title: PDE4 inhibitor, roflumilast protects cardiomyocytes against NO-induced apoptosis via activation of PKA and Epac dual pathways.
abstract: Myocyte apoptosis plays an important role in myocardial infarction and cAMP is crucial in the regulation of myocyte apoptosis. Phosphodiesterase-4 (PDE4) inhibitor blocks the hydrolysis of cAMP via inhibition of PDE4 and is attractive candidate for novel anti-inflammatory drugs. However, its function in cardiovascular diseases and cardiomyocyte apoptosis is unclear. Therefore, we investigated whether roflumilast, a PDE4 inhibitor, exerts protective effect against NO-induced apoptosis in both of H9c2 cells and neonatal rat cardiomyocytes (NRCMs), focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). According to our data, intracellular cAMP was increased by roflumilast treatment in H9c2 cells and NRCMs. Roflumilast inhibited SNP-induced apoptosis and this effect was reversed by PKA specific inhibitor H-89 and KT-5720. In addition, PKA specific activator N(6)-benzoyladenosine 3',5-cyclic monophosphate (N(6)Bz-cAMP) mimicked the effects of roflumilast. CREB phosphorylation by roflumilast was also inhibited by H-89, indicating that roflumilast protects SNP-induced apoptosis via PKA-dependent pathway. Roflumilast increased Epac1/GTP-Rap1 and the protective effect was abolished by Epac1 siRNA transfection, demonstrating that Epac signaling was also involved in this protective response. In support, Epac specific activator 8-(4-chlrorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) protected SNP-induced apoptosis. PI3K/Akt inhibitor LY294002 blocked roflumilast-induced Akt phosphorylation and protective effect. Furthermore, inhibition of Epac1 with siRNA had no effect on roflumilast-induced CREB phosphorylation, whereas inhibited Akt phosphorylation, implicating that Akt phosphorylation was regulated by Epac pathway. In addition, it was also observed that rolipram and cilomilast exert similar effects as roflumilast. In summary, our data indicate that roflumilast protects NO-induced apoptosis via both cAMP-PKA/CREB and Epac/Akt-dependent pathway. Our study suggests a possibility of PDE4 inhibitor roflumilast as a potential therapeutic agent against myocardial ischemia/reperfusion (I/R) injury.
authors.affiliation: Division of Cardiovascular Diseases, Department of Biomedical Sciences, National Institutes of Health, 194 Tongillo, Eunpyung-gu, Seoul 122-701, Republic of Korea.
authors.forename: Hyun-Jeong
authors.initials: HJ
authors.lastname: Kwak
authors.forename: Kyoung-Mi
authors.initials: KM
authors.lastname: Park
authors.forename: Hye-Eun
authors.initials: HE
authors.lastname: Choi
authors.forename: Kyung-Sook
authors.initials: KS
authors.lastname: Chung
authors.forename: Hyun-Joung
authors.initials: HJ
authors.lastname: Lim
authors.forename: Hyun-Young
authors.initials: HY
authors.lastname: Park
mesh_terms: D000631:Aminopyridines / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D001549:Benzamides / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003521:Cyclopropanes / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D058988:Phosphodiesterase 4 Inhibitors; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D020889:Rolipram / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
chemical_list: D000631:Aminopyridines; D001549:Benzamides; D003521:Cyclopropanes; D020662:Guanine Nucleotide Exchange Factors; D020030:Nitric Oxide Donors; D058988:Phosphodiesterase 4 Inhibitors; D010726:Phosphodiesterase Inhibitors; D034741:RNA, Small Interfering; C526973:Rapgef3 protein, rat; C424423:Roflumilast; D009599:Nitroprusside; D009569:Nitric Oxide; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; D020827:rap1 GTP-Binding Proteins; D020889:Rolipram