Calcium overload is the key trigger in cardiac microvascular ischemia–reperfusion (I/R) injury, and calreticulin (CRT) is a calcium buffering protein located in the endoplasmic reticulum (ER). Additionally, the role of pinacidil, an antihypertensive drug, in protecting cardiac microcirculation against I/R injury has not been investigated. Hence, this study aimed to explore the benefits of pinacidil on cardiac microvascular I/R injury with a focus on endothelial calcium homeostasis and CRT signaling. Cardiac vascular perfusion and no-reflow area were assessed using FITC–lectin perfusion assay and Thioflavin-S staining. Endothelial calcium homeostasis, CRT–IP3Rs–MCU signaling expression, and apoptosis were assessed by real-time calcium signal reporter GCaMP8, western blotting, and fluorescence staining. Drug affinity-responsive target stability (DARTS) assay was adopted to detect proteins that directly bind to pinacidil. The present study found pinacidil treatment improved capillary density and perfusion, reduced no-reflow and infraction areas, and improved cardiac function and hemodynamics after I/R injury. These benefits were attributed to the ability of pinacidil to alleviate calcium overload and mitochondria-dependent apoptosis in cardiac microvascular endothelial cells (CMECs). Moreover, the DARTS assay showed that pinacidil directly binds to HSP90, through which it inhibits chaperone-mediated autophagy (CMA) degradation of CRT. CRT overexpression inhibited IP3Rs and MCU expression, reduced mitochondrial calcium inflow and mitochondrial injury, and suppressed endothelial apoptosis. Importantly, endothelial-specific overexpression of CRT shared similar benefits with pinacidil on cardiovascular protection against I/R injury. In conclusion, our data indicate that pinacidil attenuated microvascular I/R injury potentially through improving CRT degradation and endothelial calcium overload.

钙超载是心脏微血管缺血再灌注（I/R）损伤的关键触发因素，而钙网蛋白（CRT）是一种位于内质网（ER）中的钙缓冲蛋白。此外，吡那地尔（一种抗高血压药物）在保护心脏微循环免受缺血再灌注损伤方面的作用尚未得到研究。因此，本研究旨在探讨吡那地尔对心脏微血管缺血再灌注损伤的益处，重点关注内皮钙稳态和 CRT 信号传导。使用 FITC-凝集素灌注测定和硫磺素-S 染色评估心血管灌注和无复流面积。通过实时钙信号报告器 GCaMP8、蛋白质印迹和荧光染色评估内皮钙稳态、CRT-IP3Rs-MCU 信号表达和细胞凋亡。采用药物亲和响应靶稳定性（DARTS）测定法来检测直接与吡那地尔结合的蛋白质。本研究发现吡那地尔治疗可改善毛细血管密度和灌注，减少无复流和梗死面积，并改善 I/R 损伤后的心脏功能和血流动力学。这些益处归因于吡那地尔能够减轻心脏微血管内皮细胞（CMEC）中的钙超载和线粒体依赖性细胞凋亡。此外，DARTS 测定表明，吡那地尔直接与 HSP90 结合，通过 HSP90 抑制 CRT 分子伴侣介导的自噬 (CMA) 降解。 CRT 过表达抑制 IP3R 和 MCU 表达，减少线粒体钙流入和线粒体损伤，并抑制内皮细胞凋亡。重要的是，CRT 的内皮特异性过度表达与吡那地尔在预防心血管损伤方面具有相似的益处。总之，我们的数据表明吡那地尔可能通过改善 CRT 降解和内皮钙超载来减轻微血管 I/R 损伤。