Pseudo-Phosphorylation Mediated Rescue of R58Q-Linked Familial Hypertrophic Cardiomyopathy Phenotype

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Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myofibrillar disarray and often caused by mutations in genes encoding for major sarcomeric proteins. This study is focused on Arginine to Glutamine substitution (R58Q) in the ventricular myosin regulatory light chain (RLC) resulting in a malignant hypertrophic phenotype. We previously showed a mutation-induced decrease in the endogenous level of cardiac RLC phosphorylation in R58Q mice, resulting in compromised communication between the functional regions of the RLC and affecting the interaction of myosin and actin and cardiac muscle contraction. In this report we explored the therapeutic potential of pseudo-phosphorylation, in which Aspartic Acid is replaced for phosphorylatable Serine-15 (S15D) in the R58Q background. In vitro actin-activated myosin ATPase activity was assessed using S15D-R58Q and R58Q vs. WT reconstituted porcine cardiac myosin. R58Q largely decreased maximal ATPase activity, Vmax (∼30% lower) compared to WT. S15D-R58Q rescued the maximal ATPase activity to the WT level. The Michaelis–Menten constant (in μM) for R58Q (Km=4.04±0.45) was significantly increased compared to WT (Km=1.68±0.19), suggesting that a higher concentration of actin is needed to activate cross-bridges. S15D-R58Q-RLC showed a small but significant increase in Km (2.56±0.32), compared with WT. Steady-state acto-myosin binding was studied using fluorescently labelled actin to quantify the dissociation constants (Kd) and stoichiometry of binding (n) between myosin and actin in the absence of nucleotide. Binding of WT-RLC to actin was strong (Kd value =5.5 nM, n=0.89). A large decrease in binding (∼67-fold increase in Kd) was observed for R58Q-RLC. Kd of S15D-R58Q-RLC was only slightly increased (∼3-fold) compared with WT indicating a rescue in binding affinity by pseudo-phosphorylated RLC mutant. Our findings on S15D-R58Q support the idea that myosin phosphorylation may have important translational applications for the treatment of RLC induced hypertrophic cardiomyopathy. Supported by NIH-HL123255 (DSC).