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. 2017 Jul 20;2(14):e93885.
doi: 10.1172/jci.insight.93885.

Nicotinamide Mononucleotide Requires SIRT3 to Improve Cardiac Function and Bioenergetics in a Friedreich's Ataxia Cardiomyopathy Model

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Free PMC article

Nicotinamide Mononucleotide Requires SIRT3 to Improve Cardiac Function and Bioenergetics in a Friedreich's Ataxia Cardiomyopathy Model

Angelical S Martin et al. JCI Insight. .
Free PMC article

Abstract

Increasing NAD+ levels by supplementing with the precursor nicotinamide mononucleotide (NMN) improves cardiac function in multiple mouse models of disease. While NMN influences several aspects of mitochondrial metabolism, the molecular mechanisms by which increased NAD+ enhances cardiac function are poorly understood. A putative mechanism of NAD+ therapeutic action exists via activation of the mitochondrial NAD+-dependent protein deacetylase sirtuin 3 (SIRT3). We assessed the therapeutic efficacy of NMN and the role of SIRT3 in the Friedreich's ataxia cardiomyopathy mouse model (FXN-KO). At baseline, the FXN-KO heart has mitochondrial protein hyperacetylation, reduced Sirt3 mRNA expression, and evidence of increased NAD+ salvage. Remarkably, NMN administered to FXN-KO mice restores cardiac function to near-normal levels. To determine whether SIRT3 is required for NMN therapeutic efficacy, we generated SIRT3-KO and SIRT3-KO/FXN-KO (double KO [dKO]) models. The improvement in cardiac function upon NMN treatment in the FXN-KO is lost in the dKO model, demonstrating that the effects of NMN are dependent upon cardiac SIRT3. Coupled with cardio-protection, SIRT3 mediates NMN-induced improvements in both cardiac and extracardiac metabolic function and energy metabolism. Taken together, these results serve as important preclinical data for NMN supplementation or SIRT3 activator therapy in Friedreich's ataxia patients.

Keywords: Cardiology; Metabolism.

Conflict of interest statement

Conflict of interest: RMP has intellectual property, income, research support, and ownership in a company related to gene therapy in Friedreich’s ataxia.

Figures

Figure 1
Figure 1. FXN-KO cardiac mitochondria have protein hyperacetylation, redox imbalance, and altered NAD+ homeostasis.
(A) Difference in mitochondrial protein acetylation relative occupancy sites (i.e., acetyl peptides quantification corrected for change in protein abundance) between WT and FXN-KO at 5, 8, and 13 weeks of age. Black represents statistically significant (Padjusted ≤ 0.05) acetyl sites with FCs above 2- or below –2-fold; dark gray represents FCs in between; and light gray represents acetyl sites with no statistically significant FC. (B) Proteomic measurements of OXPHOS complex I subunit levels in WT and FXN-KO heart mitochondria at 5, 8, and 13 weeks of age. Bar line represents sample mean. Subunits shown are statistically significant from respective WT (at 8 and 13 weeks). (C–D) Proteomic measurements of NAD+ biosynthesis proteins NAMPT (C) and NMNAT3 (D) in WT and FXN-KO heart mitochondria at 5, 8, and 13 weeks of age. Bar line represents sample mean. (E) Statistically significant acetyl-lysine sites in common between the SIRT3-KO whole hearts (green; ^ from ref. 28); and FXN-KO heart mitochondria at 5, 8, and 13 weeks of age (shades of red). Purple and pink shading represents 1 SD from the mean of the bottom and top, respectively, 25% SIRT3-KO sites. (F) Schematic summarizing shifts in the mitochondrial NAD+ metabolome in the FXN-KO heart. *P < 0.05, difference from respective WT values at each age group by Student’s t test and Benjamini-Hochberg FDR correction; n = 3 mice per age group per genotype. W: WT, F: FXN-KO and S: SIRT3-KO.
Figure 2
Figure 2. SIRT3 ablation in the FXN-KO heart induces sustained left ventricular wall thickening.
(A) Targeting strategy used to generate the cardiac and skeletal muscle–specific Sirt3-KO and Sirt3/Fxn-KO (dKO) mice. A map of the Fxn (left) and Sirt3 (right) genomic locus, which shows the conditional allele (upper panels) and the KO allele (lower panels). Arrowheads indicate LoxP sites. Vertical bars depict respective exons. (B) Cardiac Fxn and Sirt3 mRNA expression. Values are means ± SEM (n = 3–5 mice/group). (C) Cardiac protein acetylation, complex I (NDUFB8) and II (SDHB) subunits, SIRT3, and GAPDH protein expression assessed via Western blotting. (D and E) Western blot quantification of acetylation (D) and SIRT3 expression (E); values are means ± SEM. (F) Body weight from 6 weeks to 10 weeks of age. Values are means ± SEM (n = 11–18 mice/group). (G) Wall thickness measured via echocardiography at 9–10 weeks of age. Values are means ± SEM (n = 7–12 mice/group). (H) Left ventricle end-systolic diameter measured via echocardiography at 9–10 weeks of age. Values are means ± SEM (n = 7–12 mice/group). (I) Hemodynamic measure ejection fraction assessed by PV-loop analysis. Values are means ± SEM (n = 5–12 mice/group). *P < 0.05, difference from saline-treated WT as determined by two-way ANOVA for 8 groups (4 genotypes, 2 treatment) and Bonferroni correction. W, WT; F, FXN-KO; S, SIRT3-KO; and D, dKO.
Figure 3
Figure 3. NMN in the FXN-KO improves diastolic and normalizes systolic function in a SIRT3-dependent manner.
(A) Experimental schedule of treatment and clinical phenotyping protocols. E, echocardiography; C, noninvasive monitoring by CLAMS; P, PV-loop analysis; and †, sacrifice and collection of tissues for further analysis. Terminal procedures represented by olive-colored box. (B) Metabolomics profiling of cardiac NAD+ levels. Values are means ± SEM (n = 3–5 mice/group). (C) Wall thickness in NMN-treated animals measured via echocardiography at 9–10 weeks of age. Values are means ± SEM (n = 7–12 mice/group). (D–F) Hemodynamic measures assessed by PV-loop analysis: ejection fraction (D), active relaxation (τ, E), passive filling (linear end diastolic pressure volume relation [EDPVR], F), and contractility (linear ESPVR, G; dP/dtmax vs. end-diastolic volume [EDV], H; maximal elastance [Emax], I). Values are means ± SEM (E and G–I, n = 5–10 mice/group; D and F, n = 7–10 mice/group). Hashed line at 50% represents normal mouse ejection fraction. *P < 0.05, difference from saline-treated WT as determined by two-way ANOVA for 8 groups (4 genotypes, 2 treatment) and Bonferroni correction. W, WT; F, FXN-KO; S, SIRT3-KO; D, dKO; subscript S, saline; and subscript N, nicotinamide mononucleotide (NMN).
Figure 4
Figure 4. NMN reduces energy wasting and improves energy utilization in the FXN-KO heart.
(A and B) High-energy phosphate-bearing metabolite ratios, PCr/Cr (A) and PCr/ATP (B). Bar line represents sample mean (n = 3–5 mice/group). (C) Cardiac efficiency and (D) ventriculoarterial energy transfer ratio. Ea, effective arterial elastance; Ees, ventricular end-systolic elastance. Values are means ± SEM (n = 5–12 mice/group). (E) Energy contribution diagrams (n = 5–12 mice/group). Mechanical energy (olive green) and potential energy (dark brown) contribute to overall available energy (PVA). (F) Quantification of the energy contribution diagrams in E. *P < 0.05, difference from saline-treated WT as determined by two-way ANOVA for 8 groups (4 genotypes, 2 treatment) and Bonferroni correction. W, WT; F, FXN-KO; S, SIRT3-KO; D, dKO; subscript S, saline; and subscript N, NMN.
Figure 5
Figure 5. NMN reduces daily energy expenditure in the FXN-KO mouse in a SIRT3-dependent manner.
(A) Energy expenditure, (B) fat oxidation, (C) carbohydrate oxidation, (D) oxygen consumption (VO2) and carbon dioxide production (VCO2), and (E) respiratory exchange ratio (RER). Values are means ± SEM (n = 3–6 mice/group). *P < 0.05, difference from saline-treated WT as determined by two-way ANOVA for 8 groups (4 genotypes, 2 treatment) and Bonferroni correction. W, WT; F, FXN-KO; S, SIRT3-KO; D, dKO; subscript S, saline; and subscript N, NMN.
Figure 6
Figure 6. NMN glucose utilization in the FXN-KO mouse in a SIRT3-dependnet manner.
(A) Serum lactate levels. Values are means ± SEM (n = 6–12 mice/group). (B) 2-14C-pyruvate oxidation measured in cardiac tissue lysates. Values are means ± SEM (n = 3–6 mice/group). (C–H) Metabolomic profiling from cardiac tissue of metabolites involved in glucose metabolism. Ratio of glucose committed to glucose metabolism in the heart (G6P/glucose ratio, C), glycolysis intermediates (D), glycogen intermediate (E), hexosamine biosynthesis pathway (HBP) and sialic acid biosynthesis pathway (SBP) intermediates (F), pentose phosphate pathway (PPP) intermediates (G), and glutathione/GSSG ratio (H). Bar line represents sample mean (n = 3–5 mice/group). *P < 0.05, difference from saline-treated WT as determined by two-way ANOVA for 8 groups (4 genotypes, 2 treatment) and Bonferroni correction. W, WT; F, FXN-KO; S, SIRT3-KO; D, dKO; subscript S, saline; and subscript N, NMN.

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