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Expression, activity, and pro-hypertrophic effects of PDE5A in cardiac myocytes

机译：PDE5A在心肌细胞中的表达，活性和促肥厚效应

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Cyclic GMP-selective phosphodiesterase type 5 (PDE5) has been traditionally thought to play a little role in cardiac myocytes, yet recent studies using selective inhibitors such as sildenafil suggest it can potently modulate acute and chronic cardiac stress responses. To date, evidence for myocyte PDE5 expression and regulation has relied on small-molecule inhibitors and anti-sera, leaving open concerns regarding nonspecific immune-reactivity, and off-target drug effects. To directly address both issues, we engineered a robust PDE5-gene silencing shRNA (inserted into miRNA-155 cassette) and DsRed-PDE5 fusion protein, both coupled to a CMV promoter and incorporated into adenoviral vectors. PDE5 mRNA and protein knock-down eliminated anti-sera positivity on immunoblots and fluorescent immuno-histochemistry in neonatal and adult cardiomyocytes, and suppressed PDE5 enzyme activity. Stimulation of myocyte hypertrophy by phenylephrine was blunted by PDE5 gene silencing in a protein kinase G dependent manner, and this effect was similar to that from sildenafil with no additive response by both combined. DsRed-PDE5 fusion protein expression showed normal z-band localization in adult myocytes but was diffused in eNOS(-/-) myocytes; echoing reported findings with anti-sera. PDE5 overexpression increased enzyme activity and amplified natriuretic peptide gene expression from phenylephrine stimulation. These data confirm PDE5 expression, activity, and targeted inhibition by sildenafil in cardiomyocytes, as well as the role of this PDE in cardiomyocyte hypertrophy modulation. (c) 2008 Elsevier Inc. All rights reserved.
机译：环状GMP选择性磷酸二酯酶类型5（PDE5）传统上被认为在心脏肌细胞中发挥一点作用，然而，最近使用Sildenafil等选择性抑制剂的研究表明它可以效果地调节急性和慢性心脏应激反应。迄今为止，肌细胞PDE5表达和调节的证据依赖于小分子抑制剂和抗血清，留下关于非特异性免疫反应性和脱靶药物作用的开放担忧。为了直接解决这两个问题，我们设计了一种稳健的PDE5-基因沉默ShRNA（插入miRNA-155盒）和DSRED-PDE5融合蛋白，均偶联至CMV启动子并掺入腺病毒载体中。 PDE5 mRNA和蛋白质敲低消除了新生儿和成人心肌细胞中免疫印迹和荧光免疫组织化学的抗血清积极性，并抑制了PDE5酶活性。通过PDE5基因沉默于蛋白激酶G依赖性方式刺激苯妥的肌细胞肥大，并且这种效果与来自Sildenafil的这种效果类似于通过组合的添加剂反应。 DSRED-PDE5融合蛋白表达显示成人肌细胞的正常Z波段定位，但在eNOS（ - / - ）肌细胞中扩散;回显报告的抗血清的发现。 PDE5过表达酶活性和来自苯妥肾上腺素刺激的扩增的利尿尿肽基因表达。这些数据证实了Sildenafil在心肌细胞中的表达，活性和靶向抑制，以及该PDE在心肌细胞肥大调制中的作用。 （c）2008年elestvier Inc.保留所有权利。

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