In this funding period, we continued to compare human myobundles made from 3 dysferlin-deficient and 3 healthy hiPSC lines for their drug response, metabolic function, and transcriptomic differences. Previously, we found that LGMD2B myobundles display decreased contractile force generation, impaired membrane repair capacity, lipid droplet accumulation, and altered mitochondrial structure and function. Inhibiting calcium leakage from the sarcoplasmic reticulum with dantrolene or facilitating membrane repair with vamorolone restored contractile function, membrane repair capacity, and mitochondrial function, and significantly reduced lipid droplet accumulation in LGMD2B myobundles. Additionally, we found that inducing calcium leakage in healthy myobundles resulted in several disease signatures such as decreased force generation, lipid droplet accumulation, and mitochondrial dysfunction. Interestingly, membrane repair capacity was not compromised, suggesting that altered calcium homeostasis alone does not drive the LGMD2B phenotype. As our metabolomic studies confirmed that cholesterol esters accumulate within LGMD2B myobundles, we explored the effects of decreasing intracellular cholesterol biosynthesis with statins and reducing cholesterol esterification/storage with small molecule inhibitors. These cholesterol-lowering drugs improved LGMD2B contractile force and membrane repair capacity. Lastly, for the first time, we optimized cell culture conditions and injury protocols to induce intramuscular adipose tissue (IMAT) formation in tissue-engineered skeletal muscle. Our ongoing work in myobundles will further determine the role of dysferlin in regulating intramuscular cholesterol homeostasis and reveal mechanisms of IMAT formation and immune cell infiltration in LGMD2B. We anticipate that these studies will identify novel pharmacological interventions aimed at alleviating the pathology of dysferlin deficiency.