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 have found that LGMD2B myobundles display decreased contractile force generation, impaired membrane repair capacity, lipid droplet accumulation, and altered mitochondrial structure and function. As cholesterol esters accumulate within LGMD2B myobundles, we explored the functional consequences of inducing cholesterol accumulation in healthy myobundles by inhibiting lysosomal cholesterol export with the small molecule U18666A. Increased cholesterol accumulation in healthy myobundles resulted in no changes in force generation but significantly impaired membrane repair capacity and increased lipid droplet accumulation. To explore the role of cholesterol trafficking, we acutely decreased or increased plasma membrane (PM) cholesterol with methyl beta cyclodextrin (MβCD) or MβCD-cholesterol, respectively. Decreasing PM cholesterol levels impaired membrane repair capacity in healthy but had no effect in LGMD2B myobundles. In contrast, increasing PM cholesterol levels increased membrane repair capacity in LGMD2B myobundles to healthy levels. Together, this suggests that dysferlin deficiency impairs PM cholesterol trafficking which subsequently impairs membrane repair, without impacting force generation. During this funding period we also explored the role of dysferlin in macrophage function by generating hiPSC-derived macrophages (iMPs) from 3 healthy and 3 LGMD2B lines. Our findings suggest that dysferlin deficiency in IMPs alters secretion of cytokines related to muscle regeneration, immune cell chemoattraction, and chemotaxis, as well as impairs endocytosis and phagocytosis which are required for clearance of damaged muscle, all of which may contribute to LGMD2B progression. In the next funding period, we will continue to explore the roles of dysferlin in regulating intramuscular cholesterol homeostasis, macrophage function and metabolism, and function and stress response of engineered cardiac muscle (“cardiobundles”). We anticipate these studies will help identify novel disease mechanisms and pharmacological interventions in dysferlinopathy.