Functional consequences of the human cardiac troponin I hypertrophic cardiomyopathy mutation R145G in transgenic mice

In this study, we addressed the functional consequences of the human cardiac troponin I (hcTnI) hypertrophic cardiomyopathy R145G mutation in transgenic mice. Simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca(2+)-activated force without changes in the maximal ATPase activity and an increase in the Ca(2+) sensitivity of both ATPase and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state), showing that changes in Ca(2+) sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared with Tg-WT fibers. The addition of 3 mm 2,3-butanedione monoxime at pCa 9.0 showed that there was approximately 2-4% of force generating cross-bridges attached in Tg-R145G fibers compared with less than 1.0% in Tg-WT fibers, suggesting that the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca(2+)] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared with Tg-WT. These results suggest that the phenotype of hypertrophic cardiomyopathy is most likely caused by the compensatory mechanisms in the cardiovascular system that are activated by 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge, 2) slowed relaxation (diastolic dysfunction) caused by prolonged [Ca(2+)] and force transients, and 3) an inability of the cardiac TnI to completely inhibit activation in the absence of Ca(2+) in Tg-R145G mice.     

Predicting Cardiomyopathic Phenotypes by Altering Ca2+ Affinity of Cardiac Troponin C

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 Ca²⁺ sensitivity of contraction. Mutations in the Ca²⁺ sensor, troponin C (TnC), were generated to increase/decrease the Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ sensitivity, partial loss of ATPase inhibition, and increased basal force). Two mutations were identified (E40A and I61Q) with DCM properties (decreased Ca²⁺ sensitivity, maximal force recovery, and activation of the ATPase at high [Ca²⁺]). Steady-state fluorescence was utilized to assess Ca²⁺ affinity in isolated cardiac (c)TnCs containing F27W and did not necessarily mirror the fiber Ca²⁺ sensitivity. Circular dichroism of mutant cTnCs revealed a trend where increased α-helical content correlated with increased Ca²⁺ 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 Ca²⁺ sensitivity in skinned fibers was identified; and 3) the F27W reporter mutation affected Ca²⁺ sensitivity, maximal force, and ATPase activation of some mutants.     

Prolonged Ca2+ and force transients in myosin RLC transgenic mouse fibers expressing malignant and benign FHC mutations

Clinical studies have revealed that mutations in the ventricular myosin regulatory light chain (RLC) lead to the development of familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. While mutations in other contractile proteins have been studied widely by others, there is no report elucidating the mechanism(s) associated with FHC-linked RLC mutations. In this study, we have assessed the functional consequences of two RLC mutations, R58Q and N47K, in transgenic mice. Clinical phenotypes associated with these mutations included inter-ventricular hypertrophy, abnormal ECG findings and the R58Q mutation caused multiple cases of premature sudden cardiac death. Simultaneous measurements of the ATPase and force in transgenic skinned papillary muscle fibers from mutated versus control mice showed an increase in the Ca(2+) sensitivity of ATPase and steady-state force only in R58Q fibers. The calculated energy cost or rate of dissociation of force generating myosin cross-bridges (ATPase/force ratio) plotted as a function of activation state was the same in all groups of fibers. Both mutations caused prolonged [Ca(2+)] transients in electrically stimulated intact papillary muscles; however, the R58Q mutation also resulted in a significantly prolonged force transient. Our results suggest that the phenotypes of FHC observed in patients harboring these RLC mutations correlate with the extent of physiological changes monitored in transgenic fibers. Cardiac hypertrophy observed in patients is most likely caused by the activation of compensatory mechanisms ensuing from higher workloads due to incomplete relaxation as evidenced by prolonged [Ca(2+)] transients for both N47K and R58Q fibers. Furthermore, the poor prognosis of the R58Q patients may be associated with more severe diastolic dysfunction due to the slower off-rate of Ca(2+) from troponin C leading to longer force and [Ca(2+)] transients and increased Ca(2+) sensitivity of ATPase and force.     

Regulation of expression of cardiac sarcoplasmic reticulum proteins under pathophysiological conditions

Congestive heart failure presents a significant medical problem and accumulating evidence indicates that slow relaxation during diastole maybe at least in part be medlated by decreased expression of the gene coding for the Ca²⁺ ATPase of the sarcoplasmic reticulum (SR). In order to determine if increased expression of the SR Ca²⁺ ATPase gene leads to alterations in calcium transients and in contractile behavior we constructed transgenic mice overexpressing the SERCA2 gene. Measuring dP/dt_max and dpPdt_min with a 2 French Milar catheter we found a significant Increase in systolic contraction and diastolic relaxation in transgene positive versus transgene negative mice. In addition we constructed adenoviruses overexpressing the gene coding for the Ca²⁺ ATPase of the sarcoplasmic reticulum. Infacting cardiac myocytes with the adenovirus expressing this transgene led to an accelerated calcium transient. Determining cell shortening and relengthening with a edge detection method indicated that increased expression of the SERCA2 transgene mediated by adenovirus Infection accelerated contractile parameters. In summary increased expression of the SERCA2 transgene leads to an enhancement of cardiac contrectile parameters under in vivo conditions in transgenic mice and in myocytes in cell culture using an adenovirus based approach to increase expression of the SERCAX gene.     

Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca²⁺]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061–1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca²⁺-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca²⁺ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca²⁺ signaling support that CeTnT-1 phenotypes are dependent on Ca²⁺ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca²⁺] does not require TnT.     

Familial hypertrophic cardiomyopathy related E180G mutation increases flexibility of human cardiac α-tropomyosin

α-Tropomyosin (αTm) is central to Ca²⁺-regulation of cardiac muscle contraction. The familial hypertrophic cardiomyopathy mutation αTm E180G enhances Ca²⁺-sensitivity in functional assays. To investigate the molecular basis, we imaged single molecules of human cardiac αTm E180G by direct probe atomic force microscopy. Analyses of tangent angles along molecular contours yielded persistence length corresponding to ∼35% increase in flexibility compared to wild-type. Increased flexibility of the mutant was confirmed by fitting end-to-end length distributions to the worm-like chain model. This marked increase in flexibility can significantly impact systolic and possibly diastolic phases of cardiac contraction, ultimately leading to hypertrophy.     

Role of mitochondrial calcium transport in the control of substrate oxidation

This paper reviews the model of the control of mitochondrial substrate oxidation by Ca²⁺ ions. The mechanism is the activation by Ca²⁺ of four mitochondrial dehydrogenases, viz: glycerol 3-phosphate dehydrogenase, the pyruvate dehydrogenase multienzyme complex (PDH), NAD-linked isocitrate dehydrogenase (NAD-IDH) and 2-oxoglutarate dehydrogenase (OGDH). This results in the increase, or near-maintenance, of mitochondrial NADH/NAD ratios in the activated state, depending upon the tissue and the degree of "downstream" activation by Ca²⁺, likely at the level of the F₁F₀ ATP-ase. Higher values of the redox span of the respiratory chain allow for greatly increased fluxes through oxidative phosphorylation with a minimal drop in protonmotive force and phosphorylation potential. As PDH, NAD-IDH and OGDH are all located within the inner mitochondrial membrane, it is changes in matrix free Ca²⁺ ( [Ca²⁺]m ) which act as a signal to these activities. In this article, we review recent work in which ([Ca²⁺]m) is measured in cells and tissues, using different techniques, with special emphasis on the question of the degree of damping of ([Ca²⁺]m) relative to changes in cytosol free Ca²⁺ in cells with rapid transients in cytosol Ca²⁺, e.g. cardiac myocytes. Further, we put forward the point of view that the failure of mitochondrial energy transduction to keep pace with cellular energy needs in some forms of heart failure may involve a failure of ([Ca²⁺]m) to be raised adequately to allow the activation of the dehydrogenases. We present new data to show that this is so in cardiac myocytes isolated from animals suffering from chronic, atreptozocin-induced diabetes. This raises the possibility of therapy based upon partial inhibition of mitochondrial Ca²⁺ efflux pathways, thereby raising ([Ca²⁺]m) at a given, time-average value of cytosol free Ca²⁺.     

Mutations in human cardiac troponin I that are associated with restrictive cardiomyopathy affect basal ATPase activity and the calcium 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.     

Cardiac sarcolemmal Na+-Ca2+ exchange and Na+-K+ ATPase activities and gene expression in alloxan-induced diabetes in rats

To determine the sequence of alterations in cardiac sarcolemmal (SL) Na⁺-Ca²⁺ exchange, Na⁺-K⁺ ATPase and Ca²⁺-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na⁺-K⁺ ATPase and Ca²⁺-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg²⁺ ATPase activity was not depressed throughout the experimental periods. Both Na⁺-Ca²⁺ exchange and ATP-dependent Ca²⁺-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na⁺-K⁺ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of _--subunit of Na⁺-K⁺ ATPase and Na⁺-Ca²⁺ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na⁺-K⁺ ATPase _-- subunit mRNA was noted in diabetic heart. A significant increase in the Na⁺-Ca²⁺ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na⁺-K⁺ ATPase, Na⁺Ca²⁺ exchange and Ca²⁺-pump activities may lead to the occurrence of intracellular Ca²⁺ overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.     

Modulation of cardiac troponin C function by the cardiac-specific N-terminus of troponin I: influence of PKA phosphorylation and involvement in cardiomyopathies

The cardiac-specific N-terminus of cardiac troponin I (cTnI) is known to modulate the activity of troponin upon phosphorylation with protein kinase A (PKA) by decreasing its Ca²⁺ affinity and increasing the relaxation rate of the thin filament. The molecular details of this modulation have not been elaborated to date. We have established that the N-terminus and the switch region of cTnI bind to cNTnC [the N-domain of cardiac troponin C (cTnC)] simultaneously and that the PKA signal is transferred via the cTnI N-terminus modulating the cNTnC affinity toward cTnI_147-163 but not toward Ca²⁺. The K_d of cNTnC for cTnI_147-163 was found to be 600 μM in the presence of cTnI_1-29 and 370 μM in the presence of cTn1_1-29PP, which can explain the difference in muscle relaxation rates upon the phosphorylation with PKA in experiments with cardiac fibers. In the light of newly found mutations in cNTnC that are associated with cardiomyopathies, the important role played by the cTnI N-terminus in the development of heart disorders emerges. The mutants studied, L29Q (the N-domain of cTnC containing mutation L29Q) and E59D/D75Y (the N-domain of cTnC containing mutation E59D/D75Y), demonstrated unchanged Ca²⁺ affinity per se and in complex with the cTnI N-terminus (cTnI_1-29 and cTnI_1-29PP). The affinity of L29Q and E59D/D75Y toward cTnI_147-163 was significantly perturbed, both alone and in complex with cTnI_1-29 and cTnI_1-29PP, which is likely to be responsible for the development of malfunctions.     

The mobility of troponin C and troponin I in muscle

In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca²⁺ concentration. The binding of Ca²⁺ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (τ_r increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg²⁺ by Ca²⁺ at these sites (τ_r increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca²⁺ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl. © 1997 John Wiley & Sons, Ltd.     

Calcium dynamics in the ventricular myocytes of SERCA2 knockout mice: A modeling study

We describe a simulation study of Ca²⁺ dynamics in mice with cardiomyocyte-specific conditional excision of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) gene, using an experimental data-driven biophysically-based modeling framework. Previously, we reported a moderately impaired heart function measured in mice at 4 weeks after SERCA2 gene deletion (knockout (KO)), along with a >95% reduction in the level of SERCA2 protein. We also reported enhanced Ca²⁺ flux through the L-type Ca²⁺ channels and the Na⁺/Ca²⁺ exchanger in ventricular myocytes isolated from these mice, compared to the control Serca2^flox/flox mice (flox-flox (FF)). In the current study, a mathematical model-based analysis was applied to enable further quantitative investigation into changes in the Ca²⁺ handling mechanisms in these KO cardiomyocytes. Model parameterization based on a wide range of experimental measurements showed a 67% reduction in SERCA activity and an over threefold increase in the activity of the Na⁺/Ca²⁺ exchanger. The FF and KO models were then validated against experimentally measured [Ca²⁺]_i transients and experimentally estimated sarco(endo)plasmic reticulum (SR) function. Simulation results were in quantitative agreement with experimental measurements, confirming that sustained [Ca²⁺]_i transients could be maintained in the KO cardiomyocytes despite severely impaired SERCA function. In silico analysis shows that diastolic [Ca²⁺]_i rises sharply with progressive reductions in SERCA activity at physiologically relevant pacing frequencies. Furthermore, an analysis of the roles of the compensatory mechanisms revealed that the major combined effect of the compensatory mechanisms is to lower diastolic [Ca²⁺]_i. Finally, by using a comprehensive sensitivity analysis of the role of all cellular calcium handling mechanisms, we show that the combination of upregulation of the Na⁺/Ca²⁺ exchanger and increased L-type Ca²⁺ current is the most effective means to maintain diastolic and systolic calcium levels after loss of SERCA function.     

Cellular and molecular aspects of familial hypertrophic cardiomyopathy caused by mutations in the cardiac troponin I gene

Mutations in the cardiac troponin I (CTnI) gene occur in 5% of families with familial hypertrophic cardiomyopathy (FHC) and 20 mutations in this gene that cause FHC have now been described. The clinical manifestations of CTnI mutations that cause FHC are diverse, ranging from asymptomatic with high life expectancy to severe heart failure and sudden cardiac death. Most of these FHC mutations in CTnI result in cardiac hypertrophy unlike cardiac troponin T FHC mutations. All CTnI FHC mutations investigated in vitro affect the physiological function of CTnI, but other factors such as environmental or genetic factors (other genes that may affect the CTnI gene) are likely to be involved in influencing the severity of the phenotype produced by these mutations, since the distribution of hypertrophy among affected individuals varies within and between families. CTnI mutations mainly alter myocardial performance via changes in the Ca²⁺-sensitivity of force development and in some cases alter the muscle relaxation kinetics due to haemodynamic or physical obstructions of blood flow from the left ventricle. (Mol Cell Biochem 263: 99–114, 2004)     

Increased calcium leak associated with reduced calsequestrin expression in hyperthyroid cardiomyocytes

IntroductionCalcium (Ca²⁺) leak during cardiac diastole is chiefly mediated by intracellular Ca²⁺ channel/Ryanodine Receptors. Increased diastolic Ca²⁺ leak has been proposed as the mechanism underlying the appearance of hereditary arrhythmias. However, little is known about alterations in diastolic Ca²⁺ leak and the specific roles played by key intracellular Ca²⁺-handling proteins in hyperthyroidism, a known arrhythmogenic condition.AimWe sought to determine whether there were modifications in diastolic Ca²⁺ leak, based on the recording of Ca²⁺ sparks and Ca²⁺ waves; we also investigated changes in the expression and activity of key Ca²⁺ handling proteins, including ryanodine receptors, Sarco-Endoplasmic Reticulum Ca²⁺ ATPase pump and calsequestrin in isolated left-ventricular cardiomyocytes isolated from hyperthyroid rats.Materials and methodsElectrocardiography (ECG) recordings were performed in control and hyperthyroid rats. Ca²⁺ sparks, Ca²⁺ waves, and electrically-stimulated Ca²⁺ transients were recorded in Fluo-3-loaded cardiomyocytes from both experimental groups using confocal microscopy. In addition, left-ventricular homogenates and Ryanodine Receptor-enriched membrane fractions were prepared for assessing [³H]-ryanodine binding, hydrolytic ATPase activity of SERCA pump and expression levels of key proteins by Western blot, and cDNA for real-time qPCR.Results and conclusionsExtrasystoles were observed in hearts of hyperthyroid rats by ECG recordings. Arrhythmogenic activity, high incidence of Ca²⁺ waves, and de novo Ca²⁺ wavelets −in the absence of sarcoplasmic reticulum Ca²⁺ overload- were recorded in these cardiomyocytes. The exacerbated diastolic Ca²⁺ leak and arrhythmogenic activities were related to a diminished expression of calsequestrin along with increased SERCA pump activity, which, in effect, promoted a gain-of-function in RyRs without alterations in SR Ca²⁺ load, RyR expression or its Ca²⁺ sensitivity.     

Mg-ATPase and CA2+ activated myosin ATPase activity in ventricular myofibrils from non-failing and diseased human hearts – effects of calcium sensitizing agents MCI-154, DPI 201–106, and caffeine

We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201–106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca²⁺ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca²⁺ sensitivity. Effects of DPI 201–106 were, however, different. Only at the 10^–6 M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201–106 caused a concentration-dependent increase in myofibrillar ATPase Ca²⁺ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201–106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca²⁺ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca²⁺ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

Store-operated Ca entry and depolarization explain the anomalous behaviour of myometrial SR: Effects of SERCA inhibition on electrical activity,Ca and force

In the myometrium SR Ca²⁺ depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca²⁺ sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca²⁺-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca²⁺ transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca²⁺]_i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca²⁺ spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca²⁺ and depolarization continued a point was reached when electrical and Ca²⁺ spikes and phasic contractions ceased, and a maintained, tonic force and Ca²⁺ was produced. Lanthanum, a non-selective blocker of store-operated Ca²⁺ entry, but not the L-type Ca²⁺ channel blocker nifedipine (1–10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca²⁺ depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca²⁺ release is coupled to marked Ca²⁺ entry, via store operated Ca²⁺ channels, leading to depolarization and enhanced electrical and mechanical activity.     

Dilated cardiomyopathy mutations in α-tropomyosin inhibit its movement during the ATPase cycle

The Glu40Lys and Glu54Lys mutations in α-tropomyosin cause dilated cardiomyopathy (DCM). Functional analysis has demonstrated that both mutations decrease thin filament Ca²⁺-sensitivity and that Glu40Lys reduces maximum activation. To understand the molecular mechanism underlying these changes, we labeled wild type α-tropomyosin and both mutants at Cys190 with 5-iodoacetamide-fluorescein and incorporated the labeled proteins into ghost muscle fibers. Using the polarized fluorimetry, the position of the labeled tropomyosins on the thin filament and their affinity for actin were measured and the change in these parameters at different stages of the ATPase cycle determined. Both DCM mutations were found to shift tropomyosin towards the periphery of thin filament and to change the affinity of tropomyosin for actin; during the ATPase cycle the amplitude of tropomyosin movement was reduced and at some stages of the cycle even reversed. The correlation of these structural changes with the observed function effects is discussed.     

Mitochondrial Ca2+ Transients in Cardiac Myocytes During the Excitation–Contraction Cycle: Effects of Pacing and Hormonal Stimulation

Using laser scanning confocal microscopy, our objective was to measure mitochondrial, nuclear, and cytosolic free ionized Ca²⁺ in adult rabbit cardiac myocytes loaded with Ca²⁺-indicating fluorophores. When myocytes were loaded with Fluo 3 at 37°C, the fluorophore was loaded extensively into the cytosol and nucleus, but poorly into mitochondria, and Fluo 3 fluorescence transients after field stimulation were confined to the cytosol and nucleus. In contrast, after loading at 4°C, Fluo 3 also entered mitochondria, and large transients of mitochondrial Fluo 3 fluorescence then occurred after stimulation. Isoproterenol (1 M) increased the magnitude of Ca²⁺ transients and their subsequent rate of decay, an effect more marked in the cytosol and nucleus than in mitochondria. As pacing frequency was increased from 0.5 to 2 Hz, diastolic mitochondrial Ca²⁺ rose markedly in the absence but not in the presence of isoproterenol. Resting Ca²⁺ estimated by Indo 1 ratio imaging using UV/visible laser scanning confocal microscopy was about 200 nM in all compartments. During field stimulation, Ca²⁺ transiently increased to 671, 522, and 487 nM in cytosol, interfibrillar mitochondria, and perinuclear mitochondria, respectively. Isoproterenol increased these respective peak values to 1280, 750, and 573 nM. These results were consistent with those obtained in Fluo 3 experiments. We conclude that rapid mitochondrial Ca²⁺ transients occur during excitation–contraction coupling in adult rabbit cardiac myocytes, which may be important in matching mitochondrial metabolism to myocardial ATP demand during changes in cardiac output.     

Decreased Ca2+-binding and Ca2+-ATPase activities in heart sarcolemma upon phospholipid methylation

Heart sarcolemma has been shown to possess three catalytic sites (I, II and III) for methyl transferase activity (Panagia V, Ganguly PK and Dhalla NS. Biochim Biophys Acta 792: 245–253, 1984). In this study we examined the effect of phosphatidylethanolamine N-methylation on ATP-independent Ca²⁺ binding and ATPase activities in isolated rat heart sarcolemma. Both low affinity (1.25 mM Ca²⁺) and high affinity (50 µM Ca²⁺) Ca²⁺ binding activities were decreased following incubation of sarcolemmal membranes with AdoMet under optimal conditions for site II and III. Similarly, Ca²⁺ ATPase activities measured at 1.25 mM and 4 mM Ca²⁺ were depressed by phospholipid N-methylation. S-adenosyl homocysteine, a specific inhibitor of phospholipid N-methylation, prevented the depression of low affinity Ca²⁺ binding and Ca²⁺ ATPase activities, whereas the methylation-induced effect on the high affinity Ca²⁺ binding was not influenced by this agent. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino group blocking agent, also prevented the methylation-induced inhibition of both Ca²⁺ binding and Ca²⁺ ATPase. A further decrease in Ca²⁺ binding and Ca²⁺ ATPase activities together with a marked increase in the intramembranal level of PC was seen when membranes were methylated under the site III conditions in the presence of phosphatidyldimethylethanolamine as exogenous substrate. There was no effect of phospholipid methylation on sarcolemmal Na⁺-K⁺ ATPase and Mg²⁺ ATPase activities. These results indicate a role of phospholipid N-methylation in the regulation of sarcolemmal Ca²⁺ ATPase and low affinity ATP-independent Ca²⁺ binding.