PITX2 Deficiency Leads to Atrial Mitochondrial Dysfunction

Aims Reduced left atrial PITX2 is associated with atrial cardiomyopathy and atrial fibrillation (AF). PITX2 is restricted to left atrial cardiomyocytes (aCMs) in the adult heart. The links between PITX2 deficiency, atrial cardiomyopathy, and AF are not fully understood.Methods and results To identify mechanisms linking PITX2 deficiency to AF, we generated and characterized PITX2-deficient human aCMs derived from human induced pluripotent stem cells (hiPSC) and their controls. PITX2-deficient hiPSC-derived atrial cardiomyocytes showed shorter and disorganized sarcomeres and increased mononucleation. Electron microscopy found an increased number of smaller mitochondria compared with isogenic controls. Mitochondrial protein expression was altered in PITX2-deficient hiPSC-derived atrial cardiomyocytes. Single-nuclear RNA-sequencing found differences in cellular respiration pathways and differentially expressed mitochondrial and ion channel genes in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2 repression in hiPSC-derived atrial cardiomyocytes replicated dysregulation of cellular respiration. Mitochondrial respiration was shifted to increased glycolysis in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2-deficient human hiPSC-derived atrial cardiomyocytes showed higher spontaneous beating rates. Action potential duration was more variable with an overall prolongation of early repolarization, consistent with metabolic defects. Gene expression analyses confirmed changes in mitochondrial genes in left atria from 42 patients with AF compared with 43 patients with sinus rhythm. Dysregulation of left atrial mitochondrial (COX7C) and metabolic (FOXO1) genes was associated with PITX2 expression in human left atria.Conclusion PITX2 deficiency causes atrial mitochondrial dysfunction and a metabolic shift to glycolysis in human aCMs. PITX2-dependent metabolic changes can contribute to the structural and functional defects found in PITX2-deficient atria. Graphical Abstract Deficiency in PITX2, a gene with left atrial and skeletal muscle expression in adults leads to mitochondrial dysfunction. PITX2 deficiency is likely to underlie the genomic basis for atrial fibrillation (AF). Reduced PITX2 in atrial cardiomyocytes (aCMs) conveys electrical changes and structural alterations. The cellular mechanisms linking PITX2 deficiency to AF are not fully understood. PITX2 deficiency increases cellular and functional heterogeneity in human iPSC-derived aCMs. These experiments show that PITX2 alters mitochondrial function and metabolism by altering gene and protein expression in aCMs, creating a metabolic shift away from respiration towards glycolysis. Left atrial tissue from patients with AF shows similar changes in gene expression patterns of mitochondrial genes and their association with PITX2. Figure was generated using BioRender.com.