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

Basic Res Cardiol. 2010 Sep;105(5):643-50. doi: 10.1007/s00395-010-0097-0. Epub 2010 Mar 28.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Calcium-Binding Proteins / metabolism
  • Cell Survival / drug effects
  • Cell Survival / physiology
  • Cells, Cultured
  • Cyclic GMP / analogs & derivatives
  • Cyclic GMP / pharmacology
  • Cyclic GMP-Dependent Protein Kinases / metabolism*
  • Enzyme Activation / drug effects
  • Enzyme Activation / physiology
  • Myocardial Reperfusion Injury / metabolism*
  • Myocardial Reperfusion Injury / pathology*
  • Myocytes, Cardiac / cytology*
  • Myocytes, Cardiac / metabolism*
  • Natriuretic Peptide, Brain / metabolism
  • Nitric Oxide / metabolism
  • Nitric Oxide Donors / pharmacology
  • Phosphorylation / drug effects
  • Phosphorylation / physiology
  • Rats
  • Rats, Wistar
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*


  • Calcium-Binding Proteins
  • Nitric Oxide Donors
  • phospholamban
  • Natriuretic Peptide, Brain
  • 8-bromocyclic GMP
  • Nitric Oxide
  • Cyclic GMP-Dependent Protein Kinases
  • Cyclic GMP