Role of cGMP-PKG signaling in the protection of neonatal rat cardiac myocytes subjected to simulated ischemia/reoxygenation

Abstract

Nitric oxide (NO) and B-type natriuretic peptide (BNP) are protective against ischemia-reperfusion injury as they increase intracellular cGMP level via activation of soluble (sGC) or particulate guanylate cyclases (pGC), respectively. The aim of the present study was to examine if the cGMP-elevating mediators, NO and BNP, share a common downstream signaling pathway via cGMP-dependent protein kinase (PKG) in cardiac cytoprotection. Neonatal rat cardiac myocytes in vitro were subjected to 2.5 h simulated ischemia (SI) followed by 2 h reoxygenation. Cell viability was tested by trypan blue exclusion assay. PKG activity of cardiac myocytes was assessed by phospholamban (PLB) phosphorylation determined by western blot. Cell death was 34 +/- 2% after SI/reoxygenation injury in the control group. cGMP-inducing agents significantly decreased irreversible cell injury: the cGMP analog 8-bromo-cGMP (8-Br-cGMP, 10 nM) decreased it to 13 +/- 1% (p < 0.001), the direct NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1 microM) to 18 +/- 6% (p < 0.05) and BNP (10 nM) to 12 +/- 2% (p < 0.001), respectively. This protective effect was abolished by the selective PKG inhibitor KT-5823 (600 nM) in each case. As PLB is not a unique reporter for PKG activity since it is also phosphorylated by protein kinase A (PKA), we examined PLB phosphorylation in the presence of the PKA inhibitor KT-5720 (1 microM). The ratio of pPLB/PLB significantly increased after administration of both BNP and 8-Br-cGMP under ischemic conditions, which was abolished by the PKG inhibitor. This is the first demonstration that elevated cGMP produced either by the sGC activator SNAP or the pGC activator BNP exerts cytoprotective effects via a common downstream signaling pathway involving PKG activation.

cGMP-Dependent Protein Kinase in the Regulation of Cardiovascular Functions

“…These effects are accompanied by an increased PKG activity and attenuated by PKG inhibitors or by selective knockdown of PKG in cardiomyocytes (Das, 2008 et al;Gorbe et al, 2010). It is generally recognized that ischemia/reperfusion injury arises primarily from the opening of the mitochondrial permeability transition pore (mPTP) in the first minutes of reperfusion.…”

Cytoprotective approaches to protect myocardium against ischemia/reperfusion injury

“…We have previously shown that the exogenously administered NO donor S-nitroso-nacetyl-penicillamine (SNAP) and the particulate guanylate cyclase activator B-type natriuretic peptide (BNP) exert cytoprotective effect against simulated ischemia/reoxygenation (SI/R) injury in primary neonatal rat cardiomyocytes [58]. This protection acts via the activation of cGMP/PKG signaling pathway, thereby stimulating its multiple downstream signal transduction cascade which leads to increased cell viability against SI/R injury [59,58,60].…”

“…This protection acts via the activation of cGMP/PKG signaling pathway, thereby stimulating its multiple downstream signal transduction cascade which leads to increased cell viability against SI/R injury [59,58,60].…”

“…could influence the action of matrix metalloproteinases [61,62]. Cell isolation, culturing method and the SI/R drug screening platform were described before in detail [58,63]. Briefly, neonatal rats were sacrificed by cervical dislocation and hearts were cell/well.…”

“…We used a combination of hypoxic chamber and hypoxic solution to simulate tissue ischemia [58]. In the simulated ischemic group, the medium of the cultures were replaced with a hypoxic solution [58,64] and plates were kept in a hypoxic chamber (gassed with 95% N 2 and 5% CO 2 at 37°C) for 240 min in the presence or absence of ilomastat (5 nM, 50 nM 500 nM and 5 µM).…”