The Role of the N-Terminus of the Myosin Essential Light Chain in Cardiac Muscle Contraction

Abstract

SummaryTo study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43 amino acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per crosssectional area of muscle, which is likely caused by a reduced number of force generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of non-pathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.

Discrete effects of A57G-myosin essential light chain mutation associated with familial hypertrophic cardiomyopathy

“…Slides of whole mouse hearts were prepared at the Histology Laboratory (University of Miami Miller School of Medicine, Miami FL). The paraffin-embedded longitudinal sections of whole mouse hearts stained with hematoxylin and eosin (H&E) and Masson's trichrome were examined for overall morphology and fibrosis using a Dialux 20 microscope, ϫ40/0.65 NA (numerical aperture) Leitz Wetzlar objective and an AxioCam HRc (Zeiss) as described previously (24,40). Slides with H&E-stained sections from Tg-WT and Tg-A57G mouse hearts were analyzed for the number of nuclei.…”

“…Myosin was isolated from the left and right ventricles of 4-to 6 mo-old and 10-to 12 mo-old Tg-A57G and Tg-WT mice as described previously (24,49). Briefly, after euthanasia, whole hearts were isolated and the atria were removed.…”

“…Rabbit skeletal F-actin was prepared according to Kazmierczak et al (24). Actin-activated myosin ATPase activity was measured as a function of actin concentration and the data analyzed as described in Kazmierczak et al (23,24). Briefly, 0.5 M myosin dissolved in 0.4 M KCl was added to a 96-well microplate containing increasing concentrations of F-actin (in M): 0.1, 0.5, 1.5, 3.0, 5, 7.5, 10, and 15.…”

“…A wealth of evidence suggests that it may play a role in force development and muscle contraction (17,24,36,40,54). The functional importance of myosin ELC in cardiac muscle has emerged through the identification of several mutations in the ELC gene (MYL3) shown to cause familial hypertrophic cardiomyopathy (FHC).…”

“…40) and its effects were compared with those seen in Tg-wild-type (WT) mice expressing the full-length nonmutated human ventricular ELC (24). Previously performed histological examination of mouse hearts showed that the human phenotype including extensive disorganization of myocytes and interstitial fibrosis could be recapitulated in Tg-A57G mice (40).…”