Sex-Specific Effect of CARM1 in Skeletal Muscle Adaptations to Exercise.

PURPOSE: The purpose of this study was to determine how the intersection of coactivator-associated arginine methyltransferase 1 (CARM1) and biological sex affects skeletal muscle adaptations to chronic physical activity. METHODS: Twelve-week-old female (F) and male (M) wild-type (WT) and CARM1 skeletal muscle-specific knockout (mKO) mice were randomly assigned to sedentary (SED) or voluntary wheel running (VWR) experimental groups. For 8 wk, the animals in the VWR cohort had volitional access to running wheels. Subsequently, we performed whole-body functional tests, and 48 h later muscles were harvested for molecular analysis. Western blotting, enzyme activity assays, as well as confocal and transmission electron microscopy were used to examine skeletal muscle biology. RESULTS: Our data reveal a sex-dependent reduction in VWR volume caused by muscle-specific ablation of CARM1, as F CARM1 mKO mice performed less chronic, volitional exercise than their WT counterparts. Regardless of VWR output, exercise-induced adaptations in physiological function were similar between experimental groups. A broad panel of protein arginine methyltransferase (PRMT) biology measurements, including markers of arginine methyltransferase expression and activity, were unaffected by VWR, except for CARM1 and PRMT7 protein levels, which decreased and increased with VWR, respectively. Changes in myofiber morphology and mitochondrial protein content showed similar trends among animals. However, a closer examination of transmission electron microscopy images revealed contrasting responses to VWR in CARM1 mKO mice compared with WT littermates, particularly in mitochondrial size and fractional area. CONCLUSIONS: The present findings demonstrate that CARM1 mKO reduces daily running volume in F mice, as well as exercise-evoked skeletal muscle mitochondrial plasticity, which indicates that this enzyme plays an essential role in sex-dependent differences in exercise performance and mitochondrial health.

CARM1 drives mitophagy and autophagy flux during fasting-induced skeletal muscle atrophy.

CARM1 (coactivator associated arginine methyltransferase 1) has recently emerged as a powerful regulator of skeletal muscle biology. However, the molecular mechanisms by which the methyltransferase remodels muscle remain to be fully understood. In this study, carm1 skeletal muscle-specific knockout (mKO) mice exhibited lower muscle mass with dysregulated macroautophagic/autophagic and atrophic signaling, including depressed AMP-activated protein kinase (AMPK) site-specific phosphorylation of ULK1 (unc-51 like autophagy activating kinase 1; Ser555) and FOXO3 (forkhead box O3; Ser588), as well as MTOR (mechanistic target of rapamycin kinase)-induced inhibition of ULK1 (Ser757), along with AKT/protein kinase B site-specific suppression of FOXO1 (Ser256) and FOXO3 (Ser253). In addition to lower mitophagy and autophagy flux in skeletal muscle, carm1 mKO led to increased mitochondrial PRKN/parkin accumulation, which suggests that CARM1 is required for basal mitochondrial turnover and autophagic clearance. carm1 deletion also elicited PPARGC1A (PPARG coactivator 1 alpha) activity and a slower, more oxidative muscle phenotype. As such, these carm1 mKO-evoked adaptations disrupted mitophagy and autophagy induction during food deprivation and collectively served to mitigate fasting-induced muscle atrophy. Furthermore, at the threshold of muscle atrophy during food deprivation experiments in humans, skeletal muscle CARM1 activity decreased similarly to our observations in mice, and was accompanied by site-specific activation of ULK1 (Ser757), highlighting the translational impact of the methyltransferase in human skeletal muscle. Taken together, our results indicate that CARM1 governs mitophagic, autophagic, and atrophic processes fundamental to the maintenance and remodeling of muscle mass. Targeting the enzyme may provide new therapeutic approaches for mitigating skeletal muscle atrophy.

Impact of short-term, pharmacological CARM1 inhibition on skeletal muscle mass, function, and atrophy in mice.

Coactivator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of arginine residues on target proteins critical for health and disease. The purpose of this study was to characterize the effects of short-term, pharmacological CARM1 inhibition on skeletal muscle size, function, and atrophy. Adult mice (n = 10 or 11/sex) were treated with either a CARM1 inhibitor (150 mg/kg EZM2302; EZM) or vehicle (Veh) via oral gavage for 11-13 days and muscle mass, function, and exercise capacity were assessed. In addition, we investigated the effect of CARM1 suppression on unilateral hindlimb denervation (DEN)-induced muscle atrophy (n = 8/sex). We report that CARM1 inhibition caused significant reductions in the asymmetric dimethylation of known CARM1 substrates but no change in CARM1 protein or mRNA content in skeletal muscle. Reduced CARM1 activity did not affect body or muscle mass, however, we observed a decrease in exercise capacity and muscular endurance in male mice. CARM1 methyltransferase activity increased in the muscle of Veh-treated mice following 7 days of DEN, and this response was blunted in EZM-dosed mice. Skeletal muscle mass and myofiber cross-sectional area were significantly reduced in DEN compared with contralateral, non-DEN limbs to a similar degree in both treatment groups. Furthermore, skeletal muscle atrophy and autophagy gene expression programs were elevated in response to DEN independent of CARM1 suppression. Collectively, these results suggest that short-term, pharmacological CARM1 inhibition in adult animals affects muscle performance in a sex-specific manner but does not impact the maintenance and remodeling of skeletal muscle mass during conditions of neurogenic muscle atrophy.NEW & NOTEWORTHY Short-term pharmacological inhibition of coactivator-associated arginine methyltransferase 1 (CARM1) was effective at significantly reducing CARM1 methyltransferase function in skeletal muscle. CARM1 inhibition did not impact muscle mass, but exercise capacity was impaired, particularly in male mice, whereas morphological and molecular signatures of denervation-induced muscle atrophy were largely maintained in animals administered the inhibitor. Altogether, the role of CARM1 in neuromuscular biology remains complex and requires further investigation of its therapeutic potential in muscle-wasting conditions.

The CARM1 transcriptome and arginine methylproteome mediate skeletal muscle integrative biology.

OBJECTIVE: Coactivator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of arginine residues on target proteins to regulate critical processes in health and disease. A mechanistic understanding of the role(s) of CARM1 in skeletal muscle biology is only gradually emerging. The purpose of this study was to elucidate the function of CARM1 in regulating the maintenance and plasticity of skeletal muscle. METHODS: We used transcriptomic, methylproteomic, molecular, functional, and integrative physiological approaches to determine the specific impact of CARM1 in muscle homeostasis. RESULTS: Our data defines the occurrence of arginine methylation in skeletal muscle and demonstrates that this mark occurs on par with phosphorylation and ubiquitination. CARM1 skeletal muscle-specific knockout (mKO) mice displayed altered transcriptomic and arginine methylproteomic signatures with molecular and functional outcomes confirming remodeled skeletal muscle contractile and neuromuscular junction characteristics, which presaged decreased exercise tolerance. Moreover, CARM1 regulates AMPK-PGC-1α signalling during acute conditions of activity-induced muscle plasticity. CONCLUSIONS: This study uncovers the broad impact of CARM1 in the maintenance and remodelling of skeletal muscle biology.

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

Coactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

The emergence of protein arginine methyltransferases in skeletal muscle and metabolic disease.

Protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the methylation of arginine residues on target proteins and thus alter the stability, localization, or activity of the substrate. In doing so, PRMTs mediate a variety of intracellular functions that are essential for survival. Additionally, PRMT dysregulation is involved in a number of the most prevalent health disorders, including cancer and neurodegenerative and cardiovascular diseases, as well as in the aging process. Investigations of PRMT biology in skeletal muscle cells began in 2002, and since then these enzymes have emerged as regulators of skeletal muscle phenotype determination, maintenance, and remodeling. Specifically, more recent in vivo studies have revealed that PRMTs impact multiple aspects of skeletal muscle biology, including satellite cell function and phenotypic plasticity in response to exercise and disuse. Skeletal muscle plays critically important roles in regulating whole body metabolism, and recent investigations have also begun elucidating PRMT expression and function under conditions of metabolic dysfunction. The goals of this review are to 1) summarize the literature on PRMT biology in skeletal muscle with a particular emphasis on the in vivo evidence and 2) survey PRMTs in metabolic disorders, namely, obesity and type 2 diabetes mellitus. We also identify notable knowledge gaps therein and present opportunities to further expand our understanding of these enzymes so critical to health and disease.

Protein arginine methyltransferase biology in humans during acute and chronic skeletal muscle plasticity.

Protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the methylation of arginine residues on target proteins. While dysregulation of PRMTs has been documented in a number of the most prevalent diseases, our understanding of PRMT biology in human skeletal muscle is limited. This study served to address this knowledge gap by exploring PRMT expression and function in human skeletal muscle in vivo and characterizing PRMT biology in response to acute and chronic stimuli for muscle plasticity. Fourteen untrained, healthy men performed one session of sprint interval exercise (SIE) before completing four bouts of SIE per week for 6 wk as part of a sprint interval training (SIT) program. Throughout this time course, multiple muscle biopsies were collected. We found that at basal, resting conditions PRMT1, PRMT4, PRMT5, and PRMT7 were the most abundantly expressed PRMT mRNAs in human quadriceps muscle. Additionally, the broad subcellular distribution pattern of PRMTs suggests methyltransferase activity throughout human myofibers. A spectrum of PRMT-specific inductions, and decrements, in expression and activity were observed in response to acute and chronic cues for muscle plasticity. In conclusion, our findings demonstrate that PRMTs are present and active in human skeletal muscle in vivo and that there are distinct, enzyme-specific responses and adaptations in PRMT biology to acute and chronic stimuli for muscle plasticity. This work advances our understanding of this critical family of enzymes in humans.NEW & NOTEWORTHY This is the first report of protein arginine methyltransferase (PRMT) biology in human skeletal muscle in vivo. We observed that PRMT1, -4, -5, and -7 were the most abundant PRMT mRNAs in human muscle and that PRMT proteins exhibited a broad subcellular localization that included myonuclear, cytosolic, and sarcolemmal compartments. Acute exercise and chronic training evoked PRMT-specific alterations in expression and activity. This study reveals a hitherto unknown complexity to PRMT biology in human muscle.

Exercise-induced Protein Arginine Methyltransferase Expression in Skeletal Muscle.

PURPOSE: This study aimed to determine protein arginine methyltransferase 1 (PRMT1), -4 (also known as coactivator-associated arginine methyltransferase 1 [CARM1]), and -5 expression and function during acute, exercise-induced skeletal muscle remodeling in vivo. METHODS: C57BL/6 mice were assigned to one of three experimental groups: sedentary, acute bout of exercise, or acute exercise followed by 3 h of recovery. Mice in the exercise groups performed a single bout of treadmill running at 15 m·min for 90 min. Hindlimb muscles were collected, and quantitative real-time polymerase chain reaction and Western blotting were used to examine exercise-induced gene expression. RESULTS: The PRMT gene expression and global enzyme activity were muscle-specific, generally being higher (P < 0.05) in slow, oxidative muscle, as compared with faster, more glycolytic tissue. Despite the significant activation of canonical exercise-induced signaling involving AMP-activated protein kinase and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), PRMT expression and activity at the whole muscle level were unchanged. However, subcellular analyses revealed a significant exercise-evoked myonuclear translocation of PRMT1 before the nuclear accumulation of PGC-1α. Acute physical activity also augmented (P < 0.05) the targeted methyltransferase activities of the PRMT in the myonuclear compartment, suggesting that PRMT-mediated histone arginine methylation is part of the early signals that drive muscle plasticity. Finally, basal PGC-1α asymmetric dimethylarginine status, as well as constitutive interactions between PGC-1α and PRMT1 or CARM1 may contribute to the exercise-induced muscle remodeling process. CONCLUSIONS: The present study provides the first evidence that PRMT activity is selectively augmented during the initial activation of exercise-induced skeletal muscle remodeling in vivo. These data support the emergence of PRMTs as important players in the regulation of skeletal muscle plasticity.

Regulation of Skeletal Muscle Plasticity by Protein Arginine Methyltransferases and Their Potential Roles in Neuromuscular Disorders.

Protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the methylation of arginine residues on target proteins, thereby mediating a diverse set of intracellular functions that are indispensable for survival. Indeed, full-body knockouts of specific PRMTs are lethal and PRMT dysregulation has been implicated in the most prevalent chronic disorders, such as cancers and cardiovascular disease (CVD). PRMTs are now emerging as important mediators of skeletal muscle phenotype and plasticity. Since their first description in muscle in 2002, a number of studies employing wide varieties of experimental models support the hypothesis that PRMTs regulate multiple aspects of skeletal muscle biology, including development and regeneration, glucose metabolism, as well as oxidative metabolism. Furthermore, investigations in non-muscle cell types strongly suggest that proteins, such as peroxisome proliferator-activated receptor-γ coactivator-1α, E2F transcription factor 1, receptor interacting protein 140, and the tumor suppressor protein p53, are putative downstream targets of PRMTs that regulate muscle phenotype determination and remodeling. Recent studies demonstrating that PRMT function is dysregulated in Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS) suggests that altering PRMT expression and/or activity may have therapeutic value for neuromuscular disorders (NMDs). This review summarizes our understanding of PRMT biology in skeletal muscle, and identifies uncharted areas that warrant further investigation in this rapidly expanding field of research.