Ryanodine receptor‐associated myopathies: What's myosin got to do with it?

The ryanodine receptor (RyR) in skeletal muscle—the protein product of the RYR1 gene—is the gatekeeper for release of Ca2+ from where it is stored in the sarcoplasmic reticulum (SR) into the cytoplasm (Figure 1).1, 2 As such, it is a central element in excitation–contraction coupling (EC-coupling). Normal release of Ca2+ through RyR1 results in a transient elevation of cytoplasmic Ca2+ concentration that triggers contraction when Ca2+ binds to troponin on thin filaments, enabling actomyosin crossbridge cycling and muscle contraction.3 Genetic variants of RYR1 may affect Ca2+ release from the SR, thus modifying EC-coupling and skeletal muscle contraction.4 RYR1 variants are the most common cause of congenital myopathies with prevalence estimated to be at least 1:90 000.4, 5 Because there are a number of distinct RYR1 variants, clinical manifestations may be mild to severe; particularly problematic when respiratory muscles are affected. In this issue of Acta Physiologica, Sonne et al.6 report on results from an innovative study on skeletal muscle biopsies from five human patients with RYR1-related congenital (early onset) myopathies (RYR1-CM) compared with five controls (CTL) and five patients with RYR1-related, adult-onset rhabdomyolysis/myalgia (RYR1-RM). Sequencing identified one or two base changes in an RYR1 gene of each subject in the RYR1-CM and RYR1-RM groups; all patients carried distinct genetic variants. Female and male subjects were included in each group. Altered cytoplasmic Ca2+—at rest and/or during stimulation—associated with RYR1 variants, can affect force production by the myofilaments (Figure 1) and myriad other Ca2+-dependent processes. Sonne et al. asked whether specific changes in myosin occur in RYR1-CM patient muscles. First, they used mass spectroscopy to examine post-translational modifications (PTMs) of myosin heavy chain proteins (gene products of MYH2 and MYH7) with focus on phosphorylation (transfer of the terminal phosphate from ATP to serine, threonine or tyrosine sidechains) and acetylation (covalent addition of a 2-carbon acetyl group to a lysine sidechain). A well-known PTM of the myosin molecule is phosphorylation of myosin's light chain 2 (LC2), commonly referred to as the regulatory light chain (RLC), because it regulates contraction of vertebrate smooth muscle7 and modulates contraction of vertebrate striated muscles.8, 9 There is a growing catalog of PTMs of the larger myosin heavy chains (MHCs), but relatively few of these MHC PTMs have been studied in detail.10, 11 In MyHC type IIa (gene product of MYH2), Sonne et al. identified eight lysines that were acetylated in the CTL group but not in the RYR1-CM group, with two of those eight also missing in the RYR1-RM group. These eight lysines are distributed throughout the MYH2 polypeptide, with half in the motor domain and half in the rod of MHC. In β/slow MyHC (gene product of MYH7), two phosphorylations and one acetylation were found only in the RYR1-CM group, and a third phosphorylation that was found in both RYR1-CM and RYR1-RM groups but not CTL. These four PTMs are located in the rod portion of the MYH7 polypeptide that comprises the thick filament backbone, suggesting that some could influence the likelihood of the two motor domains of a myosin molecule forming the interacting heads motif (IHM)12-14 where myosin heads are sequestered onto the thick filament backbone (and could interact with the modified residues), and the disordered state where myosin heads have been released from the thick filament backbone and thus have a higher probability of forming crossbridges. Sonne et al. selected two of the phosphorylated residues in the MYH7 polypeptide (Ser1362 in RYR1-CM and Tyr1375 in both RYR1-CM and RYR1-RM) for closer examination using molecular dynamics (MD) simulations. MD has been widely used to examine molecular processes that are challenging, if not impossible to assess in muscle with current experimental approaches.11, 15-19 Dynamic simulations of portions of MYH7 rod suggest that phosphorylation could negatively impact structural dynamics of the rod—and by implication the thick filament backbone and interaction with IHM—in myopathy patients. IHM may be related to the biochemically defined super-relaxed state (SRX) that is associated with ultra slow hydrolysis of MgATP by myosin in the relaxed sarcomere.20, 21 Quantitation of the relative proportions of SRX and the faster disordered-relaxed state (DRX) has been widely used to examine energetics of relaxed muscle.20-23 Some of the RYR1-associated changes in PTMs that were identified by Sonne et al. could alter the relative proportions of SRX and DRX through altered interactions between the thick filament backbone and the interacting heads motif that is thought to be associated with the SRX state. Using muscle fiber preparations—that do not allow distinction among the individual PTMs—Sonne et al. showed that the proportions of SRX/DRX were the same in all three populations. Surprisingly, the fast phase of ATP turnover (reflecting the DRX state) was faster for fibers from the RYR1-CM samples despite the presence of more slow fibers containing β/slow MyHC (gene produce of MYH7), a conundrum that deserves further investigation. These results lead to the conclusion that RYR1 mutations have secondary, negative consequences on myosin structure and function that most likely contribute to the congenital myopathic pathophysiology. The authors propose an interesting hypothesis that changes in the intracellular environment resulting from altered Ca2+-handling by pathological RYR1 variants lead to differentially altered PTMs on molecules beyond RyR. These secondary effects on myosin in turn alter contractility in skeletal muscles and contribute to RYR1 pathology. The signal(s) that triggers changes in PTMs could be cytoplasmic Ca2+ (Figure 1),24, 25 but could also be associated with altered ATP hydrolysis and cellular energetics,26 and could additionally involve changes in force with corresponding effects on thick filament structure.22 The study by Sonne et al. provides a significant roadmap for wider exploration of genetic diseases beyond the molecule coded for by the affected gene. P. Bryant Chase: Conceptualization; supervision; writing – original draft; writing – review and editing. Arianna N. Coons: Visualization; writing – review and editing. The authors thank Maria Larsson for expert assistance with finalizing the artwork. The authors declare that there are no conflicts of interest.