Impact of mitochondrial nitrite reductase on hemodynamics and myocardial contractility

Figures

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  Figure 1. (A–D) Experimental design and measurement of hematocrit (Hct), arterial oxygen partial pressure (pO2) and arterial oxygen saturation (sO2) over time. In vivo experiments were performed in three groups: with normal Hct under (a) normoxic (Hct40-NOX: blue bars) and (b) hypoxic (Hct40-HOX: red bars) conditions and (c) halved Hct under hypoxic (Hct20-HOX: green bars) conditions. Experimental design includes anesthesia and catheter placement for Hct adjustment (HCT ADJ), pO2 adjustment (NOX: normoxic group 100 mmHg; HOX: hypoxic groups: 50 mmHg), hemodynamic monitoring and blood sampling. A bolus of nitrite (15 µmol/kg) was infused intra venously after pO2 adjustment. Blood samples were taken at baseline (BL), after HCT ADJ, after NOX/HOX adjustment, 10 and 20 minutes after nitrite infusion (A). Changes on Hct (B), pO2 (C) and sO2 (D) values over time in all three groups determined by blood gas analysis. Data are expressed as mean ± SEM of at least n = 8, ***p < 0.001. One animal died upon adjustment of Hct.

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  Figure 2. Plasma levels of nitritre (left) and nitrate (right). Both nitrite and nitrate peaked 10 min after a bolus injection of 15 µmol/kg sodium nitrite and were markedly decreased after 10 min.

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  Figure 3. (A–D) Effect of nitrite infusion on the formation of methemoglobin (metHb) and nitric oxide hemoglobin (NO-Hb) in red blood cells (RBCs). Schematic illustration of nitrite metabolism in oxygenated RBCs; the predominant pathway is the formation of biologically inactive NO3 − and metHb (A). Schematic illustration of nitrite metabolism in deoxygenated RBCs; the predominant pathway is the formation of NO-Hb complexes and metHb (B). Nitrite mediated formation of metHb (C) determined by EPR and NO-Hb (D) over time in RBCs determined by electron paramagnetic resonance spectroscopy in Hct40-NOX (blue bars), Hct40-HOX (red bars) and Hct20-HOX (green bars) group. Blood samples were taken at baseline (BL), after HCT ADJ, after NOX/HOX, 10 and 20 minutes after nitrite infusion. Data are expressed as mean ± SEM of at least n = 8, **p < 0.01, ***p < 0.001. One animal died upon adjustment of Hct.

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  Figure 4. (A–D) Effect of nitrite infusion on hemodynamic parameters examined by pulse wave analysis. Illustration and calculation of pulse wave analysis: heart rate (HR), mean arterial blood pressure (MAP), pulse wave velocity (PWV), indicating aorta stiffness and systolic slope (SYS), indicating myocardial contractility. Reflection index (RI) was calculated by dividing B/A, indicating peripheral resistance. Solid line represents a typical pulse wave; dotted line is the first derivative from pulse wave identifying the diastolic phase (A). Effect of nitrite infusion on the hemodynamic parameters HR (B), MAP (C), aorta stiffness (D), myocardial contractility (E) and peripheral resistance (F) in Hct40-NOX (blue bars), Hct40-HOX (red bars) and Hct20-HOX (green bars) group. Pulse waves were analysed before nitrite infusion (BN) and after 2, 10 and 20 minutes of observation. Data are expressed as mean ± SEM of n = 9, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 5. (A–F) Effect of different oxygen concentrations on nitrite reduction in red blood cells (RBCs) and heart homogenates determined by NO analysis. Time curve of NO release in RBCs (A) and heart homogenates (C,E) measured at 0.0 µM (blue line), 3.1 µM (red line) and 42.0 µM (green line) oxygen (O2) concentration. The contribution of dilution buffer is represented at 0 µM (black line) and 42 µM (grey line) oxygen (O2) concentration (A). Statistical evaluation of NO release in RBCs (B) and heart homogenates (D). Mitochondrial contribution to NO release in heart homogenates determined by addition of myxothiazol (Myxo, grey lines), a specific inhibitor of mitochondrial complex III at 0.0 µM and 42 µM O2 concentration (E,F). Arrow indicates addition of potassium cyanide, a specific inhibitor of mitochondrial complex IV. Nitrite was added at 0 min. Data are expressed as mean ± SEM of at least n = 5, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 6. (A,B) Intracellular effects contributing to hemodynamic parameters assessed by NO-Hb formation and cGMP synthesis in different cell cultures. Contribution of nitrite (30 µM, final concentration), myxodiazol (Myxo) and potassium cyanide (KCN) on NO-Hb formation in cell cultures of red blood cells (RBCs) and cardiomyocyte (HL-1) (A). Contribution of SNAP, nitrite, myxodiazol (Myxo) and potassium cyanide (KCN) on cGMP formation in cell cultures of RBCs and cardiomyocyte (HL-1) (B). Controls are displayed with open bars. Data are expressed as mean ± SEM of three independent experiments with 4 replicates each, *p < 0.05, ***p < 0.001.

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  Figure 7. (A–C) Intracellular effects contributing to hemodynamic parameters assessed by cGMP synthesis in different cell cultures. Effect of RBCs on nitrite mediated cGMP synthesis on HL1-cardiomyocytes (A). Effect of myxothiazol (Myxo), a specific inhibitor of mitochondrial complex III, allopurinol (Allo), a specific inhibitor xanthine oxireductase and N (G)-nitro-L- arginine methyl ester (L-Name), a specific inhibitor of NOS on nitrite mediated cGMP synthesis on HL-1 cardiomyocytes (B) and BRL hepatocytes (C). Data are expressed as mean ± SEM of at least n = 3, *p < 0.05, ***p < 0.001.