Project Grant F30HL189131

MITOCHONDRIAL COMPLEX IV DEFICIENCIES IN HUMAN CARDIOMYOCYTES - MITOCHONDRIAL DISEASES ARE CAUSED BY DISRUPTIONS IN OXIDATIVE PHOSPHORYLATION (OXPHOS), THE PRIMARY MEANS OF GENERATING ENERGY (ATP) TO FUEL CELLULAR ACTIVITIES. NO THERAPIES OR CURES ARE APPROVED FOR THE TREATMENT OF MITOCHONDRIAL DISEASES. OXPHOS COMPLEX IV DEFICIENCY IS ONE OF THE MOST COMMON MITOCHONDRIAL DISEASES, CAUSING SEVERE CARDIOMYOPATHIES WITH STRIKING CLINICAL HETEROGENEITY - SOME PATIENTS PRESENT WITH FATAL INFANTILE CARDIOMYOPATHY WHILE OTHERS DEVELOP ADULT-ONSET HEART FAILURE. THE PHENOTYPIC HETEROGENEITY OF COMPLEX IV DEFICIENCY REMAINS MECHANISTICALLY UNEXPLAINED, LIMITING PROGNOSTIC ACCURACY AND THERAPEUTIC DEVELOPMENT. I HYPOTHESIZE THAT THE CLINICAL HETEROGENEITY OF COMPLEX IV DEFICIENCIES IS DETERMINED BY THE ASSEMBLY STAGE IN WHICH THE AFFECTED SUBUNIT IS INCORPORATED INTO COMPLEX IV. COMPLEX IV IS THE RATE-LIMITING ENZYME OF OXPHOS AND IS ASSEMBLED FROM 13 PROTEIN SUBUNITS, RANGING FROM EARLYSTAGE ASSEMBLY SUBUNITS THAT FORM CORE INTERMEDIATE STRUCTURES TO LATE-STAGE ASSEMBLY SUBUNITS INCORPORATED LATER FOR FUNCTIONAL OPTIMIZATION. DIFFERENT COMPLEX IV PATHOGENIC VARIANTS DISRUPT COMPLEX IV ASSEMBLY AT DISTINCT STAGES, LIKELY RESULTING IN VARIABLE RESIDUAL COMPLEX IV ACTIVITY AND POSSIBLY EXPLAINING CLINICAL HETEROGENEITY. I HYPOTHESIZE THAT SUPPRESSION OF EARLY-STAGE ASSEMBLY SUBUNITS OF COMPLEX IV DISRUPTS COMPLEX IV ASSEMBLY, CAUSING SEVERE METABOLIC DYSFUNCTION AND CONTRACTILE IMPAIRMENTS IN CARDIOMYOCYTES. MOREOVER, I HYPOTHESIZE THAT SUPPRESSION OF LATE-STAGE ASSEMBLY SUBUNITS OF COMPLEX IV RESULTS IN INTACT COMPLEX IV ASSEMBLY AND MILDER CARDIOMYOCYTE DYSFUNCTION. TO TEST MY HYPOTHESES, I WILL USE ANTISENSE OLIGONUCLEOTIDES TO ACHIEVE ~50% SUPPRESSION OF INDIVIDUAL NUCLEAR-ENCODED COMPLEX IV SUBUNITS (2 EARLY-STAGE, 2 LATE-STAGE SUBUNITS) IN HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES (HIPSC-CMS, N=4 VALIDATED LINES) TO MODEL THE HETEROZYGOUS INHERITANCE PATTERN OF SUBUNIT-SPECIFIC COMPLEX IV DEFICIENCIES. USING THIS MODEL, I WILL COMPREHENSIVELY DEFINE SUBUNIT-SPECIFIC METABOLIC DYSFUNCTION FOR MY FOUR TARGETS (AIM 1). I WILL MEASURE COMPLEX IV ACTIVITY AND INTERMEDIATE ASSEMBLY LEVELS TO ASSESS COMPLEX IV DEFECTS. TO ASSESS GENERAL METABOLIC DYSFUNCTION, I WILL QUANTIFY MITOCHONDRIAL RESPIRATION, GLYCOLYSIS, MITOCHONDRIAL MEMBRANE POTENTIAL, OXIDANT PRODUCTION AND METABOLITES. IN AIM 2, I WILL DETERMINE SUBUNIT-SPECIFIC EFFECTS ON CARDIOMYOCYTE PHYSIOLOGY AND GENE EXPRESSION PROFILES. THROUGH MY STUDY, I WILL CONDUCT THE FIRST INVESTIGATION OF INDIVIDUAL COMPLEX IV SUBUNITS IN HUMAN CARDIOMYOCYTES TO OFFER MECHANISTIC INSIGHTS INTO THE CLINICAL HETEROGENEITY OF CARDIOMYOPATHIES DUE TO COMPLEX IV DEFICIENCY. IMPORTANTLY, I WILL GAIN RIGOROUS TRAINING IN MITOCHONDRIAL BIOLOGY, HIPSC-CM MODELS, ADVANCED MITOCHONDRIAL AND CARDIOMYOCYTE PHYSIOLOGY MEASURES, AND GENE REGULATION. THESE EXPERIENCES WILL HELP DEVELOP MY SCIENTIFIC REASONING, TECHNICAL EXPERTISE, AND TRANSLATIONAL RESEARCH SKILLS, ADVANCING MY LONGTERM GOAL OF BECOMING AN INDEPENDENTLY FUNDED PHYSICIAN-SCIENTIST DEDICATED TO TREATING AND INVESTIGATING CARDIOVASCULAR DISEASES.