Horváth Csaba, Ravingerová Tanya, Suleiman M Saadeh, Adameová Adriana
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, 83232 Bratislava, Slovak Republic.
Centre of Experimental Medicine, Institute for Heart Research, Slovak Academy of Sciences, 81438 Bratislava, Slovak Republic.
Rev Cardiovasc Med. 2022 Jun 9;23(6):213. doi: 10.31083/j.rcm2306213. eCollection 2022 Jun.
The role of cardiac autophagy during ischemia and reperfusion (I/R) remains controversial. Furthermore, whether this cell death during I/R is also interconnected with other cell damaging event, such as necroptosis, is insufficiently known. Thus, the aim of this study was to investigate possible links between autophagy and necroptosis in the hearts under conditions of acute I/R injury.
Langendorff-perfused male Wistar rat hearts were subjected to 30-min global ischemia followed by 10-min reperfusion in the presence of either vehicle or a drug inhibiting the pro-necroptotic receptor-interacting protein kinase 3 (RIP3). Hemodynamic parameters and lactate dehydrogenase (LDH) release were measured to assess heart function and non-specific cell death due to the disruption of plasma membrane.
Immunoblot analysis of left ventricles revealed that early reperfusion suppressed the activation of autophagy as evidenced by the decreased protein expression of Beclin-1, pSer555-ULK1, pSer555-ULK1/ULK1 ratio, and LC3-II/LC3-I ratio. On the other hand, the molecular signalling responsible for autophagy inhibition did not appear to be affected in these I/R settings. RIP3 inhibition during reperfusion significantly mitigated the loss of the plasma membrane integrity but did not improve cardiac function. This pharmacological intervention targeting necroptosis-mediating protein decreased LC3-II expression in I/R hearts, suggesting some effect on autophagosome processing, but it did not significantly alter other signalling pathways involved in autophagy activation or inhibition.
In summary, we showed for the first time that an early reperfusion phase does not promote autophagy and that there may be an interplay between pro-necroptotic protein RIP3 and autophagy with respect to the regulation of autophagosome processing.
心脏自噬在缺血再灌注（I/R）过程中的作用仍存在争议。此外，I/R期间的这种细胞死亡是否也与其他细胞损伤事件（如坏死性凋亡）相互关联，目前尚不清楚。因此，本研究的目的是探讨急性I/R损伤条件下心脏自噬与坏死性凋亡之间的可能联系。
采用Langendorff灌注法，对雄性Wistar大鼠心脏进行30分钟全心缺血，随后在存在溶剂或抑制促坏死性凋亡受体相互作用蛋白激酶3（RIP3）的药物的情况下进行10分钟再灌注。测量血流动力学参数和乳酸脱氢酶（LDH）释放，以评估心脏功能和由于质膜破坏导致的非特异性细胞死亡。
左心室的免疫印迹分析显示，早期再灌注抑制了自噬的激活，这可通过Beclin-1、pSer555-ULK1的蛋白表达降低、pSer555-ULK1/ULK1比值以及LC3-II/LC3-I比值降低来证明。另一方面，在这些I/R环境中，负责自噬抑制的分子信号似乎未受影响。再灌注期间RIP3抑制显著减轻了质膜完整性的丧失，但并未改善心脏功能。这种针对坏死性凋亡介导蛋白的药物干预降低了I/R心脏中LC3-II的表达，表明对自噬体加工有一定影响，但并未显著改变参与自噬激活或抑制的其他信号通路。
总之，我们首次表明早期再灌注阶段不会促进自噬，并且在自噬体加工的调节方面，促坏死性凋亡蛋白RIP3与自噬之间可能存在相互作用。
