An Ex Vivo Muscle Physiology Method for Robust Measurement of Supraspinatus Muscle Function in Mouse Models

Abstract The supraspinatus is the most frequently injured rotator cuff muscle, but its anatomical characteristics such as larger size, complex fiber architecture, and a single exposed tendon have limited the development of reproducible ex vivo contractility assays. In this study, we establish a robust method for ex vivo assessment of murine supraspinatus contractile function and characterize its physiological properties across age and injury conditions. We additionally adapt a barium chloride (BaCl2)–induced injury protocol for the supraspinatus, an approach not previously described, to evaluate how acute myofiber degeneration affects muscle performance. Male C57BL/6 mice (4 months) underwent 1.2% BaCl2 injection directly into the supraspinatus to induce controlled myofiber necrosis, allowing comparison of contractile behavior between injured and uninjured muscles. Using our injury ex vivo physiological testing protocol, we quantified optimal length (L0), twitch kinetics, force–frequency responses, peak tetanic force, and preliminary fatigue–recovery dynamics. Our protocol consistently generated fused tetanic contractions and reproducible force–frequency curves in the supraspinatus. We observed differences in supraspinatus contractility between young and old mice, consistent with well-established age-related changes in hindlimb muscle contractility. In addition, BaCl2 injury produced significant impairments in contractility 48 hours post-injection, demonstrating the sensitivity of this method to acute muscle damage. This study provides a novel and reliable method for evaluating the contractile function of the murine supraspinatus muscle ex vivo, overcoming previous anatomical challenges. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵* Baylah Mazonson and Hana Kalčo, co-first authors