Sodium nitrate supplementation alters mitochondrial H2O2 emission but does not improve mitochondrial oxidative metabolism in the heart of healthy rats

Abstract

Supplementation with dietary inorganic nitrate ([Formula: see text]) is increasingly recognized to confer cardioprotective effects in both healthy and clinical populations. While the mechanism(s) remains ambiguous, in skeletal muscle oral consumption of NaNO₃ has been shown to improve mitochondrial efficiency. Whether NaNO₃ has similar effects on mitochondria within the heart is unknown. Therefore, we comprehensively investigated the effect of NaNO₃ supplementation on in vivo left ventricular (LV) function and mitochondrial bioenergetics. Healthy male Sprague-Dawley rats were supplemented with NaNO₃ (1 g/l) in their drinking water for 7 days. Echocardiography and invasive hemodynamics were used to assess LV morphology and function. Blood pressure (BP) was measured by tail-cuff and invasive hemodynamics. Mitochondrial bioenergetics were measured in LV isolated mitochondria and permeabilized muscle fibers by high-resolution respirometry and fluorometry. Nitrate decreased ( P < 0.05) BP, LV end-diastolic pressure, and maximal LV pressure. Rates of LV relaxation (when normalized to mean arterial pressure) tended ( P = 0.13) to be higher with nitrate supplementation. However, nitrate did not alter LV mitochondrial respiration, coupling efficiency, or oxygen affinity in isolated mitochondria or permeabilized muscle fibers. In contrast, nitrate increased ( P < 0.05) the propensity for mitochondrial H₂O₂ emission in the absence of changes in cellular redox state and decreased the sensitivity of mitochondria to ADP (apparent K_m). These results add to the therapeutic potential of nitrate supplementation in cardiovascular diseases and suggest that nitrate may confer these beneficial effects via mitochondrial redox signaling.

Keywords: bioenergetics; heart; hemodynamics; mitochondria; nitrate.

Fig. 1.

Blood pressure measurements by noninvasive hemodynamics in control and nitrate-supplemented rats. In conscious healthy rats, nitrate supplementation decreased systolic blood pressure (A) and trended to decrease mean arterial pressure (B) but did not significantly alter diastolic blood pressure (C) and heart rate (D) after 7 days. Values are means ± SE; n = 6–7 rats/group. *P < 0.05 vs. control.

Fig. 2.

Left ventricular (LV) pressure characteristics measured by invasive hemodynamics in control and nitrate-supplemented rats. A–D: nitrate supplementation significantly decreased maximal LV pressure (LVP_max) and LV end-diastolic pressure (LVEDP) but did not alter maximal rates of pressure development (dP/dt_max) or maximal rates of pressure decline (dP/dt_min). E: nitrate trended to increase maximal rates of pressure decline normalized to mean arterial pressure (dP/dt_min/MAP). Values are means ± SE; n = 8–11 rats/group. *P < 0.05 vs. control.

Fig. 3.

Nitrate supplementation does not lead to increases in left ventricular (LV) mitochondrial oxidative capacity or coupling efficiency in subsarcolemmal (SS; A and B) and intermyofibrillar (IMF; C and D) mitochondria. Mitochondrial respiration was measured in state IV respiration [addition of 10 mM pyruvate + 2 mM malate (PM) in the absence of ADP (−ADP)] followed by state III respiration [addition of 5 mM ADP (+ADP)] with final sequential additions of glutamate (10 mM; PMG) and succinate (10 mM; PMGS). Maximal complex I- and II-supported respiration was measured in SS (A) and IMF (C) mitochondria. Mitochondrial coupling efficiency (P/O ratio) was calculated following an initial submaximal bolus of ADP (100 µM) in SS (B) and IMF (D) mitochondria. Jo₂, mitochondrial oxygen flux; RCR, respiratory control ratio (state III/state IV). Values are means ± SE; n = 9–14 rats/group.

Fig. 4.

Nitrate supplementation does not alter left ventricular (LV) mitochondrial oxidative capacity or leak respiration in permeabilized cardiac muscle fibers (PmFBs). A: mitochondrial respiration was measured using the protocol used for isolated mitochondria. B: oligomycin (oligo; 2 μg/ml) was added during state IV respiration [10 mM pyruvate + 2 mM malate (PM) in the absence of ADP]. C: protein content of oxidative phosphorylation cocktail (OXPHOS), expressed as percentage of control, with representative blots of OXPHOS and cytochrome c oxidase subunit IV (COXIV-4), and with α-tubulin as loading control (right). Con, control; Jo₂, mitochondrial oxygen flux; Nit, nitrate; PM, state IV respiration with 10 mM pyruvate + 2 mM malate in the absence of ADP (–ADP); PmFBs, permeabilized muscle fibers; PMG, 10 mM pyruvate + 2 mM malate + 10 mM glutamate in the presence of maximal ADP (+ADP); PMGS, 10 mM pyruvate + 2 mM malate + 10 mM glutamate + 10 mM succinate in the presence of maximal ADP (+ADP); RCR, respiratory control ratio (state III/state IV). Values are means ± SE; n = 8 rats/group for PmFBs and 7–8 rats/group for Western blot data.

Fig. 5.

In vitro mitochondrial oxygen affinity in control and nitrate-supplemented rats. A: representative trace of the simultaneous decrease in oxygen concentration (right y-axis, dashed line) and mitochondrial oxygen flux (Jo₂; left y-axis, solid line) during the transition to anoxia in isolated mitochondria with saturating complex I and complex II substrates during state III respiration (nonlimiting ADP). Time to reach anoxia was calculated immediately after addition of mitochondria to the chambers (set as time 0). B: individual data points of Jo₂ in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria and corresponding sigmoidal fits calculated with Prism and plotted as a function of oxygen concentration. C and D: nitrate supplementation did not affect EC₅₀ or cooperativity in SS or IMF mitochondria. LV, left ventricle. Values are means ± SE; n = 5 rats/group.

Fig. 6.

Nitrate supplementation alters mitochondrial ADP kinetics in cardiac permeabilized muscle fibers (PmFBs). A and D: ADP titrations in the presence of 10 mM pyruvate and 2 mM malate generated typical Michaelis-Menten kinetics in the absence of creatine (Cr; A) and in the presence of phosphocreatine (PCr) and Cr (D) in PmFBs. B and E: apparent K_m remained unchanged in the absence of PCr/Cr (B) but was increased in the presence of PCr/Cr (E) with nitrate supplementation. C and F: maximal respiration (V_max) was not different in any group. G: increase in the apparent K_m occurred in the absence of changes in protein content of adenosine nucleotide translocase (ANT) and mitochondrial creatine kinase (miCK), expressed as percentage of control, with α-tubulin as a loading control. H: representative blots. Con, control; Jo₂, mitochondrial oxygen flux; Nit, nitrate. Values are means ± SE; n = 7–8 rats/group. *P < 0.05 vs. control.

Fig. 7.

Nitrate supplementation increases mitochondrial reactive oxygen species (ROS) production in cardiac permeabilized muscle fibers (PmFBs) and isolated mitochondria but does not lead to oxidative stress. A: succinate (10 mM)-supported H₂O₂ emission potential was increased in the nitrate group and decreased with addition of 50 µM ADP to PmFBs in both groups. B: the majority of increased H₂O₂ emission potential with nitrate stemmed from subsarcolemmal (SS) mitochondria, as this remained unchanged in intermyofibrillar (IMF) mitochondria. C: nitrate supplementation did not reduce protein content of the antioxidant enzymes catalase and SOD2, expressed as percentage of control; representative Western blots for catalase and SOD2 with α-tubulin as a loading control are shown. D: nitrate supplementation did not alter markers of overt of oxidative stress, expressed as percentage of control; representative blots of lipid peroxidation (4-HNE) and nitrosylation (N₃) are shown. E–G: redox state, measured as reduced glutathione (GSH)-to-oxidized glutathione (GSSH) ratio, was not altered with nitrate supplementation. Con, control; Nit, nitrate. Values are means ± SE; n = 7 rats/group for PmFBs, 10–14 rats/group for isolated mitochondria, 7–8 rats/group for Western blot data, and 5 rats/group for GSH:GSSG. *P < 0.05 vs. control.