基于TAAR1/CaMKⅡ途径研究外源性T1AM在心肌缺血再灌注损伤中抑制细胞凋亡及程序性坏死的作用机制

CaMKII activation plays a nodal role in apoptosis and necroptosis induced by myocardial ischemia reperfusion (I/R). The activity of CaMKⅡis reduced during animal hibernation. A hibernation inducing agent 3-iodothyronamine (T1AM), synthesized autonomously by our team, has been found to inhibit myocardial I/R-induced apoptosis and necroptosis, but its mechanism remains to be elucidated. To address the issue, we will carry out serial experiments to construct myocardial I/R injury model and to up/down-regulate T1AM receptor TAAR1 expression and CaMKII activity. The differences and similarities between T1AM and low temperature affecting I/R-induced myocardial apoptosis and necroptosis will be examined in vivo, ex vivo and in vitro. The underlying mechanism by which T1AM inhibits I/R-induced myocardial cell death will be investigated, as to the CaMKⅡ-related intracellular calcium handling and opening of mitochondria permeability transition pore（PTP）. The findings of this study should elucidate if hibernation mechanism of myocardial protection exists in warm blooded animal, provide a novel therapeutic strategy to reduce the damage of I/R, deepen the understanding of myocardial necrosis death, especially necroptosis, and add the experimental evidence for the development of T1AM and related drugs for myocardial protection.

蛋白激酶CaMKⅡ激活是心肌缺血再灌注(I/R)诱导细胞凋亡及程序性坏死的中心环节。动物冬眠时CaMKII活性降低。我们自主合成的冬眠诱导剂3-碘甲状腺胺（T1AM）抑制I/R诱导细胞死亡，但机制不明。推测外源性T1AM唤醒机体潜在的冬眠机制，抑制细胞凋亡及程序性坏死，减轻心肌I/R损伤。为此，我们构建心肌I/R损伤模型、上/下调T1AM受体TAAR1表达以及CaMKII的活性，在整体、离体心脏、细胞水平研究T1AM及低温对I/R损伤诱导心肌细胞凋亡与程序性坏死的作用异同，从CaMKII相关的细胞内钙离子调控、线粒体PTP开放方面研究T1AM抑制细胞死亡的调控机制。本研究可望明确恒温动物是否存在类似冬眠保护心肌机制，拓展对心肌死亡特别是程序性坏死调控的认识，为寻求减轻I/R损伤的新治疗策略提供思路，为开发T1AM相关药物提供实验证据。

缺血性心脏病是危害人类健康的重要疾病。早期血运重建是目前治疗缺血性心脏病最有效的治疗手段，但是血运重建常伴发心肌缺血/再灌注损伤，后者常导致心肌顿抑、心律失常及细胞死亡，因此深入探讨缺血再灌注损伤的机制对保护心肌有重要的价值。治疗性低温能显著缩小缺血再灌注损伤所致梗死面积，寻找降低体温药物有望为急性心肌梗死的心肌保护提供更有效方法。项目利用我们自主合成的冬眠诱导剂3-碘甲状腺胺（T1AM）抑制I/R诱导细胞死亡，推测外源性T1AM唤醒机体潜在的冬眠机制，抑制细胞凋亡及程序性坏死，减轻心肌I/R损伤。为此，我们构建心肌I/R损伤模型，通过转录组学测序，寻找T1AM的作用靶点，在动物及其细胞水平研究T1AM及低温对I/R损伤诱导心肌细胞凋亡与程序性坏死的作用异同，从Akt/FoxO1信号通路，CaMKII相关的细胞内钙离子调控、线粒体PTP开放方面研究T1AM抑制细胞死亡的调控机制。结果提示在动物水平，T1AM可以减轻大鼠心肌缺血再灌注损伤，保护心肌细胞；在细胞水平，与常氧组比较，缺氧/复氧损伤对心肌细胞差异基因显著，T1AM可以通过代谢相关通路减少心肌细胞（AC-16细胞）缺氧/复氧损伤；T1AM可通过调控Akt/FoxO1信号通路降低心肌细胞代谢从而减少细胞凋亡。此外，课题组通过缺氧复氧心肌细胞（H9C2细胞）发现TIAM可以减轻心肌细胞的程序性坏死和凋亡，其机制与抑制RIPK1和RIPK3及上下游的通路相关蛋白p62、CaMKII和VDAC表达相关，表明T1AM对缺氧复氧的H9C2心肌细胞有保护作用。本研究明确恒温动物存在类似冬眠保护心肌机制，拓展对心肌死亡特别是程序性坏死调控的认识，为寻求减轻I/R损伤的新治疗策略提供思路，为开发T1AM相关药物提供实验证据。