title: 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.
authors.affiliation: Division of Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205, USA.
authors.forename: Manling
authors.initials: M
authors.lastname: Zhang
authors.forename: Norimichi
authors.initials: N
authors.lastname: Koitabashi
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nagayama
authors.forename: Ryan
authors.initials: R
authors.lastname: Rambaran
authors.forename: Ning
authors.initials: N
authors.lastname: Feng
authors.forename: Eiki
authors.initials: E
authors.lastname: Takimoto
authors.forename: Trisha
authors.initials: T
authors.lastname: Koenke
authors.forename: Brian
authors.initials: B
authors.lastname: O'Rourke
authors.forename: Hunter C
authors.initials: HC
authors.lastname: Champion
authors.forename: Michael T
authors.initials: MT
authors.lastname: Crow
authors.forename: David A
authors.initials: DA
authors.lastname: Kass
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000235:genetics / Q000378:metabolism*; D019332:Endothelin-1 / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D045265:Natriuretic Peptides / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D058986:Phosphodiesterase 5 Inhibitors; D010879:Piperazines / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D000068677:Sildenafil Citrate; D013450:Sulfones / Q000494:pharmacology
chemical_list: D019332:Endothelin-1; D008164:Luminescent Proteins; D035683:MicroRNAs; D045265:Natriuretic Peptides; D058986:Phosphodiesterase 5 Inhibitors; D010879:Piperazines; D011687:Purines; D012333:RNA, Messenger; D013450:Sulfones; C413663:fluorescent protein 583; D010656:Phenylephrine; D000068677:Sildenafil Citrate; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D017869:Cyclic GMP-Dependent Protein Kinases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517384:Pde5a protein, rat
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