Project Summary Mutations in nuclear envelope proteins (NEPs) cause devastating genetic diseases, known as envelopathies, which primarily affect the heart and skeletal muscle. The LEM domain nuclear envelope protein 2 (LEMD2) is a ubiquitously expressed inner nuclear membrane protein. In vitro studies reported that LEMD2 interacts with DNA-binding proteins, implicating LEMD2 in genome regulation and chromatin organization processes. Importantly, a missense mutation in the LEMD2 coding sequence causes severe cardiomyopathy in humans. In a recent study, we reported that “humanized” mice carrying the Lemd2 c.T38>G human mutation as well as cardiac-specific Lemd2 KO (cKO) mice develop dilated cardiomyopathy (DCM) and die prematurely due to heart failure. Moreover, Lemd2- deficient cardiomyocytes (CMs) display high levels of DNA damage. Despite its association with cardiac phenotypes in both human and mice, the role of LEMD2 in the mammalian heart and the pathological mechanisms responsible for its association with cardiac disease are far from being understood. Based on my preliminary findings, I hypothesize that high levels of DNA damage in LEMD2-mutant CMs cause the development of cardiomyopathy. To test this hypothesis, I will first perform a comprehensive characterization of the DNA damage and DNA damage response (DDR) in both mice and human CMs carrying LEMD2 mutations. The second aim is focused on the mechanistic link between LEMD2 loss-of-function, DNA damage and cardiomyopathy. I will determine if LEMD2 interacts with the chromatin-binding protein BAF, and whether the LEMD2 c.T38>G mutation disrupts this interaction. I will also detect nuclear envelope ruptures, cytosolic DNA leakage, and the activation of the pro-inflammatory pathway cGAS in LEMD2-mutant CMs. The third aim will determine the therapeutic potential of LEMD2 gene therapy for envelopathies characterized by the presence of DNA damage. I will overexpress LEMD2 in CMs and mice carrying mutations in the lamin A gene and assess the extent of rescue at the structural and functional levels. By accomplishing the objectives of this proposal, we will reveal important mechanistic insights regarding LEMD2 functions and its associated cardiomyopathy. With this new knowledge, we hope to ultimately design new therapeutic strategies and preventive methods for genetic cardiomyopathies.