Liposomal tetrahydrobiopterin preserves eNOS coupling in the post-ischemic heart conferring in vivo cardioprotection

Abstract

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS), and reduced BH4 availability leads to endothelial NOS (eNOS) uncoupling and increased reactive oxygen species (ROS) generation. Questions remain regarding the functional state of eNOS and role of BH4 availability in the process of in vivo myocardial ischemia-reperfusion (I/R) injury. Rats were subjected to 60min of in vivo left coronary artery occlusion and varying periods of reperfusion with or without pre-ischemic liposomal BH4 supplementation (1mg/kg, iv). Myocardial infarction was correlated with cardiac BH4 content, eNOS protein level, NOS enzyme activity, and ROS generation. In the vehicle group, 60-min ischemia drastically reduced myocardial BH4 content in the area at risk (AAR) compared to non-ischemic (NI) area and the level remained lower during early reperfusion followed by recovery after 24-h reperfusion. Total eNOS, activated eNOS protein level (eNOS Ser1177 phosphorylation) and NOS activity were also significantly reduced during ischemia and/or early reperfusion, but recovered after 24-h reperfusion. With liposomal BH4 treatment, BH4 levels were identical in the AAR and NI area during ischemia and/or early reperfusion, and were significantly higher than with vehicle. BH4 pre-treatment preserved eNOS Ser1177 phosphorylation and NOS activity in the AAR, and significantly reduced myocardial ROS generation and infarction compared to vehicle. These findings provide direct evidence that in vivo I/R induces eNOS dysfunction secondary to BH4 depletion, and that pre-ischemic liposomal BH4 administration preserves eNOS function conferring cardioprotection with reduced oxidative stress.

Keywords: Ischemia/reperfusion; Myocardial BH(4) content; Myocardial protection; NOS activity; Oxidative stress; Tetrahydrobiopterin.

Figure 1

A–B, Myocardial infarction in rat hearts subjected to 60-min LCA ligation and 24-hour reperfusion with and without vehicle, BH₄ (6R-BH₄), and 6S-BH₄; and the effect of NOS inhibitor L-NAME on myocardial infarction of vehicle (L+Vehicle) or BH₄ (L+6R-BH₄) groups. Representative sections of the hearts stained with Evan’s blue and TTC after I/R are shown above the bar graphs. Area at risk (AAR) is expressed as a percentage of left ventricle (LV), AAR/LV. LV infarct area (IA) is expressed as a percentage of AAR. Values are means ± SEM. N=6–8/group. ***P<0.001, **P<0.01 vs. Control; ^&&&P<0.001, ^&&P<0.01 vs. Vehicle; ^###P<0.001 vs. 6S-BH₄; ⁺⁺⁺P<0.001 vs. L+Vehicle or L+6R-BH₄.

Figure 2

BH₄ concentration in the area at risk (AAR) and non-ischemic (NI) area as determined by HPLC method. A, BH₄ levels in the AAR and NI area of the heart after vehicle treatment. Data are mean ± SEM of 5 rats in each group. ***P<0.001 vs. 0-min I+0-min R (baseline) in AAR; ^†††P<0.001 vs. NI area. B, BH₄ levels in the AAR and NI areas of the heart after BH₄ (1mg/kg, iv) treatment. Values are means ± SEM of 5 rats in each group. ***P<0.001 vs. 0-min I+0-min R (baseline) in AAR; ^§§§P<0.001 vs. 0-min I+0-min R (baseline) in NI area.

Figure 3

In vitro NOS activity in the area at risk (AAR) of rat hearts was measured by L-[¹⁴C]-arginine to L-[¹⁴C]-citrulline conversion in absence (open symbols) and presence (closed symbols) of added BH₄ (10 μM). Values are means ± SEM. ^*P<0.05, ^***P=0.001 vs. 0-min I/0-min R (baseline) without BH₄ supplement; ^†P<0.05, ^††P<0.01, ^†††P=0.001 vs. treatment with added BH₄ (N=4/group).

Figure 4

Immunoblots of total eNOS (A) and its activated phosphorylated form (eNOS Ser1177 phosphorylation) (B) in the cardiac homogenates of area at risk. Top: Representative Western blots for eNOS proteins. Bottom: graphical presentation of the densitometric data. Open bars for vehicle-treated and filled bars for BH₄-treated groups. At equal protein loading, post-ischemic total eNOS and eNOS Ser1177 phosphorylation were well-preserved with BH₄ treatment compared to vehicle. Values are means ± SEM. N = 5/group. All data are compared with non-treated control hearts. *P<0.05 vs. 0-min I+0-min R (baseline) in BH₄-treated group, ^†P<0.05, ^††P<0.01, ^†††P<0.001 vs. 0-min I+0-min R (baseline) in vehicle-treated group; ^§§§P<0.001 vs. time-matched vehicle-treated group.

Figure 5

In situ myocardial superoxide generation following ischemia and/or reperfusion. A, Representative phase contrast (left column) and matching fluorescent photomicrographs (right column) of confocal microscopic sections of LV myocardium from vehicle- or BH₄-treated groups labeled with the redox dye dihydroethidium (DHE). Each representative panel shows small blood vessel(s) and adjacent myocardium. Panels from top to bottom: 0-min I+0-min R; 60-min I+0-min R; 60-min I+10-min R; 60-min I+20-min R; 60-min I+24-hr R. Magnification 60x; bar, 40 μm. B, Bar graphs for the total intensity of ethidium fluorescence (in arbitrary units, AU) measured from the corresponding LV sections of vehicle- or BH₄-treated hearts. Values are means ± SEM. N=3/group. ^**P<0.01, ^***P<0.001 vs. 0-min I+0-min R (baseline) in vehicle-treated group; ^§§P<0.01, ^§§§P<0.001 vs. 0-min I+0-min R (baseline) in BH₄-treated group; ^†††P<0.001 vs. time-matched BH₄-treated group.

Figure 6

In situ myocardial superoxide and NO generation following 60-min ischemia and 10-min reperfusion. A and B, Representative confocal fluorescent photomicrographs of LV myocardial sections labeled with the redox dye dihydroethidium (DHE) and NO-sensitive dye CuFL, respectively. Magnification 60x; bar, 40 μm. C, Bar graphs showing effects of NOS inhibitor L-NAME and SOD-mimetic MnTBAP on myocardial superoxide generation (in arbitrary units, AU). D, Bar graphs showing effects of NOS inhibitor L-NAME and NO scavenger PTIO on myocardial NO (in arbitrary units, AU). Values are means ± SEM. N=3/group. ^*P<0.05 vs. vehicle I/R; ^†P<0.05, ^†††P<0.001 vs. vehicle I/R; P<0.01, P<0.001 vs. BH₄ I/R.

Figure 7

Nitrotyrosine formation following ischemia-reperfusion. A, Detection of nitrotyrosine (NT) in myocardium of rat subjected to in vivo ischemia reperfusion. Representative sections of the vehicle- and BH₄-treated hearts probed for nitrotyrosine (green fluorescence). Each representative panel shows small blood vessel(s) and adjacent myocardium. Panels from top to bottom: 0-min I+0-min R; 60-min I+0-min R; 60-min I+10-min R; 60-min I+20-min R; 60-min I+24-hr R. Magnification 60x; bar, 40 μm. B, Bar graphs for the total intensity of nitrotyrosine formation (green fluorescence in arbitrary units, AU) measured from the corresponding LV ± SEM. N=3/group. ^*P<0.001 vs. sections of vehicle- or BH₄-treated hearts. Values are means time matched vehicle I/R.