Protein Kinase G-dependent Cardioprotective Mechanism of Phosphodiesterase-5 Inhibition Involves Phosphorylation of ERK and GSK3β

Abstract

Sildenafil, a potent inhibitor of phosphodiesterase-5 (PDE-5) induces powerful protection against myocardial ischemiareperfusion injury. PDE-5 inhibition increases cGMP levels that activate cGMP-dependent protein kinase (PKG). However, the cause and effect relationship of PKG in sildenafil-induced cardioprotection and the downstream targets of PKG remain unclear. Adult ventricular myocytes were treated with sildenafil and subjected to simulated ischemia and reoxygenation. Sildenafil treatment significantly decreased cardiomyocyte necrosis and apoptosis. The PKG inhibitors, KT5823, guanosine 3,5-cyclic monophosphorothioate, 8-(4-chloro-phenylthio) (R p -8-pCPT-cGMPs), or DT-2 blocked the anti-necrotic and anti-apoptotic effect of sildenafil. Selective knockdown of PKG in cardiomyocytes with adenoviral vector containing short hairpin RNA of PKG also abolished sildenafil-induced protection. Furthermore, intra-coronary infusion of sildenafil in Langendorffisolated mouse hearts prior to ischemia-reperfusion significantly reduced myocardial infarct size after 20 min ischemia and 30 min reperfusion, which was abrogated by KT5823. Sildenafil significantly increased PKG activity in intact hearts and cardiomyocytes. Sildenafil also enhanced the Bcl-2/Bax ratio, phosphorylation of Akt, ERK1/2, and glycogen synthase kinase 3␤. All these changes (except Akt phosphorylation) were significantly blocked by KT5823 and short hairpin RNA of PKG. These studies provide the first evidence for an essential role of PKG in sildenafil-induced cardioprotection. Moreover, our results demonstrate that sildenafil activates a PKG-dependent novel signaling cascade that involves activation of ERK and inhibition of glycogen synthase kinase 3␤ leading to cytoprotection. cGMP-dependent protein kinase or protein kinase G (PKG)2 is one of the major intracellular receptors for cGMP. This serine/threonine protein kinase has two isozymes (type I and type II; i.e. PKGI and PKGII) that are presented in eukaryotic cells. The N terminus of PKGI is encoded by two alternatively spliced exons that produce the isoforms, ␣ and ␤ (1-3). PKGI␣ is mainly found in lung, heart, platelets, and cerebellum, whereas PKGI␤ is highly expressed with PKGI␣ in smooth muscles of uterus, vessels, intestine, and trachea (4, 5). More importantly, the activation of PKG phosphorylates numerous intracellular proteins that in turn regulate many important physiological functions such as control of vascular tone, cell differentiation and proliferation, and platelet aggregation (4). The role of PKG in cardioprotection was identified based on the findings that indicated a possible link between PKG activation, the opening of mitochondrial ATP-sensitive potassium (mito-K ATP ) channels (6 -8), and blockade of pharmacological preconditioning by PKG inhibitors in cardiomyocytes (9, 10). Furthermore, we recently demonstrated that adenoviral gene transfer of PKGI␣ inhibited necrosis and apoptosis following simulated ischemia and reoxygenation (SI-RO) in adult rat cardiomyocytes (11). To trans...

Overexpression of cGMP-dependent protein kinase I (PKG-I) attenuates ischemia-reperfusion-induced kidney injury

“…In addition to regulating the expression of antiapoptotic genes, other mechanisms may also contribute to the antiapoptotic effect of PKG on renal IR, such as PKG-mediated direct phosphorylation of NF-B proteins (19,24) or PKG-mediated opening of mitochondria K ATP channels and decreases in calcium influx (36). Recently, the survival signaling such as ERK or Akt has been shown to be phosphorylated by PKG in cardiomyocytes and plays a role in PKG-induced cytoprotection (13). Consistent with these reports, we found that the levels of phosphorylated ERK in the kidney were increased to a greater extent in the IR group of transgenic mice than in wild-type mice.…”

“…PKG-I is expressed in vascular smooth muscle cells, cardiomyocytes, endothelial cells, mesangial cells, renal tubular cells, macrophages, and other cell types (9). Recent studies demonstrated the protective effect of the cGMP/PKG signaling pathway on IR injury in the heart as well as in cardiomyocytes through several mechanisms such as regulation of mitochondria K ATP channels or enhanced phosphorylation of Akt, ERK, and glycogen synthase kinase (GSK)-3␤ (5,11,13) . However, whether PKG displays a protective effect on kidney IR injury is unknown.…”

Dexmedetomidine protects the heart against ischemia-reperfusion injury by an endothelial eNOS/NO dependent mechanism

“…For example, evidence shows that PKG can protect cells from IR injury by activating signaling cascades that delay mPTP opening [60][61][62].…”

Pivotal effects of phosphodiesterase inhibitors on myocyte contractility and viability in normal and ischemic hearts

“…cGMP then activates PKG, which opens the mitoKATP that prevents the loss of ionic gradients and allows for continued ATP production and calcium transport [199,200] . The protective effects of sildenafil are also associated with PKG-dependent phosphorylation of ERK and GSK3β [212] . The role of mitoKATP is essential since the cardioprotective effects of PDE5 inhibitors can be abrogated by the putative blocker of mitoKATP, 5-HD [197,202] .…”