This study investigates the mechanisms by which calcium (Ca²⁺) and reactive oxygen species (ROS) individually and synergistically induce mitochondrial dysfunction during myocardial ischemia/reperfusion (I/R) injury. Using isolated mitochondrial assays and in vivo I/R surgery in mice, the researchers found that ROS does not directly trigger the mitochondrial permeability transition pore (mPTP) but instead induces mitochondrial dysfunction through lipid peroxidation (LIPOX). Subtoxic levels of ROS and Ca²⁺ synergistically induce mPTP-dependent mitochondrial swelling. This synergistic effect is independent of cyclophilin D (CypD), suggesting a CypD-independent mechanism for ROS sensitization of the mPTP. Dual inhibition of the mPTP and LIPOX with cyclosporine A (CsA) and MitoQ, respectively, significantly reduces infarct size in mice subjected to I/R injury, indicating that targeting both pathways is an effective therapeutic approach to mitigate I/R injury. The findings highlight the importance of understanding the distinct roles of Ca²⁺ and ROS in mitochondrial dysfunction and suggest that dual inhibition of these pathways may be a promising strategy for reducing cardiac damage following ischemic events.This study investigates the mechanisms by which calcium (Ca²⁺) and reactive oxygen species (ROS) individually and synergistically induce mitochondrial dysfunction during myocardial ischemia/reperfusion (I/R) injury. Using isolated mitochondrial assays and in vivo I/R surgery in mice, the researchers found that ROS does not directly trigger the mitochondrial permeability transition pore (mPTP) but instead induces mitochondrial dysfunction through lipid peroxidation (LIPOX). Subtoxic levels of ROS and Ca²⁺ synergistically induce mPTP-dependent mitochondrial swelling. This synergistic effect is independent of cyclophilin D (CypD), suggesting a CypD-independent mechanism for ROS sensitization of the mPTP. Dual inhibition of the mPTP and LIPOX with cyclosporine A (CsA) and MitoQ, respectively, significantly reduces infarct size in mice subjected to I/R injury, indicating that targeting both pathways is an effective therapeutic approach to mitigate I/R injury. The findings highlight the importance of understanding the distinct roles of Ca²⁺ and ROS in mitochondrial dysfunction and suggest that dual inhibition of these pathways may be a promising strategy for reducing cardiac damage following ischemic events.