Search

Recombinant cTnC proteins with single Cys residues designed for binding of fluorescent probes that would aid in the distinction of myosin cross bridges from direct Ca+2 binding to cTnC were prepared and characterized. The experiment yielded two monocysteine derivatives of cTnC which were labeled specifically with IAANS bound to Cys 35 or Cys 84. The reaction rate with sulfhydryl reagents confirmed that the resulting fluorescent activities are dependent on the position of the label, other troponin subunits and protein-protein reactions in situ and in vitro.

Transgenic (Tg) mice expressing ~95% of the D166V (aspartic acid to valine) mutation in the ventricular myosin regulatory light chain (RLC) shown to cause a malignant familial hypertrophic cardiomyopathy (FHC) phenotype were generated, and the skinned and intact papillary muscle fibers from the Tg-D166V mice were examined using a Guth muscle research system. A large increase in the Ca2+ sensitivity of force and ATPase (ΔpCa50>0.25) and a significant decrease in maximal force and ATPase were observed in skinned muscle fibers from Tg-D166V mice compared with control mice. The cross-bridge dissociation rate g was dramatically decreased, whereas the energy cost (ATPase/ force) was slightly increased in Tg-D166V fibers compared with controls. The calculated average force per D166V cross-bridge was also reduced. Intact papillary muscle data demonstrated prolonged force transients with no change in calcium transients in Tg-D166V fibers compared with control fibers. Histopathological e xamination revealed fibrotic lesions in the hearts of the older D166V mice. Our results suggest that a charge effect of the D166V mutation and/or a mutation-dependent decrease in RLC phosphorylation could initiate the slower kinetics of the D166V cross-bridges and ultimately affect the regulation of cardiac muscle contraction. Profound cellular changes observed in Tg-D166V myocardium when placed in vivo could trigger a series of pathological responses and result in poor prognosis for D166V-positive patients. [ABSTRACT FROM AUTHOR]

Abstract: 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 Ca2+ 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 [Ca2+] 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 [Ca2+] 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 Ca2+ from troponin C leading to longer force and [Ca2+] transients and increased Ca2+ sensitivity of ATPase and force. [Copyright &y& Elsevier]

We have formulated a three-compartment model of muscle activation that includes both strong cross-bridge (XB) and Ca2+-activated regulatory-unit (RU) mediated nearest-neighbor cooperative influences. The model is based on the tight coupling premise—that XB retain activating Ca2+ on the thin filament. Using global non-linear least-squares, the model produced excellent fits to experimental steady-state force-pCa and ATPase-pCa data from skinned rat soleus fibers. In terms of the model, nearest-neighbor influences over the range of Ca2+ required for activation cause the Ca2+ dissociation rate from regulatory-units (koff) to decrease and the cross-bridge association rate (f) to increase each more than ten-fold. Moreover, the rate variations occur in separate Ca2+ regimes. The energy of activation governing f is strongly influenced by both neighboring RU and XB. In contrast, the energy of activation governing koff is less affected by neighboring XB than by neighboring RU. Nearest-neighbor cooperative influences provide both an overall sensitization to Ca2+ and the well-known steep response of force to free Ca2+. The apparent sensitivity for Ca2+-activation of force and ATPase is a function of cross-bridge kinetic rates. The model and derived parameter set produce simulated behavior in qualitative agreement with steady-state experiments reported in the literature for partial TnC replacement, increased [Pi], increased [ADP], and MalNEt-S1 addition. The model is an initial attempt to construct a general theory of striated muscle activation—one that can be consistently used to interpret data from various types of muscle manipulation experiments. [Copyright &y& Elsevier]

Abstract: The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an ∼13–16% increase in maximal Ca2+-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate (g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca2+ sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca2+] transients and increased time to peak force. Analysis of force and Ca2+ transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca2+ transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca2+ transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype. [Copyright &y& Elsevier]

In this study, we addressed the functional consequences of the human cardiac troponin I (hcTnl) hypertrophic cardiomyopathy R145G mutation in transgenic mice. Simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnl R145G transgenic mice (Tg-R145G) versus hcTnl wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca2+-activated force without changes in the maximal ATPase activity and an increase in the Ca2+ 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 Ca2+ 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 ∼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 [Ca2+] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared with Tg-WT. These results suggest that the phenotype of hyper- trophic 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 [Ca2+] and force transients, and 3) an inability of the cardiac TnI to completely inhibit activation in the absence of Ca2+ in Tg-R145G mice. [ABSTRACT FROM AUTHOR]

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. Ca2+ 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 Ca2+ transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca2+ [³H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca2+ activation or Ca2+ and Mg2+ 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 Ca2+ homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling. [ABSTRACT FROM AUTHOR]