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.

目前的证据表明，冠状动脉微循环是防止心脏缺血再灌注 (I/R) 损伤的关键目标。线粒体钙单向转运体 (MCU) 复合物激活和线粒体钙 ([Ca ²⁺ ] _m ) 超载是心血管疾病的潜在机制。据报道，组氨酸三联体核苷酸结合 2 (HINT2) 通过 MCU 复合物调节 [Ca ²⁺ ] _m，我们之前的工作表明，HINT2 可改善心肌细胞存活率并保持心肌缺血小鼠的心脏功能。本研究旨在探讨 HINT2 在 I/R 损伤中对心脏微循环的益处，重点关注线粒体、MCU 复合物和 [Ca ²⁺ ] _m内皮细胞超载。目前的工作表明，HINT2 过表达显着减少了 I/R 损伤小鼠心脏的无复流区域并改善了微血管灌注，这可能是通过促进内皮一氧化氮合酶 (eNOS) 表达和磷酸化来实现的。微血管屏障功能因再灌注损伤而受损，但通过抑制 VE-Cadherin 在 Tyr ^{731处的磷酸化而通过 HINT2 过表达修复}并增强 VE-Cadherin/β-Catenin 的相互作用。此外，HINT2 过表达通过抑制血管细胞粘附分子-1 (VCAM-1) 和细胞间粘附分子-1 (ICAM-1) 来抑制炎症反应。线粒体裂变发生在心脏微血管内皮细胞 (CMEC) 中，受到氧 - 葡萄糖剥夺/复氧 (OGD/R) 损伤并导致线粒体功能障碍和线粒体依赖性细胞凋亡，其影响在很大程度上因 HINT2 过表达而得到缓解。其他实验证实 [Ca ²⁺ ] _m 超载是线粒体裂变的起始因素，HINT2 抑制 [Ca ²⁺ ] _m通过直接与 CMEC 中的 MCU 交互来调制 MCU 复合体来实现过载。通过 MCU 激动剂精胺恢复 [Ca ²⁺ ] _m过载，消除了 HINT2 对 OGD/R 损伤的 CMEC 和 I/R 损伤的心脏微循环的所有保护作用。总之，本报告表明，HINT2 过表达抑制 MCU 复合体-线粒体钙超载-线粒体分裂和细胞凋亡通路，从而减轻心脏微血管缺血再灌注损伤。