The genome is inherently unstable. It is estimated that ~ 105 DNA lesions occur every day in each mammalian cell, the vast majority of which are quickly repaired. Cell stress leads to the accumulation of DNA lesions, including double-stranded DNA breaks (DSBs). Unrepaired DSBs are deleterious, induce genomic instability, and result in the release of nuclear DNA (nDNA) into the cytosol. The latter activates the CyDNA-sensing proteins (CDSPs), involving the CGAS-STING1 pathway, which activates IRF3 and NFkB1, the transcriptional regulators of genes involved in inflammation, senescence, cell death, and fibrosis. In the last 3 decades, PI has focused on studying the molecular genetics and pathogenesis of hereditary cardiomyopathies, which are the genetic forms of cell stress and the prototypic forms of heart failure. Studies supported by the R01 award subject to this renewal, indicated activation of the CDSP and the DNA damage response (DDR) pathways in the heart samples from patients and mouse models of lamin (LMNA)-associated cardiomyopathy (LAC). The pathogenic role of the activation of these pathways was illustrated upon deletion of the Mb21d1 gene, encoding CGAS, which prolonged survival, improved cardiac function, reduced fibrosis, and mitigated cell death in a mouse model of LAC. Moreover, for the first time, to our knowledge, PI and colleagues have identified, by END-Seq, and partially characterized genome-wide DSBs at the nucleotide level in the mouse cardiac myocytes (CMs). The team has also defined the lamin-associated domains (LADs) in mouse CMs and shown that LMNA protects the genome against DSBs. In addition, the roles of chromatin remodeler CTCF, repair protein XRCC6, and topoisomerase TOP2B in the induction and repair of DSBs have been partially defined. We will test the hypothesis that transcription/replication stress (TRS) increases DSBs. Thus, we identify and characterize DSBs in CMs and cardiac fibroblasts (CFs) in two mouse models representing the human dominant- negative (LMNAD300N knock-in) and haploinsufficiency (inducible heterozygous Lmna deletion in CMs) mechanisms in LAC. We will define the roles of LADs and topographically associated domains (TADs), identified by the CTCF and SMC1A loops; TOP2B, and selected repair proteins in the DSBs generation and transcription (TRS). To test the hypothesis that LMNA regulates chromatin accessibility and hence, susceptibility to DSBs, chromatin accessibility will be assessed by ATAC-Seq, and susceptibility of the open chromatin regions (OCRs) to DSBs will be determined. Finally, the phenotypic effects of replenishing the depleted cellular NAD+ in LAC by administering -nicotinamide mononucleotide (NMN) and deleting the Sting1 gene to reduce NAD+ use in inflammation, will be determined in the LAC mice. The complementary approaches are expected to replenish the depleted NAD+, enhance repair protein PARylation, increase OCRs, and make the DSBs accessible to the repair proteins. The find...