071 FHC-related cardiac troponin c l29q mutation affects the contractility of skinned cardiac myocytes and exacerbates the functional effects of the phosphorylation of the N-terminus of troponin I

Abstract

A Familial Hypertrophic Cardiomyopathy (FHC)-related cardiac troponin C (cTnC) mutation, L29Q, was found in a patient with symptoms of concentric hypertrophy of the left ventricle. Leu-29 is located at the transition of helix A to the non-functional Ca2+-binding site I of cTnC. Our previous work showed that amino acid substitutions in this region affect the structure and the Ca2+ affinity of site II of cTnC. Furthermore, this domain of cTnC interacts with the non-phosphorylated N-terminal arm of cardiac troponin I (cTnI) during Ca2+ activation. Studies have suggested the possibility of the L29Q cTnC mutation affecting Ca2+ activation and the functional transduction of PKA-dependent phosphorylation of cTnI to Ca2+-activated force; however, functional studies have yielded highly contradictory results as to its effects on Ca2+ sensitivity of force generation in skinned fibers. The purpose of this study was therefore to investigate the effects of recombinant mouse cTnC L29Q mutation on the contractile parameters in triton-skinned cardiac myocytes. With the skinned cardiac myocyte technique one can precisely control the sarcomere length and get better troponin subunit exchange than those with skinned muscle fiber bundle and more accurately determine Ca2+ sensitivity. In particular, we investigated whether the L29Q cTnC mutation abrogates the effects of PKA-dependent phophorylation of cTnI on the Ca2+ sensitivity of force generation by replacing endogenous cardiac troponin (cTn) with the cTn that contained L29Q cTnC and a phosphomimetic mutant cTnI(S23/24D). The results showed that: (1)myocytes reconstituted with L29Q cTn had higher Ca2+ sensitivity of force generation than those with WT cTn. (2)S22/23D cTnI mutations decreased the Ca2+ sensitivity of force generation. This effect of S22/23D cTnI mutation was reduced by about 44.5% by the L29Q cTnC mutation. (3)S22/23D cTnI mutations enhanced the length-dependent Ca2+ sensitivity. This effect was significantly affected by cTnC L29Q mutation in the skinned cardiac myocytes. In conclusion, FHC-related L29Q cTnC mutation caused a small but significant increase in the Ca2+ sensitivity of force generation in skinned mouse cardiac myocytes; the functional effects of the phosphorylation of the N-terminus of cTnI were exacerbated by L29Q cTnC mutation.