Pinacidil ameliorates cardiac microvascular ischemia–reperfusion injury by inhibiting chaperone-mediated autophagy of calreticulin

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2024年1月2日

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Muyin Liu,
Su Li,
Ming Yin,
Youran Li,
Jinxiang Chen,
Yuqiong Chen,
You Zhou,
Qiyu Li,
Fei Xu,
Chunfeng Dai,
Yan Xia,
Ao Chen,
Danbo Lu,
Zhangwei Chen,
Juying Qian,
Junbo Ge

摘要

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.