Ventricular ELC Structural Changes induced by Skeletal RLC and an A57G Cardiomyopathy mutation

Abstract

Time-resolved fluorescence quenching measurements were used to measure the conformational changes in the ventricular essential light chain (vELC) for two specific aims. First, is to investigate whether the interactions between cardiac vELC and the ventricular regulatory light chain (vRLC) are isoform-specific, and second, is to determine the conformational effects of the A57G-vELC cardiomyopathy mutation on the ELC structure. Both the ELC and the RLC play essential roles in cardiac muscle contraction, and the specific interactions between ELC and RLC of scallop and smooth muscle are important for the regulation of contraction. The effect of different isoforms of RLC on vELC aids us to investigate the specific requirement of certain RLC isoforms in cardiac muscle. Familial hypertrophic cardiomyopathy (FHC) is a human cardiac disease in which the left ventricle and inter-ventricular septum of the myocardium is hypertrophied. A57G is an ELC mutation that causes FHC, and occurs in the middle of the first α-helix in ELC. In this study, a single cysteine was engineered with ELC at C81 for labeling with a fluorescent probe called IAEDANS. The endogenous RLC and ELC were removed from skinned rabbit psoas muscle fibers. For the first experimental goal, labeled rat wildtype vELC with either rat wildtype vRLC or rabbit skeletal rRLC were exchanged back into the fibers, which were subsequently named as the WT and the rRLC. For the second experimental purpose, either labeled rat wildtype vELC or labeled A57G vELC with rat wildtype vRLC were exchanged back on the fibers and these were subsequently named as the WT and the A57G. The time-resolved fluorescence quenching experiments were performed in rigor, relaxation and contraction solutions, and the quenching of the lifetimes of the probe IAEDANS, was determined as a function of [Acrylamide]. The existence of rRLC and A57G mutation caused the  (fluorescence lifetime in the absence of the quencher) of the rRLC and the A57G to be shorter than the WT, respectively. The Stern-Volmer constants for the rRLC and the A57G were also higher to different extents compared to the WT in all solutions, suggesting that the WT has a more closed configuration in which the probe is more buried and less exposed to the solvent. These indicated that both rRLC and A57G could induce ELC conformational changes that may open the domain. The powerstroke during contraction caused less conformational change due to the existence of the skeletal RLC; this suggested that the interaction between RLC and ELC could affect force generation. A potential reason for this change is the different amino acids in the sequence between rRLC and vRLC linker area for the c-terminal domain. The Ksv constants for A57G in states were similar to each other. It is possible that the A57G cardiomyopathy mutation disrupted the helix structure so that the labeled site in vELC were constantly open.