MAPK signaling consists of an array of successively acting kinases. ERK1 and -2 (ERK1/2) are major components of the greater MAPK cascade that transduce growth factor signaling at the cell membrane. Here, we investigated ERK1/2 signaling in skeletal muscle homeostasis and disease. Using mouse genetics, we observed that the muscle-specific expression of a constitutively active MEK1 mutant promotes greater ERK1/2 signaling that mediates fiber-type switching to a slow, oxidative phenotype with type I myosin heavy chain expression. Using a conditional and temporally regulated Cre strategy, as well as Mapk1 (ERK2) and Mapk3 (ERK1) genetically targeted mice, MEK1-ERK2 signaling was shown to underlie this fast-to-slow fiber-type switching in adult skeletal muscle as well as during development. Physiologic assessment of these activated MEK1-ERK1/2 mice showed enhanced metabolic activity and oxygen consumption with greater muscle fatigue resistance. In addition, induction of MEK1-ERK1/2 signaling increased dystrophin and utrophin protein expression in a mouse model of limb-girdle muscle dystrophy and protected myofibers from damage. In summary, sustained MEK1-ERK1/2 activity in skeletal muscle produces a fast-to-slow fiber-type switch that protects from muscular dystrophy, suggesting a therapeutic approach to enhance the metabolic effectiveness of muscle and protect from dystrophic disease.

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2^−/−) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2^−/− mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2^−/− mice. Small-angle X-ray diffraction of C2^−/− EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2^−/− EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca^2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2^−/− mice. Finally, C2^−/− muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle. Significance Myosin-binding protein-C (MyBP-C) is a thick filament regulatory protein found exclusively in the C-zone of the A-band in the sarcomeres of vertebrate striated muscle. Cardiac, slow skeletal, and fast skeletal MyBP-C (fMyBP-C) paralogs perform different functions. All three paralogs share similar protein structures but likely differ substantially in terms of expression and function, which may serve the distinct physiologies of fast and slow muscle fibers. However, the functional role of fMyBP-C in fast skeletal muscle is completely unknown. Genetic mutations in human fMyBP-C lead to skeletal myopathies. In this study, we used knockout mice to define the molecular basis for fMyBP-C paralog diversity. We demonstrate that fMyBP-C modulates the speed and force of fast skeletal muscle contraction.

There is a growing awareness that both thick-filament and classical thin-filament regulations play central roles in modulating muscle contraction. Myosin ATPase assays have demonstrated that under relaxed conditions, myosin may reside either in a high-energy-consuming disordered-relaxed (DRX) state available for binding actin to generate force or in an energy-sparing super-relaxed (SRX) state unavailable for actin binding. X-ray diffraction studies have shown that the majority of myosin heads are in a quasi-helically ordered OFF state in a resting muscle and that this helical ordering is lost when myosin heads are turned ON for contraction. It has been assumed that myosin heads in SRX and DRX states are equivalent to the OFF and ON states, respectively, and the terms have been used interchangeably. In this study, we use X-ray diffraction and ATP turnover assays to track the structural and biochemical transitions of myosin heads, respectively, induced with either omecamtiv mecarbil (OM) or piperine in relaxed porcine myocardium. We find that while OM and piperine induce dramatic shifts of myosin heads from the OFF to the ON state, there are no appreciable changes in the population of myosin heads in the SRX and DRX states in both unloaded and loaded preparations. Our results show that biochemically defined SRX and DRX can be decoupled from structurally defined OFF and ON states. In summary, while SRX/DRX and OFF/ON transitions can be correlated in some cases, these two phenomena are measured using different approaches, reflect different properties of the thick filament, and should be investigated and interpreted separately.

Muscle progenitor cell fusion is required for the formation and regeneration of multinucleated skeletal muscle fibers. Chronic muscle regeneration in Duchenne muscular dystrophy (DMD) is characterized by ongoing fusion of satellite cell (SC) progeny, but the effects of fusion on disease and the mechanisms by which fusion is accomplished in this setting are not fully understood. Using the mdx mouse model of DMD, we deleted the fusogenic protein Myomaker in SCs or myofibers. Following deletion in SCs, mice displayed a complete lack of myocyte fusion, resulting in severe muscle loss, enhanced fibrosis, and significant functional decline. Reduction of Myomaker in mature myofibers in mdx mice, however, led to minimal alterations in fusion dynamics. Unexpectedly, myofiber-specific deletion of Myomaker resulted in improvement of disease phenotype, with enhanced function and decreased muscle damage. Our data indicate that Myomaker has divergent effects on dystrophic disease severity depending upon its compartment of expression. These findings show that myocyte fusion is absolutely required for effective regeneration in DMD, but persistent Myomaker expression in myofibers due to ongoing fusion may have unintended deleterious consequences for muscle integrity. Thus, sustained activation of a component of the myogenic program in dystrophic myofibers exacerbates disease.

In this study, we investigated the role of DNA methylation [5-methylcytosine (5mC)] and 5-hydroxymethylcytosine (5hmC), epigenetic modifications that regulate gene activity, in dilated cardiomyopathy (DCM). A MYBPC3 mutant mouse model of DCM was compared with wild type and used to profile genomic 5mC and 5hmC changes by Chip-seq, and gene expression levels were analyzed by RNA-seq. Both 5mC-altered genes (957) and 5hmC-altered genes (2,022) were identified in DCM hearts. Diverse gene ontology and KEGG pathways were enriched for DCM phenotypes, such as inflammation, tissue fibrosis, cell death, cardiac remodeling, cardiomyocyte growth, and differentiation, as well as sarcomere structure. Hierarchical clustering of mapped genes affected by 5mC and 5hmC clearly differentiated DCM from wild-type phenotype. Based on these data, we propose that genomewide 5mC and 5hmC contents may play a major role in DCM pathogenesis. NEW & NOTEWORTHY Our data demonstrate that development of dilated cardiomyopathy in mice is associated with significant epigenetic changes, specifically in intronic regions, which, when combined with gene expression profiling data, highlight key signaling pathways involved in pathological cardiac remodeling and heart contractile dysfunction.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca ²⁺ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca ²⁺ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament’s relaxed state resulting in an increased number of crossbridges during Ca ²⁺ activation. Additionally, in the low Ca ²⁺ -relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca ²⁺ , and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.