Manivasagam Senthamizharasi, Vellaichamy Elangovan
Department of Biochemistry, University of Madras, Guindy Campus, Chennai, 600025, India.
Department of Biochemistry, University of Madras, Guindy Campus, Chennai, 600025, India.
Biochem Biophys Res Commun. 2017 Sep 16;491(2):250-256. doi: 10.1016/j.bbrc.2017.07.123. Epub 2017 Jul 22.
Npr1 gene (coding for NPR-A) and Npr2 gene (coding for NPR-B) are identified as intrinsic anti-hypertrophic genes that opposes abnormal cardiac remodeling. However, the functional role of Npr1 and Npr2 genes during cardiac hypertrophic growth is not well understood. Hence, the present investigation was aimed to study the effect of Npr1 and Npr2 gene silencing, respectively on β-AR activation induced cardiac hypertrophic growth in H9c2 cells in vitro. The control, Npr1, and Npr2 gene suppressed H9c2 cells, respectively were treated with ISO (10 M) for 48 h. The mRNA and protein expression profile of NPR-A, NPR-B, PKG-I and cGMP were analyzed by qPCR, Western blotting, ELISA, and immunofluorescence methods, respectively. A marked increase in cell size (30.10 ± 0.51 μm vs 61.83 ± 0.43 μm, 2-fold) accompanied by elevated hypertrophic marker genes (α-sk and β-MHC 3-fold, respectively) expression was observed in Npr1 gene suppressed H9c2 cells as compared with control cells. In contrast, the Npr2 gene suppression in H9c2 cells neither altered the cell size nor the level of hypertrophic marker genes expression. Upon exposure to Isoproterenol, the Npr1 suppressed H9c2 cells exhibited further increase in cell size (1.5 fold), whereas, no significant increase in cell size or marker genes expression was noticed in Npr2 suppressed cells. Moreover, the intracellular cGMP level was down-regulated by 2-fold in Npr1 suppressed cells, while, no significant change was observed in Npr2 suppressed cells. Together, these results suggest that Npr1, not Npr2 gene function is positively associated with the initiation of cardiac fetal gene program and development of cardiac hypertrophic growth.
Npr1基因（编码NPR - A）和Npr2基因（编码NPR - B）被确定为对抗异常心脏重塑的内在抗肥厚基因。然而，Npr1和Npr2基因在心脏肥厚生长过程中的功能作用尚未完全明确。因此，本研究旨在分别研究Npr1和Npr2基因沉默对体外培养的H9c2细胞中β - AR激活诱导的心脏肥厚生长的影响。将对照、Npr1和Npr2基因抑制的H9c2细胞分别用ISO（10 μM）处理48小时。分别通过qPCR、蛋白质印迹、ELISA和免疫荧光方法分析NPR - A、NPR - B、PKG - I和cGMP的mRNA和蛋白质表达谱。与对照细胞相比，在Npr1基因抑制的H9c2细胞中观察到细胞大小显著增加（30.10±0.51μm对61.83±0.43μm，增加2倍），同时肥厚标记基因（α - sk和β - MHC分别增加3倍）表达升高。相反，H9c2细胞中Npr2基因抑制既未改变细胞大小，也未改变肥厚标记基因的表达水平。暴露于异丙肾上腺素后，Npr1抑制的H9c2细胞的细胞大小进一步增加（1.5倍），而Npr2抑制的细胞中未观察到细胞大小或标记基因表达的显著增加。此外，Npr1抑制的细胞中细胞内cGMP水平下调2倍，而Npr2抑制的细胞中未观察到显著变化。总之，这些结果表明，与心脏胎儿基因程序启动和心脏肥厚生长发展呈正相关的是Npr1基因功能，而非Npr2基因功能。
