Abstract
Abstract
Ischemia/reperfusion injury limits functional recovery after myocardial infarction through mitochondrial dysfunction, oxidative stress, energetic failure, impaired calcium handling, and contractile dysfunction. We previously identified the acetyltransferase TIP60/KAT5 as a maladaptive regulator of post-ischemic cardiac injury and showed that pharmacologic TIP60 inhibition with TH1834 mitigates myocardial infarction injury in mice. Here, we tested whether TH1834 promotes recovery in complementary 2D and 3D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) and cardioid (hiPSC-CO) models. Maturation-enhanced hiPSC-CMs subjected to hypoxia/reoxygenation (H/R) reproduced key features of ischemia/reperfusion injury, including mitochondrial loss, oxidative stress, cell death, impaired calcium handling, and contractile dysfunction. RNA sequencing further showed activation of stress programs and suppression of oxidative-metabolic pathways. At a non-arrhythmogenic dose, TH1834 inhibited TIP60 acetyltransferase activity, improved post-H/R survival and beating recovery, preserved mitochondrial and myofibrillar integrity, reduced reactive oxygen species, restored calcium transient kinetics, normalized sarcoplasmic reticulum calcium reserve, and improved contraction and relaxation. A central effect of TH1834 was enhancement of metabolic flexibility: following H/R, treatment restored mitochondrial respiratory capacity while simultaneously increasing glycolytic capacity and reserve. Because CM calcium cycling and mechanical work are highly dependent on energetic supply, this recovery of complementary oxidative and glycolytic capacity provides an energetic basis for improved calcium homeostasis and contractile recovery. Transcriptomic analysis supported this phenotype, showing preferential counter-regulation of H/R-induced stress programs and attenuation of persistent HIF1A-ARNT-PDK1 hypoxic metabolic signaling. In 3D hiPSC-COs, TH1834 similarly enhanced mitochondrial respiratory reserve and glycolytic capacity while improving calcium dynamics, contractile performance, beating recovery, and survival. Together, these findings identify enhanced metabolic flexibility as a central feature of TH1834-mediated protection against H/R, linking restoration of energetic capacity to preservation of calcium signaling and contractile function, supporting TIP60 as a therapeutic target for ischemic heart disease.