Drp1介导的线粒体分裂异常参与脓毒症心肌损伤机制的研究

Myocardial injury is a major contributor to mortality and morbidity in patients with sepsis. Mitochondrial dysfunction is one of the major molecular mechanisms that may be involved in myocardial dysfunction during sepsis. The new findings have altered the perception that cardiac mitochondria are static. Mitochondrial fusion and fission in cardiomyocytes are detected as that in non-cardiac systems. The dynamic equilibrium between mitochondrial fusion/fission is necessary to maintain mitochondrial integrity and normal fuction in myocardium. Our primarily study showed that increase in the dynamin-related peptide 1 (Drp1)-mediated mitochondrial fission might be the key cause which led to sepsis-induced myocardial dysfunction. However the exact mechanism is still unknown. The aims of present study are to identify which of the critical phosphorylation sites of Drp1 are responsible for its cleavage activity during mitochondrial fission, and also actively participate in apoptosis induction, by using site directed mutagenesis method; to investigate the role of calcineurin in the dephosphorylation of Drp1 during sepsis; to explore which of the pro-apoptosis proteins are the key effectors/targets of Drp1 in this sepsis model by using pharmacological method, molecular biological method and co-immunoprecipitation assay; to elucidate whether the increase in Drp1-medicated mitochondrial fission participates in apoptosis induction during sepsis through Bax dependent manner. This study may help to elucidate the molecular mechanism and role of Drp1-mediated mitochondrial fission in sepsis-induced myocardical apoptosis and dysfunction in. The results of this study will provide new ideas and theoretical basis for the new target that preventment of septic cardiomyopathy.

线粒体功能障碍是导致脓毒症心肌损害的主要原因。最新研究表明，线粒体融合和分裂的动态平衡是维持心肌线粒体正常生理功能的关键。我们前期的预实验发现，动力相关蛋白1（dynamin-related peptide 1，Drpl）介导的线粒体分裂增加可能是导致脓毒症心肌细胞凋亡和功能障碍的主要原因，但具体分子机制尚不明了。本课题将采用DNA定点突变的方法，确定哪些磷酸化位点是导致Drp1蛋白线粒体剪切活性改变，引发脓毒症心肌细胞凋亡的关键，并探讨钙调磷酸酶在Drp1蛋白去磷酸化中的地位。进一步利用分子生物学及免疫共沉淀等实验方法，筛选并鉴定与Drp1相互作用的凋亡蛋白，阐明Drp1蛋白介导的线粒体分裂增加是否通过Bax依赖性机制发挥促凋亡作用。通过对本课题的研究将阐明Drp1介导的线粒体分裂异常参与脓毒血症心肌损伤分子机制，从而为临床上寻求对抗脓毒症心肌损伤的有效作用靶点提供新的思路和理论依据。

线粒体功能障碍是脓毒症心肌损害的主要原因。而线粒体融合和分裂的动态平衡是维持心肌线粒体正常生理功能的关键。本课题主要研究动力相关蛋白1（dynamin-related peptide 1，Drpl）介导的线粒体分裂增加是否是导致脓毒症心肌细胞凋亡和功能障碍的主要原因，并阐明其可能机制。.本课题采用腹腔注射脂多糖（lipopolysaccharide，LPS）建立雄性SD大鼠脓毒症模型，TNF-α刺激体外培养的H9C2细胞建立心肌细胞炎症模型。测定心功能指标、细胞凋亡和线粒体片段化程度。WB法测定Drpl蛋白的表达和分布、CaMKII、RhoA、Bax、caspase-3等蛋白表达情况。.研究发现：（1）大鼠腹腔注射LPS后每搏输出量和心输出量逐渐下降；线粒体片段化程度增加。心肌组织总Drp1水平无明显变化，但线粒体Drp1蛋白的表达量明显增高，且呈现浓度依赖性特征。（2）Drp1的特异性抑制剂Mdivi-1可显著降低心肌细胞线粒体片段化程度，减少心肌细胞凋亡的发生，并明显抑制脓毒症大鼠心肌收缩功能的下降。（3）TNF-α（20 ng/ml）对H9C2细胞Drp1总量无明显影响，但Drp1 Ser616位点的磷酸化和线粒体Drp1水平明显增加。（4）TNF-α处理H9C2细胞后，可明显增加CaMKⅡ的表达。但CaMKⅡ的抑制剂KN-93不能抑制TNF-α诱导的Drp1的磷酸化和线粒体转位。(5) TNF-α处理H9C2细胞后，RhoA蛋白表达明显增加。ROCK1和ROCK2的特异性抑制剂Y-27632和Fasudil可明显抑制TNF-α诱导的Drp1的Ser616磷酸化和线粒体转位，减少线粒体片段化程度。（6）抑制Drp1不仅可阻止TNF-α诱导的线粒体片段化的发生；还可抑制TNF-α诱导的凋亡蛋白Bax的线粒体转位；抑制MPT孔开放、降低capase-3活性、减少细胞色素c的释放和细胞凋亡的发生。.以上结果提示，Drp1介导的线粒体分裂增加可能是导致脓毒症心肌细胞凋亡和功能障碍的主要原因。其分子机制是通过引起RohA/ROCK通路激活，导致Drp1蛋白Ser616磷酸化增加有关。Drp1介导的线粒体分裂增加最终通过Bax依赖性机制诱导脓毒症心肌细胞凋亡的发生。本课题的研究结果将为临床上心肌保护药物的研究提供新的思路和理论依据。