类泛素蛋白FAT10调控CaMK2D介导的钙离子信号通路抵抗心肌肥厚的机制研究

Cardiac hypertrophy is one of the major risk factors leading to cardiac sudden death. Studies demonstrated that activation of the signaling transduction and calcium overload mediated by calcium/calmodulin-dependent protein kinase II delta (CaMK2D), one member of calmodulin-dependent protein kinase family, plays a significant role in the development of cardiac hypertrophy. However, its regulatory mechanisms remain unclear. Our studies previously showed that the ubiquitin-like protein FAT10 was a protective protein in heart tissue. The preliminary experiments suggested that CaMK2D was predicted as a partner playing with FAT10 by LC-MS/MS; FAT10 was down-regulated in the hypertrophic hearts; overexpression of FAT10 in cardiomyocytes attenuated the hypertrophic response to the stimuli; heart specific knock-out of FAT10 aggravated cardiac hypertrophic response induced by pressure overload and upregulated the expression level of CaMK2D. Consequently, we hypothesize that the activation of Ca2+-related signaling and Ca2+ overload mediated by up-regulation of CaMK2D is induced by the down-regulation of FAT10, which prompt the development of cardiac hypertrophy. To determine this, transverse aortic constriction in the cardiac specific knock-out of FAT10 mice and angiotensin II-derived cardiomyocyte hypertrophic model were used, in addition to adult cardiomyocytes isolation and Ca2+ imaging approach. This study will provide scientific sights into new therapeutic strategy and medication targets screening.

心肌肥厚是心源性猝死的主要危险因素之一。研究表明CaMK2D介导的钙离子信号通路活性升高与钙离子超载在心肌肥厚的发生中起重要作用，但调控机制仍不明确。本课题组前期研究证实类泛素蛋白FAT10是心肌组织的保护性蛋白。预实验结果发现：蛋白质谱预测CaMK2D是FAT10的底物蛋白；FAT10在肥厚心肌中表达下调；心肌细胞中过表达FAT10改善药物诱导的心肌肥大反应；心脏组织敲除FAT10可显著加重由压力负荷导致的心肌肥厚并上调CaMK2D的表达。由此提出假设：FAT10表达下调导致CaMK2D及其介导的信号通路活性增高与钙离子超载进而促进心肌肥厚的发展。为验证此假设，本课题拟利用FAT10心脏特异性敲除小鼠结合压力负荷性心肌肥厚与血管紧张素II诱导的体外心肌细胞肥大模型，结合成年心肌细胞分离、钙离子成像等技术探讨其保护心肌肥厚中的分子机制，为寻找新药物靶点提供科学依据。

心脏性猝死（SCD）是造成全球人口死亡的主要原因之一，且在未来的10年中显示出增加趋势。病理性心脏肥厚性重构作为引起SCD的危险因素之一可以显著增加SCD风险，因此，深入研究心肌肥厚的发病机制对控制心脏性猝死的发生及改善心脏功能具有重要的科学意义和临床价值。在小鼠和大鼠心肌肥大模型和血管紧张素II (AngII)处理的心肌细胞中，心脏FAT10蛋白水平降低。蛋白质质谱鉴定CaMK2D是心脏中FAT10的一种新型底物。在分离的成年大鼠心肌细胞中，免疫共沉淀和共定位实验证实了这一发现。在心肌细胞中，腺病毒介导的Fat10 (Ad-Fat10)表达降低了CaMK2D水平和CaMK2D依赖的兰尼碱受体2 (RyR2)磷酸化。相反，用特异性siRNA (Si-Fat10)敲低Fat10对CaMK2D-RyR2信号通路产生相反的作用。同样，心肌细胞特异性敲除Fat10 (cKO-Fat10)与CaMK2D-RyR2通路激活相关，并在TAC反应中加剧心肌肥大和纤维化。相反，过表达FAT10促进了CaMK2D与热休克蛋白70 (Hsc70)的结合，并促进了CaMK2D通过分子伴侣介导的自噬降解。此外，我们发现CMA是CaMK2D分子与PARP-1分子降解的主要机制，且在心肌氧化应激损伤中起保护作用。维持CMA活性能够通过降解PARP-1抵抗氧化应激介导的心肌细胞程序性死亡。 总之，本课题的研究的结果提供了体内和体外的证据，证明FAT10具有防止心脏肥厚的功能。FAT10的抗肥大作用机制与CMA介导的CaMK2D降解和Ca2+稳态有关。本课题揭示了FAT10在心肌肥厚中的生物学功能，并将FAT10的分子功能扩展到其调节蛋白酶体降解信号的作用之外领域。证明了CMA可能是心脏病理生理的潜在药物干预靶点。