Mitochondria play an essential role in cellular function and impact human health through varied mechanisms, including energy metabolism, cell signaling, and apoptosis. Many aging-related diseases have a mitochondrial (MT) component, with studies highlighting associations with cardiovascular disease, frailty, and overall mortality. In addition to inherited variation, mitochondria experience somatic mutations at much higher rates than the nuclear genome, introducing a state of heteroplasmy, defined as having more than 1 mtDNA allele within a cell. Using whole-genome sequence data from ~200,000 samples in the UK Biobank (UKB), we found that increased levels of MT heteroplasmy are associated with all-cause mortality, with individuals harboring a heteroplasmic nonsense mutation having a 1.7-fold increased risk of death, even after adjusting for the total number of heteroplasmies. These data indicate that the functional nature of the heteroplasmic mutation is a key driver of increased mortality risk. We propose to directly test the impact of heteroplasmic nonsense mutations across a range of variant allele fractions (VAF) on MT function through base editing of the MT genome followed by a comprehensive suite of MT functional assays. Given the strong link between primary MT disorders and cardiac function, we further propose to test the impact of these variants on cardiomyocyte electrical activity using human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs). In the ~200,000 UKB participants, we have identified 47 ultra-rare heteroplasmic nonsense single base-pair changes. Nineteen of the variants are targetable by existing MT base editing technologies. We propose to optimize constructs to target all 19 bases in HEK293T cell lines, which have served as the model system for MT genome editing, and then select up to 2 constructs per gene for downstream functional analyses in additional cell lines. To functionally characterize heteroplasmic nonsense mutations, we will first expand the range of VAF through a combination of single-cell expansion and the use of mitoTALENs and test the hypothesis of a dose-response relationship between VAF and MT function. We will then use a series of high-throughput assays to comprehensively characterize the mutant cell lines by assessing MT function (e.g., cellular respiration, glycolytic flux) and quantity (mtDNA-CN, nucleoid density, mass). We will then use hiPSC-CMs to test whether deleterious heteroplasmic variants across a range of VAFs modify electrical excitability, repolarization, or conduction, or compromise the development and maturation of cardiomyocytes. The increasing recognition of the role of MT heteroplasmic mutation in aging-related disease has created an urgent need to functionally characterize these mutations. Indeed, while this proposal focuses on heteroplasmic nonsense mutations, we have already identified ~1000 ultra-rare heteroplasmic missense and non-coding mutations at highly constrained sites th...