Archive for July 16th, 2018


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

on July 16, 2018 in Uncategorized Comments Off on 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+-asso- ciated 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 di- rect 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.

Introduction

 

The adult mammalian heart requires a continuous supply of ATP to sustain contraction. The majority of the heart’s ATP (> 95%) is pro- duced from mitochondria through oxidative phosphorylation (OXPHOS) [1,2]. Given the fact that cardiac ATP reserve is very limited, a robust uninterrupted ATP supply from mitochondrial is critical to maintain cardiac function and failure to do so contributes to disease states such as heart failure [3–5]. The importance of this adaptation is underscored by the severe cardiomyopathy seen in patients with inborn errors of the mitochondrial fatty acid oxidation (FAO) pathway [6]. In adulthood, abnormalities in FAO and other mitochondrial functions also occur with aging and a variety of acquired pathologic conditions (ischemia, hypertension, valvular disease, etc.) leading to heart failure [2,7,8]. Accumulating experimental evidence from animal studies identifies reduced cardiac oxidative metabolism as a signature of heart dysfunction and failure [9]. Clinical studies also find the myocardial [PCr]/[ATP] ratio correlates with heart failure severity and is a strong predictor of cardiovascular mortality [10]. To date, mitochondrial

dysfunction has been recognized as critical for the pathogenesis of cardiac diseases and a promising therapeutic target [11,12].

In addition to inborn genetic defects, mitochondrial dysfunction can result from accumulated damage at the mitochondrial genomic DNA (mtDNA), protein, and lipid levels due to high oxidative environment inside the mitochondria. However, clinical trials with a variety of an- tioxidant strategies have failed to show beneficial effects [13]. In the last decade, mitochondrial protein acetylation has emerged as an im- portant mechanism that affects mitochondrial homeostasis and organ function. Lysine-acetylation is a revisable posttranslational modifica- tion of proteins that is determined by the balance between acetyl- transferase and deacetylase activity. In mitochondria, there are three NAD+-dependent deacetylases, namely Sirt3, Sirt4 and Sirt5, forming a Sirtuin network that mediates the acetylation of mitochondrial proteins and subsequently regulates mitochondrial function [14]. Given that the reducing equivalents NADH (and FADH2) drives the mitochondrial electron transfer chain (ETC), the equilibrium of NADH/NAD+ is cri- tical for mitochondrial OXPHOS. Therefore, NAD+ is at center of OX- PHOS and protein acetylation, making it an attractive target for heart failure treatment [12].

Kruppel-like factors (KLF) are a subclass of the zinc-finger tran-scription factors that bind a consensus 5′-C(A/T)CCC-3′ motif in the promoters and enhancers of various genes and regulate critical cellular processes, such as cell proliferation, differentiation, survival, apoptosis, metabolism and stemness. Members of the KLF family play important roles in multiple cell types within the cardiovascular system [15–20]. In particular, our recent studies have identified Kruppel-like factor 4 (KLF4) as a critical regulator of cardiac mitochondrial homeostasis [20]. We previously showed that mice bearing cardiac deficiency of KLF4 were sensitized to pressure overload-induced heart failure due to disruption of mitochondrial homeostasis upon stress [18,20]. However, the underlying initial defects leading to such rapid cardiac dysfunction remain unknown. Although mitochondrial function is reduced in the KLF4-deficient hearts at baseline, transcriptomic studies did not reveal big changes at transcriptional level (82 genes with FDR < 0.05 at baseline) [20]. These results strongly suggest that the posttranslational mechanisms are important contributors to the mitochondrial defects. Here we show that hyperacetylation of mitochondrial proteins in the KLF4-deficient hearts might be the underlying defects at baseline and administration of Nicotinamide Mononucleotide (NMN), a precursor of NAD+, successfully preserved mitochondrial homeostasis and rescued heart function during pressure overload.

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Fig. 1. Hyperacetylation of mitochondrial proteins in KLF4-deficient hearts.
(A) Blue-Native PAGE showing mitochondrial ETC complexes.
(B–D) Mitochondrial 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.

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