Expression, activity, and pro-hypertrophic effects of PDE5A in cardiac myocytes.
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.
Zhang
Manling
M
Division of Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205, USA.
Koitabashi
Norimichi
N
Nagayama
Takahiro
T
Rambaran
Ryan
R
Feng
Ning
N
Takimoto
Eiki
E
Koenke
Trisha
T
O'Rourke
Brian
B
Champion
Hunter C
HC
Crow
Michael T
MT
Kass
David A
DA
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
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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