CRTC2通过抑制心肌细胞铁死亡减轻心肌细胞肥大

CRTC2 attenuates cardiomyocyte hypertrophy by inhibiting cardiomyocyte ferroptosis

摘要:

目的]探究CREB调节转录共激活因子2(CRTC2)在心肌细胞肥大中的作用及其调控机制。 [方法]通过腹腔注射异丙肾上腺素(ISO)构建C57BL/6小鼠病理性心肌细胞肥大模型,并采用Western blot检测心脏组织中CRTC2的表达。构建CRTC2基因敲除小鼠模型,利用小动物心动超声仪检测小鼠心功能,通过Masson染色检测小鼠心脏组织中胶原纤维含量,采用Western blot检测小鼠心脏组织中心肌细胞肥大相关蛋白——骨骼肌α1-肌动蛋白(ACTA1)、脑钠肽(BNP)和铁死亡相关蛋白——酰基辅酶A合成酶长链家族成员4(ACSL4)、溶质载体家族7成员11(SLC7A11)及谷胱甘肽过氧化物酶4(GPX4)表达,使用铁离子试剂盒检测小鼠心脏组织中铁离子含量,评估CRTC2与心肌细胞肥大和铁死亡的相关性。用ISO诱导H9c2细胞构建心肌细胞肥大体外模型,使用铁死亡抑制剂Ferrostatin-1(Fer-1)干预后,检测CRTC2、ACTA1、BNP、ACSL4、SLC7A11及GPX4的蛋白表达。通过ISO诱导CRTC2过表达的H9c2细胞构建心肌细胞肥大体外模型,检测与心肌细胞肥大及铁死亡相关的指标,以探究CRTC2在心肌细胞肥大中的作用机制。 [结果]与对照组相比,ISO诱导的心肌细胞肥大小鼠心脏组织中CRTC2蛋白的表达增加(P＜0.05)。与野生型小鼠相比,CRTC2－/－小鼠心功能恶化,表现为左心室舒张末内径(LVEDD)、左心室收缩末内径(LVESD)、左心室后壁厚度(LVPWT)、心重/胫骨长度(HW/TL)和心重/体重(HW/BW)增加,短轴缩短率(FS)和射血分数(EF)降低,心脏组织中胶原纤维含量增加,心肌细胞肥大相关蛋白ACTA1和BNP的表达升高,铁死亡相关蛋白ACSL4的mRNA和蛋白表达升高,SLC7A11和GPX4的mRNA和蛋白表达降低,心脏组织中铁离子含量升高(P＜0.05或P＜0.01)。体外实验显示,与ISO组相比,ISO＋Fer-1组CRTC2蛋白表达无明显变化(P＞0.05),ACTA1和BNP蛋白的表达降低,心肌细胞表面积减少,ACSL4蛋白的表达降低,SLC7A11和GPX4蛋白的表达升高(P＜0.05或P＜0.01)；与ISO组相比,LV-CRTC2＋ISO组心肌细胞表面积减少(P＜0.01),ACTA1、BNP和ACSL4蛋白的表达降低,SLC7A11和GPX4蛋白的表达增加,ROS和铁离子含量降低(P＜0.05或P＜0.01)。 [结论]CRTC2可通过抑制心肌细胞铁死亡减轻心肌细胞肥大,保护心功能。

Abstract:

Aim To investigate the role and regulatory mechanism of CREB regulated transcription coactivator 2 (CRTC2) in cardiomyocyte hypertrophy. Methods A pathological cardiomyocyte hypertrophy model was established in C57BL/6 mice by intraperitoneal injection of isoproterenol (ISO), the expression of CRTC2 in cardiac tissue was detected by Western blot. The CRTC2 knockout mice model was constructed, the cardiac function of mice was detected by small animal echocardiography, the collagen fiber content in mice cardiac tissue was detected by Masson staining, the cardiomyocyte hypertrophy related proteins:skeletal muscle α1-actin (ACTA1) and brain natriuretic peptide (BNP), as well as ferroptosis related proteins:acyl-CoA synthetase long chain family member 4 (ACSL4), solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in mice cardiac tissue were detected by Western blot, the iron ion content in mice cardiac tissue was detected by iron ion kit, to evaluate the correlation between CRTC2 and cardiomyocyte hypertrophy and ferroptosis. H9c2 cells were induced by ISO to construct an in vitro model of cardiomyocyte hypertrophy, the protein expressions of CRTC2, ACTA1, BNP, ACSL4, SLC7A11 and GPX4 were detected after intervention with ferroptosis inhibitor ferrostatin-1 (Fer-1). H9c2 cells with CRTC2 overexpression induced by ISO were used to construct an in vitro model of cardiomyocyte hypertrophy, the related indicators of cardiomyocyte hypertrophy and ferroptosis were detected to explore the mechanism of CRTC2 in cardiomyocyte hypertrophy. Results Compared with the control group, the expression of CRTC2 protein in the cardiac tissue of ISO induced cardiomyocyte hypertrophy mice was increased (P＜0.05). Compared with wild-type mice, CRTC2－/－ mice showed worsened cardiac function, manifested as increased left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular posterior wall thickness (LVPWT), heart weight/tibia length (HW/TL) and heart weight/body weight (HW/BW), decreased short axis shortening (FS) and ejection fraction (EF), increased collagen fiber content in cardiac tissue, upregulated expression of cardiomyocyte hypertrophy-related proteins ACTA1 and BNP, increased mRNA and protein expression of ferroptosis-related protein ACSL4, decreased mRNA and protein expression of SLC7A11 and GPX4, and elevated iron ion content in cardiac tissue (P＜0.05 or P＜0.01). In vitro experiments showed that compared with ISO group, the ISO＋Fer-1 group had no significant change in CRTC2 protein expression (P＞0.05), the expression of ACTA1 and BNP protein decreased, the surface area of cardiomyocyte reduced, the expression of ACSL4 protein decreased, and the expression of SLC7A11 and GPX4 proteins increased (P＜0.05 or P＜0.01). Compared with the ISO group, the LV-CRTC2＋ISO group showed a decrease in surface area of cardiomyocytes (P＜0.01), a decrease in ACTA1, BNP and ACSL4 protein expression, an increase in SLC7A11 and GPX4 protein expression, and a decrease in ROS and iron ion content (P＜0.05 or P＜0.01). Conclusion CRTC2 alleviates cardiomyocyte hypertrophy and protect cardiac function by suppressing ferroptosis in cardiomyocytes.