目的:糖尿病心肌病(DCM)是2型糖尿病(T2DM)最常见的心血管并发症。与其他人群相比,患有DCM的患者面临进展为充血性心力衰竭的风险明显更高。心肌肥厚,明确证实的DCM病理变化,在DCM的发展中发挥着重要作用,以异常的Ca2+稳态作为诱发心肌肥大的关键信号。因此,研究Ca2+转运机制对防治T2DM心肌肥厚具有重要意义。
方法:实验大鼠分为野生型(WT)组和T2DM组。采用高脂高糖饲料喂养大鼠3个月,联合低剂量链脲佐菌素(100mg/kg)建立2型糖尿病大鼠模型。之后,分离培养原代大鼠心肌细胞,通过高糖治疗诱导心肌细胞肥大。随后,通过si-STIM1和oe-STIM1转染进行了机理研究。Westernblot(WB)用于检测STIM1,Orai1和p-CaMKII的表达。qRT-PCR用于检测心肌肥厚标志物蛋白的mRNA水平。使用TRITC-Phalloidin染色检测细胞表面积,使用Fluo-4荧光染色测量心肌细胞中的细胞内Ca2+浓度。
结果:通过动物实验,在T2DM诱导的大鼠心肌肥厚模型中发现Orai1和STIM1上调。同时,通过细胞实验,发现在高糖(HG)诱导的肥大心肌细胞中,STIM1、Orai1和p-CaMKII的表达上调,随着商店操作的Ca2进入(SOCE)和异常的Ca2稳态水平的增加。然而,当STIM1在HG诱导的心肌细胞中下调时,SOCE水平下降,p-CaMKII下调,导致心肌肥大的改善。进一步阐明SOCE和CaMKII在T2DM心肌肥厚中的作用机制,分别用BTP2(SOCE阻断剂)和KN-93(CaMKII抑制剂)处理高糖心肌细胞,结果表明,STIM1可以介导SOCE,从而影响CaMKII的磷酸化水平,增进心肌细胞肥大。
结论:STIM1/Orai1介导的SOCE调节p-CaMKII水平,从而诱导T2DM心肌肥大。
OBJECTIVE: Diabetic cardiomyopathy (DCM) is the most common cardiovascular complication of type 2 diabetes mellitus (T2DM). Patients affected with DCM face a notably higher risk of progressing to congestive heart failure compared to other populations. Myocardial hypertrophy, a clearly confirmed pathological change in DCM, plays an important role in the development of DCM, with abnormal Ca2+ homeostasis serving as the key signal to induce myocardial hypertrophy. Therefore, investigating the mechanism of Ca2+ transport is of great significance for the prevention and treatment of myocardial hypertrophy in T2DM.
METHODS: The rats included in the experiment were divided into wild type (WT) group and T2DM group. The T2DM rat model was established by feeding the rats with high-fat and high-sugar diets for three months combined with low dose of streptozotocin (100mg/kg). Afterwards, primary rat cardiomyocytes were isolated and cultured, and cardiomyocyte hypertrophy was induced through high-glucose treatment. Subsequently, mechanistic investigations were carried out through transfection with si-STIM1 and oe-STIM1. Western blot (WB) was used to detect the expression of the STIM1, Orai1 and p-CaMKII. qRT-PCR was used to detect mRNA levels of myocardial hypertrophy marker proteins. Cell surface area was detected using TRITC-Phalloidin staining, and intracellular Ca2+ concentration in cardiomyocytes was measured using Fluo-4 fluorescence staining.
RESULTS: Through animal experiments, an upregulation of Orai1 and STIM1 was revealed in the rat model of myocardial hypertrophy induced by T2DM. Meanwhile, through cell experiments, it was found that in high glucose (HG)-induced hypertrophic cardiomyocytes, the expression of STIM1, Orai1, and p-CaMKII was upregulated, along with increased levels of store-operated Ca2+ entry (
SOCE) and abnormal Ca2+ homeostasis. However, when STIM1 was downregulated in HG-induced cardiomyocytes,
SOCE levels decreased and p-CaMKII was downregulated, resulting in an improvement in myocardial hypertrophy. To further elucidate the mechanism of action involving SOCE and CaMKII in T2DM-induced myocardial hypertrophy, high-glucose cardiomyocytes were respectively treated with BTP2 (
SOCE blocker) and KN-93 (CaMKII inhibitor), and the results showed that STIM1 can mediate
SOCE, thereby affecting the phosphorylation level of CaMKII and improving cardiomyocyte hypertrophy.
CONCLUSIONS: STIM1/Orai1-mediated
SOCE regulates p-CaMKII levels, thereby inducing myocardial hypertrophy in T2DM.