Inorganic nitrate, hypoxia, and the regulation of cardiac mitochondrial respiration-probing the role of PPARα

Abstract

Dietary inorganic nitrate prevents aspects of cardiac mitochondrial dysfunction induced by hypoxia, although the mechanism is not completely understood. In both heart and skeletal muscle, nitrate increases fatty acid oxidation capacity, and in the latter case, this involves up-regulation of peroxisome proliferator-activated receptor (PPAR)α expression. Here, we investigated whether dietary nitrate modifies mitochondrial function in the hypoxic heart in a PPARα-dependent manner. Wild-type (WT) mice and mice without PPARα (Ppara^-/-) were given water containing 0.7 mM NaCl (control) or 0.7 mM NaNO₃ for 35 d. After 7 d, mice were exposed to normoxia or hypoxia (10% O₂) for the remainder of the study. Mitochondrial respiratory function and metabolism were assessed in saponin-permeabilized cardiac muscle fibers. Environmental hypoxia suppressed mass-specific mitochondrial respiration and additionally lowered the proportion of respiration supported by fatty acid oxidation by 18% (P < 0.001). This switch away from fatty acid oxidation was reversed by nitrate treatment in hypoxic WT but not Ppara^-/- mice, indicating a PPARα-dependent effect. Hypoxia increased hexokinase activity by 33% in all mice, whereas lactate dehydrogenase activity increased by 71% in hypoxic WT but not Ppara^-/- mice. Our findings indicate that PPARα plays a key role in mediating cardiac metabolic remodeling in response to both hypoxia and dietary nitrate supplementation.-Horscroft, J. A., O'Brien, K. A., Clark, A. D., Lindsay, R. T., Steel, A. S., Procter, N. E. K., Devaux, J., Frenneaux, M., Harridge, S. D. R., Murray, A. J. Inorganic nitrate, hypoxia, and the regulation of cardiac mitochondrial respiration-probing the role of PPARα.

Keywords: fatty acids; heart; metabolism; mitochondria.

Figure 1

Study design. Each stage of the study took place within the ages shown ±4 d, and the length of each stage was identical for each mouse. The left-hand section represents mice with PPARα receptor (Ppara^+/+), whereas the right-hand section represents mice without (Ppara^−/−). The number in brackets indicates the number of mice per group. Chloride, 0.7 mM NaCl in distilled water ad libitum; NO₃⁻, 0.7 mM NaNO₃ in distilled water ad libitum; normoxia, 21% atmospheric O₂; hypoxia, 10% atmospheric O₂.

Figure 2

Mitochondrial respiratory function (J_O2) from assay 1 normalized to mass. A) Malate and palmitoyl CoA stimulated LEAK respiration (CPT1_L). B) CPT1-limited oxphos (CPT1_P). C) Oxphos supported by the F-pathway via β-oxidation (PalM_P) in permeabilized cardiac muscle fibers from Ppara^+/+ and Ppara^−/− mice following normoxia (white bars, 21% O₂) or hypoxia (blue bars, 10% O₂) and chloride (open bars, 0.7 mM NaCl) or NO₃⁻ (striped bars, 0.7 mM NaNO₃) supplementation. Error bars indicate sem. Black asterisks indicate main/PPARα effect; blue asterisks indicate hypoxia effect; n = 8–11/group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Mitochondrial respiratory function (J_O2) from assay 2 normalized to mass. A) Malate and octanoyl carnitine stimulated LEAK respiration (OctM_L). B) Oxphos supported by the F-pathway via β-oxidation (OctM_P). C) Oxphos supported by pyruvate and malate through the N-pathway via complex I (PM_P). D) Oxphos supported by glutamate and malate through the N-pathway via complex I (GM_P). E) Oxphos supported by glutamate, malate, and succinate through the NS-pathway via complexes I and II (GMS_P). F) Oxphos supported by succinate following the addition of rotenone (S_P) in permeabilized cardiac muscle fibers from Ppara^+/+ and Ppara^−/− mice following normoxia (white bars, 21% O₂) or hypoxia (blue bars, 10% O₂) and chloride (open bars, 0.7 mM NaCl) or NO₃⁻ (striped bars, 0.7 mM NaNO₃) supplementation. Error bars indicate sem. Black asterisks indicate main/PPARα effect; blue asterisks indicate hypoxia effect; n = 8–11/group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Mitochondrial respiratory function (J_O2) from assay 1 normalized to CS activity (J_AcoA). A) Malate and palmitoyl CoA stimulated LEAK respiration (CPT1_L). B) CPT1-limited oxphos (CPT1_P). C) Oxphos supported by the F-pathway via β-oxidation (PalM_P) in permeabilized cardiac muscle fibers from Ppara^+/+ and Ppara^−/− mice following normoxia (white bars, 21% O₂) or hypoxia (blue bars, 10% O₂) and chloride (open bars, 0.7 mM NaCl) or NO₃⁻ (striped bars, 0.7 mM NaNO₃) supplementation. Error bars indicate sem. Black Δ, 2-way interaction; orange Δ, NO₃⁻ effect; black Δ, PPARα effect; n = 4–6/group. ^ΔP < 0.05, ^ΔΔP < 0.01, ^ΔΔΔP < 0.001.

Figure 5

Mitochondrial respiratory function (J_O2) from assay 2 normalized to CS activity (J_AcoA). A) Malate and octanoyl carnitine stimulated LEAK respiration (OctM_L). B) Oxphos supported by the F-pathway via β-oxidation (OctM_P). C) Oxphos supported by pyruvate and malate through the N-pathway via complex I (PM_P). D) Oxphos supported by glutamate and malate through the N-pathway via complex I (GM_P). E) Oxphos supported by glutamate, malate, and succinate through the NS-pathway via complexes I and II (GMS_P). F) Oxphos supported by by succinate following the addition of rotenone (S_P) in permeabilized cardiac muscle fibers from Ppara^+/+ and Ppara^−/− mice following normoxia (white bars, 21% O₂) or hypoxia (blue bars, 10% O₂) and chloride (open bars, 0.7 mM NaCl) or NO₃⁻ (striped bars, 0.7 mM NaNO₃) supplementation. Error bars indicate sem. Black asterisks indicate PPARα effect; n = 4–6/group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Substrate control ratios. Ratios indicate CPT1 control over β-oxidation (FCR_CPT1) (A); the contribution of the F-pathway via β-oxidation to maximal oxphos (FCR_F) (B), and the capacity for oxphos supported by octanoyl carnitine plus malate relative to pyruvate plus malate (FCR_Oct/P) (C) in permeabilized cardiac muscle fibers from Ppara^+/+ and Ppara^−/− mice, following normoxia (white bars, 21% O₂) or hypoxia (blue bars, 10% O₂) and chloride (open bars, 0.7 mM NaCl) or NO₃⁻ (striped bars, 0.7 mM NaNO₃) supplementation. Error bars indicate sem. Two-way interaction (Δ); 3-way interaction (†); NO₃⁻ effect (orange †); hypoxia effect (blue Δ, †); PPARα effect (black Δ, †); n = 8–10/group. ^†P < 0.05, ^ΔΔΔP < 0.001, ^†††P < 0.001.

Figure 7

Enzyme activities in mouse heart tissue homogenates. Maximal activity of CS (A), HADH (B), hexokinase (C), and LDH (D) from Ppara^+/+ and Ppara^−/− mice, following normoxia (white bars, 21% O₂) or hypoxia (blue bars, 10% O₂) and chloride (open bars, 0.7 mM NaCl) or NO₃⁻ (striped bars, 0.7 mM NaNO₃) supplementation. Error bars indicate sem. Symbols in brackets denote significance of a test of a combination of groups (i.e., main effects or 2-way interactions); n = 4–5/group. ***P < 0.001 (hypoxia main effect), ^ΔΔP < 0.01 (NO₃⁻ effect following NO₃⁻/PPARα interaction), ^ΔΔΔP < 0.001 (hypoxia effect following hypoxia/PPARα interaction), ^ΔΔP < 0.01 (PPARα effect following NO₃⁻/PPARα interaction), ^ΔΔΔP < 0.01, (PPARα effect following hypoxia/PPARα interaction).

Figure 8

Total PDH levels and phosphorylation. A) Protein levels of total PDH. B–D) Phosphorylation of PDH at E1α site serine 232 (pPDH S232) (B), serine 293 (pPDH S293) (C), and serine 300 (pPDH S300) (D) in Ppara^+/+ and Ppara^−/− mice, following normoxia (white bars, 21% O₂) or hypoxia (blue bars, 10% O₂) and chloride (open bars, 0.7 mM NaCl) or NO₃⁻ (striped bars, 0.7 mM NaNO₃) supplementation. Error bars indicate sem. Two-way interaction (Δ); 3-way interaction (†); hypoxia effect (blue Δ, †); PPARα effect (black Δ, †); n = 8–10/group. ^†P < 0.05, ^ΔΔP < 0.01; ^††P < 0.01, ^†††P < 0.001.