'(De-)sensitization' vs. 'Uncoupling': what drives cardiomyopathies in the thin filament? Reply

Heart muscle contraction is regulated via the β-adrenergic response that leads to phosphorylation of Troponin I (TnI) at Ser22/23, which changes the Ca(2+) sensitivity of the cardiac myofilament. Mutations in thin filament proteins that cause dilated cardiomyopathy (DCM) and some mutations that cause hypertrophic cardiomyopathy (HCM) abolish the relationship between TnI phosphorylation and Ca(2+) sensitivity (uncoupling). Small molecule Ca(2+) sensitizers and Ca(2+) desensitizers that act upon troponin alter the Ca(2+) sensitivity of the thin filament, but their relationship with TnI phosphorylation has never been studied before.Quantitative in vitro motility assay showed that 30 µM EMD57033 and 100 µM Bepridil increase Ca(2+) sensitivity of phosphorylated cardiac thin filaments by 3.1- and 2.8-fold, respectively. Additionally they uncoupled Ca(2+) sensitivity from TnI phosphorylation, mimicking the effect of HCM mutations. Epigallocatechin-3-gallate (EGCG) decreased Ca(2+) sensitivity of phosphorylated and unphosphorylated wild-type thin filaments equally (by 2.15 ± 0.45- and 2.80 ± 0.48-fold, respectively), retaining the coupling. Moreover, EGCG also reduced Ca(2+) sensitivity of phosphorylated but not unphosphorylated thin filaments containing DCM and HCM-causing mutations; thus, the dependence of Ca(2+) sensitivity upon TnI phosphorylation of uncoupled mutant thin filaments was restored in every case. In single mouse heart myofibrils, EGCG reduced Ca(2+) sensitivity of force and kACT and also preserved coupling. Myofibrils from the ACTC E361G (DCM) mouse were uncoupled; EGCG reduced Ca(2+) sensitivity more for phosphorylated than for unphosphorylated myofibrils, thus restoring coupling.We conclude that it is possible to both mimic and reverse the pathological defects in troponin caused by cardiomyopathy mutations pharmacologically. Re-coupling by EGCG may be of potential therapeutic significance for treating cardiomyopathies.