A human two-hit platform modeling post-ischemic sterile inflammation and diastolic dysfunction in hiPSC-derived cardiac models.

Myocardial ischemia/reperfusion (MI/R) injury exposes the heart to a sequential surge of oxidative stress and sterile inflammation, driving maladaptive remodeling and lethal arrhythmias. However, the human-specific mechanisms linking post-ischemic inflammation to electromechanical dysfunction remain poorly defined. Here, we established a human Two-Hit model using human umbilical vein endothelial cells (HUVECs), human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and three-dimensional heart organoids (hHOs). This system integrates acute oxidative priming with the secretome generated by macrophages undergoing secondary necrosis (SN-Sec), thereby recapitulating a redox-active sterile inflammatory microenvironment relevant to the post-ischemic heart. The Two-Hit stress elicited striking cell-type-specific responses. HUVECs exhibited NF-κB activation, exhaustion of inducible antioxidant defenses, and irreversible cell death. In contrast, hiPSC-CMs survived oxidative and inflammatory stress but underwent maladaptive remodeling characterized by stress-induced cytoskeletal remodeling and disruption of gap junction integrity. Mechanistically, sterile inflammation was accompanied by a critical imbalance in calcium handling through selective accumulation of phospholamban (PLN) without a concomitant reduction in SERCA2a expression. The consequent stoichiometric shift markedly correlated with calcium decay kinetics and induced diastolic dysfunction. In hHOs, high-speed optical mapping revealed profound electromechanical discordance, where preserved electrical automaticity became uncoupled from delayed calcium cycling, creating a highly arrhythmogenic substrate. Collectively, these findings identify the ROS-sterile inflammation-PLN axis as a prominent molecular feature correlated with post-ischemic diastolic failure and arrhythmogenesis in human cardiac models. This Two-Hit platform provides a robust framework for dissecting inflammation-driven cardiac remodeling and for evaluating therapeutic strategies targeting post-ischemic electromechanical dysfunction.