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 µM) 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 μM). 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.

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Abbreviations

8-Br-cGMP:

8-Bromo-cGMP

BNP:

B-type natriuretic peptide

CNG:

Cyclic nucleotide-gated ion channel

KATP :

ATP-sensitive potassium channel

KT-5720:

Selective PKA inhibitor

KT-5823:

Selective PKG inhibitor

NO:

Nitric oxide

NOS:

Nitric oxide synthase

PDE:

Phosphodiesterase

pGC:

Particulate guanylate cyclase

PKA:

cAMP-dependent protein kinase

PKG:

cGMP-dependent protein kinase

PLB:

Phospholamban

pPLB:

Phosphorylated phospholamban

SERCA:

Sarcoplasmic reticulum Ca2+-ATPase

sGC:

Soluble guanylate cyclase

SI:

Simulated ischemia

SNAP:

S-Nitroso-N-acetylpenicillamine

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Acknowledgments

This work was supported by Wellcome Trust Collaborative Initiative project grant (GR074567MA) awarded to PF and GFB and grants from the Hungarian Scientific Research Fund (OTKA F046810), Hungarian Ministry of Health Grants ETT 597/2006, National Office for Research and Technology Grants NKTH-Jedlik-MMP (OM-00066/2007), Jedlik-ES (OM-00206/2007) and Jedlik MEDFOOD (OM-00131/2008).

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The authors state no conflict of interest.

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Correspondence to Peter Ferdinandy.

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Gorbe, A., Giricz, Z., Szunyog, A. et al. Role of cGMP-PKG signaling in the protection of neonatal rat cardiac myocytes subjected to simulated ischemia/reoxygenation. Basic Res Cardiol 105, 643–650 (2010). https://doi.org/10.1007/s00395-010-0097-0

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Keywords

  • Simulated ischemia
  • Cardiomyocyte
  • NO
  • BNP
  • cGMP
  • Protein kinase G