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

Abstract

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 non-specific 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.

Figure 1

A) Schematic of shRNAmiR construct. See method for details. B) AdV-gfp-shRNA^PDE5a suppresses PDE5a mRNA in RNCM. C) Mouse PDE5a overexpression in HEK293 cells is blocked by two different shRNA^PDE5a ; D) Endogenous PDE5 expression in RNCM are inhibited AdV-gfp-shRNA^PDE5a but not control AdV-gfp transfection E) Basal PDE5a enzyme activity is suppressed by AdV-gfp-shRNA^PDE5a ; F) Adult mouse myocytes infected with AdV-gfp-shRNA^PDE5a show reduced protein expression.

Figure 2

A) fluorescent immunohistochemistry of RNCM infected with AdV containing gfp or gfp- shRNA^PDE5a . The latter shows suppression of anti-sera PDE5a immunostaining; B) Adult myocytes infected in vivo with AAV encoding DsRed-shRNA^PDE5a were isolated and stained for PDE5a. Transfected cells (red) no longer co-stained for PDE5a (green).

Figure 3

Cardiomyocyte hypertrophic signaling reflected by natriuretic peptide gene expression induced by phenylephrine (PE) is suppressed similarly by PDE5a gene silencing and by sildenafil. The effect of gene silencing is blocked by co-incubation with a PKG inhibitor; A) Results for BNP stimulation, B) Results for ANP gene expression.

Figure 4

Myocyte ³H-leucine incorporation from endothelin-1 stimulation is blocked by PDE5a gene silencing.

Figure 5

A) Localization of DsRed-PDE5a is at the z-band of the cardiac myocyte, similar to results reported using anti-sera, while it became diffuse in eNOS^-/- cardiomyocytes. B) Over-expression of DsRed-PDE5a in neonatal myocytes increases enzyme activity. C) Enhanced PDE5a expression augments PE-induced stimulation of ANP and BNP gene expression.