Abnormal myosin post‐translational modifications and ATP turnover time associated with human congenital myopathy‐related RYR1 mutations
Alexander Sonne, Anna Katarina Antonovic, Elise Melhedegaard, Fariha Akter, Jesper L. Andersen, Heinz Jungbluth, Nanna Witting, John Vissing, Edmar Zanoteli, Arianna Fornili, Julien Ochala - Physiology
Abstract
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.