Regulation of Contractile Function by cMLC2v Phosphorylation in Heart Failure

Loyola University Chicago, Maywood IL

Investigators

Abstract

? DESCRIPTION (provided by applicant): Heart failure (HF) is a major clinical problem with extremely poor prognosis. Though altered phosphorylation of thin filament proteins is known to contribute to HF progression, much less is known about phosphorylation of the thick filament protein cardiac myosin regulatory light chain (cMLC2v). cMLC2v modulates force production by altering myosin head orientation. Its phosphorylation status is controlled by myosin light chain kinase (cMLCK) and phosphatase (MLCP). Decreased cMLC2v phosphorylation and cMLCK expression are observed during HF in patients and animal models, though increased phosphorylation has also been reported and may be an adaptive mechanism. In mice, cMLCK overexpression ameliorates HF; conversely, cMLCK disruption exacerbates HF. However, mice have two cMLC2v phosphorylation sites, each of which may have a separate function, whereas human cMLC2v has only one phosphorylation site. Thus, though cMLC2v phosphorylation is a key regulator of contraction, its role in human HF is largely unknown. Defining the role of cMLC2v phosphorylation in human health and HF will open new therapeutic avenues. Current HF therapies, such as beta-blockers and diuretics, are non-specific and largely palliative; in contrast, therapies targeting cMLC2v would be cardiac-specific and could improve heart function at the fundamental level of the contractile proteins. We hypothesize that cMLC2v phosphorylation is a critical regulator of human cardiomyocyte function, and manipulation of cMLC2v phosphorylation may be a novel therapy for treatment of HF. In Aim 1, we will use skinned cardiac preparations to study regulation of contractile function by cMLC2v phosphorylation in failing and non-failing human heart. We predict that cMLC2v phosphorylation will be decreased during HF, and that experimentally increasing cMLC2v phosphorylation will improve contractile function, especially in failing versus non-failing hearts. Because cMLC2v may also impact Ca2+ transients and may preferentially alter dynamic and loaded contractions, Aim 2 will investigate regulation of dynamic contractile function and Ca2+ handling by cMLC2v phosphorylation in intact electrically stimulated cardiomyocytes from failing and non-failing rabbit hearts (which have high cMLC2v homology with human and identical phosphorylation site). We expect that increasing cMLC2v phosphorylation by cMLCK transfection will improve contractile function as assessed by simulated PV loops, and will increase amplitude but decrease duration of the Ca2+ transient; we expect that cMLCP overexpression will have the opposite effect. In summary, we hypothesize that reduced cMLC2v phosphorylation is a major mechanism underlying contractile dysfunction in human HF, and restoration of cMLC2v phosphorylation may be a novel therapeutic target. Future studies will use pharmacological and/or gene therapy to treat HF in animal models and eventually in patients.