TY - JOUR

T1 - Abnormal myosin post-translational modifications and ATP turnover time associated with human congenital myopathy-related RYR1 mutations

AU - Sonne, Alexander

AU - Antonovic, Anna Katarina

AU - Melhedegaard, Elise

AU - Akter, Fariha

AU - Andersen, Jesper L.

AU - Jungbluth, Heinz

AU - Witting, Nanna

AU - Vissing, John

AU - Zanoteli, Edmar

AU - Fornili, Arianna

AU - Ochala, Julien

N1 - Publisher Copyright: © 2023 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.

PY - 2023

Y1 - 2023

N2 - Aim: Conditions related to mutations in the gene encoding the skeletal muscle ryanodine receptor 1 (RYR1) are genetic muscle disorders and include congenital myopathies with permanent weakness, as well as episodic phenotypes such as rhabdomyolysis/myalgia. Although RYR1 dysfunction is the primary mechanism in RYR1-related disorders, other downstream pathogenic events are less well understood and may include a secondary remodeling of major contractile proteins. Hence, in the present study, we aimed to investigate whether congenital myopathy-related RYR1 mutations alter the regulation of the most abundant contractile protein, myosin. Methods: We used skeletal muscle tissues from five patients with RYR1-related congenital myopathy and compared those with five controls and five patients with RYR1-related rhabdomyolysis/myalgia. We then defined post-translational modifications on myosin heavy chains (MyHCs) using LC/MS. In parallel, we determined myosin relaxed states using Mant-ATP chase experiments and performed molecular dynamics (MD) simulations. Results: LC/MS revealed two additional phosphorylations (Thr1309-P and Ser1362-P) and one acetylation (Lys1410-Ac) on the β/slow MyHC of patients with congenital myopathy. This method also identified six acetylations that were lacking on MyHC type IIa of these patients (Lys35-Ac, Lys663-Ac, Lys763-Ac, Lys1171-Ac, Lys1360-Ac, and Lys1733-Ac). MD simulations suggest that modifying myosin Ser1362 impacts the protein structure and dynamics. Finally, Mant-ATP chase experiments showed a faster ATP turnover time of myosin heads in the disordered–relaxed conformation. Conclusions: Altogether, our results suggest that RYR1 mutations have secondary negative consequences on myosin structure and function, likely contributing to the congenital myopathic phenotype.

AB - Aim: Conditions related to mutations in the gene encoding the skeletal muscle ryanodine receptor 1 (RYR1) are genetic muscle disorders and include congenital myopathies with permanent weakness, as well as episodic phenotypes such as rhabdomyolysis/myalgia. Although RYR1 dysfunction is the primary mechanism in RYR1-related disorders, other downstream pathogenic events are less well understood and may include a secondary remodeling of major contractile proteins. Hence, in the present study, we aimed to investigate whether congenital myopathy-related RYR1 mutations alter the regulation of the most abundant contractile protein, myosin. Methods: We used skeletal muscle tissues from five patients with RYR1-related congenital myopathy and compared those with five controls and five patients with RYR1-related rhabdomyolysis/myalgia. We then defined post-translational modifications on myosin heavy chains (MyHCs) using LC/MS. In parallel, we determined myosin relaxed states using Mant-ATP chase experiments and performed molecular dynamics (MD) simulations. Results: LC/MS revealed two additional phosphorylations (Thr1309-P and Ser1362-P) and one acetylation (Lys1410-Ac) on the β/slow MyHC of patients with congenital myopathy. This method also identified six acetylations that were lacking on MyHC type IIa of these patients (Lys35-Ac, Lys663-Ac, Lys763-Ac, Lys1171-Ac, Lys1360-Ac, and Lys1733-Ac). MD simulations suggest that modifying myosin Ser1362 impacts the protein structure and dynamics. Finally, Mant-ATP chase experiments showed a faster ATP turnover time of myosin heads in the disordered–relaxed conformation. Conclusions: Altogether, our results suggest that RYR1 mutations have secondary negative consequences on myosin structure and function, likely contributing to the congenital myopathic phenotype.

KW - acetylation

KW - ATP

KW - congenital myopathy

KW - myosin heavy chain

KW - phosphorylation

KW - relaxed state

KW - skeletal muscle

U2 - 10.1111/apha.14035

DO - 10.1111/apha.14035

M3 - Journal article

C2 - 37602753

AN - SCOPUS:85168496361

VL - 239

JO - Acta Physiologica

JF - Acta Physiologica

SN - 1748-1708

IS - 2

M1 - e14035

ER -