Zheng Zequn, Cai Dihui, Fu Yin, Wang Ying, Song Yongfei, Lian Jiangfang
Department of Cardiovascular, Lihuili Hospital Facilitated to Ningbo University, Ningbo University, Ningbo, China.
Ningbo Institute of Innovation for Combined Medicine and Engineering, Ningbo, China.
Front Pharmacol. 2022 Jul 7;13:889713. doi: 10.3389/fphar.2022.889713. eCollection 2022.
Old drugs for new indications in the novel coronavirus disease of 2019 (COVID-19) pandemic have raised concerns regarding cardiotoxicity, especially the development of drug-induced QT prolongation. The acute blocking of the cardiac hERG potassium channel is conventionally thought to be the primary mechanism of QT prolongation induced by COVID-19 drugs fluvoxamine (FLV) and lopinavir (LPV). The chronic impact of these medications on the hERG expression has yet to be determined. To investigate the effect of long-term incubation of FLV and LPV on the hERG channel, we used electrophysiological assays and molecular experiments, such as Western blot, RT-qPCR, and immunofluorescence, in HEK-293 cells stably expressing hERG and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Compared to the acute effects, chronic incubation for FLV and LPV generated much lower half-maximal inhibitory concentration (IC50) values, along with a left-shifted activation curve and retarded channel activation. Inconsistent with the reduction in current, we unexpectedly found that the chronic effects of drugs promoted the maturation of hERG proteins, accompanied by the high expression of Hsp70 and low expression of Hsp90. Targeting Hsp70 using siRNA was able to reverse the effects of these drugs on hERG proteins. In addition, FLV and LPV resulted in a significant reduction of APD90 and triggered the early after-depolarizations (EADs), as well as inhibited the protein level of the L-type voltage-operated calcium channel (L-VOCC) in hiPSC-CMs. Chronic incubation with FLV and LPV produced more severe channel-blocking effects and contributed to altered channel gating and shortened action potential duration by inhibiting hERG and Cav1.2.
用于2019年新型冠状病毒病（COVID-19）大流行新适应症的老药引发了人们对心脏毒性的担忧，尤其是药物诱导的QT间期延长的发生。传统上认为，心脏hERG钾通道的急性阻断是COVID-19药物氟伏沙明（FLV）和洛匹那韦（LPV）诱导QT间期延长的主要机制。这些药物对hERG表达的长期影响尚未确定。为了研究FLV和LPV长期孵育对hERG通道的影响，我们在稳定表达hERG的HEK-293细胞和人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）中使用了电生理测定和分子实验，如蛋白质免疫印迹法、逆转录定量聚合酶链反应（RT-qPCR）和免疫荧光法。与急性效应相比，FLV和LPV的慢性孵育产生了低得多的半数最大抑制浓度（IC50）值，同时激活曲线左移且通道激活延迟。与电流降低不一致的是，我们意外地发现药物的慢性效应促进了hERG蛋白的成熟，同时伴有热休克蛋白70（Hsp70）的高表达和热休克蛋白90（Hsp90）的低表达。使用小干扰RNA（siRNA）靶向Hsp70能够逆转这些药物对hERG蛋白的影响。此外，FLV和LPV导致hiPSC-CMs中90%动作电位时程（APD90）显著缩短并引发早期后去极化（EADs），同时抑制L型电压门控钙通道（L-VOCC）的蛋白水平。FLV和LPV的慢性孵育产生了更严重的通道阻断效应，并通过抑制hERG和Cav1.2导致通道门控改变和动作电位时程缩短。
