Mutations in transmembrane protein dysferlin lead to LGMDR2/2B, a progressive muscle disease with no available treatment. Studies in mouse models of disease have been limited by altered lipid profiles, cellular interactions, and mild functional deficit. We therefore developed an in vitro 3D tissue-engineered human skeletal muscle model (“myobundle”) of LGMDR2/2B using hiPSCs from patients’ and healthy (HLT) donors. Despite normal muscle mass and structure, LGMDR2/2B myobundles exhibited reduced contractile force, impaired calcium handling, and defective membrane repair. These deficits were accompanied by mitochondrial dysfunction evident from reduced mitochondrial potential, decreased oxygen consumption, and LGMDR2/2B-specific lipid droplet (LD) accumulation. Similar to mouse studies. dantrolene or vamorolone treatment successfully restored LGMDR2/2B myobundle contractility, calcium handling, and membrane repair while reducing LDs, supporting the human myobundle model as a therapeutic testing platform.
Further investigations revealed altered cholesterol homeostasis, including increased cholesterol ester accumulation and perturbed HMGCR/SREBP2/LDLR feedback in LGMDR2/2B versus HLT myobundles. Inhibiting cholesterol biosynthesis, via pravastatin or pharmacological HMGCR degradation, improved force generation, membrane repair, mitochondrial respiration, and reduced LDs in LGMDR2/2B myobundles, without affecting HLT myobundles. To determine if altered cholesterol compartmentalization contributes to impaired membrane repair in LGMDR2/2B, we manipulated plasma membrane (PM) cholesterol levels. Decreasing PM cholesterol with methyl-beta-cyclodextrin (MβCD) impaired membrane repair in HLT but not LGMDR2/2B myobundles, while increasing PM cholesterol with MBCD-cholesterol specifically improved membrane repair in LGMDR2/2B tissues. Collectively, these studies highlighted PM cholesterol content as a critical determinant of muscle membrane repair capacity and cholesterol modulation as a therapeutic target for LGMDR2/2B.
The human myobundle model provides a valuable platform for studying dysferlin function in skeletal muscle and offers a foundation for developing targeted therapies for LGMDR2/2B. Future studies will explore dysferlin’s regulatory roles in: 1) LDLR homeostasis, and 2) mevalonate pathway intermediates, with subsequent validation in mouse models.