Phosphomimetic-mediated in vitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain

Sunil Yadav, Katarzyna Kazmierczak, Jingsheng Liang, Yoel H. Sitbon, Danuta Szczesna-Cordary

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Myosin regulatory light chain (RLC) phosphorylation is important for cardiac muscle mechanics/function as well as for the Ca2+-troponin/tropomyosin regulation of muscle contraction. This study focuses on the arginine to glutamine (R58Q) substitution in the human ventricular RLC (MYL2 gene), linked to malignant hypertrophic cardiomyopathy in humans and causing severe functional abnormalities in transgenic (Tg) R58Q mice, including inhibition of cardiac RLC phosphorylation. Using a phosphomimic recombinant RLC variant where Ser-15 at the phosphorylation site was substituted with aspartic acid (S15D) and placed in the background of R58Q, we aimed to assess whether we could rescue/mitigate R58Q-induced structural/functional abnormalities in vitro. We show rescue of several R58Q-exerted adverse phenotypes in S15D-R58Q-reconstituted porcine cardiac muscle preparations. A low level of maximal isometric force observed for R58Q- versus WT-reconstituted fibers was restored by S15D-R58Q. Significant beneficial effects were also observed on the Vmax of actin-activated myosin ATPase activity in S15D-R58Q versus R58Q-reconstituted myosin, along with its binding to fluorescently labeled actin. We also report that R58Q promotes the OFF state of myosin, both in reconstituted porcine fibers and in Tg mouse papillary muscles, thereby stabilizing the super-relaxed state (SRX) of myosin, characterized by a very low ATP turnover rate. Experiments in S15D-R58Q-reconstituted porcine fibers showed a mild destabilization of the SRX state, suggesting an S15D-mediated shift in disordered-relaxed (DRX)↔SRX equilibrium toward the DRX state of myosin. Our study shows that S15D-phosphomimic can be used as a potential rescue strategy to abrogate/alleviate the RLC mutation-induced phenotypes and is a likely candidate for therapeutic intervention in HCM patients.

Original languageEnglish (US)
JournalFEBS Journal
DOIs
StateAccepted/In press - Jan 1 2018

Fingerprint

Myosin Light Chains
Hypertrophic Cardiomyopathy
Myosins
Phosphorylation
Muscle
Mutation
Swine
Fibers
Actins
Light
Transgenic Mice
Myocardium
Tropomyosin
Troponin
Phenotype
Glutamine
Papillary Muscles
Aspartic Acid
Arginine
Muscle Contraction

Keywords

  • muscle contraction
  • myosin regulatory light chain
  • R58Q mutation
  • rescue of function
  • S15D-phosphorylation mimic
  • super-relaxed state

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Phosphomimetic-mediated in vitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain. / Yadav, Sunil; Kazmierczak, Katarzyna; Liang, Jingsheng; Sitbon, Yoel H.; Szczesna-Cordary, Danuta.

In: FEBS Journal, 01.01.2018.

Research output: Contribution to journalArticle

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abstract = "Myosin regulatory light chain (RLC) phosphorylation is important for cardiac muscle mechanics/function as well as for the Ca2+-troponin/tropomyosin regulation of muscle contraction. This study focuses on the arginine to glutamine (R58Q) substitution in the human ventricular RLC (MYL2 gene), linked to malignant hypertrophic cardiomyopathy in humans and causing severe functional abnormalities in transgenic (Tg) R58Q mice, including inhibition of cardiac RLC phosphorylation. Using a phosphomimic recombinant RLC variant where Ser-15 at the phosphorylation site was substituted with aspartic acid (S15D) and placed in the background of R58Q, we aimed to assess whether we could rescue/mitigate R58Q-induced structural/functional abnormalities in vitro. We show rescue of several R58Q-exerted adverse phenotypes in S15D-R58Q-reconstituted porcine cardiac muscle preparations. A low level of maximal isometric force observed for R58Q- versus WT-reconstituted fibers was restored by S15D-R58Q. Significant beneficial effects were also observed on the Vmax of actin-activated myosin ATPase activity in S15D-R58Q versus R58Q-reconstituted myosin, along with its binding to fluorescently labeled actin. We also report that R58Q promotes the OFF state of myosin, both in reconstituted porcine fibers and in Tg mouse papillary muscles, thereby stabilizing the super-relaxed state (SRX) of myosin, characterized by a very low ATP turnover rate. Experiments in S15D-R58Q-reconstituted porcine fibers showed a mild destabilization of the SRX state, suggesting an S15D-mediated shift in disordered-relaxed (DRX)↔SRX equilibrium toward the DRX state of myosin. Our study shows that S15D-phosphomimic can be used as a potential rescue strategy to abrogate/alleviate the RLC mutation-induced phenotypes and is a likely candidate for therapeutic intervention in HCM patients.",
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AU - Sitbon, Yoel H.

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