Myocardial ischemia results in tetrahydrobiopterin (BH4) oxidation with impaired endothelial function ameliorated by BH4 Cristian Dumitrescu*, Roberto Biondi*†, Yong Xia*, Arturo J. Cardounel*, Lawrence J. Druhan*, Giuseppe Ambrosio*‡, and Jay L. Zweier*§ *Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, Ohio State University, Columbus, OH 43210; and ‡Division of Cardiology and †Sezione di Immunologia e Patologia Generale, University of Perugia School of Medicine, 06156 Perugia, Italy Edited by Salvador Moncada, University of London, London, United Kingdom, and approved August 3, 2007 (received for review March 30, 2007) Coronary vasodilation is impaired in the postischemic heart with a In addition to impairing eNOS-mediated NO formation, BH4 loss of endothelial nitric oxide synthase (eNOS) activity, but the depletion may have additional detrimental effects in postischemic mechanisms underlying ischemia-induced eNOS dysfunction are hearts. We (13) and others (14) have directly demonstrated that not understood. For nitric oxide (NO) synthesis, eNOS requires the BH4 depletion not only prevents NO formation from eNOS but also • Ϫ redox-sensitive cofactor tetrahydrobiopterin (BH4); however, the results in markedly enhanced superoxide ( O2 ) formation. Thus, role of BH4 in ischemia-induced endothelial dysfunction remains BH4 has an important role in regulating eNOS function because • Ϫ unknown. Therefore, isolated rat hearts were subjected to varying BH4 depletion can switch the enzyme from synthesis of NO to O2 . durations of ischemia, and the alterations in NOS-dependent BH4 is highly redox-sensitive and readily oxidized. With oxidative vasodilation were measured and correlated with assays of stress, intracellular BH4 content might transiently fall below a eNOS activity and cardiac BH4 concentrations. Ischemia time- critical threshold (15), leading to NOS uncoupling (13) and pref- • Ϫ dependently decreased cardiac BH4 content with 85, 95, or 97% erential production of O2 rather than NO. irreversible degradation after 30, 45, or 60 min of ischemia, re- In the ischemic and reperfused heart, there is a marked increase spectively. Paralleling the decreases in BH4, reductions of eNOS in oxygen radical generation (16, 17). Radical generation occurs activity were seen of 58, 86, or 92%, and NOS-derived superoxide within both the endothelium and myocytes (18–20). EPR studies production was greatly increased. Addition of 10 ␮MBH4 enhanced have provided direct detection of free radicals in the ischemic and • Ϫ eNOS activity in nonischemic hearts and partially restored activity reperfused heart and demonstrated that O2 and hydroxyl radicals after ischemia. It also suppressed NOS-derived superoxide produc- are formed (16, 21, 22). These radicals and their secondary oxidant tion. Impaired coronary flow during postischemic reperfusion was products could readily oxidize BH4. improved by BH4 infusion. Thus, BH4 depletion contributes to Currently, it is not known how BH4 levels are altered by the postischemic eNOS dysfunction, and BH4 treatment is effective in process of ischemia and whether these alterations trigger postisch- partial restoration of endothelium-dependent coronary flow. Sup- emic vascular dysfunction and loss of NO production from eNOS. plementation of BH4 may therefore be an important therapeu- Therefore, the goal of this work is to determine the alterations in tic approach to reverse endothelial dysfunction in postischemic BH4 that occur after periods of myocardial ischemia and their role tissues. in ischemia-induced NOS dysfunction and loss of endothelial- mediated vasodilation. We observe that with ischemic periods ischemia reperfusion injury ͉ nitric oxide ͉ nitric oxide synthase longer than 30 min, near total depletion of BH4 occurs. This BH4 ͉ ͉ • Ϫ uncoupling superoxide vascular function depletion causes a loss of NOS activity and induces O2 production from the enzyme with a resultant loss of endothelial-dependent vasodilation. itric oxide synthase (NOS) converts L-arginine and O2 to nitric oxide (NO) and L-citrulline. This enzymatic process consumes N Results NADPH and requires Ca2ϩ/calmodulin, flavin adenine dinucle- Effect of Ischemia/Reperfusion on Coronary Flow (CF). After hearts otide, flavin mononucleotide, and tetrahydrobiopterin (BH4)as NOS cofactors. Endothelial NO synthase (eNOS) contributes to the were subjected to periods of global ischemia (30, 45, or 60 min) regulation of vasomotor tone and blood pressure by producing NO followed by 30 min of reflow, basal CF values were measured and that activates soluble guanylate cyclase in vascular smooth muscle, compared with the preischemic values. CF was decreased after resulting in vasorelaxation (1–3). ischemia, and this reduction in flow was proportional to the Endothelial dysfunction is a prognostic marker of cardiovas- duration of ischemia (Fig. 1A). cular disease (4). It has been suggested that limited availability of BH contributes to eNOS dysfunction in hypercholesterol- 4 Author contributions: C.D., R.B., Y.X., A.J.C., L.J.D., G.A., and J.L.Z. designed research; C.D., emia, diabetes, atherosclerosis, hypertension, and heart failure R.B., and A.J.C. performed research; Y.X. and G.A. contributed new reagents/analytic tools; (5–9). It was also observed previously that eNOS function is C.D., R.B., A.J.C., L.J.D., G.A., and J.L.Z. analyzed data; and C.D., L.J.D., G.A., and J.L.Z. wrote impaired in ischemic hearts (10). In vivo coronary artery occlu- the paper. sion triggers endothelial dysfunction and decreased eNOS- The authors declare no conflict of interest. MEDICAL SCIENCES dependent vasoreactivity, although reactivity is preserved to This article is a PNAS Direct Submission. exogenous NO (11, 12). Endothelial-dependent coronary vaso- Abbreviations: B, biopterin; BH2, 7,8-dihydrobiopterin; BH4, tetrahydrobiopterin; CF, cor- reactivity is impaired in hearts subjected to periods of global onary flow; DHFR, dihydrofolate reductase; DHPR, dihydropterin reductase; eNOS, endo- thelial nitric oxide synthase; L-NAME, nitro-L-arginine methyl ester; NOS, nitric oxide ischemia and reperfusion (10). Endothelium-dependent vasodi- • Ϫ synthase; O2 , superoxide; P, pterin; SOD, superoxide dismutase; XO, xanthine oxidase; lators induce a relatively high increase in coronary flow in XPH2, dihydroxanthopterin. control hearts or in those made ischemic for short times, but §To whom correspondence should be addressed at: Davis Heart and Lung Research Insti- longer periods of ischemia result in an abrupt decline in vaso- tute, 473 West 12th Avenue, Columbus, OH 43210. E-mail: [email protected]. dilatory response. © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702986104 PNAS ͉ September 18, 2007 ͉ vol. 104 ͉ no. 38 ͉ 15081–15086 Downloaded by guest on September 30, 2021 Fig. 1. Effect of ischemia/reperfusion on total and NOS-dependent CF. (A) CF, calculated as a percentage of basal, preischemic flow, was measured in hearts before ischemia and after periods of ischemia followed by 30 min of reperfusion. Longer periods of ischemia were associated with more severe Fig. 2. Effect of the duration of ischemia on cardiac eNOS activity. Hearts reductions of CF. *, P Ͻ 0.0001 vs. preischemic control. (B) NOS-dependent CF were homogenized at the end of normal perfusion or after different dura- was measured in control hearts (0 min of ischemia) with infusion of 1 mM tions of ischemia, and eNOS activity was measured by L-[14C]arginine to L-NAME for 10 min after the 15-min equilibration period. NOS-dependent CF L-[14C]citrulline conversion. Loss of NOS activity paralleled the duration of was similarly measured in hearts subjected to 30, 45, or 60 min of ischemia ischemia and was fully restored by BH4 with shorter ischemic times but only followed by 30 min of reperfusion with infusion of 1 mM L-NAME for 10 min partially with longer durations. , P Ͻ 0.001 ϩ BH vs. basal eNOS activity, n ϭ at the end of the 30-min reperfusion period. NOS-dependent flow was calcu- * 4 5 hearts per point. lated as the percentage of basal, preischemic, CF inhibited by L-NAME. NOS- dependent CF was decreased in the postischemic heart, which was a function of the ischemic duration. *, P Ͻ 0.001 vs. preischemic NOS-dependent flow; #, P Ͻ 0.01 vs. 30 min of ischemia, n ϭ 5–7 hearts per group. Effect of Ischemia on Cardiac BH4 Content. The results above suggest that eNOS activity was impaired in ischemic hearts and BH4 completely or partially restored this activity when added back to the NOS Dependence of Coronary Flow in the Normal and Postischemic heart homogenates. Subsequently, we measured cardiac BH4 con- Heart. To evaluate whether the loss of CF in the postischemic heart tent and its degradation after ischemia. BH4 and its metabolites correlates with the onset of NOS dysfunction, a series of studies 7,8-dihydrobiopterin (BH2), biopterin (B), pterin (P), and dihy- were performed measuring NOS-dependent CF. For these studies, droxanthopterin (XPH2) were assayed by HPLC with electrochem- the NOS inhibitor nitro-L-arginine methyl ester (L-NAME) (1 mM) ical detection (Fig. 3). These studies demonstrate that BH4 is was infused for 10 min after the equilibration period in hearts not depleted during ischemia (Figs. 4 and 5). BH4 levels were slightly subjected to ischemia or after 30 min of reperfusion in hearts lower in hearts subjected to 15 min of ischemia compared with subjected to 30, 45, or 60 min ischemia. controls. However, ischemic durations longer than 15 min caused a Although L-NAME infusion decreased CF before the onset of ischemia by 25 Ϯ 2% (n ϭ 7), a less prominent decrease was induced by this NOS inhibitor after ischemia and reperfusion. After 30 min of ischemia followed by 30 min of reperfusion, L-NAME decreased CF by 10.5 Ϯ 1% (n ϭ 5) and had a modest effect on basal postischemic CF when hearts were subjected to longer ischemic times (45 min, 4 Ϯ 0.6% decrease, n ϭ 5; 60 min, 3.4 Ϯ 1.4% decrease, n ϭ 5).