The effect of myosin RLC phosphorylation in normal and cardiomyopathic mouse hearts

Abstract

Phosphorylation of the myosin regulatory light chain (RLC) by Ca2+-calmodulin–activated myosin light chain kinase (MLCK) is known to be essential for the inotropic function of the heart. In this study, we have examined the effects of MLCK-phosphorylation of transgenic (Tg) mouse cardiac muscle preparations expressing the D166V (aspartic acid to valine)–RLC mutation, identified to cause familial hypertrophic cardiomyopathy with malignant outcomes. Our previous work with Tg-D166V mice demonstrated a large increase in the Ca2+ sensitivity of contraction, reduced maximal ATPase and force and a decreased level of endogenous RLC phosphorylation. Based on studies demonstrating the beneficial and/or protective effects of cardiac myosin phosphorylation for heart function, we hypothesized that an ex vivo phosphorylation of Tg-D166V cardiac muscle may rescue the detrimental contractile phenotypes observed earlier at the level of single myosin molecules and in Tg-D166V papillary muscle fibres. We showed that MLCK-induced phosphorylation of Tg-D166V cardiac myofibrils and muscle fibres was able to increase the reduced myofibrillar ATPase and reverse an abnormally increased Ca2+ sensitivity of force to the level observed for Tg-wild-type (WT) muscle. However, in contrast to Tg-WT, which displayed a phosphorylation-induced increase in steady-state force, the maximal tension in Tg-D166V papillary muscle fibres decreased upon phosphorylation. With the exception of force generation data, our results support the notion that RLC phosphorylation works as a rescue mechanism alleviating detrimental functional effects of a disease causing mutation. Further studies are necessary to elucidate the mechanism of this unexpected phosphorylation-induced decrease in maximal tension in Tg-D166V–skinned muscle fibres.

Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

“…To follow up on our in vitro experiments (6,17) and to test the hypothesis that pseudo-RLC phosphorylation is capable of preventing the development of detrimental D166V phenotypes in vivo, we produced three lines of the α-MHC-driven transgenic "rescue mice" expressing a constitutively phosphorylated RLC-S15D (serine mutated to aspartic acid) in a background of RLC-D166V. Transgene expression and the incorporation of the S15D-D166V mutant into the mouse myocardium were determined in ventricular and atrial lysates (four to six hearts per group) or in myofibrils purified from three to seven hearts per line (L1, L3, and L5).…”

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

“…38). The measurement of diameter was taken at approximately three points along the muscle strip length with an SZ6045 Olympus microscope (zoom ratio of 6.3:1, up to 189 ϫ maximum magnification) and averaged (38). For the measurement of passive tension (in pCa 8 solution) in response to muscle stretch, the muscle strip was first released and stretched until it began generating tension (53).…”

“…Maximal steadystate tension measured in pCa 4 solution was expressed in newtons per cross section of the muscle strip (N/m 2 ; Ref. 38). The measurement of diameter was taken at approximately three points along the muscle strip length with an SZ6045 Olympus microscope (zoom ratio of 6.3:1, up to 189 ϫ maximum magnification) and averaged (38).…”

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

“…DCMlinked D94A-RLC mutation was shown to significantly alter the N-terminal α-helical region of the RLC, and trigger intramolecular rearrangements in the RLC molecule that ultimately resulted in alterations of RLC function . Other studies from Szczesna et al's laboratory have also revealed compromised RLC phosphorylation in several different HCM models (Tg-R58Q and Tg-D166V), occurring concurrently with diminished maximal tension and altered Ca 2+ sensitivity of contraction (Abraham et al 2009;Kerrick et al 2009a;Muthu et al 2012b). Studies with HCM causing mutations in the myosin RLC suggest a correlation between the severity of cardiomyopathy phenotype and the level of RLC phosphorylation in vivo (Huang et al 2014;Kerrick et al 2009b;Muthu et al 2012b).…”

“…Other studies from Szczesna et al's laboratory have also revealed compromised RLC phosphorylation in several different HCM models (Tg-R58Q and Tg-D166V), occurring concurrently with diminished maximal tension and altered Ca 2+ sensitivity of contraction (Abraham et al 2009;Kerrick et al 2009a;Muthu et al 2012b). Studies with HCM causing mutations in the myosin RLC suggest a correlation between the severity of cardiomyopathy phenotype and the level of RLC phosphorylation in vivo (Huang et al 2014;Kerrick et al 2009b;Muthu et al 2012b). The compromised phosphorylation of RLC mutants compared to WT suggests that diminished RLC phosphorylation observed in HCM hearts may have an important physiological role and is central to the understanding of the mutation-elicited detrimental cardiomyopathy phenotype.…”