Galectin-1通过剪接特异性调控Cav1.2钙离子通道缓解心肌细胞肥大

发布时间：2018-06-09 22:03

  本文选题：Galectin-1 + 钙离子通道　； 参考：《南京医科大学》2017年硕士论文

【摘要】：背景:由慢性压力后负荷增大而引起的病理性心肌肥大是一种较常见的心肌疾病,细胞内钙离子稳态对于细胞正常功能的维持起到重要作用,尤其是在心血管系统中,经L型钙离子通道(L-type calcium channel,LTCC)Cav1.2介导的钙离子内流影响心肌细胞兴奋-收缩耦联(excitation-contraction coupling,ECC)、平滑肌收缩等重要生理功能。在此前的研究中我们发现,钙离子通道α亚基中可变剪接外显子9*与通道电生理性质有关。半乳糖凝集素1(Galectin-1,Gal-1)作为一种半乳糖结合蛋白,能够在血管中通过与不含9*的Cav1.2可变剪接体Cav1.2 CM△9*结合发挥调节血管收缩的作用。然而,Gal-1在心肌细胞中与Cav1.2的作用尚不明确。目的:本实验探究Gal-1与外显子9*在心肌肥大过程中的表达变化,阐明Gal-1在心肌肥大过程中的作用。方法:运用Western blot和RT-PCR检测在不同周龄WKY/SHR大鼠和胸主动脉缩窄术后大鼠心脏中Gal-1与外显子9*的表达情况;运用全细胞膜片钳检测分离的原代心肌细胞以及表达不同可变剪接亚型Cav1.2通道的HEK293细胞钙电流,分别观察过表达Gal-1对其的影响;使用Fluo-4动态监测心肌细胞内钙离子浓度变化;使用real-time PCR检测过表达Gal-1对异丙肾上腺素(Isoproterenol,ISO)诱导的原代心肌细胞肥大的影响;使用免疫荧光染色检测过表达Gal-1对ISO诱导的肥大心肌细胞表面积的影响;使用KN93、H89及ISO等药物研究Gal-1对CaMKII-HDAC4通路的影响。结果:我们发现1)Gal-1在心肌肥大组织中表达明显增高,同时它与肥大心肌中外显子9*的表达升高有相关性;2)在乳鼠原代心肌细胞中过表达Gal-1能够降低钙电流,能够缓解短时程的由胞外刺激所引发的细胞内钙浓度异常升高;此外,3)过表达Gal-1能够减少由ISO或Bay K8644引起的CaMKII-HDAC4通路的激活从而抑制肥大基因的表达,减小细胞表面积,抑制心肌肥大。Gal-1可能能够作为治疗心肌肥大的一个靶点。结论:Gal-1能够通过与不含剪接外显子9*的Cav1.2 α1C亚基Ⅰ-Ⅱ loop结合发挥降低[Ca2+]i水平的功能,继而影响δCaMKII-HDAC4通路,最终导致肥大相关基因转录降低,改善了心肌肥大过程。Gal-1在肥大心脏中表达上调可能是病理过程中的一种代偿机制。
[Abstract]:Background: pathological myocardial hypertrophy caused by chronic pressure overload is a common myocardial disease. Intracellular calcium homeostasis plays an important role in the maintenance of normal cell function, especially in the cardiovascular system. Calcium influx mediated by L-type calcium channel (L-type calcium channel) affects the excitation-contractile coupling of cardiomyocytes, smooth muscle contraction and other important physiological functions. In previous studies, we have found that the variable splicing exon 9 * in a subunit of calcium channel is related to the electrophysiological properties of the channel. As a galactose-binding protein, galactosamine 1 Galectin-1 (Gal-1) can regulate vasoconstriction by binding with Cav1.2 alternative splice Cav1.2 CM9 *, which does not contain 9 *. However, the role of Gal-1 and Cav1.2 in cardiomyocytes is unclear. Aim: to investigate the expression of Gal-1 and exon 9 * in the process of myocardial hypertrophy and to elucidate the role of Gal-1 in the process of myocardial hypertrophy. Methods: Western blot and RT-PCR were used to detect the expression of Gal-1 and exon 9 * in the hearts of WKY / SHR rats and rats after thoracic aortic coarctation. The effects of Gal-1 expression on calcium current in isolated primary cardiomyocytes and HEK293 cells expressing different alternative splicing subtypes of Cav1.2 channel were detected by whole-cell patch clamp method, and the changes of intracellular calcium concentration in cardiac myocytes were monitored dynamically by Fluo-4. The effects of overexpression of Gal-1 on primary cardiomyocyte hypertrophy induced by isoprenaline isoproterenolISO and the effect of overexpression of Gal-1 on the surface area of ISO-induced hypertrophic cardiomyocytes were detected by real-time PCR and immunofluorescence staining. The effects of Gal-1 on CaMKII-HDAC4 pathway were studied by using KN93 H89 and ISO. Results: we found that the expression of Gal-1 was significantly increased in hypertrophic myocardium, and it was correlated with the expression of exon 9 * in hypertrophic myocardium.) overexpression of Gal-1 in primary neonatal rat cardiomyocytes decreased calcium current. The overexpression of Gal-1 reduced the activation of CaMKII-HDAC4 pathway induced by ISO or Bay K8644, which inhibited the expression of hypertrophic genes and reduced the surface area of cells. Inhibition of myocardial hypertrophy. Gal-1 may be a target for the treatment of myocardial hypertrophy. Conclusion: Genus Gal-1 can reduce [Ca 2] I level by binding to Cav1.2 伪 1C subunit 鈪，

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