Abstract
Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4–6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.
概要
目的
观察组蛋白脱乙酰化酶 3(HDAC3) 抑制剂 RGFP966 是否能够促进低温保存心脏复灌期心功能的恢复, 并探讨 Hippo-YAP 信号通路是否参与了 RGFP966 的心肌保护作用.
创新点
首次证实了在 Celsior 保存液中添加 RGFP966 可以减轻长时程低温保存引起的心脏功能障碍. 该研究结果为临床实践中的心脏移植提供有价值且可行的策略, 为患者获得远距供体心脏提供了可能.
方法
将大鼠离体心脏置于含有或不含有 RGFP966 的 Celsior 保存液中低温保存 12h 后, 于 Langendorff 灌流系统中复灌 60min, 测定复灌期各项心功能指标. 用 Western blotting 法分析 Mst1 和 YAP 蛋白表达和磷酸化水平. 用 TUNEL 法测定心肌细胞凋亡情况.
结论
RGFP966 可以促进低温保存心脏复灌期心功能的恢复, 其作用机制可能是通过抑制 Hippo-YAP 信号传导通路的激活, 增强心肌抗氧化能力, 抑制心肌细胞凋亡而实现的.
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Xiao-he ZHENG, Lin-lin WANG, Ming-zhi ZHENG, and Jin-jie ZHONG performed the experimental research and data analysis, wrote and edited the manuscript. Ying-ying CHEN and Yue-liang SHEN contributed to the study design, data analysis, writing and editing of the manuscript. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Xiao-he ZHENG, Lin-lin WANG, Ming-zhi ZHENG, Jin-jie ZHONG, Ying-ying CHEN, and Yue-liang SHEN declare that they have no conflict of interest.
The research was conducted in accordance with the Declaration of Helsinki and/or with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health. All experiments were approved by the Ethics Committee on Animal Experimentation of Zhejiang University (Hangzhou, China).
Project supported by the National Natural Science Foundation of China (No. 81871541)
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Zheng, Xh., Wang, Ll., Zheng, Mz. et al. RGFP966 inactivation of the YAP pathway attenuates cardiac dysfunction induced by prolonged hypothermic preservation. J. Zhejiang Univ. Sci. B 21, 703–715 (2020). https://doi.org/10.1631/jzus.B2000026
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DOI: https://doi.org/10.1631/jzus.B2000026