Short-term administration of Nicotinamide Mononucleotide preserves cardiac mitochondrial homeostasis and prevents heart failure

Abstract

Heart failure is associated with mitochondrial dysfunction so that restoring or improving mitochondrial health is of therapeutic importance. Recently, reduction in NAD⁺ levels and NAD⁺-mediated deacetylase activity has been recognized as negative regulators of mitochondrial function. Using a cardiac specific KLF4 deficient mouse line that is sensitive to stress, we found mitochondrial protein hyperacetylation coupled with reduced Sirt3 and NAD⁺ levels in the heart before stress, suggesting that the KLF4-deficient heart is predisposed to NAD⁺-associated defects. Further, we demonstrated that short-term administration of Nicotinamide Mononucleotide (NMN) successfully protected the mutant mice from pressure overload-induced heart failure. Mechanically, we showed that NMN preserved mitochondrial ultrastructure, reduced ROS and prevented cell death in the heart. In cultured cardiomyocytes, NMN treatment significantly increased long-chain fatty acid oxidation despite no direct effect on pyruvate oxidation. Collectively, these results provide cogent evidence that hyperacetylation of mitochondrial proteins is critical in the pathogenesis of cardiac disease and that administration of NMN may serve as a promising therapy.

Keywords: Heart failure; Mitochondria; NAD; Pressure overload; Protein hyperacetylation.

Fig. 1.

Hyperacetylation of mitochondrial proteins in KLF4-deficient hearts. (A) Blue-Native PAGE showing mitochondrial ETC complexes. (B–D) Mitochondxrial protein acetylation assessed by anti-acetyl-Lysine Western blot. Cardiac mitochondria isolated from baseline 4-month old animals (B), animals that received 5 days TAC (C) and animals that developed heart failure (EF < 30%, data not shown) after 5-weeks of high intensity TAC (D). (E) Acetylation of SOD2, CypD and LCAD in cardiac mitochondria. Mitochondrial protein was immunoprecipitated with anti-acetyl-Lysine antibody and probed for specific proteins as indicated.

Fig. 2.

Myocardial Sirt3, NAD⁺ and NAMPT levels. (A) Expression of mammalian Sirtuin genes in the heart. n = 5–7, *p < 0.05. (B) Sirt3 protein in cardiac mitochondria isolated from 4-month old animals. (C) Myocardial NAD⁺ levels before and after 3-day TAC. n = 4, *p < 0.05. (D) Outline of NAD⁺ synthesis pathway. (E) NAMPT gene expression in the heart. n = 5–7, *p < 0.05.

Fig. 3.

Administration of NMN corrected mitochondrial acetylation and rescued KLF4-deficient heart from TAC-induced heart failure. (A) Administration of NMN increased myocardial NAD + levels. n = 5, *p < 0.05. NMN administration: 3 days. (B) Administration of NMN reduced mitochondrial protein acetylation in the heart. (C) Echocardiography analysis showing cardiac contractility. Left ventricular contractile function shown as fractional shortening (FS). (D) Expression of hypertrophy marker genes and inflammatory genes in the heart. n = 5–9, *p < 0.05. TAC and NMN administration: 5 days. NMN was injected intraperitoneally at 500 mg/kg/day. PBS was injected as vehicle control.

Fig. 4.

NMN improves mitochondrial fatty acid oxidation. (A, B) Mitochondrial respiration function in NRVM assessed by Seahorse Mito Stress test using (A) Pyruvate + Glucose and (B) Palmitate (BSA-conjugated palmitate) as substrates, respectively. OCR: oxygen consumption rate. Oligomycin: ATP synthase inhibitor. FCCP: mitochondrial uncoupler. Rot/AA: rotenone and antimycin A, specific inhibitors for ETC complex I and III respectively. Arrows indicate time for drug injection. Basal rate was calculated as OCR before addition of Oligomycin. Maximal rate was calculated as the highest OCR after addition of FCCP. NRVMs were treated with 2 mmol/L NMN for 24 h (NMN) or left untreated (Control). n = 3, *p < 0.05. (C) The expression of FAO and mitochondrial biogenesis genes in NRVM treated with 2 mmol/L NMN for 24 h. n = 6, p ≥ 0.43. (D) Mitochondrial protein acetylation and Sirt3 levels in NRVMs that were infected for 72 h with empty virus (Sh-EV) or virus expressing a ShRNA against rat KLF4 (Sh-KLF4). (E) Mitochondrial respiration assessed using Pyruvate + Glucose and Palmitate (BSA-conjugated palmitate) as substrates, respectively. NMN groups were treated with 2 mmol/L NMN starting at 24 h post-infection and Seahorse assay was performed at 72 h post-infection. n = 3, *p < 0.05.

Fig. 5.

Administration of NMN protected cardiac mitochondria from TAC-induced damage. (A) EM images from PBS (vehicle) treated hearts after 5 days of TAC. (B) EM images from NMN (500 mg/kg/day) treated hearts after 5 days of TAC. Scale bar: 1 um. Arrows indicate damaged or abnormal mitochondria. Each image was from individual animal but different areas were chosen to display different phenotype. n = 3 in each group.

Fig. 6.

Administration of NMN reduced TAC-induced ROS generation in KLF4-deficient hearts. (A) Representative images showing myocardial DHE staining. (B) ROS level was calculated as DHE positive cells per field. TAC and NMN administration: 5 days. n = 3–5 animals in each group. *p < 0.05.

Fig. 7.

Administration of NMN reduced TAC-induced cell death in KLF4-deficient hearts. (A) Representative TUNEL staining images from n = 3–5 animals in each group. TAC: 5 days. Apoptotic nuclei were stained in brown by DAB and normal nuclei were stained in light blue by Methyl green counter stain. (B) Cell death index was calculated as percentage of brown nuclei in all nuclei. n = 3–5, *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)