Role of neuronal nitric oxide synthase in lipopolysaccharide-induced tumor necrosis factor-alpha expression in neonatal mouse cardiomyocytes

Cardiovasc Res. 2007 Jul 15;75(2):408-16. doi: 10.1016/j.cardiores.2007.03.020. Epub 2007 Mar 30.


Objective: Neuronal nitric oxide synthase (nNOS) has been shown to regulate intracellular calcium in cardiomyocytes. Calcium in turn modulates extracellular signal-related kinase (ERK) signaling, which is important in tumor necrosis factor-alpha (TNF-alpha) expression during lipopolysaccharide (LPS) stimulation. However, the role of nNOS in LPS-induced TNF-alpha expression is not known. We hypothesized that nNOS suppresses LPS-induced TNF-alpha expression by inhibiting the calcium/ERK signaling pathway.

Methods and results: Cultured neonatal mouse cardiomyocytes were challenged with LPS for 4 h. While there was no change in the basal Ca(2+) concentration, LPS increased peak Ca(2+) levels. LPS stimulation increased TNF-alpha mRNA and protein levels in wild-type cells however, the responses were enhanced in nNOS(-/-) cardiomyocytes. Treatment with an antisense oligonucleotide against nNOS also significantly enhanced TNF-alpha expression during LPS stimulation. Furthermore, LPS-induced ERK phosphorylation was significantly increased in the nNOS(-/-) compared to wild-type cardiomyocytes. The enhanced TNF-alpha expression in nNOS(-/-) cardiomyocytes was abrogated by an L-type calcium channel blocker verapamil or ERK1 siRNA. Finally, myocardial ERK phosphorylation and TNF-alpha expression were increased while cardiac function was decreased in endotoxemia in nNOS(-/-) compared to wild-type mice.

Conclusions: nNOS inhibits LPS-induced TNF-alpha expression in cardiomyocytes and improves cardiac function in endotoxemia. The inhibitory role of nNOS is mediated by a reduction in L-type calcium channel-dependent ERK signaling in cardiomyocytes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Calcium / analysis
  • Calcium / metabolism
  • Calcium Signaling / drug effects*
  • Endotoxemia / immunology*
  • Endotoxemia / metabolism
  • Lipopolysaccharides / pharmacology
  • MAP Kinase Signaling System / drug effects
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mitogen-Activated Protein Kinase Kinases / antagonists & inhibitors
  • Mitogen-Activated Protein Kinase Kinases / genetics
  • Myocytes, Cardiac / immunology*
  • Myocytes, Cardiac / metabolism
  • Nitric Oxide Synthase Type I / analysis
  • Nitric Oxide Synthase Type I / genetics
  • Nitric Oxide Synthase Type I / metabolism*
  • Oligonucleotides, Antisense / pharmacology
  • Perfusion
  • RNA Interference
  • RNA, Small Interfering / pharmacology
  • Tumor Necrosis Factor-alpha / analysis
  • Tumor Necrosis Factor-alpha / metabolism*


  • Lipopolysaccharides
  • Oligonucleotides, Antisense
  • RNA, Small Interfering
  • Tumor Necrosis Factor-alpha
  • Nitric Oxide Synthase Type I
  • Mitogen-Activated Protein Kinase Kinases
  • Calcium