PKM2巯基亚硝基修饰在心肌纤维化中的作用与机制研究

Fibrosis in the heart is a common feature in patients with advanced cardiac failure, regardless of the etiology of cardiomyopathy. Specific anti-fibrotic therapies are not currently available in the clinic. Pyruvate kinase 2(PKM2) is involved in cell metabolism regulation and plays important role in cell fate reprogramming. By applying Biotin-Switch combined proteomics, we discovered S-nitrosylation of three Cysteine residues in PKM2 in hearts of mice subjected to transverse aortic constriction (TAC). We also found that mutation of the Cysteine 49 in PKM2 inhibited activation of cardiac fibroblasts exposed to AngII. Moreover, iNOS level was increased with AngII stimulation. We further found that during fibrosis, the activity of PKM2 was decreased, and its interaction with P-ERK1/2 was enhanced. Based on these findings, we proposed that iNOS-mediated PKM2 S-nitrosylation may influence PKM2/P-ERK1/2 interaction, therefore activating transcription of fibrosis-related genes and transition of cardiac fibroblasts to myofibroblasts. We try to verify this hypothesis by using multiple molecular biological techniques including RNA interference, site mutation, adenovirus transfection and et al, as well as in vivo and in vitro fibrotic models such as iNOS-/- mice and primary cardiac fibroblasts. This work might discover novel mechanism of cardiac fibrosis and provide new therapeutic targets for treatment of cardiac fibrosis.

心肌纤维化是多种心脏疾病的共同病理基础，其发生机制尚不明确。丙酮酸激酶2（PKM2）是调控细胞代谢的关键酶，通过代谢重编程方式影响细胞存活和表型转化。预实验中，我们发现主动脉缩窄小鼠心脏组织中PKM2上3个半胱氨酸位点发生巯基亚硝基修饰，且半胱氨酸49位点突变能够抑制AngII诱导的心肌成纤维细胞活化。此外，AngII能够诱导心肌成纤维细胞中巯基亚硝基修饰关键调控蛋白iNOS表达升高。进一步研究发现，AngII刺激下PKM2酶活性降低，同时与P-ERK1/2结合作用增强。由此我们提出假设：心肌纤维化过程中，iNOS介导的PKM2巯基亚硝基修饰影响其活性及与P-ERK1/2的结合，进而促进下游纤维化相关基因转录。本研究将运用多种抑制剂，联合基因沉默、半胱氨酸位点突变、病毒转染等手段，利用原代心肌成纤维细胞和iNOS-/-小鼠共同验证此假说，为心肌纤维化的机制探索和药物治疗提供新思路。

心肌纤维化是大多数心血管疾病的共同病理生理基础，主要由心肌成纤维细胞(CFs)活化为肌成纤维细胞引起，其特征是心脏间质胶原纤维的过度沉积。心肌成纤维细胞的持续激活会引起心脏重塑并最终导致心力衰竭。亚硝基修饰是一种蛋白翻译后修饰，即在蛋白质和多肽中的半胱氨酸残基上添加NO残基。亚硝基修饰与细胞氧化还原状态相关，可以转导NO生物活性。最近研究表明亚硝基修饰调节一系列生理和病理过程，包括干细胞分化、神经元损伤和心肌病，但其在心肌纤维化中的作用研究较少。为了揭示蛋白质亚硝基修饰在心脏纤维化中的作用并阐明其潜在机制，我们对主动脉结扎（TAC）诱导的小鼠心肌纤维化模型和SHR大鼠的心脏进行了亚硝基修饰蛋白质组学进行了联合分析，发现2型丙酮酸激酶巯基亚硝基修饰水平显著升高。我们在心肌肥大临床样本和不同的心脏纤维化小鼠模型中均发现了PKM2巯基亚硝基修饰(SNO-PKM2)的增加，并在乳大鼠心肌成纤维细胞中进一步证实了SNO-PKM2的促纤维化作用。机制研究发现， PKM2巯基亚硝基修饰后，四聚减少，其与凝溶胶蛋白（GSN）的互作降低，进而导致GSN与Drp1互作增强，促进Drp1的线粒体转位并介导线粒体分裂增加，线粒体功能障碍，最终促进心脏纤维化的发生。在此基础上，利用PKM2的激动剂TEPP-46提高PKM2的四聚，或敲低GSN能够显著减少线粒体分裂，改善线粒体功能并抑制纤维化的发生。最后，在TAC小鼠模型中，TEPP-46能够剂量依赖性的改善小鼠心脏舒张功能，减少胶原沉积，抑制纤维化相关基因的表达。该研究从蛋白翻译后修饰层面发现了线粒体形态功能调控的新机制，发现了线粒体功能损伤与心肌纤维化间的作用。同时，发现PKM2是心肌纤维化进程的关键靶标，针对其的药物设计，为心肌纤维化的治疗提供新的手段。