Evaluation of quantitative muscle MRI and an intelligent phenotyping housing system as advanced phenotyping methods in a mouse model of calpain 3‐deficient muscular dystrophy

Calpainopathy is a rare genetic myopathy without causal treatment available. Recent advances have produced promising treatment strategies, including genetic treatment and immunomodulation, that are currently being tested pre-clinically in murine models. Traditional behavioral assays frequently fail to detect early motor deficits in corresponding mouse models. Thus, this study investigated whether more sophisticated measures, such as quantitative magnetic resonance imaging (qMRI) and automated motor assessments can reveal early pathological changes in a mouse model of calpainopathy, particularly in the subclinical stages. In this study, Calpain 3-deficient mice (Capn3-deficient) and wild-type controls underwent a standardized qMRI protocol on a 7T scanner at 5 and 15 months of age. Fat fraction (FF), water T2 relaxation time (wT2), and diffusion metrics were analyzed across selected hindlimb muscles. Additionally, voluntary motor activity was monitored using an intelligent phenotyping housing system. Motor activity assays revealed minimal and inconsistent genotype effects. Sex significantly affected qMRI measures, altering fat distribution and wT2 at 5 and 15 months. At 5 months, no significant differences were detected between genotypes. At 15 months, Capn3-deficient mice exhibited elevated wT2 in the soleus and gastrocnemius muscles, correlating with endomysial immune cell infiltration. Diffusion parameters and FF remained unchanged. Taken together, the results show that muscle qMRI, particularly T2 mapping, can sensitively detect subclinical muscle changes in aging Capn3-deficient mice, pointing towards inflammatory processes. Pronounced sex differences and measurement variability underscore the need for larger, sex-balanced cohorts. Extending follow-up beyond 15 months will likely increase sensitivity for later-stage pathology.