Current evidence indicates that coronary microcirculation is a key target for protecting against cardiac ischemia–reperfusion (I/R) injury. Mitochondrial calcium uniporter (MCU) complex activation and mitochondrial calcium ([Ca2+]m) overload are underlying mechanisms involved in cardiovascular disease. Histidine triad nucleotide-binding 2 (HINT2) has been reported to modulate [Ca2+]m via the MCU complex, and our previous work demonstrated that HINT2 improved cardiomyocyte survival and preserved heart function in mice with cardiac ischemia. This study aimed to explore the benefits of HINT2 on cardiac microcirculation in I/R injury with a focus on mitochondria, the MCU complex, and [Ca2+]m overload in endothelial cells. The present work demonstrated that HINT2 overexpression significantly reduced the no-reflow area and improved microvascular perfusion in I/R-injured mouse hearts, potentially by promoting endothelial nitric oxide synthase (eNOS) expression and phosphorylation. Microvascular barrier function was compromised by reperfusion injury, but was repaired by HINT2 overexpression via inhibiting VE-Cadherin phosphorylation at Tyr731 and enhancing the VE-Cadherin/β-Catenin interaction. In addition, HINT2 overexpression inhibited the inflammatory response by suppressing vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Mitochondrial fission occurred in cardiac microvascular endothelial cells (CMECs) subjected to oxygen–glucose deprivation/reoxygenation (OGD/R) injury and resulted in mitochondrial dysfunction and mitochondrion-dependent apoptosis, the effects of which were largely relieved by HINT2 overexpression. Additional experiments confirmed that [Ca2+]m overload was an initiating factor for mitochondrial fission and that HINT2 suppressed [Ca2+]m overload via modulation of the MCU complex through directly interacting with MCU in CMECs. Regaining [Ca2+]m overload by spermine, an MCU agonist, abolished all the protective effects of HINT2 on OGD/R-injured CMECs and I/R-injured cardiac microcirculation. In conclusion, the present report demonstrated that HINT2 overexpression inhibited MCU complex-mitochondrial calcium overload-mitochondrial fission and apoptosis pathway, and thereby attenuated cardiac microvascular ischemia–reperfusion injury.

Aim To explore the beneficial effects of L‐carnitine on cardiac microvascular dysfunction in diabetic cardiomyopathy from the perspectives of mitophagy and mitochondrial integrity. Methods Male db/db and db/m mice were randomly assigned to groups and were treated with L‐carnitine or a solvent for 24 weeks. Endothelium‐specific PARL overexpression was attained via adeno‐associated virus serotype 9 (AAV9) transfection. Adenovirus (ADV) vectors overexpressing wild‐type CPT1a, mutant CPT1a, or PARL were transfected into endothelial cells exposed to high glucose and free fatty acid (HG/FFA) injury. Cardiac microvascular function, mitophagy, and mitochondrial function were analyzed by immunofluorescence and transmission electron microscopy. Protein expression and interactions were assessed by western blotting and immunoprecipitation. Results L‐carnitine treatment enhanced microvascular perfusion, reinforced endothelial barrier function, repressed the endothelial inflammatory response, and maintained the microvascular structure in db/db mice. Further results demonstrated that PINK1‐Parkin‐dependent mitophagy was suppressed in endothelial cells suffering from diabetic injury, and these effects were largely alleviated by L‐carnitine through the inhibition of PARL detachment from PHB2. Moreover, CPT1a modulated the PHB2‐PARL interaction by directly binding to PHB2. The increase in CPT1a activity induced by L‐carnitine or amino acid mutation (M593S) enhanced the PHB2‐PARL interaction, thereby improving mitophagy and mitochondrial function. In contrast, PARL overexpression inhibited mitophagy and abolished all the beneficial effects of L‐carnitine on mitochondrial integrity and cardiac microvascular function. Conclusion L‐carnitine treatment enhanced PINK1‐Parkin‐dependent mitophagy by maintaining the PHB2‐PARL interaction via CPT1a, thereby reversing mitochondrial dysfunction and cardiac microvascular injury in diabetic cardiomyopathy.