of the B helix by interaction with TnI was observed. Both Ca(2+) and TnI were also shown to be essential for the full opening of the N-domain in cardiac TnC.

Inhibition of the cardiac alpha-actin gene in embryonic cardiac myocytes by dominant-negative serum response factor.

Serum response factor (SRF), a transcription factor ubiquitously involved in the processes of cellular proliferation and differentiation, has been implicated in cardiac and skeletal muscle development because of its strong expression in embryonic muscle lineages, and its necessity for the transcription of transiently transfected muscle genes that contain SRF binding sites. This study was designed to ascertain whether SRF is required for the expression of an endogenous SRF-dependent gene during differentiation of early embryonic cardiac myocytes by introducing a dominant-negative SRF construct via retroviral delivery. Although no effect on overt cellular differentiation was detected, semi-quantitative RT-PCR revealed that expression of the SRF-dependent gene cardiac alpha-actin was inhibited, whereas expression of the non-SRF-dependent genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cardiac troponin-C was unaffected. No effect on myocyte proliferation was detected. Curiously, immunohistochemical localization of SRF protein suggested that whereas endogenous SRF was homogeneously dispersed throughout the cytoplasm and nucleus, the dominant-negative SRF protein was concentrated in the nucleus. These results extend previous findings using transiently transfected genes to the endogenous level, indicating that SRF is required for the full expression of muscle genes that contain SRF binding sites during cardiac myocyte differentiation.

Temperature dependence of dynamics and thermodynamics of the regulatory domain of human cardiac troponin C.

Binding of Ca(2+) to the regulatory domain of troponin C (TnC) in cardiac muscle initiates a series of protein conformational changes and modified protein-protein interactions that initiate contraction. Cardiac TnC contains two Ca(2+) binding sites, with one site being naturally defunct. Previously, binding of Ca(2+) to the functional site in the regulatory domain of TnC was shown to lead to a decrease in conformational entropy (TDeltaS) of 2 and 0.5 kcal mol(-1) for the functional and nonfunctional sites, respectively, using (15)N nuclear magnetic resonance (NMR) relaxation studies [Spyracopoulos, L., et al. (1998) Biochemistry 37, 18032-18044]. In this study, backbone dynamics of the Ca(2+)-free regulatory domain are investigated by backbone amide (15)N relaxation measurements at eight temperatures from 5 to 45 degrees C. Analysis of the relaxation measurements yields an order parameter (S(2)) indicating the degree of spatial restriction for a backbone amide H-N vector. The temperature dependence of S(2) allows estimation of the contribution to protein heat capacity from pico- to nanosecond time scale conformational fluctuations on a per residue basis. The average heat capacity contribution (C(p,j)) from backbone conformational fluctuations for regions of secondary structure for the regulatory domain of cardiac apo-TnC is 6 cal mol(-1) K(-1). The average heat capacity for Ca(2+) binding site 1 is larger than that for site 2 by 1.3 +/- 0.8 cal mol(-1) K(-1), and likely represents a mechanism where differences in affinity between Ca(2+) binding sites for EF hand proteins can be modulated.

The role of the Ca(2+) regulatory sites of skeletal troponin C in modulating muscle fibre reactivity to the Ca(2+) sensitizer bepridil.

1. The Ca(2+)-sensor protein troponin C (TnC) exerts a key role in the regulation of muscle contraction, and constitutes a target for Ca(2+) sensitizer compounds, such as bepridil, known to increase its apparent Ca(2+) affinity. Moreover, bepridil has been reported to exert a differential effect in slow and fast skeletal muscle fibres, which express the slow/cardiac and fast TnC isoform, respectively. 2. The role of the TnC isoform in establishing the differential effect of bepridil was assessed in slow soleus and fast tibialis rat skinned fibres, by extraction of endogenous TnC and consecutive reconstitution with either slow or fast recombinant TnC. A mutant (VG2), lacking the regulatory site II, was also used to distinguish the role of each regulatory site. 3. Fast tibialis fibres reconstituted with cardiac TnC exhibited a typical slow bepridil reactivity, while slow soleus fibres reincorporated with fast TnC displayed a typically fast reactivity to bepridil. These results indicated that the differential effect of bepridil in slow and fast fibres is related to the TnC isoform predominantly expressed in a fibre. 4. Experiments with the VG2 mutant demonstrated that BPD can achieve an increase in the Ca(2+) affinity in the absence of a functional site II. Thus, site I was necessary for the BPD effect to be independent of the Ca(2+) concentration. Moreover, the amplitude of the reinforcement in the Ca(2+) affinity, induced by the binding of bepridil to the TnC molecule, is dependent on the number of functional regulatory sites, the larger affinity reinforcement being detected when only one regulatory site (either site I or II) is functional.

Biological Function and Site II Ca2+-induced Opening of the Regulatory Domain of Skeletal Troponin C Are Impaired by Invariant Site I or II Glu Mutations

To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.

Evolutionary and physiological variation in cardiac troponin C in relation to thermal strategies of fish.

Striated muscle contraction is initiated when troponin C (TnC) binds Ca(2+), which activates actinomyosin ATPase. We investigated (i) the variation between cardiac TnC (cTnC) primary structure within teleost fish and (ii) the pattern of TnC expression in response to temperature acclimation. There were few differences between rainbow trout (Oncorhynchus mykiss), yellowfin tuna (Thunnus albacares), yellow perch (Perca flavescens), goldfish (Carassius auratus), white sucker (Catostomus commersoni), and icefish (Chaenocephalus aceratus) in cTnC amino acid sequence. No variation existed in the regulatory Ca(2+)-binding site (site 2). The site 3 and 4 substitutions were limited to residues not directly involved in Ca(2+) coordination. Fish cTnC primary structure was highly conserved between species (93%-98%) and collectively divergent from the highly conserved sequence seen in birds and mammals. Northern blots and polymerase chain reaction showed that thermal acclimation of trout (3 degrees, 18 degrees C) did not alter the TnC isoform pattern. While cardiac and white muscle had the expected isoforms-cTnC and fast troponin C (fTnC), respectively-red muscle unexpectedly expressed primarily ftnC. Cold acclimation did not alter myofibrillar ATPase Ca(2+) sensitivity, but maximal velocity increased by 60%. We found no evidence that TnC variants, arising between species or in response to thermal acclimation, play a major role in mitigating the effects of temperature on contractility of the adult fish heart.

Regulatory Domain Conformational Exchange and Linker Region Flexibility in Cardiac Troponin C Bound to Cardiac Troponin I

Previously, we utilized (15)N transverse relaxation rates to demonstrate significant mobility in the linker region and conformational exchange in the regulatory domain of Ca(2+)-saturated cardiac troponin C bound to the isolated N-domain of cardiac troponin I (Gaponenko, V., Abusamhadneh, E., Abbott, M. B., Finley, N., Gasmi-Seabrook, G., Solaro, R.J., Rance, M., and Rosevear, P.R. (1999) J. Biol. Chem. 274, 16681-16684). Here we show a large decrease in cardiac troponin C linker flexibility, corresponding to residues 85-93, when bound to intact cardiac troponin I. The addition of 2 m urea to the intact cardiac troponin I-troponin C complex significantly increased linker flexibility. Conformational changes in the regulatory domain of cardiac troponin C were monitored in complexes with troponin I-(1-211), troponin I-(33-211), troponin I-(1-80) and bisphosphorylated troponin I-(1-80). The cardiac specific N terminus, residues 1-32, and the C-domain, residues 81-211, of troponin I are both capable of inducing conformational changes in the troponin C regulatory domain. Phosphorylation of the cardiac specific N terminus reversed its effects on the regulatory domain. These studies provide the first evidence that the cardiac specific N terminus can modulate the function of troponin C by altering the conformational equilibrium of the regulatory domain.

Binding of cardiac troponin-I147-163 induces a structural opening in human cardiac troponin-C.

The interaction of troponin-C (TnC) with troponin-I (TnI) plays a central role in skeletal and cardiac muscle contraction. We have recently shown that the binding of Ca2+ to cardiac TnC (cTnC) does not induce an "opening" of the regulatory domain in order to interact with cTnI [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221; Spyracopoulos et al. (1997) Biochemistry 36, 12138-12146], which is in contrast to the regulatory N-domain of skeletal TnC (sTnC). This implies that the mode of interaction between cTnC and cTnI may be different than that between sTnC and sTnI. In sTnI, a region downstream from the inhibitory region (residues 115-131) has been shown to bind the exposed hydrophobic pocket of Ca2+-saturated sNTnC [McKay, R. T., et al. (1997) J. Biol. Chem. 272, 28494-28500]. The present study demonstrates that the corresponding region in cTnI (residues 147-163) binds to the regulatory domain of cTnC only in the Ca2+-saturated state to form a 1:1 complex, with an affinity approximately six times weaker than that between the skeletal counterparts. Thus, while Ca2+ does not cause opening, it is required for muscle regulation. The solution structure of the cNTnC.Ca2+.cTnI147-163 complex has been determined by multinuclear multidimensional NMR spectroscopy. The structure reveals an open conformation for cNTnC, similar to that of Ca2+-saturated sNTnC. The bound peptide adopts a alpha-helical conformation spanning residues 150-157. The C-terminus of the peptide is unstructured. The open conformation for Ca2+-saturated cNTnC in the presence of cTnI (residues 147-163) accommodates hydrophobic interactions between side chains of the peptide and side chains at the interface of A and B helices of cNTnC. Thus the mechanistic differences between the regulation of cardiac and skeletal muscle contraction can be understood in terms of different thermodynamics and kinetics equilibria between essentially the same structure states.

Dynamics and thermodynamics of the regulatory domain of human cardiac troponin C in the apo- and calcium-saturated states.

The contraction of cardiac and skeletal muscles is triggered by the binding of Ca2+ to their respective troponin C (TnC) proteins. Recent structural data of both cardiac and skeletal TnC in both the apo and Ca2+ states have revealed that the response to Ca2+ is fundamentally different for these two proteins. For skeletal TnC, binding of two Ca2+ to sites 1 and 2 leads to large changes in the structure, resulting in the exposure of a hydrophobic surface. For cardiac TnC, Ca2+ binds site 2 only, as site 1 is inactive, and the structures show that the Ca2+-induced changes are much smaller and do not result in the exposure of a large hydrophobic surface. To understand the differences between regulation of skeletal and cardiac muscle, we have investigated the effect of Ca2+ binding on the dynamics and thermodynamics of the regulatory N-domain of cardiac TnC (cNTnC) using backbone 15N nuclear magnetic resonance relaxation measurements for comparison to the skeletal system. Analysis of the relaxation data allows for the estimation of the contribution of changes in picosecond to nanosecond time scale motions to the conformational entropy of the Ca2+-binding sites on a per residue basis, which can be related to the structural features of the sites. The results indicate that binding of Ca2+ to the functional site in cNTnC makes the site more rigid with respect to high-frequency motions; this corresponds to a decrease in the conformational entropy (TdeltaS) of the site by 2.2 kcal mol(-1). Although site 1 is defunct, binding to site 2 also decreases the conformational entropy in the nonfunctional site by 0.5 kcal mol(-1). The results indicate that the Ca2+-binding sites in the regulatory domain are structurally and energetically coupled despite the inability of site 1 to bind Ca2+. Comparison between the cardiac and skeletal isoforms in the apo state shows that there is a decrease in conformational entropy of 0.9 kcal mol(-1) for site 1 of cNTnC and little difference for site 2.

Troponin C modulates the activation of thin filaments by rigor cross-bridges

Extraction of troponin C (TnC) from skinned muscle fibers reduces maximum Ca2+ and rigor cross-bridge (RXB)-activated tensions and reduces cooperativity between neighboring regulatory units (one troponin-tropomyosin complex and the seven associated actins) of thin filaments. This suggests that TnC has a determining role in RXB, as well as in Ca(2+)-dependent activation processes. To investigate this possibility further, we replaced fast TnC (fTnC) of rabbit psoas fibers with either CaM[3,4TnC] or cardiac TnC (cTnC) and compared the effects of these substitutions on Ca2+ and RXB activation of tension. CaM[3,4TnC] substitution has the same effect on Ca(2+)- and RXB-activated tensions; they are reduced 50%, and cooperativity between regulatory units is reduced 40%. cTnC substitution also reduces the maximum Ca(2+)-activated tension and cooperativity. But with RXB activation the effects on tension and cooperativity are opposite; cTnC substitution potentiates tension but reduces cooperativity. We considered whether tension potentiation could be explained by increased activation by cycling cross-bridges (CXBs), but the concerted transition formalism predicts fibers will fail to relax in high substrate and high pCa when CXBs are activator ligands. It predicts resting tension, which is not observed in either control or cTnC-substituted fibers. Rather, it appears that cTnC facilitates RXB activation of fast fibers more effectively than fTnC. The order of RXB-activated tension facilitation is cTnC > fTnC > CaM[3,4TnC] > empty TnC-binding sites. Comparison of the structures of fTnC, CaM[3,4TnC], and cTnC indicates that the critical region for this property lies in the central helix or N-terminal domain, including EF hand motifs 1 and 2.

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 2.4μm. 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 Cin situis 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.

The effect of pH on the Ca2+ affinity of the Ca2+ regulatory sites of skeletal and cardiac troponin C in skinned muscle fibres.

It is known that intracellular pH drops rapidly after the onset of ischemia in cardiac muscle and may play some role in the rapid drop in force that ensues. It is also known that alpha 1-adrenoceptor agonists alkalinize intracellular pH by stimulating Na+/H+ exchange and may represent a mechanism which facilitates recovery of intracellular pH from acidosis. Lowering or raising pH shifts the Ca2+ dependence of force development in muscle fibres to higher or lower free Ca2+ concentrations, respectively, yet the precise mechanism is unknown. To investigate this phenomenon we have used skinned skeletal or cardiac muscle fibres whose endogenous troponin C (TnC) has been replaced with chicken skeletal TnC labelled with DANZ (STnCDANZ) or recombinant cardiac TnC labelled with IAANS (CTnC3(C84)[AANS), respectively. The fluorescence of the STnCDANZ or CTnC3(C84)IAANS was enhanced by Ca2+ binding to the Ca(2+)-specific (regulatory) site(s) of STnC or CTnC when incorporated into skinned fibres, and was measured simultaneously with force. When the pH was changed from 7.0 to 6.5 or 7.5 the shift in the Ca2+ dependence of force paralleled the shift in fluorescence. Since the force and fluorescence shift in parallel as the pH is lowered or raised, it can be concluded that these changes in Ca2+ sensitivity are caused by a decrease or increase, respectively, in the Ca2+ affinity of the Ca(2+)-specific site(s) of TnC. Since lowering or raising the pH also resulted in lower or higher, respectively, maximal Ca2+ activated force while maximal fluorescence remained unchanged, it is possible that H+ may act indirectly, as well, by reducing or increasing, respectively, the number or type of crossbridges attached to actin and thereby alter the crossbridge induced depression or elevation, respectively of the observed TnC Ca2+ affinity. Experiments with 2,3-butanedione monoxime, however, where force-generating crossbridges were greatly reduced, indicated that the pH effect may be primarily related to a direct change in the Ca2+ affinity to the regulatory sites of TnC.

Molecular basis of depression of Ca 2+ sensitivity of tension by acid pH in cardiac muscles of the mouse and the rat

Background: Acid pH decreases the Ca 2+ 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 frommice 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 Ca 2+ 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 inthe 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 Ca 2++ 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.

Effect of developed tension on the time courses of Ca2+ transients and tension in twitch contraction in ferret myocardium.

The aim of the study is to test the hypothesis that tension-dependent change in the affinity of cardiac troponin-C influences the time courses of Ca2+ transients and tension in twitch contraction. The Ca(2+)-sensitive photoprotein, aequorin, was microinjected into superficial cells of ferret papillary muscles and the Ca2+ transients and tension were simultaneously measured. The peak of developed tension was altered by changing the extracellular Ca2+ concentration, initial muscle length, and the application of 2,3-butanedione monoxime. In each maneuver, the decay time of Ca2+ transients was prolonged and the tension relaxation time was shortened when the peak of developed tension was decreased. In contrast, when the peak of developed tension was increased, the decay time of Ca2+ transients was shortened and the tension relaxation time was prolonged. The decay time of Ca2+ transients measured with different maneuvers was negatively correlated with the peak tension and the tension relaxation time was positively correlated with the tension peak. The changes in the decay time of Ca2+ transients and the tension relaxation time indicate that developed tension modulates the affinity of troponin-C for Ca2+ in normal twitch contraction.