醛固酮上调心脏TRPC通道致心肌肥厚机制研究

Cardiac hypertrophy increases the risk for atrial fibrillation, heart failure, and sudden death in patients with hypertension. Several studies have shown that excess aldosterone is associated with the development of cardiac hypertrophy. TRPC channels in general are nonselective Ca2+ permeable cation channels. However, whether TRPC channels mediate aldosterone-induced cardiac hypertrophy is still unclear. Our previous results have shown that the expression levels of TRPC1 and TRPC3 significantly increased in aldosterone-induced cardiac hypertrophy in rat. Knocking down the gene expression levels of TRPC3 significantly attenuated aldosterone-induced cardiomycytes hypertrophy. Herein, we suppose that aldosterone induces cardiac hypertrophy through upregulation of the expression level of TRPC1 and TRPC3 channels. The project was divided into two parts: first, primary cultured rat cardiomyocytes combined with molecular biology approaches will be used to determine whether aldosterone upregulated TRPC channels expression and the role of TRPC channels in aldosterone induced cardiac hypertrophy; second, TRPC1 or TRPC3 gene knockout mice will be used to further determine the role of TRPC1 and TRPC3 channels in aldosterone-induced cardiac hypertrophy. We try to clarify the role and mechanism of TRPC1 and TRPC3 channels in aldosterone-induced cardiac hypertrophy to provide new molecular target for prevention and treatment of cardiac hypertrophy.

心肌肥厚显著增加高血压患者房颤、心衰及猝死的风险。研究发现，醛固酮异常升高可导致心肌肥厚。TRPC通道是一个非选择性阳离子通道超家族，主要通透钙离子。然而，TRPC通道是否介导醛固酮诱导心肌肥厚还未见报道。我们的结果表明，醛固酮诱导大鼠心肌肥厚模型中TRPC1及TRPC3通道表达水平显著升高。体外基因敲减TRPC3通道显著抑制醛固酮诱导心肌肥厚。因此，我们假设醛固酮异常升高上调心脏TRPC通道而介导心肌肥厚发生。本课题分为两部分：第一部分，运用分子生物学技术和原代培养大鼠心肌细胞，在细胞水平探讨醛固酮对心脏TRPC通道上调的具体分子机制。第二部分，运用TRPC1及TRPC3基因敲除小鼠通过体内包埋醛固酮微量泵诱导心肌肥厚模型，进一步研究其在醛固酮诱导心肌肥厚中的作用及分子机制。本课题将首次阐明TRPC通道介导醛固酮诱导心肌肥厚的作用机制，为心肌肥厚的防治提供新的分子靶点。

1）越来越多的证据表明，经典瞬时受体电位通道TRPC1，TRPC3和TRPC6在不同病理刺激的反应中促进心肌肥厚。然而，醛固酮是否调控这些通道仍不清楚。本研究的目的是研究醛固酮是否通过调控TRPC通道介导小鼠心肌肥厚。通过大鼠皮下埋入微型泵将醛固酮（0.75μg/ h）6周。通过超声心动图和心肌细胞横截面积的测量来检测心脏肥大。TRPC1和TRPC3表达显着增加，而盐皮质激素受体拮抗剂依普利酮可逆转这个作用。醛固酮处理体外培养小鼠心室肌细胞24小时后，TRPC1和TRPC3的表达呈时间和剂量依赖性增加，并增加细胞[Ca2+]i，细胞表面积和β-肌球蛋白重链（β-MHC）。通过转染特异性TRPC1和TRPC3的siRNA显着抑制心肌细胞肥大。用细胞外信号调节激酶（ERK）途径特异性抑制剂U0126处理细胞可降低TRPC1和TRPC3表达，并抑制醛固酮诱导的心肌细胞肥大。醛固酮通过增加TRPC通道表达诱导心肌肥厚。抑制TRPC1和TRPC3可能缓解醛固酮诱导心肌肥厚。 2）发现CsA导致大鼠，野生型和ApoE敲除鼠的血压显著升高；CsA可引起上述模型鼠CCD主细胞内胆固醇蓄积、ENaC表达及活性升高。洛伐他汀可逆转上述过程。MβCD可通过降低胆固醇水平，减少PIP2与ENaC的结合，抑制ENaC活性；外源性给予胆固醇可恢复ENaC活性；衰老引起小鼠肾脏CCD主细胞中ABCA1表达水平降低，引起细胞内胆固醇蓄积而异常激活ENaC，提示ABCA1表达下降与老年高血压的发生相关。 3）小鼠肾脏CCD主细胞ABCA1基因特异性敲除后可引起细胞内胆固醇水平升高，使主细胞内氧化应激水平增加，激活SGK1/Nedd4-2信号通路，使full-length ENaC表达增加；增加furin蛋白水平，使cleaved γ-ENaC蛋白表达水平显著升高；纤毛长度变短，尿液中ATP含量降低，最终上调ENaC活性，导致高血压。洛伐他汀可通过抑制胆固醇水平缓解肾主细胞ABCA1基因特异性敲除诱导的高血压。