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 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 knockdown 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 diffuse in eNOS -/-myocytes; echoing reported findings with anti-sera. PDE5 over-expression 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.

Phosphodiesterase inhibition with tadalafil provides longer and sustained protection of stem cells

“…Inhibition of PDE5 in the hypertrophied myocardium significantly improved myocardial contractility (17). Unlike previous reports, more recent studies have shown that PDE5A was expressed in the cardiomyocytes, and targeted inhibition of its activity with sildenafil modulated the cardiomyocyte hypertrophy response to phenylephrine treatment in vitro (27). Extrapolating these observations of cytoprotective effects to the bone marrow-derived mesenchymal stem cells (MSCs), we hypothesized that inhibition of PDE5A activity with tadalafil would promote cGMP/protein kinase G (PKG) activity to condition stem cells for improved survival and proliferation postengraftment in the infarcted heart.…”

Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury

“…The EmGFP linked to the artificial miRNA region was then transferred into pAd.CMV-DEST by recombinase cloning (Invitrogen). A control adenovirus (Ad.Neg-shRNA) containing EmGFP and a control shRNAmiR that was predicted not to target any known vertebrate gene was reported earlier (54). The initial viral amplification and purification was performed in our laboratory using an AdenopureLS adenovirus purification kit (Puresyn; Malvern, PA) according to the manufacturer's instructions.…”

Regulator of G protein signaling 2 mediates cardiac compensation to pressure overload and antihypertrophic effects of PDE5 inhibition in mice

“…Second, PDE5 inhibition suppresses Cn/nuclear factor of activated T cell (Cn/NFAT) signaling and expression of the genes encoding atrial and brain natriuretic peptides in isolated myocytes stimulated with a G q agonist (21,44). Third, knockdown of the gene encoding PDE5 in cardiac myocytes is antihypertrophic, having an impact similar to that of sildenafil, and the effect of combining both is similar to that of each alone (44). This last finding supports specificity of sildenafil for inhibiting PDE5.…”