Nitro-oleic acid enhances mitochondrial metabolism and ameliorates heart failure with preserved ejection fraction in mice

Figures

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  Fig. 1. Nitro-oleic acid mitigates heart failure in mice with HFpEF. Mice received a high-fat diet (HFD) and the endothelial nitric oxide synthase inhibitor L-NAME (HFD + L-NAME) or a standard chow diet (Chow) for 15 weeks (wk). They were treated with vehicle or nitro-oleic acid (NO2-OA) during the last 4 wk. Echocardiography (Echo), glucose tolerance test (GTT), hemodynamics (PV-loop), exercise, transmission electron microscopy (TEM), proteomics of left ventricular tissue (LV proteome) and respirometry of isolated cardiomyocytes was conducted at the final time point. a Experimental protocol. b Left ventricular ejection fraction (LVEF) and left ventricular posterior wall thickness (LVPW, d) (N = 19/14/18). c Representative ultrasound images of 4-chamber B-Mode (upper panel) and LV tissue doppler at mitral annulus (lower panel) to assess diastolic function. d Ratio of early diastolic mitral inflow velocity to early diastolic mitral annulus velocity (MV E/e´) and isovolumetric relaxation time (IVRT) (N = 19/14/18). e LV global longitudinal strain (GLS) (N = 9/9/9). f Compliance index shown as E/e´ in ratio to the individual end diastolic volume (EDV) (N = 19/14/18). g, h LV end-diastolic…

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  Fig. 2. Nitro-oleic acid treatment alters myocardial proteome and glucose metabolism in HFpEF mice. a Principal component analysis (Chow: N = 9; HFD + L-NAME vehicle: N = 8; HFD + L-NAME NO2-OA: N = 6). Volcano-plots comparing high-fat diet (HFD) + L-NAME vehicle with chow mice (b) and NO2-OA-treated with vehicle-treated HFD + L-NAME mice (d). Significantly changed proteins are marked (significance ≥ -log10(0.05); red: fold change > log2(1.5); blue: fold change < -log2(1.5)). Protein names, log fold changes and p-values are listed in the source data file. c KEGG pathway enrichment analysis of significantly increased proteins of HFD + L-NAME vehicle compared to chow mice (protein count ≥ 2; significance ≥ -log10(0.05)). e Venn diagram of proteins significantly decreased in the HFD + L-NAME vehicle compared to the chow group and proteins significantly increased in NO2-OA-treated compared to vehicle-treated HFD + L-NAME mice. f Body weight (N = 12/12/12) and glucose tolerance test (N = 7/7/7). g Protein abundances of solute carrier family 2, member 4 (GLUT4) and pyruvate dehydrogenase kinase 4 (PDK4) relative to cytochrome c oxidase 4 (COX4) derived from liquid chromatography–mass…

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  Fig. 3. Mitochondrial dynamics is stimulated by nitro-oleic acid in LV of mice with HFpEF. Identification of mitochondrial proteins (a) and localization (b) by mitoCarta29. a Heatmap of mitochondrial proteins detected by liquid chromatography–mass spectrometry in left ventricular tissue of mice after chow diet, 15 weeks of high-fat diet (HFD) and L-NAME with 4 weeks of vehicle (HFD + L-NAME vehicle) or NO2-OA treatment (HFD + L-NAME NO2-OA). Protein names and z-scores are in the source data file. b Shown is the percentage of identified mitochondrial proteins (grey) and the percentage of mitochondrial proteins with significantly enhanced abundance after administration of NO2-OA in HFD + L-NAME mice compared to vehicle-treated HFD + L-NAME mice (red). c Protein levels of highly abundant mitochondrial proteins (-log10(p-value) > 4.5; log2(fold change) > 1.9) (N = 9/8/6), ABCD3: ATP-binding cassette subfamily D member 3; COX: cytochrome c oxidase; TOM70: translocase of outer mitochondrial membrane 70; TM177: transmembrane protein 177. d Representative immunoblots (IB) and quantification of COX4 and TOM70 (N = 9/8/6). Protein levels were normalized to the total protein amount assessed on IB…

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  Fig. 4. Activation of the AMPK signaling pathway is induced by nitro-oleic acid in LV of HFpEF mice. a KEGG pathway enrichment analysis of proteins derived from liquid chromatography followed by mass spectrometry (LC-MS) with significantly enhanced abundance after administration of NO2-OA in high-fat diet (HFD) + L-NAME treated mouse hearts compared to vehicle-treated mouse hearts (HFD + L-NAME vehicle) (inclusion criteria: protein count ≥ 10; pathway enrichment factor ≥ 30%; significance ≥ -log10(0.05)). The most significant KEGG pathway is marked with a red arrow. b Heatmap of all proteins associated with the AMP-activated protein kinase (AMPK) signaling pathway and identified by LC-MS analysis. Protein names and z-scores are in the source data file. c Representative immunoblots (IB) and quantification of AMPK and phosphorylation level of AMPK at threonine residue 172 (p-AMPK), sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3) (N = 9/8/6). Protein levels were normalized to the total protein amount assessed on IB by fluorescence labeling (Figs. S12 and S14). Data in c are shown relative to the mean of the chow group as mean ± standard deviation. Statistical significance was calculated by…

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  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…

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  Fig. 6. Nitro-oleic acid enhances cardiac fatty acid metabolism in HFpEF mice. a Heatmap of all proteins associated with fatty acid metabolism and identified by liquid chromatography mass spectrometry (LC-MS) after administration of NO2-OA in high-fat diet (HFD) + L-NAME treated mouse hearts compared to vehicle-treated mouse hearts. Protein names and z-scores are in the source data file. b Protein abundances of carnitine palmitoyltransferase 1B (CPT1B) relative to cytochrome c oxidase 4 (COX4) and long chain fatty acid transporter (FAT/CD36) derived from LC-MS. Data are shown relative to the mean of the chow group (N = 9/8/6). c Representative images of adult murine cardiomyocytes stained for neutral lipids (left). Quantification of lipid droplet area normalized to cardiomyocyte area shown for all individual analyzed images (right) (9 to 10 images at a magnification of 63x per animal, N = 6/5/6). Data are presented as mean ± standard deviation. Statistical significance was calculated by One-way ANOVA followed by Bonferroni´s post-hoc test. Only statistically significant differences are indicated. p: *<0.05, **<0.01, ***<0.001, ****<0.0001.