Abstract Similar to skeletal muscle myofibers, cardiomyocytes in the heart appear to be particularly susceptible to membrane instability and rupture during disease, in part because of their contractile status that produces ongoing mechanical deformation. Mutations in genes that disrupt or weaken the membrane anchoring proteins of the dystrophin-glycoprotein complex (DGC) or the integrin adhesion network causes a wide range of muscular dystrophies that also cause cardiomyopathy. We have shown that the thrombospondin gene family (Thbs1-5) plays a critical role in membrane stability through both effects on the ER stress response and secretory pathways, as well as controlling the integrin complexes present on the sarcolemma. In our previous cycle of funding, we showed that overexpression of Thbs3 has a remarkable effect of reducing sarcolemma stability in heart (opposite of Thbs4) by removing large arrays of integrin heterodimers from the adhesion complexes, while Thbs3 KO mice have enhanced integrin membrane levels and are protected from insults that would otherwise cause cardiomyopathy, like overexpression of Thbs4 that also increases membrane stability by increasing membrane attachment protein complexes in the sarcolemma. However, several critical mechanistic questions remain to be addressed in attempting to translate our findings into therapeutic approaches. Here we propose the unifying hypothesis that the cardiac expressed Thbs proteins primarily function from within the secretory pathway in mediating the stability, or recycling of membrane attachment complexes, which has profound effects on sarcolemmal stability and healing dynamics within the heart with both acute injury and chronic disease, which will suggest potential novel gene therapeutic ventures for cardiomyopathy in heart failure or with muscular dystrophy. To interrogate this hypothesis, we propose the following 2 Specific Aims: 1) Mechanistically define how Thbs3 and Thbs4 antithetically regulate sarcolemmal stability through integrin processing for gene therapy application in cardiomyopathy and muscular dystrophy. 2) Examine the molecular mechanism of dilated cardiomyopathy in a mouse model for the Human THBS4 D717N variant. The goal will be to better understand human heart disease through the Thbs gene family and how it regulates membrane stability and adhesion complex activity. Translational implications are that a better understanding of these molecular mechanisms will suggest why humans with mutations in Thbs4 show dilated cardiomyopathy and will suggest modified versions of Thbs4 to be used in gene therapy approaches to treat muscular dystrophy, as well as a wide array of heart diseases in which the membrane is weaker.