Fig. 5: Nitro-oleic acid enhances cardiac mitochondrial respiration.

Cardiomyocytes of mice after chow diet, 15 weeks (wk) of high-fat diet (HFD) and L-NAME with 4 wk of vehicle (HFD + L-NAME vehicle) or NO₂-OA treatment (HFD + L-NAME NO₂-OA) were isolated and subjected to high resolution respirometry. a Representative trace of oxygen flux (upper panel). Addition of components are marked with blue lines, CarnPCoA: carnitine palmitoyl-CoA; ADP: adenosine diphosphate; PGM: pyruvate glutamate malate; CytC: cytochrome C; Succ: succinate; Omy: oligomycin; FCCP: mitochondrial uncoupler; Rot: rotenone; AMA: antimycin A. Quantification of complex I- and complex II-linked respiration, oxidative phosphorylation (OXPHOS), and carnitine palmitoyltransferase 1 (CPT1B)-linked respiration (lower panel) (N = 8/6/5). b Isolated adult murine cardiomyocytes were cultivated for 48 h under control conditions (Ctrl) with normal glucose levels (low Glu: low glucose levels), under metabolic stress conditions induced by high glucose levels (high Glu), endothelin-1 (ET1) and hydrocortisone (HC) with treatment of methanol (Vehicle) or nitro-oleic acid (NO₂-OA). c Representative immunoblot (IB) and phosphorylation level of AMP-activated protein kinase at threonine residue 172 (p-AMPK). Protein levels were normalized to the total protein amount assessed on IB by fluorescence labeling (Fig. S15) and shown relative to the individual Ctrl group (N = 4). d Quantification of fluorescence intensity of translocase of outer mitochondrial membrane 70 (TOM70), shown as arbitrary units (a.u.), for all analyzed individual cardiomyocytes of three experiments (N = 3). e High resolution respirometry was performed with primary murine cardiomyocytes after cultivation and treatment. Representative trace of oxygen flux. The addition of components are marked with blue lines. Oxidative phosphorylation (OXPHOS) values were extracted from oxygen flux traces (N = 9). Data are presented as mean ± standard deviation. Statistical significance was calculated by One-way ANOVA followed by Bonferroni´s post-hoc test for a and d and unpaired, two-sided Student’s t-test for e. Only statistically significant differences are indicated. p: *<0.05, ***<0.001, ****<0.0001.