Abstract
The cardiac troponin I (cTnI) R145W mutation is associated with restrictive cardiomyopathy (RCM). Recent evidence suggests that this mutation induces perturbed myofilament length-dependent activation (LDA) under conditions of maximal protein kinase A (PKA) stimulation. Some cardiac disease-causing mutations, however, have been associated with a blunted response to PKA-mediated phosphorylation; whether this includes LDA is unknown. Endogenous troponin was exchanged in isolated skinned human myocardium for recombinant troponin containing either cTnI R145W, PKA/PKC phosphomimetic charge mutations (S23D/S24D and T143E), or various combinations thereof. Myofilament Ca2+ sensitivity of force, tension cost, LDA, and single myofibril activation/relaxation parameters were measured. Our results show that both R145W and T143E uncouple the impact of S23D/S24D phosphomimetic on myofilament function, including LDA. Molecular dynamics simulations revealed a marked reduction in interactions between helix C of cTnC (residues 56, 59, and 63), and cTnI (residue 145) in the presence of either cTnI RCM mutation or cTnI PKC phosphomimetic. These results suggest that the RCM-associated cTnI R145W mutation induces a permanent structural state that is similar to, but more extensive than, that induced by PKC-mediated phosphorylation of cTnI Thr-143. We suggest that this structural conformational change induces an increase in myofilament Ca2+ sensitivity and, moreover, uncoupling from the impact of phosphorylation of cTnI mediated by PKA at the Ser-23/Ser-24 target sites. The R145W RCM mutation by itself, however, does not impact LDA. These perturbed biophysical and biochemical myofilament properties are likely to significantly contribute to the diastolic cardiac pump dysfunction that is seen in patients suffering from a restrictive cardiomyopathy that is associated with the cTnI R145W mutation.
Highlights
Introduction ofR145W ϩ protein kinase A (PKA) into cardiac troponin I (cTnI) induced reduced kinetics of force relaxation in the single myofibrils, a phenomenon that has previously been reported for the R145G hypertrophic cardiomyopathy (HCM)-associated mutation in rodent single myofibrils [26]
Impact of Isolated PKA/PKC/restrictive cardiomyopathy (RCM) on Myofilament Ca2ϩ Sensitivity—Fig. 2A summarizes force-[Ca2ϩ] relationships recorded on troponin-exchanged multicellular skinned human muscles containing wild-type (WT), RCM, PKC, or PKA cTnI
PKA/PKC/RCM Interactions—Fig. 2B summarizes force[Ca2ϩ] relationships recorded on muscles that were exchanged with troponin that contained within cTnI combinations of either the RCM disease mutation or the PKA/PKC charge mutations or PKA ϩ PKC as compared with the WT troponin exchange
Summary
Introduction ofR145W ϩ PKA into cTnI induced reduced kinetics of force relaxation in the single myofibrils, a phenomenon that has previously been reported for the R145G HCM-associated mutation in rodent single myofibrils [26]. Regardless of the underlying molecular mechanism, slowed and delayed force relaxation coupled with the drastic increase in myofilament Ca2ϩ sensitivity is likely to contribute to relaxation abnormalities at the level of the whole heart and may lead to persistent cross-bridge activity throughout the diastolic phase of the cardiac cycle. Both myofilament-based phenomena are expected to induce the diastolic cardiac pump dysfunction that is seen in RCM patients in the notable absence of ventricular hypertrophy
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