Cai ChengZhe, Wu YiQin, Feng XiaoQian, Ye XianQu, Liu PingFang, Huang XiangJin, Li ZhiJun, Xu ZhuoFan
Department of Internal Medicine-Cardiovascular, Guangzhou Twelfth People's Hospital, No.1, Tianqiang Road, Tianhe District, Guangzhou City, Guangdong Province, 510620, China.
Appl Biochem Biotechnol. 2025 May;197(5):2910-2926. doi: 10.1007/s12010-024-05171-3. Epub 2025 Jan 14.
Myocardial infarction (MI) is a coronary artery-related disease that seriously threatens human life and is the leading cause of sudden death worldwide, where a lack of nutrients and oxygen leads to an inflammatory response and death of cardiomyocytes. Ferroptosis is a form of non-apoptotic cell death associated with metabolic dysfunction, resulting in abnormal breakdown of glutamine and iron-dependent accumulation of reactive oxygen species (ROS) during metabolism. However, the molecular mechanism of ferroptosis in the pathogenesis of MI and the function of Klotho and KRAS on ferroptosis during MI remain unclear. The MI rat model was established by LAD ligation with a 6-0 suture. H9c2 cells were placed in glucose-deficient DMEM (Thermo) and cultured in an anaerobic environment (1% CO and 5% CO) to establish an in vitro OGD cell model. The damage to rat heart tissue was detected by HE staining, and Klotho and KRAS were determined by RT-qPCR, Western Blot, and IHC. TUNEL staining was used to determine apoptosis in rat heart tissue samples. The interaction between Klotho and KRAS was verified by co-immunoprecipitation and Western Blot. The cardiomyocyte activity was measured by CCK-8 assay. LDH, CK-MB, cTnT, and Fe markers were detected by the kits. For the assessment of ferroptosis, GSH and ROS in cardiomyocytes and serum were detected by kits, and PTSG was detected by Western Blot. IL-1β and IL-6 in cardiomyocytes and serum were determined by ELISA. Klotho was downregulated in MI. Downregulation of Klotho promoted myocardial injury; increased apoptosis of cardiomyocytes; promoted LDH, CK-MB, and cTnT concentrations; inhibited GSH; and promoted ROS levels, PTGS2 expression, and ferroptosis in rats. The same results were obtained in vitro. Klotho and KRAS had endogenous interactions. KRAS knockdown can reverse Klotho knockdown-mediated MI and ferroptosis. RAP1/ERK pathway was highly expressed in MI, and inhibiting RAP1/ERK pathway activation can reverse the promoting effect of overexpressed KRAS on MI progression and ferroptosis. Klotho interacts with KRAS and inhibits ferroptosis after MI by regulating the RAP1/ERK pathway.
心肌梗死（MI）是一种与冠状动脉相关的疾病，严重威胁人类生命，是全球范围内猝死的主要原因，其中心肌细胞缺乏营养和氧气会导致炎症反应和死亡。铁死亡是一种与代谢功能障碍相关的非凋亡性细胞死亡形式，导致谷氨酰胺异常分解以及代谢过程中铁依赖性活性氧（ROS）积累。然而，铁死亡在MI发病机制中的分子机制以及Klotho和KRAS在MI期间对铁死亡的作用仍不清楚。通过用6-0缝线结扎左冠状动脉前降支（LAD）建立MI大鼠模型。将H9c2细胞置于葡萄糖缺乏的DMEM（赛默飞世尔科技公司）中，并在厌氧环境（1%二氧化碳和5%二氧化碳）中培养，以建立体外氧糖剥夺（OGD）细胞模型。通过苏木精-伊红（HE）染色检测大鼠心脏组织损伤，通过逆转录定量聚合酶链反应（RT-qPCR）、蛋白质免疫印迹法（Western Blot）和免疫组织化学法（IHC）检测Klotho和KRAS。采用末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）染色法测定大鼠心脏组织样本中的细胞凋亡。通过免疫共沉淀和蛋白质免疫印迹法验证Klotho和KRAS之间的相互作用。通过细胞计数试剂盒（CCK-8）测定心肌细胞活性。使用试剂盒检测乳酸脱氢酶（LDH）、肌酸激酶同工酶（CK-MB）、心肌肌钙蛋白T（cTnT）和铁标记物。为了评估铁死亡，使用试剂盒检测心肌细胞和血清中的谷胱甘肽（GSH）和ROS，并通过蛋白质免疫印迹法检测前列腺素内过氧化物合酶2（PTGS2）。通过酶联免疫吸附测定法（ELISA）测定心肌细胞和血清中的白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）。在MI中Klotho表达下调。Klotho下调促进心肌损伤；增加心肌细胞凋亡；促进LDH、CK-MB和cTnT浓度升高；抑制GSH；并促进大鼠体内ROS水平、PTGS2表达和铁死亡。在体外也获得了相同的结果。Klotho和KRAS存在内源性相互作用。敲低KRAS可以逆转敲低Klotho介导的MI和铁死亡。在MI中，RAS相关蛋白1（RAP1）/细胞外信号调节激酶（ERK）通路高表达，抑制RAP1/ERK通路激活可以逆转过表达KRAS对MI进展和铁死亡的促进作用。Klotho与KRAS相互作用，并通过调节RAP1/ERK通路抑制MI后的铁死亡。
