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Background: People with a DNA mismatch repair (MMR) gene mutation have a substantially elevated risk of colorectal cancer (CRC) but the modifiers of this risk are not well established. We investigated the association between dietary supplement intake and CRC risk for carriers., Methods: This study included 1966 (56% female) carriers of an MMR gene mutation (719 MLH1, 931 MSH2, 211 MSH6 and 105 PMS2) who were recruited from the USA, Canada, Australia and New Zealand into the Colon Cancer Family Registry between 1997 and 2012. Information on lifestyle factors including supplement intake was collected at the time of recruitment. Using Cox proportional hazards regression weighted to correct for ascertainment bias, we estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for associations between self-reported multivitamin, calcium and folic acid supplement intake and CRC risk., Results: Of 744 carriers with CRC, 18%, 6% and 5% reported intake of multivitamin, calcium and folic acid supplements for at least 1 month, respectively, compared with 27%, 11% and 10% of 1222 carriers without CRC. After adjusting for identified confounding variables, a decreased CRC risk was associated with multivitam inintake for at least 3 years (HR 0.47, 95% CI 0.32-0.69) and calcium intake for at least 3 years(HR 0.42, 95% CI 0.23-0.74), compared with never users. There was no evidence of an association between folic acid supplement intake and CRC risk (P = 0.82)., Conclusion: Intake of multivitamin and calcium supplements might be associated with a decreased risk of CRC for MMR gene mutation carriers., (© The Author 2016; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.)

EF-hand proteins are ubiquitous in cell signaling. Parvalbumin (Parv), the archetypal EF-hand protein, is a high-affinity Ca(2+) buffer in many biological systems. Given the centrality of Ca(2+) signaling in health and disease, EF-hand motifs designed to have new biological activities may have widespread utility. Here, an EF-hand motif substitution that had been presumed to destroy EF-hand function, that of glutamine for glutamate at position 12 of the second cation binding loop domain of Parv (ParvE101Q), markedly inverted relative cation affinities: Mg(2+) affinity increased, whereas Ca(2+) affinity decreased, forming a new ultra-delayed Ca(2+) buffer with favorable properties for promoting cardiac relaxation. In therapeutic testing, expression of ParvE101Q fully reversed the severe myocyte intrinsic contractile defect inherent to expression of native Parv and corrected abnormal myocardial relaxation in diastolic dysfunction disease models in vitro and in vivo. Strategic design of new EF-hand motif domains to modulate intracellular Ca(2+) signaling could benefit many biological systems with abnormal Ca(2+) handling, including the diseased heart.

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca(2+) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca(2+) dissociation (k(off)) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca(2+) affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca(2+) off-rate is most likely to be affected rather than the Ca(2+) on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca(2+)-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.

Cardiac diseases associated with mutations in troponin subunits include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM). Altered calcium handling in these diseases is evidenced by changes in the Ca(2+) sensitivity of contraction. Mutations in the Ca(2+) sensor, troponin C (TnC), were generated to increase/decrease the Ca(2+) sensitivity of cardiac skinned fibers to create the characteristic effects of DCM, HCM, and RCM. We also used a reconstituted assay to determine the mutation effects on ATPase activation and inhibition. One mutant (A23Q) was found with HCM-like properties (increased Ca(2+) sensitivity of force and normal levels of ATPase inhibition). Three mutants (S37G, V44Q, and L48Q) were identified with RCM-like properties (a large increase in Ca(2+) sensitivity, partial loss of ATPase inhibition, and increased basal force). Two mutations were identified (E40A and I61Q) with DCM properties (decreased Ca(2+) sensitivity, maximal force recovery, and activation of the ATPase at high [Ca(2+)]). Steady-state fluorescence was utilized to assess Ca(2+) affinity in isolated cardiac (c)TnCs containing F27W and did not necessarily mirror the fiber Ca(2+) sensitivity. Circular dichroism of mutant cTnCs revealed a trend where increased alpha-helical content correlated with increased Ca(2+) sensitivity in skinned fibers and vice versa. The main findings from this study were as follows: 1) cTnC mutants demonstrated distinct functional phenotypes reminiscent of bona fide HCM, RCM, and DCM mutations; 2) a region in cTnC associated with increased Ca(2+) sensitivity in skinned fibers was identified; and 3) the F27W reporter mutation affected Ca(2+) sensitivity, maximal force, and ATPase activation of some mutants.

Several cardiac troponin I (cTnI) mutations are associated with restrictive cardiomyopathy (RCM) in humans. We have created transgenic mice (cTnI(193His) mice) that express the corresponding human RCM R192H mutation. Phenotype of this RCM animal model includes restrictive ventricles, biatrial enlargement and sudden cardiac death, which are similar to those observed in RCM patients carrying the same cTnI mutation. In the present study, we modified the overall cTnI in cardiac muscle by crossing cTnI(193His) mice with transgenic mice expressing an N-terminal truncated cTnI (cTnI-ND) that enhances relaxation. Protein analyses determined that wild type cTnI was replaced by cTnI-ND in the heart of double transgenic mice (Double TG), which express only cTnI-ND and cTnI R193H in cardiac myocytes. The presence of cTnI-ND effectively rescued the lethal phenotype of RCM mice by reducing the mortality rate. Cardiac function was significantly improved in Double TG mice when measured by echocardiography. The hypersensitivity to Ca(2+) and the prolonged relaxation of RCM cTnI(193His) cardiac myocytes were completely reversed by the presence of cTnI-ND in RCM hearts. The results demonstrate that myofibril hypersensitivity to Ca(2+) is a key mechanism that causes impaired relaxation in RCM cTnI mutant hearts and Ca(2+) desensitization by cTnI-ND can correct diastolic dysfunction and rescue the RCM phenotypes, suggesting that Ca(2+) desensitization in myofibrils is a therapeutic option for treatment of diastolic dysfunction without interventions directed at the systemic beta-adrenergic-PKA pathways., (Copyright 2010 Elsevier Ltd. All rights reserved.)

In human cardiomyopathy, anatomical abnormalities such as hypertrophy and fibrosis contribute to the risk of ventricular arrhythmias and sudden death. Here we have shown that increased myofilament Ca2+ sensitivity, also a common feature in both inherited and acquired human cardiomyopathies, created arrhythmia susceptibility in mice, even in the absence of anatomical abnormalities. In mice expressing troponin T mutants that cause hypertrophic cardiomyopathy in humans, the risk of developing ventricular tachycardia was directly proportional to the degree of Ca2+ sensitization caused by the troponin T mutation. Arrhythmia susceptibility was reproduced with the Ca2+-sensitizing agent EMD 57033 and prevented by myofilament Ca2+ desensitization with blebbistatin. Ca2+ sensitization markedly changed the shape of ventricular action potentials, resulting in shorter effective refractory periods, greater beat-to-beat variability of action potential durations, and increased dispersion of ventricular conduction velocities at fast heart rates. Together these effects created an arrhythmogenic substrate. Thus, myofilament Ca2+ sensitization represents a heretofore unrecognized arrhythmia mechanism. The protective effect of blebbistatin provides what we believe to be the first direct evidence that reduction of Ca2+ sensitivity in myofilaments is antiarrhythmic and might be beneficial to individuals with hypertrophic cardiomyopathy.

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.

The troponin complex was discovered over thirty years ago and since then much insight has been gained into how this complex senses fluctuating levels of Ca(2+) and transmits this signal to the myofilament. Advances in genetics methods have allowed identification of mutations that lead to the phenotypically distinct cardiomyopathies: hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM) and dilated cardiomyopathy (DCM). This review serves to highlight key in vivo studies of mutation effects that have followed many years of functional studies and discusses how these mutations alter energetics and promote the characteristic remodeling associated with cardiomyopathic diseases. Studies have been performed that examine alterations in signaling and genomic methods have been employed to isolate upregulated proteins, however these processes are complex as there are multiple roads to hypertrophy or dilation associated with genetic cardiomyopathies. This review suggests future directions to explore in the troponin field that would heighten our understanding of the complex regulation of cardiac muscle contraction.

Restrictive cardiomyopathy (RCM) is a rare disorder characterized by impaired ventricular filling with decreased diastolic volume. We are reporting the functional effects of the first cardiac troponin T (CTnT) mutation linked to infantile RCM resulting from a de novo deletion mutation of glutamic acid 96. The mutation was introduced into adult and fetal isoforms of human cardiac TnT (HCTnT3-DeltaE96 and HCTnT1-DeltaE106, respectively) and studied with either cardiac troponin I (CTnI) or slow skeletal troponin I (SSTnI). Skinned cardiac fiber measurements showed a large leftward shift in the Ca(2+) sensitivity of force development with no differences in the maximal force. HCTnT1-DeltaE106 showed a significant increase in the activation of actomyosin ATPase with either CTnI or SSTnI, whereas HCTnT3-DeltaE96 was only able to increase the ATPase activity with CTnI. Both mutants showed an impaired ability to inhibit the ATPase activity. The capacity of the CTnI.CTnC and SSTnI.CTnC complexes to fully relax the fibers after TnT displacement was also compromised. Experiments performed using fetal troponin isoforms showed a less severe impact compared with the adult isoforms, which is consistent with the cardioprotective role of SSTnI and the rapid onset of RCM after birth following the isoform switch. These data indicate that troponin mutations related to RCM may have specific functional phenotypes, including large leftward shifts in the Ca(2+) sensitivity and impaired abilities to inhibit ATPase and to relax skinned fibers. All of this would account for and contribute to the severe diastolic dysfunction seen in RCM.

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca(2+) imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca(2+) transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca(2+).[(3)H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca(2+) activation or Ca(2+) and Mg(2+) inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca(2+) homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.

Troponin T (TnT) mutations that cause familial hypertrophic cardiomyopathy (FHC) and sudden cardiac death frequently increase myofilament Ca2+ sensitivity, suggesting that their Ca2+-sensitizing effect contributes importantly to the FHC pathogenesis. To test this hypothesis, we compared transgenic mice expressing the Ca2+-sensitizing TnT-I79N mutant (I79N), which causes a high rate of sudden cardiac death in patients, with mice expressing the more benign TnT-R278C mutant (R278C) that does not affect myofilament Ca2+ sensitivity. Acutely increasing myofilament Ca2+ sensitivity with EMD57033 served as a positive control. Isovolumically contracting hearts were compared over a range of loading conditions (Frank-Starling curve). Consistent with their increased myofilament Ca2+ sensitivity, I79N-Tg hearts demonstrated significantly higher systolic performance at low perfusate [Ca2+] compared with R278C-Tg hearts, which were not statistically different from control hearts expressing either human wild-type TnT or no transgene (CON). Diastolic function was impaired in both FHC mutants (time to 90% relaxation: I79N 48 +/- 1.0 ms, n = 10 or R278C 47 +/- 0.4 ms, n = 7, versus CON 44 +/- 1.0 ms, n = 20, P < 0.05). In the presence of isoproterenol, almost all contractile parameters of R278C hearts became indistinguishable from control hearts, whereas both systolic and diastolic function of I79N hearts significantly worsened (end-diastolic pressure: I79N 20 +/- 4 mmHg versus CON 13 +/- 2 mmHg or R278C 11 +/- 2 mmHg, P < 0.05). The Ca2+ sensitizer EMD57033 produced an even greater contractile dysfunction than the I79N mutation at fast pacing rates. In vivo, maximal exercise tolerance was significantly impaired only in I79N mice. Pretreatment with beta-adrenergic receptor antagonists abolished differences in exercise tolerance. In conclusion, the Ca2+-sensitizing effects of TnT mutations may reduce the responsiveness of mouse hearts to inotropic stimuli.

Many biological systems use ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) to regulate the free calcium concentration ([Ca(2+)](free)) in the presence of physiological levels of free Mg(2+) ([Mg(2+)](free)). Frequently, it is necessary to work at [Ca(2+)](free) beyond EGTA's buffering capabilities. Therefore, we have developed methods to extend the buffering range by adding nitrilotriacetic acid (NTA) to solutions containing EGTA. This extension results from NTA having a lower K'(dCa) than EGTA. Such equilibria are solved by pCa Calculator, a computer program designed to aid in the study of Ca(2+)-dependent physiological processes while accounting for the effects of pH, temperature, and ionic strength. With multiple chelators and pH buffers from which to choose, pCa Calculator calculates the total concentration of each species required to achieve specified free concentrations of Ca(2+), ATP, and Mg(2+). The program is intuitive, user-friendly, and flexible enough to fix or vary the [Mg-ATP(2-)] and ionic strength. Moreover, it can account for increases in experimental volume from calcium addition. A comparative analysis is reported for testing solutions in the presence and absence of NTA by measuring the calcium binding affinity of fluorescent cardiac troponin C. These findings demonstrate that EGTA, when used in conjunction with NTA, improves and expands the regulation of free calcium in solution.

The first human cardiac troponin I (hcTnI) mutation in the N-terminal 32 residue region, R21C (arginine residue number 21 mutated to cysteine), which has been linked to hypertrophic cardiomyopathy (HCM), has recently been reported. The effect of this mutation on the physiological function of hcTnI was investigated. Human cTnI R21C (in the absence or presence of troponin T and troponin C) was phosphorylated by protein kinase A (PKA) at a significantly slower rate than wild-type hcTnI. In skinned fiber studies, the TnI R21C mutant showed a large increase in Ca(2+)-sensitivity of force development when compared to wild-type TnI (DeltapCa(50)=0.33). Phosphorylation of skinned fibers containing TnI R21C by PKA resulted in a significantly smaller decrease in the Ca(2+)-sensitivity of force development when compared to phosphorylation of fibers containing wild-type TnI. The decreased sensitivity of TnI R21C to PKA is most likely due to a decreased ability of PKA to phosphorylate this TnI rather than conformational problems within this TnI. In addition, skinned fibers were found to contain an endogenous kinase that is capable of phosphorylating wild-type TnI. However, the endogenous kinase activity did not affect the Ca(2+)-sensitivity of force development, the Hill coefficient or maximal force of these skinned fibers. Actomyosin ATPase assays showed that the R21C mutation did not affect the inhibitory properties of TnI or the maximal ATPase activity. TnI R21C was also found to be more susceptible to proteolysis by calpain II than wild-type TnI. These results suggest that this R21C mutation in TnI affects the Ca(2+)-sensitizing effect of Tn, the ability of TnI to be readily phosphorylated by PKA and the stability of TnI to calpain. The results also suggest that the N-terminal region may have important roles such as modulating the Ca(2+)-sensitivity of force-development.

Human cardiac Troponin I (cTnI) is the first sarcomeric protein for which mutations have been associated with restrictive cardiomyopathy. To determine whether five mutations in cTnI (L144Q, R145W, A171T, K178E, and R192H) associated with restrictive cardiomyopathy were distinguishable from hypertrophic cardiomyopathy-causing mutations in cTnI, actomyosin ATPase activity and skinned fiber studies were carried out. All five mutations investigated showed an increase in the Ca2+ sensitivity of force development compared with wild-type cTnI. The two mutations with the worst clinical phenotype (K178E and R192H) both showed large increases in Ca2+ sensitivity (deltapCa50 = 0.47 and 0.36, respectively). Although at least one of these mutations is not in the known inhibitory regions of cTnI, all of the mutations investigated caused a decrease in the ability of cTnI to inhibit actomyosin ATPase activity. Mixtures of wild-type and mutant cTnI showed that cTnI mutants could be classified into three different groups: dominant (L144Q, A171T and R192H), equivalent (K178E), or weaker (R145W) than wild-type cTnI in actomyosin ATPase assays in the absence of Ca2+. Although most of the mutants were able to activate actomyosin ATPase similarly to wild-type cTnI, L144Q had significantly lower maximal ATPase activities than any of the other mutants or wild-type cTnI. Three mutants (L144Q, R145W, and K178E) were unable to fully relax contraction in the absence of Ca2+. The inability of the five cTnI mutations investigated to fully inhibit ATPase activity/force development and the generally larger increases in Ca2+ sensitivity than observed for most hypertrophic cardiomyopathy mutations would likely lead to severe diastolic dysfunction and may be the major physiological factors responsible for causing the restrictive cardiomyopathy phenotype in some of the genetically affected individuals.

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca(2+) sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca(2+) was released from site II of cTnC in the cTnI.cTnC complex (122/s) was 12.5-fold faster than for the ssTnI.cTnC complex (9.8/s). Addition of cTnT3 to the cTnI.cTnC complex resulted in a 3.6-fold decrease in the Ca(2+) dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI.cTnC complex resulted in a 1.9-fold increase in the Ca(2+) dissociation rate from site II of cTnC. The rate at which Ca(2+) dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca(2+) dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.

To understand the molecular function of troponin T (TnT) in the Ca(2+) regulation of muscle contraction as well as the molecular pathogenesis of familial hypertrophic cardiomyopathy (FHC), eight FHC-linked TnT mutations, which are located in different functional regions of human cardiac TnT (HCTnT), were produced, and their structural and functional properties were examined. Circular dichroism spectroscopy demonstrated different secondary structures of these TnT mutants. Each of the recombinant HCTnTs was incorporated into porcine skinned fibers along with human cardiac troponin I (HCTnI) and troponin C (HCTnC), and the Ca(2+) dependent isometric force development of these troponin-replaced fibers was determined at pH 7.0 and 6.5. All eight mutants altered the contractile properties of skinned cardiac fibers. E244D potentiated the maximum force development without changing Ca(2+) sensitivity. In contrast, the other seven mutants increased the Ca(2+) sensitivity of force development but not the maximal force. R92L, R92W, and R94L also decreased the change in Ca(2+) sensitivity of force development observed on lowering the pH from 7 to 6.5, when compared with wild type TnT. The examination of additional mutants, H91Q and a double mutant H91Q/R92W, suggests that mutations in a region including residues 91-94 in HCTnT can perturb the proper response of cardiac contraction to changes in pH. These results suggest that different regions of TnT may contribute to the pathogenesis of TnT-linked FHC through different mechanisms.

The cardiac troponin T (TnT) I79N mutation has been linked to familial hypertrophic cardiomyopathy and high incidence of sudden death, despite causing little or no cardiac hypertrophy in patients. Transgenic mice expressing mutant human TnT (I79N-Tg) have increased cardiac contractility, but no ventricular hypertrophy or fibrosis. Enhanced cardiac function has been associated with myofilament Ca2+ sensitization, suggesting altered cellular Ca2+ handling. In the present study, we compare cellular Ca2+ transients and electrophysiological parameters of 64 I79N-Tg and 106 control mice in isolated myocytes, isolated perfused hearts, and whole animals. Ventricular action potentials (APs) measured in isolated I79N-Tg hearts and myocytes were significantly shortened only at 70% repolarization. No significant differences were found either in L-type Ca2+ or transient outward K+ currents, but inward rectifier K+ current (IK1) was significantly decreased. More critically, Ca2+ transients of field-stimulated ventricular I79N-Tg myocytes were reduced and had slow decay kinetics, consistent with increased Ca2+ sensitivity of I79N mutant fibers. AP differences were abolished when myocytes were dialyzed with Ca2+ buffers or after the Na+-Ca2+ exchanger was blocked by Li+. At higher pacing rates or in presence of isoproterenol, diastolic Ca2+ became significantly elevated in I79N-Tg compared with control myocytes. Ventricular ectopy could be induced by isoproterenol-challenge in isolated I79N-Tg hearts and anesthetized I79N-Tg mice. Freely moving I79N-Tg mice had a higher incidence of nonsustained ventricular tachycardia (VT) during mental stress (warm air jets). We conclude that the TnT-I79N mutation causes stress-induced VT even in absence of hypertrophy and/or fibrosis, arising possibly from the combination of AP remodeling related to altered Ca2+ transients and suppression of IK1.

At least four isoforms of troponin T (TnT) exist in the human heart, and they are expressed in a developmentally regulated manner. To determine whether the different N-terminal isoforms are functionally distinct with respect to structure, Ca(2+) sensitivity, and inhibition of force development, the four known human cardiac troponin T isoforms, TnT1 (all exons present), TnT2 (missing exon 4), TnT3 (missing exon 5), and TnT4 (missing exons 4 and 5), were expressed, purified, and utilized in skinned fiber studies and in reconstituted actomyosin ATPase assays. TnT3, the adult isoform, had a slightly higher alpha-helical content than the other three isoforms. The variable region in the N terminus of cardiac TnT was found to contribute to the determination of the Ca(2+) sensitivity of force development in a charge-dependent manner; the greater the charge the higher the Ca(2+) sensitivity, and this was primarily because of exon 5. These studies also demonstrated that removal of either exon 4 or exon 5 from TnT increased the cooperativity of the pCa force relationship. Troponin complexes reconstituted with the four TnT isoforms all yielded the same maximal actin-tropomyosin-activated myosin ATPase activity. However, troponin complexes containing either TnT1 or TnT2 (both containing exon 5) had a reduced ability to inhibit this ATPase activity when compared with wild type troponin (which contains TnT3). Interestingly, fibers containing these isoforms also showed less relaxation suggesting that exon 5 of cardiac TnT affects the ability of Tn to inhibit force development and ATPase activity. These results suggest that the different N-terminal TnT isoforms would produce different functional properties in the heart that would directly affect myocardial contraction.

The role of phosphorylation of the myosin regulatory light chains (RLC) is well established in smooth muscle contraction, but in striated (skeletal and cardiac) muscle its role is still controversial. We have studied the effects of RLC phosphorylation in reconstituted myosin and in skinned skeletal muscle fibers where Ca2+ sensitivity and the kinetics of steady-state force development were measured. Skeletal muscle myosin reconstituted with phosphorylated RLC produced a much higher Ca2+ sensitivity of thin filament-regulated ATPase activity than nonphosphorylated RLC (change in -log of the Ca2+ concentration producing half-maximal activation = approximately 0.25). The same was true for the Ca2+ sensitivity of force in skinned skeletal muscle fibers, which increased on reconstitution of the fibers with the phosphorylated RLC. In addition, we have shown that the level of endogenous RLC phosphorylation is a crucial determinant of the Ca2+ sensitivity of force development. Studies of the effects of RLC phosphorylation on the kinetics of force activation with the caged Ca2+, DM-nitrophen, showed a slight increase in the rates of force development with low statistical significance. However, an increase from 69 to 84% of the initial steady-state force was observed when nonphosphorylated RLC-reconstituted fibers were subsequently phosphorylated with exogenous myosin light chain kinase. In conclusion, our results suggest that, although Ca2+ binding to the troponin-tropomyosin complex is the primary regulator of skeletal muscle contraction, RLC play an important modulatory role in this process.

Laboratory studies and epidemiological investigations suggest that vitamin D plays a role in the etiology of colorectal adenomas, possibly through a mechanism mediated by the vitamin D receptor (VDR). We conducted a clinic-based case-control study to examine the association between VDR polymorphisms and colorectal adenomas. We selectively identified a random subset of 393 cases of colorectal adenomas and 406 colonoscopy-negative controls from a clinic-based case-control study conducted in the metropolitan Minneapolis/St. Paul area during 1991-1994. A self-administered questionnaire was used to collect data on dietary and supplement intake of vitamin D and calcium, as well as on demographics, physical activity, medical information, lifestyle factors, reproductive history, and anthropometry. DNA was extracted from whole blood and assayed for the BsmI VDR polymorphism using an ABI 7700 TaqMan assay. Adjusted odds ratios (OR) and 95% confidence intervals (CIs) were evaluated using logistic regression. Compared with the bb genotype (33% of controls), neither the Bb (48.8% of controls) nor the BB (18.2% of controls) genotypes was strongly associated with risk of colorectal adenomas (OR = 0.86, CI = 0.63-1.19 and OR = 0.77, CI = 0.50-1.18, respectively). However, those with the lowest tertile of vitamin D intake and the BB genotype had a lower risk of colorectal adenoma (OR = 0.24, CI = 0.08-0.76) than those with the highest tertile of intake and the bb genotype. Similarly, those with the lowest tertile of calcium intake and the BB genotype had a reduced risk of colorectal adenoma (OR = 0.34, CI = 0.11-1.06). Although it has generally been shown that higher calcium and vitamin D intake are associated with a modestly reduced risk of colorectal neoplasia, our data suggest that those with the BB BsmI VDR genotype may be at reduced risk of colorectal adenoma in the presence of lower calcium and vitamin D intake.

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.

Two genetically engineered, recombinant versions of native barnacle troponin C (TnC) (BTnC,) were created from the bacterially expressed, recombinant, wild-type BTnC (BTnCWT) to investigate the role of the Ca(2+)-specific sites in force regulation. The mutant BTnC4- contains a single amino acid mutation in site IV which results in the inactivation of site IV Ca2+ binding; the mutant BTnCTrunc lacks the last II amino acids of the C-terminal, and hence most of site IV. Both mutant proteins, which retain an active site II, bind to native TnC-depleted myofibrillar bundles and restore approximately 40% of the tension-generating capacity, about half that seen with purified native BTnC1 or BTnC2. This observation implies that the Mg(2+)-dependent interaction with troponin I (TnI) is at a location on TnC other than the C-terminal Ca(2+)-binding sites of BTnC2. Replacement with BTnCTrunc increases the sensitivity of the myofibrillar bundle to changes in ionic strength. Decreasing the ionic strength from 0.15 to 0.075 M increased force by 34%, a value much greater that the 8% increase seen in control bundles or bundles substituted with BTnC4-. These findings implicate TnC in determining this fibre characteristic, although this cannot be simply due to the alteration in the numbers of Ca2+ ions bound by the troponin complex since both BTnC4- and BTnCTrunc bind only 1 mol Ca2+/mol protein.

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.

In order to study the role of the Ca2+-specific sites (I and II) and the high affinity Ca2+-Mg2+ sites (III and IV) of TnC in the regulation of muscle contraction, we have constructed four mutants and the wild type (WTnC) of chicken skeletal TnC, with inactivated Ca2+ binding sites I and II (TnC1,2-), site III (TnC3-), site IV (TnC4-), and sites III and IV (TnC3,4C-). All Ca2+ binding site mutations were generated by replacing the Asp at the X-coordinating position of the Ca2+ binding loop with Ala. The binding of these mutated proteins to TnC-depleted skinned skeletal muscle fibers was investigated as well as the rate of their dissociation from these fibers. The proteins were also tested for their ability to restore steady state force to TnC-depleted fibers. We found that although the NH2-terminal mutant of TnC (TnC1,2-) bound to the TnC-depleted fibers (with a lower affinity than wild type TnC (WTnC)), it was unable to reactivate Ca2+-dependent force. This supports earlier findings that the low affinity Ca2+ binding sites (I and II) in TnC are responsible for the Ca2+-dependent activation of skeletal muscle contraction. All three COOH-terminal mutants of TnC bound to the TnC-depleted fibers, had different rates of dissociation, and could restore steady state force to the level of unextracted fibers. Although both high affinity Ca2+ binding sites (III and IV) are important for binding to the fibers, site III appears to be the primary determinant for maintaining the structural stability of TnC in the thin filament. Moreover, our results suggest an interaction between the NH2- and COOH-terminal domains of TnC, since alteration of sites I and II lowers the binding affinity of TnC to the fibers, and mutations in sites III and IV affect the Ca2+ sensitivity of force development.

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.

Troponin (Tn), containing three subunits: Ca2+ binding (TnC), inhibitory (TnI), and tropomyosin binding (TnT), plays a crucial role in the Ca2+ regulation of vertebrate striated muscle contraction. These three subunits function by interacting with each other and with the other thin filament proteins. Previous studies suggested that the primary role of TnT is to anchor the TnI.TnC complex to the thin filament, primarily through its interactions with TnI and tropomyosin. We propose here a new role for TnT. Our results indicate that, when TnT is combined with the TnI.TnC complex, there is an activation of actomyosin ATPase that is Ca(2+)-dependent. To determine whether the latter results from a direct effect of TnC on TnT or indirectly from an effect of TnC on TnI which is transmitted to TnT, we prepared a deletion mutant (deletion of residues 1-57) of TnI, TnId57 (Sheng et al. (1992) J. Biol. Chem. 267, 25407-25413), which interacts with TnC but not TnT. Both wild type (TnI.TnC.TnT) and mutant (TnId57.TnC.TnT) Tn complexes demonstrated equivalent activity in the Ca2+ regulation of actomyosin-S1 ATPase activity. Similarly, both TnI and TnId57 could equally reconstitute TnI-depleted skinned muscle fibers. Therefore, since TnId57 does not interact with TnT, these results suggest that TnT reconstitutes native Ca2+ sensitivity via direct interaction with TnC. Thus Ca2+ binding to TnC would have a dual role: 1) release of the ATPase inhibition by TnI and 2) activation of the ATPase through interaction with TnT.

The complete amino acid sequence of troponin C (ETnC) from the white muscle of the European eel has been determined by Edman degradation procedures. Its single tryptophan residue is situated in helix H at amino acid position 152 of the aligned sequence; the tryptophan is the first residue on the C-terminal side of Ca2+ binding loop IV. The increase of tryptophan fluorescence emission intensity occurring upon titration of ETnC with Ca2+ has been used to determine the affinity constants of ETnC for Ca2+. The calculated affinity of ETnC for Ca2+ results in a K(Ca) of 1.3 10(7) M-1, typical of the Ca(2+)-Mg2+ sites of the second domain of fast skeletal muscle TnCs. Moreover, a direct competition between Ca2+ and Mg2+ was also observed. The calculated affinity of ETnC for Mg2+ is K(Mg) = 1.2 10(3) M-1. In order to probe the affinity constants of the Ca2+ binding sites of the regulatory domain, ETnC was labelled with dansylaziridine (Danz). The Danz fluorescent signal was used to estimate the affinity constants of ETnC-Danz for Ca2+ and also for Mg2+ (assuming a competitive behaviour between these two metal ions). The calculated affinity constants are K(Ca) = 9.4 10(5) M-1 and K(Mg) = 2.0 10(2) M-1, respectively. These values are typical of the Ca(2+)-specific sites of the regulatory domain of fast skeletal muscle TnCs.

Background: Colonic epithelial cell proliferation is increased in patients at high risk for colon cancer. Calcium administration has ameliorated the proliferative changes in rodents, and findings in small, uncontrolled clinical trials have suggested similar effects in humans., Purpose: This preliminary, double-blind, randomized clinical trial was designed 1) to investigate whether supplemental calcium will reduce colonic epithelial cell proliferation in patients with sporadic adenomas who consume a high-fat, Western-style diet; 2) to determine the sample size (number of scorable crypts per person) needed to achieve adequate statistical power; and 3) to evaluate the feasibility of full-scale clinical trials., Methods: Twenty-one sporadic adenoma patients were treated daily with placebo or 1200 mg of supplemental calcium. To determine colonic epithelial cell proliferation, we used tritiated thymidine labeling of colon crypt epithelial cells in rectal biopsy specimens and calculated the percentage of labeled cells (labeling index [LI]). Two pathology technician "readers" independently scored each specimen, and inter-reader reliability was determined. Subjects remained on their usual diet during the study, and intake of calories, calcium, total fat, and vitamin D did not differ substantially among them. We calculated curves for statistical power to determine the number of scorable crypts needed per person for detection of a statistically significant difference (P < .05) of 1.0% in mean LI., Results: The pooled baseline LI was 4.7%. In the calcium-treated group, the LI increased 0.6% (proportional increase, 12.8%); in the placebo-treated group, it decreased 0.5% (proportional decrease, 10.6%). The difference between change in the mean LI from baseline to 8 weeks' follow-up in the placebo group versus the calcium group was not statistically significant. The intraclass correlation coefficient for inter-reader reliability for the baseline LI was .66. Analyses indicated scoring eight crypts sufficient for estimates of the LI adequate for between-group comparisons, a level achieved in 81% of biopsy specimens., Conclusions: Calcium carbonate supplements delivering 1200 mg elemental calcium daily may not decrease colonic epithelial cell proliferation over an 8-week period in sporadic adenoma patients. In future trials measuring the LI, consideration should be given to ensuring adequate numbers of scorable crypts and to the impact of inadequate biopsy procedures, labeling failure, reader reliability, and participant withdrawal. Our findings support the feasibility of a full-scale clinical trial to further study the relationships among dietary calcium, colonic epithelial cell proliferation, and colorectal cancer.

The effect of Ca2+ activation on the apparent rate constant governing the dissociation of force generating myosin crossbridges was studied in skinned rabbit adductor magnus fibres (fast-twitch) at 21 +/- 1 degree C. Simultaneous measurements of Ca2(+)-activated isometric force and ATPase activity were conducted in parallel with simultaneous measurements of DANZ-labelled troponin C (TnCDANZ) fluorescence and isometric force in fibres whose endogenous troponin C had been partially replaced with TnCDANZ. The Ca2+ activation of isometric force occurred at approximately two times higher Ca2+ concentration than did actomyosin ATPase activity at 2.0 mM MgATP. Since increases in both TnCDANZ fluorescence and ATPase activity occurred over approximately the same Ca2+ concentration range at substantially lower concentrations of Ca2+ than did force, this data suggests that the TnCDANZ fluorescence is associated with the Ca2+ activation of myosin crossbridge turnover (ATPase) rather than force. According to the model of Huxley (1957) and assuming the hydrolysis of one molecule of ATP per cycle of the crossbridge, the apparent rate constant gapp for the dissociation of force generating myosin crossbridges is proportional to the actomyosin ATPase/isometric force ratio. This measure of gapp shows approximately a fivefold decrease during Ca2+ activation of isometric force. This change in gapp is responsible for separation of the Ca2+ sensitivity of the normalized ATPase activity and isometric force curves. If the MgATP concentration is reduced to 0.5 mM, the change in gapp is reduced and consequently the difference in Ca2+ sensitivity between normalized steady state ATPase and force is also reduced.

Much of our knowledge of the relationship between elevation of free sarcoplasmic [Ca2+] and skeletal muscle contraction has come from physiological studies on barnacle muscle fibers. Little is known, however, about the biochemical properties of the barnacle proteins responsible for Ca2+ regulation. In order to help rectify this unfortunate situation, we purified the two major isoforms (BTnC1 and BTnC2) of troponin C (TnC) from the giant barnacle, Balanus nubilis, and determined their amino acid sequences. BTnC2, the more abundant isoform, contains 151 amino acid residues and has a calculated molecular weight of 16,838. Due to an elongated N-terminus, BTnC1 contains 158 amino acid residues and has a calculated molecular weight of 17,984. The two sequences can be aligned from their C-termini, with no insertions or deletions, but they are only 61% identical. Sequence differences are twice as frequent in the N-terminal halves as in the C-terminal halves but occur in all regions of the polypeptides. This indicates that BTnC1 and BTnC2 are products of different genes, rather than alternative transcripts of a single gene. Both isoforms contain the usual four ancestral Ca2(+)-binding regions, numbered I-IV from the N-terminus. Analysis of the sequences predicts that functional Ca2(+)-binding sites are present only in regions II and IV and that these sites are low-affinity Ca2(+)-specific type sites. Direct Ca2(+)-binding measurements using fluorescent Ca2+ indicators show that both isoforms bind 2 Ca2+/mol with equal affinity (KCa = 1.3 x 10(5) M-1) and the sites appear to be Ca2(+)-specific.

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.

The very close interdependence of Ca2+ and hormones in the overall metabolism of cyclic nucleotides has recently been emphasized by Cheung. Clearly the results presented here show that [Ca2+] in the physiological range (less than 10(-7) M to greater than 10(-6) M) has profound effects on the activity of adenylate cyclase from both brain and cardiac muscle. Whereas both brain and cardiac cyclase exhibit a Ca2+ dependent inhibition (perhaps mediated by calmodulin), only the brain cyclase is activated by Ca2+ via calmodulin. With both cyclases there is an inverse relationship between the inhibition of cyclase and the activation of calmodulin dependent (cAMP and cGMP) phosphodiesterase as a function of Ca2+ concentration. Because the IC50's for Ca2+ are the same in both heart and brain, the possibility exists that the Ca2+ inhibitory site of both cyclases is similar and perhaps identical. Considering the ability of Ca2+ to both stimulate and inhibit cyclase, one could imagine that in different species, tissues, or regions of the same tissue, there could exist multiple populations of cyclase, that is a cyclase which would only show Ca2+ dependent inhibition, Ca2+ dependent stimulation, or the biphasic response to Ca2+ (FIGURE 7). The fact that Ca2+ still regulates adenylate cyclase after various stimuli (histamine, NaF, etc.) suggests that Ca2+ may function to regulate the cyclase over shorter time periods (regardless of its state of stimulation) and that other affectors of cyclase (e.g., hormones) would serve to regulate the cyclase over longer time periods.

According to recent views the active troponin (Tn) complex consists of three components distinguishable by their ability to bind Ca2+ (TnC), to combine with troponyosin (TnT), and to inhibit actomyosin ATPase with ot without Ca2+ (TnI). Addition of Tn to an actin-myosin-tropomyosin system produceas a Ca2+ requirement for ATPase activity. Recent developments bearing on the mechanism both in vitro and in vivo are reviewed in order to correlate biochemical, physiochemical, electron microscopic, and physiological findings. The use of various combination of isolated components considerably helps in pinpointing changes that occur within components (conformational changes) and in the relative positions of interacting moieties. Available data on differences between the skeletal and cardiac system are also discussed.

The direct binding of Ca2+ to reconstituted thin filaments containing troponin C and the 5-dimethylaminonaphthalene-1-sulphonylaziridine (DANZ) fluorescent analogue of troponin C (TnCDANZ) was measured (25 degrees C) at three Mg2+ concentrations. Biphasic Scatchard plots were found for all binding curves reflecting the binding of Ca2+ to high- and low-affinity sites of troponin. The binding of Ca2+ to the high-affinity sites had a greater sensitivity to Mg2+ (KMg = 1 x 10(4)M-1) than the low-affinity sites (KMg = 1.2 x 10(3)M-1). The fluorescence change of thin filaments reconstituted with TnCDANZ was titrated with Ca2+ in the same solutions used for binding assays. The Ca2+-dependent fluorescence change had nearly the same sensitivity to Mg2+ (KMg = 9.4 x 10(2)M-1) as did Ca2+ binding to the low-affinity sites. The Ca2+ concentration at the midpoint of the fluorescence change was about 0.3 log units less than at the midpoint for Ca2+ binding to the low-affinity sites. A similar relationship between the fluorescence change and Ca2+ binding to the low-affinity sites of isolated TnCDANZ was measured (4 degrees C). From these results the binding of Ca2+ to either low-affinity site is concluded to produce the fluorescence change. In comparison with the low-affinity sites of isolated troponin and troponin-tropomyosin complex, the low-affinity sites of reconstituted thin filaments were consistently lower in Ca2+ affinity.

Carp parvalbumin has been shown to activate rat brain phosphodiesterase in a Ca2+-dependent manner. The concentration of Ca2+ required for half-maximal stimulation is 1.4 X 10(-7) M, whereas rat testis Ca2+-dependent regulator (CDR) of phosphodiesterase required 1.2 X 10(-6) M Ca2+. The difference in the slopes of the two curves demonstrated that the activation induced by parvalbumin was not the result of a small contamination by CDR. In addition, it has been shown that Ca2+ binding to parvalbumin parallels its activation of phosphodiesterase. These data suggest that Ca2+ must bind to a single specific metal binding site before phosphodiesterase can be fully activated.

We have observed a biphasic increase in [theta]222 and the tyrosine fluorescence of troponin C (TnC) with increasing [Ca2+]. Approximately 62% of the total helical change and 81% of the total increase in tyrosine fluorescence occurred with Ca2+ binding to a class of sites with a KCa of approximately 2.7 X 10(7) M-1. The remaining spectral changes occurred with Ca2+ binding to a class of sites with KCa of approximately 3.1 X 10(5) M-1. We interpret these biphasic responses in terms of distinct structural changes occurring in TnC with Ca2+ binding to the high affinity (KCa approximately 2.1 X 10(7) M-1) Ca2+-Mg2+ sites and to the low affinity (KCa approximately 3.2 X 10(5) M-1) Ca2+-specific regulatory sites.

The binding of cadmium to the calcium binding subunit of skeletal troponin (STnC) has been reinvestigated using direct binding methods and fluorescent derivatives. These data provide straightforward explanations of the observed titration behavior in the 113Cd NMR (Ellis, P.D., Strang, P., and Potter, J.D. (1984) J. Biol. Chem. 259, 10348-10356). Further, fluorescent derivatives of skeletal troponin C provide an excellent means of establishing a sequence assignment for the resonances observed in the 113Cd NMR. The results of these experiments demonstrate that sites I and II, the Ca2+ regulatory sites, can be assigned to resonances at -108.5 and -101.5 ppm, respectively. Sites III and IV, the structural sites, are assigned to resonances -112.8 and -106.8 ppm, respectively. These data are discussed in terms of recent structural findings and speculations.

Troponin-C (TnC) from rabbit skeletal muscle contains two high affinity Ca2+ binding sites (sites 1 and 2) that bind Mg2+ competitively (Ca2+-Mg2+ sites) and two Ca2+ binding sites (sites 3 and 4) of lower affinity that do not bind Mg2+ (Ca2+-specific sites). The free energy (deltaG0i), enthalpy (delta H0i) and entropy (deltaS0i) of binding Ca2+ to each of the four sites (i = 1 to 4) on TnC have been evaluated from microcalorimetry and equilibrium dialysis. The enthalpy of Ca2+ binding to each site was identical (-7.7 kcal mol-1); the entropy of Ca2+ binding to sites 1 and 2 was deltaS01,2 approximately equal to 14.7 e.u. whereas delta S03.4 approximately equal to 8.0 e.u. The positive entropy associated with Ca2+ binding to sites 1 and 2 is probably due to displacement of water produced by the alpha-helix formation, known to accompany the binding of Ca2+ to the Ca2+-Mg2+ sites. Thus, Ca2+ binding to the Ca2+-Mg2+ sites is driven by both enthalpy and entropy and the lower Ca2+ affinity for sites 3 and 4 is reflected in the lower entropy of Ca2+-binding. The entropy associated with Ca2+ binding to sites 3 and 4 suggests that some change in protein conformation is occurring upon binding of Ca2+ to these sites.

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

The mechanism of contraction in rabbit fast-twich, and bovine and rabbit cardiac muscle was examined using functionally skinned fibers, ATPase activity of myofibrils, and cardiac or skeletal troponin-tropomyosin regulated actin heavy meromyosin. The Ca2+ and Sr2+ activation properties for the different measures of contraction were evaluated. (1) Tension in rabbit and bovine cardiac skinned fibers and rabbit cardiac myofibrillar ATPase were activated equally well by either Ca2+ or Sr2+. By contrast, rabbit adductor magnus (fast-twich) skinned fibers required substantially higher [Sr2+] than [Ca2+] for activation, as did rabbit myofibrils from back muscle (fast-twitch). (2) Substantially more Sr2+ than Ca2+ was also required for activation of skeletal muscle actin heavy meromyosin ATPase, controlled by either the skeletal or cardiac troponin-tropomyosin complex, similar to the activation of fast-twitch muscle. (3) The absence of correlation between the divalent cation selectivity properties of actin heavy meromyosin ATPase controlled by cardiac troponin-tropomyosin and cardiac muscle tension or myofibrillar ATPase activation by Ca2+ and Sr2+ suggests that troponin, if primarily responsible for the activation of cardiac muscle, has very different in vivo and in vitro binding properties. (4) The close correlation between percentage of maximal Ca2+- and Sr2+-activated myofibrillar ATPase and tension in skinned fibers strongly justifies the use of myofibrillar ATPase, in contrast to a reconstituted troponin-tropomyosin actin heavy meromyosin ATPase system, as a biochemical measure of contraction.

The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.

Porcine left ventricular cardiac myosin and rabbit white skeletal myosin were phosphorylated by rabbit skeletal myosin light chain kinase and their Ca2+ binding properties were examined by equilibrium dialysis techniques. No significant effect of phosphorylation on the Ca2+ binding properties of these myosins was observed. Both types of striated muscle myosins bound approximately 2 mol of Ca2+/mol of myosin with similar affinities of 3 x 10(7) M-1. In the presence of 3 x 10(-4) M Mg2+ the myosins bound Ca2+ with a reduced affinity of 3 to 4 x 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the binding sites on myosin, the changes in Ca2+ binding can be accounted for by a Mg2+ affinity of 2.5 to 3.0 x 10(5) M-1.