Cardiomyopathy Causing Mutations Stabilize an Intermediate State of Thin Filaments

Abstract

Congenital cardiomyopathies are initiated by changes in ATP hydrolysis and result in hypertrophy, fibrosis, and myofibrillar disarray. We studied the mechanism by which mutations in troponin and tropomyosin change ATPase rates and have linked several mutants to inappropriate switching between the inactive and active states of the actin thin filament. We have shown that troponin I mutants mimicking protein kinase C phosphorylation stabilize the inactive state of actin filaments whereas the Δ14 TnT mutant stabilizes the active state. We have now shown that two mutations on troponin I, R146G and R146W, which cause cardiomyopathy produce complex effects on ATPase activity. These TnI mutations produced increased ATPase rates in the absence of calcium and decreased rates in the presence of calcium compared to wild type. These differences were maintained at high actin concentrations. Saturating concentrations of the activator NEM-S1 equalized the rates of both the mutants and wild type. The NEM-S1 data rule out alterations in rate constants of transitions (i.e. product release) along the active pathway. The results from the R146G and R146W mutants have implications for the function of the 3 structural states of regulated actin that have been observed. That is, the results can be explained most readily if the mutants stabilize an intermediate state in both calcium and EGTA with an activity between that of the inactive and active states. In the past we have assumed that the intermediate state had properties identical to the inactive state. Our current data show that while the intermediate more closely resembles the inactive state it has unique properties. Our present results, as well as previous results, indicate that inappropriate stabilization of any state of regulated actin can result in cardiac dysfunction.