Mitochondria are essential organelles in eukaryotic cells that play a crucial role in energy production and cellular respiration. Mitochondrial function is indispensable for skeletal muscle (SkM) regeneration as it maintains the regenerative capacity of muscle stem cells (MuSCs). SkM regeneration involves a cascade of myogenic events controlled by a subset of adult MuSCs. Upon activation, MuSCs undergo proliferation and differentiation, ultimately giving rise to myotubes, which serve as precursors for mature muscle fibers. Although mitochondrial dysfunction has been associated with hindered SkM regeneration, the regulatory mechanisms responsible for this remain uncharacterized. This has led to a growing interest in understanding the pathways linking mitochondrial dysfunction and impaired MuSC regenerative capacity. Oligomycin, an inhibitor of ATP synthesis, compromises the proton gradient and mitochondrial membrane integrity. Chapter 2 demonstrates that the administration of oligomycin results in the release of mitochondrial DNA (mtDNA) into the cytosol, consequently triggering a cGAS/STING-mediated immune response within murine myoblast C2C12 cells. The folate-dependent enzyme SHMT2 plays a critical role in mitochondrial one-carbon metabolism (FOCM). FOCM is essential for various cellular processes, including formate biosynthesis for cytosolic/nuclear FOCM, mitochondrial dTMP production, and intermediates required for mitochondrial protein translation. Research on the Shmt2 gene and its correlation with mtDNA stability and mitochondrial function has attracted significant scientific attention. It has been reported that Shmt2 heterozygosity results in uracil incorporation into mtDNA, leading to mitochondrial dysfunction without affecting overall mtDNA mass. However, the effects of Shmt2 heterozygosity on other aspects of mitochondrial biology remain poorly understood. Chapter 3 demonstrates that Shmt2 heterozygosity-induced disruption of mitochondrial FOCM leads to cytosolic mtDNA leakage, which activates apoptosis. Therefore, this dissertation fills a gap in knowledge regarding disruptions of mitochondrial metabolism, inflammation, and apoptosis by examining the role of mtDNA leakage into the cytosol in mouse myoblast C2C12 cells and Shmt2 heterozygous mouse embryonic fibroblast cells, respectively.