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This Article Full Text Full Text (PDF) Supplemental Figures All Versions of this Article: 295/5/H2178    most recent 00748.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Ionova, I. A. Articles by Pieper, G. M. PubMed PubMed Citation Articles by Ionova, I. A. Articles by Pieper, G. M.

Deficient BH₄ production via de novo and salvage pathways regulates NO responses to cytokines in adult cardiac myocytes

¹Department of Surgery (Transplant Surgery), ²Department of Biophysics, ³Cardiovascular Center, ⁴Division of Pulmonary and Critical Care Medicine, ⁵Orthopaedic Surgery, and ⁶Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 17 July 2008 ; accepted in final form 25 September 2008

Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH₄)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH₄ and 7,8-dihydrobiopterin (BH₂) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH₄ synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH₄) increased BH₄ as well as BH₂, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH₄ after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.

tetrahydrobiopterin; GTP cyclohydrolase; dihydrofolate reductase; arginase; inducible nitric oxide synthase; cardiomyocyte; sepiapterin