Characterization of CARM1 Inhibition in Skeletal Muscle

Coactivator‐associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of arginine residues of target proteins critical for health and disease. CARM1 has recently emerged as an important regulator of skeletal muscle biology. The purpose of this study was to further investigate CARM1 biology in skeletal muscle. For our first experiment, we utilized 3‐month‐old male and female mice (n = 3/sex) that were housed and cared for in accordance with Canadian Council for Animal Care guidelines. Mice were treated with either a CARM1 inhibitor (150 mg/kg EZM2302; Epizyme, Inc.) or vehicle via oral gavage BID for 2, 4, or 8 days. Tissues were collected 6 hours following the final dose and muscle mass was recorded. Western blotting was performed to evaluate protein expression in the tibialis anterior (TA) muscle and liver. To assess CARM1 activity, we investigated the arginine methylation status (i.e., the methylated form of the protein relative to its total amount) of known CARM1 substrates BAF155 and PABP1, as well as the myocellular level of arginine methylated proteins preferentially marked by CARM1. We observed similar outcomes between males and females, so the data were collapsed by sex (n = 6). CARM1 inhibition significantly reduced BAF155 methylation status in the TA muscle by 70‐80% between 2‐8 days of treatment. Muscle PABP1 methylation status was also attenuated (p < 0.05) by 60‐65% after 4 and 8 days of exposure. Similarly, arginine methylation of CARM1‐specific substrates across all muscle proteins was reduced by ~50% (p < 0.05) between 2‐8 days of CARM1 inhibitor administration. The attenuation of BAF155 and PABP1 methylation status, as well as arginine methylated CARM1‐specific substrates, was greater in liver as compared to muscle. Next, we completed additional experiments to assess the effect of pharmacological CARM1 inhibition on skeletal muscle function. After 11 days of CARM1 inhibitor treatment, we observed a sex‐specific reduction (p < 0.05) in exercise capacity and muscular endurance such that muscle function in female mice was unaffected by CARM1 suppression. Collectively, these results suggest that global, pharmacological CARM1 inhibition preferentially impacts liver enzyme activity over muscle. Furthermore, CARM1 inhibitor treatment may affect muscle performance with males being more sensitive to the functional outcomes of CARM1 suppression than females. Future work in our laboratory will examine the mechanisms and functional consequences of sex‐based differences in CARM1 biology within skeletal muscle.