Department of Medicine (H.A.P., A.V.G., J.F.M., O.S., D.K.-A., M.P.M., T.K.), University of Cambridge, United Kingdom.
MRC Mitochondrial Biology Unit (H.A.P., A.V.G., G.B., J.R., M.M.P.), University of Cambridge, United Kingdom.
Circ Res. 2022 Sep 2;131(6):528-541. doi: 10.1161/CIRCRESAHA.121.320717. Epub 2022 Aug 12.
Inhibiting SDH (succinate dehydrogenase), with the competitive inhibitor malonate, has shown promise in ameliorating ischemia/reperfusion injury. However, key for translation to the clinic is understanding the mechanism of malonate entry into cells to enable inhibition of SDH, its mitochondrial target, as malonate itself poorly permeates cellular membranes. The possibility of malonate selectively entering the at-risk heart tissue on reperfusion, however, remains unexplored.
C57BL/6J mice, C2C12 and H9c2 myoblasts, and HeLa cells were used to elucidate the mechanism of selective malonate uptake into the ischemic heart upon reperfusion. Cells were treated with malonate while varying pH or together with transport inhibitors. Mouse hearts were either perfused ex vivo (Langendorff) or subjected to in vivo left anterior descending coronary artery ligation as models of ischemia/reperfusion injury. Succinate and malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS, in vivo by mass spectrometry imaging, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining.
Malonate was robustly protective against cardiac ischemia/reperfusion injury, but only if administered at reperfusion and not when infused before ischemia. The extent of malonate uptake into the heart was proportional to the duration of ischemia. Malonate entry into cardiomyocytes in vivo and in vitro was dramatically increased at the low pH (≈6.5) associated with ischemia. This increased uptake of malonate was blocked by selective inhibition of MCT1 (monocarboxylate transporter 1). Reperfusion of the ischemic heart region with malonate led to selective SDH inhibition in the at-risk region. Acid-formulation greatly enhances the cardioprotective potency of malonate.
Cardioprotection by malonate is dependent on its entry into cardiomyocytes. This is facilitated by the local decrease in pH that occurs during ischemia, leading to its selective uptake upon reperfusion into the at-risk tissue, via MCT1. Thus, malonate's preferential uptake in reperfused tissue means it is an at-risk tissue-selective drug that protects against cardiac ischemia/reperfusion injury.
抑制 SDH（琥珀酸脱氢酶），使用竞争抑制剂丙二酸，可以改善缺血/再灌注损伤。然而，关键在于理解丙二酸进入细胞的机制，以抑制其线粒体靶标 SDH，因为丙二酸本身很难穿透细胞膜。然而，丙二酸是否有可能在再灌注时选择性地进入易损心脏组织仍然未知。
使用 C57BL/6J 小鼠、C2C12 和 H9c2 成肌细胞和 HeLa 细胞来阐明丙二酸在再灌注时选择性进入缺血心脏的机制。用丙二酸处理细胞，同时改变 pH 值或与转运抑制剂一起处理。将小鼠心脏离体（Langendorff）灌流或在体内进行左前降支冠状动脉结扎，作为缺血/再灌注损伤的模型。通过液相色谱-串联质谱法（LC-MS/MS）、体内质谱成像和 TTC（2,3,5-三苯基-2H-四唑氯化物）染色评估琥珀酸和丙二酸的水平。
丙二酸对心脏缺血/再灌注损伤具有很强的保护作用，但仅在再灌注时给予，而不是在缺血前给予。丙二酸进入心脏的程度与缺血时间成正比。在与缺血相关的低 pH 值（≈6.5）下，丙二酸在体内和体外进入心肌细胞的程度显著增加。这种丙二酸的摄取增加被选择性抑制 MCT1（单羧酸转运蛋白 1）所阻断。缺血心脏区域的再灌注导致易损区域的 SDH 选择性抑制。酸制剂极大地增强了丙二酸的心脏保护效力。
丙二酸的心脏保护作用依赖于其进入心肌细胞。这是由缺血时发生的局部 pH 值下降促进的，导致其在再灌注时通过 MCT1 选择性进入易损组织。因此，丙二酸在再灌注组织中的优先摄取意味着它是一种易损组织选择性药物，可预防心脏缺血/再灌注损伤。
