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Am J Physiol Heart Circ Physiol 295: H2178-H2187, 2008. First published October 3, 2008; doi:10.1152/ajpheart.00748.2008
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Deficient BH4 production via de novo and salvage pathways regulates NO responses to cytokines in adult cardiac myocytes

Irina A. Ionova,1 Jeannette Vásquez-Vivar,2,6 Jennifer Whitsett,2 Anja Herrnreiter,2 Meetha Medhora,3,4 Brian C. Cooley,5 and Galen M. Pieper1,3,6

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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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

THE EXACT ROLE of nitric oxide (NO) on heart function is not fully established. Low levels of NO production from constitutive NO synthase (NOS) have been shown to exert a positive inotropic effect on left ventricular function (47). In contrast, high levels of NO production have been shown to have a negative impact on the heart (33). Upregulation of inducible NOS (iNOS) results in high levels of NO production in a variety of cell types and tissues. In the heart, it is generally understood that high levels of NO depress mitochondrial respiration (4) and cardiac function (33) and cause apoptosis of cardiac myocytes (1, 48). Thus, iNOS expression is generally linked to cardiac failure (36), sepsis (36), and acute cardiac transplant rejection (5, 45). It has also been shown that LPS and inflammatory cytokines increase iNOS expression leading to injury of isolated cardiac myocytes in vitro (40).

A primary determinant of NO production by iNOS is the level of protein expression. However, actual NO bioactivity can be controlled by additional mechanisms. One of the prominent factors regulating iNOS activity is the cofactor tetrahydrobiopterin (BH4). This has been indicated by a study (60) showing that coordinated increases in intracellular BH4 synthesis are essential for full activity of NO production by cells overexpressing iNOS. BH4 facilitates full catalytic activity of iNOS by several mechanisms including homodimerization.

The intracellular levels of BH4 are determined by de novo synthesis via the expression of GTP cyclohydrolase I. There is evidence that BH4 synthesis correlates well with expression of GTP cyclohydrolase I and to a lesser extent with expression of 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, the other enzymes involved in the biosynthetic pathway of BH4 (59) (as shown in Fig. 1). Alternative to de novo synthesis, intracellular BH4 can be increased via the salvage pathway using either 7,8-dihydrobiopterin (BH2) or sepiapterin as substrates. Enzymes important for the salvage pathway include sepiapterin reductase and dihydrofolate reductase.


Figure 1
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  		Fig. 1. Diagram showing the pathways and key enzymatic reactions of de novo and salvage pathway synthesis of tetrahydrobiopterin (BH4). BH2, 7,8-dihydrobiopterin.

 
More recently, it has been shown that the activity of GTP cyclohydrolase I and, thus, the production of BH4 can also be determined by the levels of its negative allosteric regulator, GTP cyclohydrolase feedback regulator protein (GFRP) (16, 22, 63). Thus, either upregulation of GTP cyclohydrolase expression or downregulation of GFRP can individually increase intracellular levels of BH4. The relative contribution of the de novo and salvage pathways and the contribution of the allosteric regulation of GTP cyclohydrolase to the cellular availability of BH4, however, varies depending on the cell type. To date, no published data exist on either the constitutive expression of the various genes regulating BH4 synthesis from both de novo and salvage pathways or the regulation of their expression under cytokine stimulation in adult cardiac myocytes.

The possibility that BH4 synthesis might be suboptimal or impaired in adult cardiac myocytes arises from two recent studies in our laboratories. First, in acute cardiac allograft rejection in vivo, we found a decline in myocardial GTP cyclohydrolase activity and production of total biopterin in the late stages of acute rejection that coincided with the onset of cardiac dysfunction (44). This coincided with a shift in iNOS expression from infiltrating macrophages to cardiac myocytes (G. M. Pieper, unpublished observations). This suggested the possibility that BH4 synthesis may not be coordinated with iNOS upregulation in cardiac myocytes (44). Second, it was independently found that total biopterin levels in isolated adult rat cardiac myocytes expressing iNOS by stimulation in vitro with LPS were a fraction of the total biopterin synthesized by endothelial cells in response to the same range of concentrations of LPS, indicating that adult cardiac myocytes exhibit atypical responses to inflammatory stimuli (22).

The initial finding that increases in total biopterin (i.e., the sum of BH4 + BH2 and biopterin) levels in response to LPS were blunted in response of adult cardiac myocytes suggests the possibility that BH4 levels might also be low or that the amount of BH4 relative to BH2 may not be sufficient to support NO production when iNOS is upregulated in cardiac myocytes by LPS or other inflammatory stimuli. In support of this concept, using an improved technique that directly measures BH4, we (61) confirmed that authentic BH4 levels in adult cardiac myocytes are not increased dramatically in response to LPS and that the consequences resulted in no or little significant increase in NO production.

Currently, there is no other information available on levels of BH4 or BH2 in adult cardiac myocytes under basal conditions or stimulated with other inflammatory agents such as cytokines important in various myocardial inflammatory conditions. After our preliminary findings, the purpose of the present study was to characterize, in detail, BH4 production as well as the expression of various genes responsible for de novo BH4 synthesis in adult cardiac myocytes stimulated to express iNOS by various cytokines. We examined BH4 and NO levels as well as the expression of various genes known to regulate BH4 production (as enumerated above) and NO bioactivity (e.g., iNOS and arginase 1 and 2) to understand their role in the regulation of iNOS activity in adult cardiac myocytes. To circumvent the low BH4 synthesis via the de novo pathway, we also examined the impact of supplementation with sepiapterin to increase intracellular BH4 via the salvage pathway.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. The experiments conducted on animals in this study were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23). All procedures were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

Isolation of adult or neonatal cardiac myocytes. Male Sprague-Dawley rats (Harlan Sprague Dawley) were used for these experiments. Adult cardiac myocytes were isolated by standard collagenase digestion procedures as originally described (62). Cardiac myocytes were plated on to 100-mm plates precoated with laminin (Invitrogen, Carlsbad, CA). For in vitro culture, media 199 (Invitrogen) supplemented with 0.2% BSA, 10 nM insulin, 5 mM creatine, 2 mM L-carnitine, 5 mM taurine, 100 U/ml penicillin, and 100 mg/ml streptomycin was used. Under the conditions of these experiments, cardiac myocytes displayed the typical rod-shaped morphology with striation typical of that seen in culture of adult cardiac myocytes (see Suppl. Fig. 1).1 Furthermore, we have shown that these adult cardiac myocytes in culture retain normal aconitase enzyme activity (61), similar in magnitude to that see in native rat hearts (43).

In some cases, for comparison of constitutive or stimulated gene expression, neonatal cardiac myocytes from 1-day-old Sprague-Dawley rats were isolated and cultured as previously described (9). Briefly, myocytes were extracted by collagenase digestion. The cells derived were suspended in DMEM supplemented with 17% media 199, 10% horse serum, 5% FBS, 0.5% penicillin-streptomycin, and 20 mM HEPES (pH 7.2) for 2 h. Faster-attaching nonmyocyte cells were allowed to adhere to plastic, and ventricular myocytes in the supernatant were collected and plated on gelatin-coated dishes. Neonatal cells were cultured in media containing 0.1 mM bromodeoxyuridine before experiments were performed. Cells displayed typical rhythmic contractions up to at least 72 h. Cells prepared in this way displayed phenotypes similar to those seen in the HL-1 cardiac cell line (9).

Cytokine stimulation. Adult or neonatal cardiac myocytes in culture were treated or not for up to 24 h with recombinant rat TNF-{alpha} (20 ng/ml), IFN-{gamma} (50 ng/ml), IL-1β (20 ng/ml), or the combination of TNF-{alpha} and IFN-{gamma} (R&D Systems, Minneapolis, MN). We used cytokines that are known in many studies to induce iNOS protein in isolated cardiac myocytes and that are also upregulated in acute cardiac allograft rejection. The important role of these cytokines on cardiac rejection and graft survival has been previously demonstrated (7, 53, 56). In vivo, these cytokines are produced mostly by infiltrating T lymphocytes or by macrophage cells. Therefore, graft parenchymal cells, including cardiac myocytes, are exposed to these cytokines. We used three different individual cytokines in the present study to show that the response of adult cardiac myocytes is not unique to a single cytokine. In additional experiments, cardiac myocytes were pretreated for 6 h with 10 µM sepiapterin (Schircks Laboratories, Jona, Switzerland). This concentration has been shown to increase intracellular biopterin by 10- to 20-fold and to increase NO in a variety of cell types (34, 38, 51). After supplementation, cytokines were added, and cells were incubated for an additional 12 h.

Rat cardiac allograft transplantation. Heterotopic abdominal aortic transplantation of Wistar-Furth donor hearts (allografts) or Lewis donor hearts (isografts) to Lewis recipient rats was performed as previously described by this laboratory (8). Cardiac grafts were harvested from pentobarbital-anesthetized (50 mg/kg ip) rats. For the determination of cardiac iNOS monomer and BH4 levels, some allograft recipient animals received daily intraperitoneal injections of 10 mg/kg sepiapterin (Schircks Laboratories) starting the day of transplantation after surgery. Transplanted hearts were harvested on postoperative day 6, and the left ventricles were evaluated for Western blot or HPLC analysis to determine iNOS monomer and BH4 levels, respectively.

Western blot analysis. For Western blot analysis, myocytes were lysed with 1x RIPA buffer (Upstate, Charlottesville, VA) supplemented with 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and protease inhibitor cocktail tablets (Roche, Basel, Switzerland). Samples were processed under reducing or nonreducing conditions (with and without β-mercaptoethanol, respectively) for iNOS homodimerization experiments after cells had been lysed in PBS buffer (pH 7.4) supplemented with 0.1% Triton X-100, 2 mM L-arginine, 20 µM BH4,1 mM PMSF, and protease inhibitor cocktail tablets. Total protein concentrations were determined using the Bio-Rad DC Protein Assay. Cell lysates were mixed with 2x Laemmli sample buffer before being loaded. Following the protocol for Western blot analysis under reducing conditions, samples were also heated at 100°C for 5 min before being loaded. Proteins were resolved by 7.5% or 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed at 4°C overnight with antibodies for GTP cyclohydrolase, iNOS (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, or Cayman Chemical, Anna Arbor, MI), dihydrofolate reductase (1:500, Abnova, Walnut, CA), or GAPDH (1:1,000, Chemicon, Temecula, CA). Immunoblots were developed with the SuperSignal West Femto Maximum Sensitivity Substrate kit (Pierce, Rockford, IL).

RT-PCR analysis. Total cellular RNA was isolated from myocytes using 1 ml of TRIzol reagent (Invitrogen) per 100-mm culture plate according to the manufacturer's protocol. Genomic DNA was digested by treatment with RNase-free DNase (Ambion, Austin, TX), and RNA concentrations were determined spectrophotometrically. cDNA was synthesized from 1 µg of total RNA using random hexamers primers and the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's directions. Primers and conditions are shown in Table 1. PCRs were performed in a 25-µl volume containing 1 µl of cDNA, 25 pmol of sequence-specific primers, and 22.5 µl of Platinum PCR SuperMix High Fidelity (Invitrogen). A 10-µl aliquot of the PCR product was resolved by 1% TAE-agarose gel electrophoresis. Densitometric analysis of specific bands was performed using an Alpha Imager (Alpha Innotech, San Leandro, CA).


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  		Table 1. Primers and conditions used for RT-PCR analyses

 
NO measurements. NO production into media was measured from individual cell preparations. Media from cell cultures were filtered through 10-kDa molecular mass cutoff filters at 14,000 rpm for 30 min. Accumulation of total nitrate plus nitrite (NOx) was determined by chemiluminescence using a NO Analyzer (Sievers Instruments, Boulder, CO) and normalized to cell protein.

HPLC measurements. Quantification of BH4 and BH2 was performed by HPLC with electrochemical detection. Values were normalized to protein content. Details of cell preparation and HPLC methods used have been described in detail elsewhere (64).

Statistical analysis. Data are expressed as mean values ± SE. Statistical analysis was performed using ANOVA followed by the Student-Newman-Keuls test for multiple group means or Student's t-test for comparison between two group means. The level of significance was set at P values of <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We established conditions in which iNOS expression was increased in response of cardiac myocytes to several cytokines known to be produced in acute cardiac rejection in vivo (7, 53, 56) and then determined the expression of genes that function to regulate BH4 synthesis in these cells. In unstimulated adult rat cardiac myocytes, expression of iNOS mRNA or protein (Fig. 2, A and B) was not detected. In contrast, iNOS mRNA was upregulated by three different cytokines after 12 h (Fig. 2A) or 24 h (Suppl. Fig. 2) of stimulation of cardiac myocytes. The largest increase was seen using the combination of TNF-{alpha} + IFN-{gamma}, which peaked by 12 h (i.e., iNOS-to-GAPDH ratio: 1.20, 1.12, and 1.20 for 12, 18, and 24 h, respectively). Levels of both mRNA and protein for GTP cyclohydrolase were found constitutively expressed and also upregulated by cytokine stimulation (Fig. 2, A and B). Expression of iNOS protein was upregulated by 12 h with individual cytokines or the combination of TNF-{alpha} + IFN-{gamma} (Fig. 2B).


Figure 2
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  		Fig. 2. A: mRNA levels for inducible nitric oxide (NO) synthase (iNOS) and GTP cyclohydrolase (GTPCH) in cytokine-stimulated cardiac myocytes for 12 h. Similar findings were observed at 18 h (not shown). Ctrl, control. B: Western blots for protein expression conducted under the same experimental conditions.

 
To determine the functional consequences of GTP cyclohydrolase expression on biopterin synthesis, we measured the intracellular levels of both BH4 and BH2. We found insignificant changes in BH4 after stimulation with various cytokines (Fig. 3A). Levels of BH2 were undetected under both resting and cytokine stimulation conditions. In agreement with the impaired BH4 production, stimulation of cardiac myocytes for 12 h with cytokines did not result in any significant increase in NO production, as determined by nitrite alone (not shown) or NOx released into media despite the upregulation of iNOS (Fig. 3B). After 24 h of stimulation, there was no significant increase in NO release (NOx) to individual cytokines but a significant (P < 0.05) but small increase using the combination of TNF-{alpha} + IFN-{gamma} (Suppl. Fig. 3). For perspective and as a positive control, NO release in neonatal cardiac myocytes stimulated with TNF-{alpha} + IFN-{gamma} was shown to be significantly increased by 12-fold compared with that seen in adult cardiac myocytes stimulated under comparable conditions (Suppl. Fig. 3B).


Figure 3
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  		Fig. 3. A: HPLC analysis showing no significant change in BH4 levels in cardiac myocytes stimulated for 12 h. B: NO production [nitrate + nitrite (NOx)] in media for cardiac myocytes stimulated for 12 h with the same cytokine groups (n = at least 3 experiments/group).

 
In addition to the increased expression of GTP cyclohydrolase, we found that stimulation with cytokines for 12 h (Fig. 4A) also induced mRNA expression of the allosteric inhibitor of GTP cyclohydrolase GFRP. GFRP expression was not detected under unstimulated conditions. mRNA expression of both 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase was not upregulated by cytokine exposure.


Figure 4
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  		Fig. 4. Effects of cytokine stimulation of cardiac myocytes for 12 h on mRNA expression of GTPCH feedback regulatory protein (GFRP), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR).

 
Production of NO from iNOS requires arginine substrate and the arginine transporter. Culture media 199 contained adequate amount of substrate at 332 µM L-arginine. We also examined the expression of the cationic amino transporter (CAT-1) by RT-PCR. Constitutive expression of high-affinity CAT-1 in unstimulated cardiac myocytes was shown as well as upregulation of CAT-1 under all conditions of cytokine exposure (Fig. 5B). We did not detect the expression of low-affinity CAT-2 under any of the experimental conditions.


Figure 5
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  		Fig. 5. A: mRNA expression of arginase 1 and arginase 2 in cardiac myocytes without or with cytokine stimulation. +, Positive control for PCR (rat liver for arginase 1 and rat kidney for arginase 2). B: mRNA expression for cationic amino transporter (CAT)-1 and CAT-2 in cardiac myocytes with and without cytokine stimulation. The rat liver and kidney were used as positive controls.

 
We also considered the possibility that arginase expression is induced by cytokine stimulation as an alternative to explain low NO production. This enzyme competes for the substrate arginine; thereby, it may potentially diminish NO production. Using RT-PCR, we found no evidence for the expression of either arginase 1 or 2 isoforms in any preparation of adult cardiac myocytes whether unstimulated or after stimulation with inflammatory cytokines compared with positive controls for arginase 1 and 2 using rat liver and kidney samples, respectively (Fig. 5A). Similar results were found in samples after 24 h of stimulation with cytokines (not shown). However, the lack of expression appears to be unique to adult cells as we were able to show mRNA for arginase 1 but not arginase 2 in neonatal rat cardiac myocytes under both constitutive and cytokine-stimulated conditions (Suppl. Fig. 4). In support of this concept, we did not detect mRNA for either arginase 1 and 2 in the rat heart compared with high expression in rat liver and kidney positive controls (Suppl. Fig. 5).

To test the idea that BH4 is the limiting factor hampering iNOS activity, unstimulated and cytokine-stimulated adult cardiac myocytes were supplemented with sepiapterin, a precursor of BH4 synthesis via the salvage pathway. First, unstimulated cardiac myocytes were incubated for 3, 6, or 12 h with sepiapterin. Under these conditions, sepiapterin caused a time-dependent increase in intracellular BH4 levels (Fig. 6A), but this was also associated with a time-dependent increase in BH2 levels (Fig. 6B), which, in most cases, was remarkably higher than BH4 concentrations. In each case, sepiapterin did not cause any significant increase in NO released into media regardless of the incubation time (not shown). In cells preincubated with sepiapterin, a relatively small but significant increase in the level of BH4 (Fig. 7A) as well as BH2 (Fig. 7B) was observed after stimulation with TNF-{alpha} + IFN-{gamma} compared with unstimulated cells preincubated with sepiapterin. However, total media NO metabolite levels were not increased by cytokine stimulation in the presence of sepiapterin (0.269 ± 0.47 vs. 0.235 ± 0.109 nmol/mg protein without and with cytokines, respectively, n = 4–5 each).


Figure 6
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  		Fig. 6. Effect of incubation time (3, 6, and 12 h) with sepiapterin in Ctrl unstimulated cardiac myocytes on intracellular BH4 (A) and BH2 (B) (n = 3–4 experiments/group). *P < 0.05 vs. without sepiapterin.

 

Figure 7
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  		Fig. 7. A and B: intracellular BH4 (A) and BH2 (B) (n = 3 experiments/group) by preincubation of cardiac myocytes with sepiapterin (S) in Ctrl or cytokine-stimulated (Cyto; TNF-{alpha} + IFN-{gamma}) cells. *P < 0.05 vs. without sepiapterin; (*)P < 0.05 vs. Ctrl.

 
In consideration of the suboptimal increase in BH4 upon exposure of unstimulated cardiac myocytes to sepiapterin along with the findings of remarkably high BH2 levels, we theorized that these results were explained by a dysfunctional capacity to convert BH2 to BH4. To address this possibility, we examined the expression of dihydrofolate reductase. Dihydrofolate reductase mRNA was not detectable in either unstimulated adult cardiac myocytes or after stimulation with inflammatory cytokines (Fig. 8A). This was in contrast to dihydrofolate reductase mRNA detected using the rat liver as a positive control that was analyzed concurrently. Furthermore, Western blot analysis also confirmed the absence of protein expression of dihydrofolate reductase in these cardiac myocytes under both unstimulated control conditions and after stimulation with various cytokines compared with the internal positive rat liver control sample (Fig. 8B). These findings contrast with the constitutive expression of dihydrofolate reductase observed in both H9c2 embryonic cardiac cells (not shown) and in neonate rat cardiac myocytes of the same Sprague-Dawley strain with and without cyotokine stimulation (Suppl. Fig. 4). Thus, the absence of dihydrofolate reductase expression was unique to fully differentiated adult cardiac myocytes.


Figure 8
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  		Fig. 8. A: RT-PCR showing mRNA expression of dihydrofolate reductase (DHFR) in the positive control liver (labeled as +) but not in adult cardiac myocytes with or without stimulation of various cytokines. B: Western blot analysis confirming the absence of DHFR protein in adult cardiac myocytes.

 
Finally, we examined the monomer-dimer distribution of cardiac myocytes stimulated with cytokines. Normally, under reducing conditions, only iNOS monomers were found upon cytokine stimulation (Figs. 2B and 9B, right). Under nonreducing conditions, we also found significant levels of inactive 130-kDa iNOS monomers in cells stimulated with individual cytokines or the combination of TNF-{alpha} + IFN-{gamma} (Fig. 9, A and B). The presence of sepiapterin did not change the pattern of iNOS monomer levels (Fig. 9B) or total iNOS protein expression (not shown).


Figure 9
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  		Fig. 9. A: Western blot analysis run under nonreducing conditions showing iNOS dimers and/or monomers in cardiac myocytes stimulated with cytokines for 12 h. B: Western blots run under nonreducing and reducing conditions indicating that sepiapterin (Sepi) does not alter iNOS expression or iNOS monomer levels. On both A and B, open arrows indicates iNOS dimers and solid arrows indicate iNOS monomers.

 
To show that this response to sepiapterin is relevant to in vivo conditions, rat cardiac allograft recipients were treated with sepiapterin. Cardiac levels of BH4 were increased by treatment with sepiapterin compared with allograft recipients not receiving sepiapterin treatment (Fig. 10). There also was a large sepiapterin-induced increase in BH2 levels (Fig. 10). Similar to the findings in vitro, treatment with sepiapterin did not alter cardiac NO content (not shown) or iNOS monomer levels (Fig. 11).


Figure 10
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  		Fig. 10. Effect of treatment cardiac allograft recipients (n = 5–6 animals/group) with sepiapterin in vivo on levels of cardiac BH4 and BH2 as determined by HPLC with electrochemical detection. Iso, isografts; Allo, untreated allografts; Sep, sepiapterin-treated allografts. *P < 0.05 vs. isografts; (*)P < 0.05 vs. untreated allografts.

 

Figure 11
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  		Fig. 11. Presence of inactive iNOS monomers in cardiac allografts at postoperative day 6 and lack of change by treatment of recipients with sepiapterin. A: representative Western blot; B: densitometric analysis (n = 3 to 4 experiments each).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we show new evidence that adult cardiac myocytes have low levels of de novo BH4 synthesis as well as impaired BH4 synthesis via the salvage pathway as revealed by experiments using sepiapterin supplementation. Consequently, adult cardiac myocytes show a phenotypic response to inflammatory cytokines that is distinctly different than that previously reported in other cell types (15, 22), including neonatal cardiac myocytes, in which the typical high BH4 and/or NO production are manifested.

Our study provides new insight for previous observations by several independent laboratories of low or undetectable NO production despite upregulation of iNOS mRNA and protein in adult cardiac myocytes stimulated with inflammatory cytokines (14, 15, 46, 52, 65) compared with the 10- to 80-fold increases in NO production in cytokine-stimulated neonatal cardiac myocytes (21, 26, 39, 58) for which there was no forthcoming explanation. The mechanism of defective NO production was found in our study to involve defective BH4 synthesis, which is related to suboptimal upregulation of GTP cyclohydrolase. The defective NO production cannot be explained by a cytokine-induced upregulation of arginase, which would compete for the arginine substrate for the production of NO by iNOS.

Regulation of BH4 synthesis by the de novo pathway. The only previous information on the regulation of biopterin synthesis in cardiac myocytes in response to cytokine stimulation arises from studies by one research group but conducted in neonatal cardiac myocyte preparations. These studies showed that stimulation with a cytokine combination of IL-1 and IFN-{gamma} increased both GTP cyclohydrolase and iNOS activity (18, 24). In this case, biopterin (an indirect measure of BH4) levels were increased by 13- to 14-fold and NO production was increased in the range of 30- to 80-fold. It is noteworthy that the magnitude of the increase in biopterin reported in neonatal cardiac myocytes was large and clearly distinguished from the previous findings in adult cardiac myocytes of small increases in LPS-stimulated total biopterin levels (22) and confirmed here by the small increases in cytokine-stimulated BH4 levels.

In the present study, we show, for the first time, that several cytokines, in general, upregulated both GTP cyclohydrolase mRNA and protein levels in adult cardiac myocytes but that the increase in protein lacked significant activity. The conclusion about GTP cyclohydrolase activity is based on the marginal increase in BH4 levels. This finding is consistent with initial data showing that GTP cyclohydrolase activity in adult rat cardiac myocytes is marginal and is not increased with either a high dose of LPS or by incubation of unstimulated cardiac myocytes with sepiapterin (22). Similar results were shown in a study (61) with adult cardiac myocytes stimulated with either LPS or the combination of IFN-{gamma} + IL-1{alpha} at doses that produced marked increases in GTP cyclohydrolase activity in RAW264.7 macrophage-like cells. Finally, no increase in GTP cyclohydrolase activity could be detected using the combination of TNF-{alpha} + IFN-{gamma} (unpublished observations). Collectively, this suggests that the protein in adult cardiac myocytes is mostly inactive.

The finding of lack of increased GTP cyclohydrolase activity and BH4 levels can explain the low or undetectable increases in NO production. Furthermore, this finding on NO production was not unique to a single cytokine treatment but a generalized result confirmed by various cytokine stimuli and over different time periods, and there was no further increase in BH4 levels when both IFN-{gamma} and TNF-{alpha} were combined as a stimulus. Recently, stimulation with the combination of IFN-{gamma} + TNF-{alpha} was reported to dramatically increase BH4 synthesis in endothelial cells over that elicited by either cytokine individually owing to differing signal transduction pathways (i.e., NF-{kappa}B and Stat1/Stat3) (19). Our finding of lack of amplified BH4 production by stimulation with the combination of IFN-{gamma} + TNF-{alpha} despite iNOS induction suggests that an issue of differences in signaling pathways is likely not a significant limiting factor to increase BH4 in for adult cardiac myocytes.

The reason for the low GTP cyclohydrolase activation remains unclear. Potentially relevant to this issue is that it is known that NO and BH4 play a key role in the proliferation of various cell types (25, 32, 59). Specific to cardiac cells, it is known that NO plays a key role in the process of eventual cardiac cell maturation (2, 3, 23, 27). In this context, it is generally accepted that adult cardiac myocytes display a poor capacity to proliferate. Accordingly, we hypothesize that the impaired activation of GTP cyclohydrolase may represent a consequence of postmitotic maturation of cardiac myocytes coinciding with the repression of certain unknown genes or possibly increasing levels of pertinent silencing mRNA. Alternatively, splice variants of GTP cyclohydrolase have been reported that lack functional activity, thereby functioning via a dominant negative effect (13, 20, 41). However, it is remains unclear whether splice variants are an operative mechanism in adult cardiac myocytes. There are other potential factors to explain the low BH4 production, as detailed below.

Increased GTP cyclohydrolase expression is believed to be a key step regulating increased BH4 synthesis by cytokines in many cell types. In general, the constitutive activity and expression in cells of downstream enzymes such as 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase are usually deemed sufficient enough to accommodate GTP cyclohydrolase activity, thereby not limiting de novo BH4 synthesis. Expression of these downstream genes for BH4 synthesis in cardiac myocytes has not previously been reported. Here, we show that these enzymes are constitutively expressed and that the expression of each gene was not induced by cytokine exposure. These findings contrast with the induction of 6-pyruvoyltetrahydropterin synthase in human endothelial cells by IL-1β given alone or combined with other cytokines (11, 29). A consequence of any limitation of BH4 synthesis downstream of GTP cyclohydrolase would be an expected increased shunting from BH4 synthesis to neopterin formation from build up in 7,8-dihydroneopterin trisphosphate. However, we did not find any increased production of neopterin, a measure of GTP cyclohydrolase activity (data not shown). This finding would argue against a rate-limiting problem of BH4 synthesis downstream of the GTP cyclohydrolase reaction in cardiac myocytes.

Regulation of GFRP expression. BH4 synthesis can also be regulated by the expression of GFRP (12, 22, 31). mRNA levels for GFRP in the heart are low compared with tissues, such as the liver and kidney (12). The present study is the first report to examine GFRP mRNA and potential changes by cytokine stimulation in isolated cardiac myocytes. Our results showing upregulation of GFRP mRNA by various different cytokines contrast with the initial findings of decreased expression upon stimulation of endothelial cells with a single cytokine, IFN-{gamma} (12). This finding may suggest the possibility that the regulation of GFRP in cardiac myocytes may be different than in vascular cells. At present, the impact of changes in GFRP expression in cardiac myocytes is not known. Given that the mechanism of action of GFRP on GTP cyclohydrolase activity requires BH4 for its inhibitory function in other cells such as hepatocytes (35), we believe that the impact of GFRP in cardiac myocytes is limited since the levels of BH4 are low compared with hepatocytes.

Substrate availability, arginine transporter, arginase expression, and NO levels. In our study, we used a stock of media 199, which contains 332 µM arginine. This concentration is threefold higher than the plasma value of 100 µM seen in many mammalian systems and is also in excess of the ~190 µM arginine concentration previously determined for plasma in Sprague-Dawley rats (42). High-affinity CAT-1 was upregulated by all cytokine treatments, in agreement with a previous report in adult cardiac myocytes (57). Therefore, lack of substrate is unlikely to account for the low NO production seen in isolated adult cardiac myocytes.

LPS and inflammatory cytokines such as IFN-{gamma} and TNF-{alpha} induce arginase expression in macrophage and endothelial cells (10, 30, 37). However, the expression of arginase in isolated rat cardiac myocytes under basal and upon cytokine-stimulated conditions was not previously known with certainty. As we found no evidence for gene expression of either arginase isoform in adult cardiac myocytes under both control and cytokine-stimulated conditions, we conclude that the low levels of NO produced after cytokines cannot be explained by competition of the substrate arginine by upregulated arginase activity.

Arginase activity results in polyamine synthesis important for cell growth and function and plays an important role in cell proliferation (28, 50). The finding of expression of arginase in neonatal cardiac myocytes, which proliferate in culture, but lack of expression of arginase in adult cardiac myocytes, which have a poor proliferative capacity, is consistent with the important role of this enzyme in cell proliferation.

Defective salvage pathway for BH4 synthesis. Supplementation of cells with sepiapterin is a common strategy to increase intracellular BH4 levels via the salvage pathway. Previously, Kasai et al. (24) showed that sepiapterin increased total biopterin levels in neonatal cardiac myocytes stimulated with cytokines, resulting in large increases in NO production. Although BH4 levels per se were not directly measured in that study, increases in BH4 were implied indirectly based on the finding that a sepiapterin-induced increase in total biopterin levels was prevented by treatment with methotrexate, an inhibitor of dihydrofolate reductase. In this regard, it is recognized that exogenous sepiapterin is reduced to BH2 via sepiapterin reductase and then converted to BH4 by the action of dihydrofolate reductase (17, 54).

In the present study, we determined new data, previously unknown, that adult rat cardiac myocytes lack the expression of both mRNA and protein for dihydrofolate reductase under both basal and cytokine-stimulated conditions. This finding contrasts with the only other known published report (49) of dihydrofolate reductase protein expression in cardiac myocyte-enriched cultures derived from the Friend leukemia virus B mouse heart. The reasons for the difference are unknown but might arise from species, mouse strain, or differences in isolation and culturing conditions and the possible contribution by nonmyocytes such as cardiac fibroblasts. The fact that we found that dihydrofolate reductase expression in neonatal cardiomyocytes and embryonic cardiac cell lines but not in adult cardiac myocytes from the same rat strain suggests that the differences are likely more related to cardiac myocyte maturation. This is consistent with the repression of expression of dihydrofolate reductase mRNA upon terminal differentiation reported previously for muscle cells (55).

This new finding regarding the absence of dihydrofolate reductase explains why adult cardiac myocytes cannot efficiently increase levels of BH4 from sepiapterin since the salvage pathway metabolism of sepiapterin requires the conversion of BH2 to BH4. It also explains recent findings in our laboratory for the build up of BH2 in adult cardiac myocytes incubated with authentic exogenous BH4 as well. The consequences are that supplementation with sepiapterin results in the build up of BH2, which cannot support increased NO production to any significant extent. This mechanism could explain the large accumulation of BH2 after sepiapterin treatment in vivo of cardiac allograft recipients also shown in the present study. The increase in BH4 seen in the heart in vivo may be explained by the additional contribution of noncardiac myocytes (e.g., cardiac fibroblasts, endothelial cells, and vascular smooth muscle cells), since these cells are known to express dihydrofolate reductase (6, 59).

The inability of adult cardiac myocytes to convert intracellular BH2 formed from sepiapterin to high BH4 levels appears to have significant consequences on NO production via NOS. Furthermore, we hypothesize that the increased levels of BH2, by competing for the lower levels of BH4, can limit NO production from allosteric modulation of iNOS enzyme activity. Thus, despite the fact that intracellular BH4 levels are modestly increased by sepiapterin, the impact on NO production remains unchanged.

Impact on iNOS monomer levels. Stuehr and colleagues (60) were the first to show the importance of intracellular BH4 levels on iNOS protein dimer-monomer distribution in a biological setting. These authors used a NIH-3T3 fibroblast cell line deficient in de novo BH4 synthesis to show that the expression of recombinant iNOS in these cells resulted in minimal NO production. This impaired NO production from iNOS was associated with significant levels of iNOS protein monomers determined under nonreducing conditions. This effect was reversed by supplementing media with authentic BH4 or sepiapterin. Similar findings have recently been shown in macrophages from phosphatidylinositol 3-kinase-deficient mice (51).

The present study is the first to our knowledge to examine the occurrence of iNOS monomers in both cytokine-stimulated cardiac myocytes in vitro and alloimmune stimulation in acute cardiac allograft rejection in vivo. Consistent with the theme that BH4 deficiency inhibits iNOS homodimerization, we found that the low levels of BH4 synthesis in cytokine-stimulated adult cardiac myocytes was reflected by low levels of NO production. This was also associated with significant levels of iNOS monomers present in Western blots under nonreducing conditions.

In theory, one would assume that sepiapterin should lower iNOS monomers and facilitate iNOS homodimerization by increasing intracellular levels of BH4. However, to our knowledge, there are no published data validating whether such a strategy necessarily increases intracellular BH4 or enhances iNOS homodimerization in adult cardiac myocytes or hearts. The failure of supplementation with sepiapterin to alter iNOS monomer levels in vitro and its confirmation in an in vivo model is consistent with our findings of the defective capacity of adult cardiac myocytes to significantly increase intracellular BH4 levels via the salvage pathway due, in a large part, to the finding that these cells lack the expression of dihydrofolate reductase. Thus, our findings indicate that iNOS is unlikely to be fully coupled to NO production when expressed in adult cardiac myocytes due to posttranslational factors involving defective BH4 synthesis by both de novo and salvage pathways resulting in the formation of inactive iNOS monomers.

Implications of the study. Our study gives new understanding that reconciles a previously outstanding issue arising from studies by various investigators as to why adult cardiac myocytes produce low quantities of NO via iNOS after cytokine stimulation (14, 15, 46, 52, 65). The findings in the present study suggest a major rethinking of the role by which upregulation of iNOS in cardiac myocytes influences various aspects of the origins of cardiac function and injury in adult cardiovascular diseases. In one sense, it reveals that adult cardiac myocytes could not be a proficient source of NO in the heart despite the expression of iNOS protein. Since less-differentiated neonatal cardiac myocytes as well as possibly cardiac fibroblasts retain the ability to increase both NO and BH4, it is anticipated that these cells instead are more likely to mediate actions of NO through iNOS during inflammatory states in intact hearts. Furthermore, the benefits of sepiapterin administration to intact hearts ex vivo and in vivo demonstrated in previous reports are likely to be mediated from increasing BH4 in noncardiac cells, such as endothelial cells, etc. A better understanding of this complex interplay may lead to the reconciliation of remaining controversies associated with inflammatory cardiac disease conditions.


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 MATERIALS AND METHODS
 RESULTS
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-078937 (to G. M. Pieper), HL-067244 (to J. Vásquez-Vivar), and HL-069996 (to M. Medhora).


   ACKNOWLEDGMENTS
 
The technical assistance of Chao-Ying Chen is greatly appreciated.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. M. Pieper, Transplant Surgery, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (e-mail: gmpieper{at}mcw.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supplemental material for this article can be found at the American Journal of Physiology-Heart and Circulatory Physiology website. Back


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