title: In vitro rescue study of a malignant familial hypertrophic cardiomyopathy phenotype by pseudo-phosphorylation of myosin regulatory light chain.
abstract: Pseudo-phosphorylation of cardiac myosin regulatory light chain (RLC) has never been examined as a rescue method to alleviate a cardiomyopathy phenotype brought about by a disease causing mutation in the myosin RLC. This study focuses on the aspartic acid to valine substitution (D166V) in the myosin RLC shown to be associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). The mutation has also been demonstrated to cause severe functional abnormalities in transgenic mice expressing D166V in the heart. To explore this novel rescue strategy, pseudo-phosphorylation of D166V was used to determine whether the D166V-induced detrimental phenotype could be brought back to the level of wild-type (WT) RLC. The S15D substitution at the phosphorylation site of RLC was inserted into the recombinant WT and D166V mutant to mimic constitutively phosphorylated RLC proteins. Non-phosphorylatable (S15A) constructs were used as controls. A multi-faceted approach was taken to determine the effect of pseudo-phosphorylation on the ability of myosin to generate force and motion. Using mutant reconstituted porcine cardiac muscle preparations, we showed an S15D-induced rescue of both the enzymatic and binding properties of D166V-myosin to actin. A significant increase in force production capacity was noted in the in vitro motility assays for S15D-D166V vs. D166V reconstituted myosin. A similar pseudo-phosphorylation induced effect was observed on the D166V-elicited abnormal Ca(2+) sensitivity of force in porcine papillary muscle strips reconstituted with phosphomimic recombinant RLCs. Results from this study demonstrate a novel in vitro rescue strategy that could be utilized in vivo to ameliorate a malignant cardiomyopathic phenotype. We show for the first time that pseudo-RLC phosphorylation can reverse the majority of the mutation-induced phenotypes highlighting the importance of RLC phosphorylation in combating cardiac disease.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
authors.forename: Priya
authors.initials: P
authors.lastname: Muthu
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
authors.forename: Jingsheng
authors.initials: J
authors.lastname: Liang
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: William
authors.initials: W
authors.lastname: Schmidt
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Moore
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL 33136, USA. Electronic address: dszczesna@med.miami.edu.
authors.forename: Danuta
authors.initials: D
authors.lastname: Szczesna-Cordary
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D024741:Cardiomyopathy, Hypertrophic, Familial / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009206:Myocardium / Q000378:metabolism; D018994:Myosin Light Chains / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D011817:Rabbits; D013552:Swine
chemical_list: D000199:Actins; D018994:Myosin Light Chains; D002118:Calcium
keywords: Cardiomyopathy; In vitro motility; Muscle contraction; Mutation; Myosin phosphorylation; Reconstituted cardiac system