The return of oxygen during reperfusion generates ROS, primarily by mitochondria (10, 11). Damage to the ETC during ischemia leads to increased ROS production on reperfusion. Complex I and complex III are the primary sites of ROS production in the mitochondria (11, 160, 202), but other sites can also contribute (203). The increase in succinate that occurs during ischemia can lead to RET through generation of ROS by complex I. Extracellular pH is rapidly restored, which promotes extrusion of intracellular H⁺ via NHE, leading to a transient increase in intracellular Na⁺. As ATP is restored, the Na⁺-K⁺ ATPase becomes active and helps to extrude intracellular Na⁺. Depending on the relative timing of ATP restoration, a sustained increase in cytosolic Na⁺ can stimulate NCX, leading to a further increase in cytosolic Ca²⁺ during early reperfusion. ROS can also lead to damage of intracellular proteins such as SERCA and RyR2, leading to altered SR Ca²⁺ homeostasis. Together, these can lead to greater Ca²⁺ accumulation in the cytosol and exacerbate reperfusion injury. Any increase in cytosolic Ca²⁺ present at the start of reperfusion would lead to an increase in mitochondrial Ca²⁺ accumulation via MCU when the Δψ is restored on reperfusion (204). This further increase in mitochondrial Ca²⁺ on reperfusion depends on how fast Δψ is restored relative to how quickly cytosolic Ca²⁺ returns to baseline. Ca²⁺ overload in the mitochondria is thought to prime the mPTP to open on reperfusion when pH is restored. It is widely cited that mPTP activation is inhibited by the acidic pH induced by ischemia (205, 206) and that upon reperfusion, intracellular and extracellular pH are rapidly corrected, allowing for mPTP opening. However, inhibition of mPTP by acidic pH only occurs in de-energized mitochondria. In energized mitochondria, low pH actually enhances mPTP opening (207). ROS is another activator of mPTP, and it is likely that the increase in ROS that occurs during reperfusion synergizes with the increase in mitochondrial Ca²⁺ (which may already be there during ischemia) to activate mPTP on reperfusion.