Administration of NMN improves mitochondrial fatty acid oxidation

July 22, 2018


It has been shown that long-term administration of NMN enhances overall metabolism at the whole organism level. We next tested if short- term administration of NMN in present study could affect mitochon- drial respiration. We first treated primary neonatal rat ventricular myocytes (NRVM) with NMN (2 mmol/L) for 24 h and assessed mi- tochondrial respiration using a Seahorse extracellular flux analyzer. With glucose and pyruvate as substrates, we observed no difference in either basal or maximal oxygen consumption rate (OCR) between NMN- treated and control groups (Fig. 4A), suggesting short-term treatment of NMN does not induce mitochondrial biogenesis or change mitochon- drial respiration capacity. However, when using Palmitate as a long- chain fatty acid substrate, both basal and maximal OCR were sig- nificantly increased by NMN treatment (Fig. 4B), suggesting that NMN improved fatty acid oxidation (FAO). We further confirmed that such short-term NMN treatment (24 h) did not significantly induce the ex- pression of FAO genes (i.e. PPARα, CD36, CPT1β, CPT2, LCAD, MCAD) or mitochondrial biogenesis genes (i.e. PGC-1α, PGC-1β, ERRα):


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.)

(Fig. 4C), suggesting a posttranslational effect of NMN on FAO rather than transcriptional metabolic reprogramming.

In another set of NRVM experiments, KLF4 was silenced using adenoviral shRNA to recapitulate the cardiac KLF4 deficiency. Similar to the CM-K4KO heart, loss of KLF4 in NRVM resulted in hyper- acetylation of mitochondrial protein (Fig. 4D). As expected, silencing KLF4 led to reduced mitochondrial respiration in NRVM (Fig. 4E, no NMN groups). Similar to studies on normal NRVM, NMN did not affect pyruvate oxidation but significantly improved FAO (Fig. 4E, NMN- treated groups).

Collectively, these NRVM-based mitochondrial respiration studies demonstrated that NMN treatment, even for a short-term, improved long-chain fatty acid oxidation. This effect of NMN can be achieved by either deacetylation of key FAO enzymes, such as LCAD (Figs. 1E & 3B) [22], or increase of NAD+ supply for the TCA cycle (Fig. 3A). Never- theless, given that long-chain fatty acid is a major fuel of the heart, NMN can thus profoundly improve cardiac energetics and heart function.

3.5. NMN administration preserved mitochondrial ultrastructure and reduced cell death in the pressure overloaded hearts

Upon pressure overload, the CM-K4KO mice developed acute heart failure with severe disruption of mitochondrial homeostasis that man- ifested as dramatic alterations in mitochondrial ultrastructure, in- cluding degeneration, fragmentation, crista swelling, and heterogeneity (Fig. 5A). In contrast, no such change was observed in the MHC-Cre groups. Strikingly, NMN administration almost completely restored mitochondrial ultrastructure to the level comparable to the MHC-Cre group (Fig. 5B). These data were consistent with the cardiac function observed by echocardiography (Fig. 3C) and indicated that NMN suc- cessfully preserved mitochondrial homeostasis in the CM-K4KO hearts during pressure overload leading to improved cardiac function.

Mitochondrial damage is often associated with ROS stress and cell death. We found that TAC induced significant increase of ROS in the CM-K4KO myocardium and NMN treatment diminished such ROS burst (Fig. 6). Further, TAC induced significant cell death in the CM-K4KO hearts as > 10% nuclei showed TUNEL positive staining (Fig. 7). However, such TAC-induced cardiac cell death was reduced to < 5% in the CM-K4KO mice that received NMN treatment (Fig. 7). There was only minimal cell death detected in the MHC-Cre groups and it was similar before and after TAC. Collectively, these data suggested that short-term administration of NMN preserved cardiac mitochondrial function, reduced TAC-induced mitochondrial damage, reduced myo- cardial ROS and prevented cell death. The combined benefit of NMN- mediated effects at aforementioned multiple levels help maintain normal functions of cardiac mitochondria, cardiomyocytes and ulti- mately the heart.

4. Discussion

In this study, we demonstrate that cardiac KLF4-deficiency leads to hyperacetylation of mitochondrial proteins that predisposes the mi- tochondria and heart sensitive to stress. Short-term administration of NMN, a precursor of NAD+, preserved mitochondrial homeostasis and rescued heart function from pressure overload-induced heart failure. Our study thus identified protein hyperacetylation as a cause of mi- tochondrial dysfunction as well as a promising therapeutic target for heart failure. We demonstrate that administration of NMN (or other NAD+ repletion reagents) is capable of rescuing the heart through preservation of mitochondrial homeostasis.

Mitochondrial dysfunction has long been associated with heart failure. The investigation of mitochondrial function in human and an- imal models of heart failure shows a variety of mitochondrial altera- tions that point to defects at specific sites, i.e. the electron transport chain (ETC), the phosphorylation apparatus, or the supercomplexes assembly [27]. Although different etiology of heart failure leads to different presentation of mitochondrial dysfunction, in general severe heart failure is associated with reduced mitochondrial metabolic ca- pacity and low ATP production [10]. Mitochondrial dysfunction is thought to be a result of accumulative injury from ROS due to its high- level oxidative metabolism. However, antioxidant therapy failed to be effective in clinical trials, suggesting novel therapies are required to target mitochondrial dysfunction.

Recently, mitochondrial protein hyperacetylation has been re- cognized as a common mechanism underlying mitochondrial dysfunc- tion in multiple organs including heart, liver, brain, kidney and muscle [28,29]. In general, hyperacetylation of mitochondrial proteins is be- lieved to be detrimental to metabolism as demonstrated by numerous studies on the Sirt3-deficient mice [21–23,25,29–34] and recently by studies in clinical heart failure samples [12,28]. Conversely, deacety- lation of mitochondrial proteins, i.e. by overexpression of Sirt3, has been shown to enhance metabolism [25,35]. Pharmacologically,

administration of NAD+ precursors, such as NMN and nicotinamide riboside (NR), could normalize the NADH/NAD+ ratio, restore protein acetylation and boost metabolic function [36]. However, there are also studies suggested a correlation between increased acetylation and in- creased FAO enzyme activity [37,38]. Such discrepancy may be due to the different metabolic models used in different studies. In the studies that showed protein acetylation increases FAO, they were studying animals that are suffering obesity, diabetes or heart failure [38], con- ditions that are known to have profound alterations in metabolism, FAO in particular.

In present study, we demonstrated that NMN rescued TAC-induced mitochondrial dysfunction and cardiac failure in the KLF4-deficient heart. Our short-term administration of NMN did not affect the mi- tochondrial biogenesis or metabolic capacity but dramatically im- proved FAO, prevented mitochondrial damage and cell death. In studies that reported increased metabolic function by NAD+ precursors, the administration duration was often long-term (weeks or months) where some reprograming of cellular metabolic machinery including mi- tochondrial biogenesis could take place in multiple organs leading to the overall metabolic rate change of the whole organism [36,39]. However, here we showed that even short-term administration of NMN could be dramatically beneficial.

Obviously NMN can have impact at multiple levels as discussed below. We think the therapeutic effect from NMN administration is a combination of all.

First, NMN administration can potentially correct metabolic defects or enhance metabolism through repletion of intracellular NAD+ pools including the mitochondrial NAD+ pool. NAD+ is a critical coenzyme for mitochondrial ATP production. NAD+ gains two electrons at mul- tiple steps of TCA cycle to form NADH, which is the major reducing equivalent driving ETC. As the TCA cycle and ETC require NAD+ and NADH, respectively, an optimal NAD+/NADH ratio is needed for effi- cient mitochondrial metabolism. Recent studies have demonstrated that NAD+ levels are rate-limiting for mitochondrial respiration [40]. As such, supplement of NMN can immediately boost mitochondrial func- tion through increasing the NAD+/NADH ratio. Further, Sirt3-NAD+- dependent deacetylation of key FAO enzymes, such as LCAD, can im- prove mitochondrial metabolic function as well [22]. NAD+ also can activate Sirt1 leading to deacetylation and activation of PGC1α, the master regulator of metabolism and mitochondrial biogenesis [40]. Our mitochondrial respiration data from NRVM demonstrated significant improvement in FAO by NMN but not in pyruvate oxidation, suggesting short-term treatment of NMN might have a quick effect on NAD+/ NADH ratio and/or FAO enzymes deacetylation but not PGC1α-medi- ated mitochondrial biogenesis. Consistently, there was no significant changes in FAO or mitochondrial biogenesis genes during the short- term NMN treatment. Finally, before entering the TCA cycle all long- chain fatty acids are oxidized to Acetyl-CoA through β-oxidation that requires NAD+ as coenzyme. As such, FAO may require higher NAD+ concentration than pyruvate oxidation and NMN therefore can have more impact on FAO.

Second, NMN administration can protect cardiomyocytes from stress-induced cell death. We observed a strikingly high rate of cell death (> 10%) in CM-K4KO myocardium after stress, which could be a key contributor to acute heart failure. Such massive cell death, how- ever, was significantly blocked by NMN administration (Fig. 7). It has been reported that, mitochondrial NAD+ levels, the highest among all intracellular NAD+ pools, dictate cell survival [41]. Depletion of mi- tochondrial NAD+ induces cell death and overexpression of NAMPT protects against genotoxic cell death. Of note, NAMPT-mediated pro- tection requires Sirt3 and Sirt4, suggesting a mitochondrial acetylation- related mechanism [41]. In present study, we found reduction in Sirt3, NAMPT and NAD+ levels in CM-K4KO myocardium, all of which could contribute to cell death. Because of no detectable cell death at baseline in CM-K4KO hearts, it is likely that the CM-K4KO heart has low but above critical threshold level of NAD+ at baseline. However, upon TAC stress, NAD+ level declines and it may reach critical threshold level in CM-K4KO heart to trigger metabolic problems and cell death. Con- versely, supplement of NMN help replete the NAD+ pools to maintain mitochondrial function and prevent cell death.

Third, NMN administration can protect mitochondrial homeostasis. The mitochondria in KLF4-deficient hearts suffered dramatic damage after TAC (Fig. 5). Consistently, there is more inflammation and ROS in the CM-K4KO myocardium as well (Figs. 3D & 6). NMN administration significantly rescued the mitochondrial ultrastructure, reduced ROS and inflammation in CM-K4KO myocardium. In part, NAD+-mediated improvement of mitochondrial metabolism could contribute here. Moreover, we found hyperacetylation of CypD and SOD2 in KLF4-de- ficient cardiac mitochondria (Fig. 1E). It has been shown that CypD hyperacetylation is associated with heart failure [12]. Acetylation of CypD promotes its binding to oligomycin-sensitive conferring protein (OSCP) and increases the sensitivity of mitochondrial permeability pore (mPTP) [12,42]. SOD2 is the most important antioxidant in the mi- tochondrial matrix, where it neutralizes the high toxic superoxide generated from ETC. Sirt3-mediated deacetylation activates SOD2, while acetylation of SOD2 impairs its function leading to ROS stress [21,43]. Both mPTP opening and ROS has been associated with cell death, inflammation and the development of heart failure [12,44]. Therefore, NMN can preserve mitochondrial homeostasis through sta- bilization of mPTP and reduction of ROS.

Finally, NMN can reduce myocardial inflammation. Damaged mi- tochondria can be very inflammatory, likely through ROS and its pro- karyotic genomic DNA [45]. Inflammation is a vicious positive feed- back cycle in which the initial injury in cardiomyocytes would induce infiltration of immune cells to further amplify myocardial inflamma- tion. By protecting mitochondria from stress-induced damage (SOD2, mPTP, etc.), NMN thus can cut off the initial inflammatory signal, leading to reduced myocardial inflammation (Fig. 3D).

A surprising observation is that NMN did not exhibit detectable benefit in the Cre group. A simple explanation could be that these Cre mice are in fact perfectly healthy having normal NAD+ and protein acetylation levels at baseline. Although TAC does reduce cardiac NAD+ levels (Fig. 2C), it may never become rate-limiting in a normal mouse heart at the early stage of pressure overload hypertrophy (1–5 days in present study). Of note, this is quite different than chronic heart failure patients who have already reached late stage of the diseases. Reduced NAD+ levels and hyperacetylation of cardiac mitochondrial proteins have been observed in experimental heart failure models and clinical heart failure samples [12,28]. Further, normalization of NAD+ has been shown to be beneficial in mitochondrial mutant and WT mice that suffered heart failure [12].

In summary, we employed the cardiac KLF4-deficient mice as a pressure overload-induced acute heart failure model for a proof of principle study to demonstrate that administration of NMN (even short- term) could be an effective therapy for heart failure.

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