SIRT3在硫化氢抗心肌肥大中的作用机制研究

Myocardial hypertrophy is an independent risk factor of cardiovascular adverse events. The mechanism of myocardial hypertrophy has not been fully elucidated until now. The mechanism of hydrogen sulfide (H2S) against myocardial hypertrophy was unknown. SIRT3 (Sirtuin3) is one kind of deacetylases, which is closely related to oxidative stress. Our previous study showed that H2S improved vascular endothelial function via a SIRT3-dependent manner. However, there was no study on whether SIRT3 was involved in the protective effect of H2S against myocardial hypertrophy. Our preliminary experiments showed that H2S enhanced SIRT3 promoter activity and SIRT3 expression in hypertrophic cardiomyocytes, which might be related to the AP-1 binding site in the SIRT3 promoter region. H2S elevated oxidative respiration ability and mitochondrial membrane potential in hypertrophic cardiomyocytes via a SIRT3-dependently manner. H2S increased AP-1 S-sulfhydration in cardiomyocytes. In the project, isolated cells and the whole animal model of myocardial hypertrophy was made, combined with specific cysteine site mutation, to verify the scientific hypothesis that H2S may increase S-sulfhydration at cysteine sites of transcription factor AP-1, enhance binding activity between AP-1 and SIRT3 promoter, promote SIRT3 transcription, protect mitochondrial function and enhance the deacetylation level of antioxidant related proteins, inhibit oxidative stress, and finally protect against myocardial hypertrophy. It will provide a new mechanism of H2S against myocardial hypertrophy via a SIRT3-dependent manner.

心肌肥大是心血管不良事件的独立危险因素，发病机制未完全阐明。硫化氢（H2S）抗心肌肥大作用机制不明。SIRT3（Sirtuin3）是一种与氧化应激密切相关的去乙酰化酶。我们前期研究发现：H2S依赖SIRT3改善血管内皮功能，但SIRT3是否参与H2S抗心肌肥大尚无报道。预实验发现：H2S提高肥大心肌中SIRT3启动子活性与SIRT3表达，可能与启动子区AP-1结合位点有关；H2S依赖SIRT3增强肥大心肌细胞氧化呼吸能力及线粒体膜电位；H2S增加心肌细胞AP-1硫巯基化修饰。本项目拟利用细胞和动物心肌肥大模型，联合特定半胱氨酸位点突变，验证科学假说：H2S通过增加AP-1半胱氨酸位点硫巯基化修饰水平，增强AP-1与SIRT3启动子区结合，促进SIRT3转录，进而保护线粒体功能和增加抗氧化相关蛋白的去乙酰化水平，抑制氧化应激，发挥抗心肌肥大作用，揭示H2S依赖SIRT3抗心肌肥大的新机制。

心肌肥大是心血管不良事件的独立危险因素，发病机制未完全阐明。硫化氢（H2S）抗心肌肥大作用机制不明。本项目细胞水平研究发现H2S供体NaHS和GYY4137改善血管紧张素II（Ang II）诱导下线粒体呼吸功能和膜电位，增加叉头蛋白O3a（FOXO3a）和超氧化物歧化酶2（SOD2）表达，降低FOXO3a和SOD2乙酰化水平，减轻氧化应激，抑制心肌细胞肥大；NaHS促进Ang II刺激下心肌细胞SIRT3转录和表达，但是，SIRT3干扰后，H2S的上述作用均显著减弱。进一步研究发现：NaHS增加激活蛋白-1（AP-1）与SIRT3启动子的亲和力，提高Ang II诱导心肌肥大过程中SIRT3启动子活性；干扰AP-1或给予AP-1抑制剂后，H2S对Ang II诱导下心肌细胞SIRT3启动子活性和表达的增强作用大大减弱，提示H2S依赖AP-1抑制SIRT3的转录。此外，H2S增加心肌细胞中AP-1蛋白的硫巯基化修饰水平，将AP-1蛋白的半胱氨酸269位点（Cys269）突变后，H2S对AP-1蛋白的硫巯基化修饰水平、AP-1与SIRT3启动子亲和力、SIRT3表达、心肌肥大等调节作用均明显减弱，但是突变AP-1其他的10个半胱氨酸位点后，H2S的上述作用没有显著影响，提示H2S通过增加AP-1半胱氨酸269位点（Cys269）硫巯基化修饰水平来调控心肌肥大中SIRT3的表达。动物水平发现：NaHS改善野生型小鼠主动脉缩窄术和异丙肾上腺素诱导后心肌线粒体结构，调节抗氧化相关蛋白的表达，降低FOXO3a和SOD2乙酰化水平，减轻氧化应激，抑制心肌肥大，但是在SIRT3基因敲除小鼠中NaHS的上述作用明显减轻。本项目阐明了H2S通过增加AP-1硫巯基化修饰水平调控心肌肥大中SIRT3转录的具体机制，从而明确了SIRT3为H2S抗心肌肥大的关键靶点，为H2S防治心肌肥大提供新的理论依据。