Copyright © 2007, European Society of Cardiology
Role of neuronal nitric oxide synthase in lipopolysaccharide-induced tumor necrosis factor-alpha expression in neonatal mouse cardiomyocytes
aCardiology Research Laboratory, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada
bDepartment of Medicine, University of Western Ontario, London, Ontario, Canada
cDepartment of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
dDepartment of Pathology, University of Western Ontario, London, Ontario, Canada
* Corresponding author. Department of Physiology and Pharmacology, University of Western Ontario, Medical Sciences Building, Rm. M254, London, Ontario, Canada N6A 5G1. Tel.: +1 519 850 2989; fax: +1 519 661 4051. qfeng{at}uwo.ca
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-
) expression during lipopolysaccharide (LPS) stimulation. However, the role of nNOS in LPS-induced TNF- expression is not known. We hypothesized that nNOS suppresses LPS-induced TNF- 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 Ca2+ concentration, LPS increased peak Ca2+ levels. LPS stimulation increased TNF- 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- expression during LPS stimulation. Furthermore, LPS-induced ERK phosphorylation was significantly increased in the nNOS–/– compared to wild-type cardiomyocytes. The enhanced TNF- expression in nNOS–/– cardiomyocytes was abrogated by an L-type calcium channel blocker verapamil or ERK1 siRNA. Finally, myocardial ERK phosphorylation and TNF- expression were increased while cardiac function was decreased in endotoxemia in nNOS–/– compared to wild-type mice.
Conclusions nNOS inhibits LPS-induced TNF- 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.
KEYWORDS Nitric oxide; Nitric oxide synthase; Calcium; TNF-; Signal transduction; Sepsis; Cardiomyocyte
1 These authors contributed equally to this work.
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