NMNAT3 Is Involved in the Protective Effect of SIRT3 in Ang II-Induced Cardiac Hypertrophy

Experimental Cell Research 347 (2016) 261–273

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Experimental Cell Research

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Research Article NMNAT3 is involved in the protective effect of SIRT3 in Ang II-induced cardiac hypertrophy

Zhongbao Yue a,1, Yunzi Ma b,1, Jia You a, Zhuoming Li a, Yanqing Ding a, Ping He a, Xia Lu c, Jianmin Jiang a, Shaorui Chen a,n, Peiqing Liu a,d,nn a Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, Guangdong Province, People's Republic of China b Department of Pharmacy, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, Guangdong Province, People's Republic of China c School of Nursing, Guangdong Pharmaceutical University, Guangzhou 510006, Guangdong Province, People's Republic of China d National and Local Joint Engineering Laboratory of Druggability Assessment and Evaluation, Sun Yat-sen University, Guangzhou 510006, Guangdong Province, People's Republic of China article info abstract

Article history: Pathological cardiac hypertrophy is a maladaptive response in a variety of organic heart disease (OHD), Received 14 February 2016 which is characterized by mitochondrial dysfunction that results from disturbed energy metabolism. Received in revised form SIRT3, a mitochondria-localized sirtuin, regulates global mitochondrial lysine acetylation and preserves 29 June 2016 mitochondrial function. However, the mechanisms by which SIRT3 regulates cardiac hypertrophy re- Accepted 12 July 2016 mains to be further elucidated. In this study, we firstly demonstrated that expression of SIRT3 was de- Available online 14 July 2016 creased in Angiotension II (Ang II)-treated cardiomyocytes and in hearts of Ang II-induced cardiac hy- Keywords: pertrophic mice. In addition, SIRT3 overexpression protected myocytes from hypertrophy, whereas SIRT3 SIRT3 silencing exacerbated Ang II-induced cardiomyocyte hypertrophy. In particular, SIRT3-KO mice exhibited NMNAT3 significant cardiac hypertrophy. Mechanistically, we identified NMNAT3 (nicotinamide mononucleotide Cardiac hypertrophy adenylyltransferase 3), the rate-limiting enzyme for mitochondrial NAD biosynthesis, as a new target and binding partner of SIRT3. Specifically, SIRT3 physically interacts with and deacetylates NMNAT3, thereby enhancing the enzyme activity of NMNAT3 and contributing to SIRT3-mediated anti-hypertrophic effects. Moreover, NMNAT3 regulates the activity of SIRT3 via synthesis of mitochondria NAD. Taken together, these findings provide mechanistic insights into the negative regulatory role of SIRT3 in cardiac hypertrophy. & 2016 Published by Elsevier Inc.

1. Introduction

Cardiac hypertrophy is characterized by re-activation of the Abbreviations: SIRT, sirtuin; NMNAT, nicotinamide mononucleotide adenylyl- fetal gene program, increase in protein synthesis, and subsequent transferase; NAMPT, nicotinamide phosphoribosyltransferase; NAD, nicotinamide increase in the size of the cardiomyocytes [1,2]. Cardiac hyper- adenine dinucleotide; Ang II, angiotensin II; ANF, atrial natriuretic factor; BNP, trophy is initially regarded as a compensatory response of the brain natriuretic polypeptide; NRCMs, neonatal rat cardiomyocytes; NAM, nicoti- heart to various physiological or pathological stimuli. However, namide; FOXO3a, forkhead box O3a; LKB1, liver kinase B1; ROS, reactive oxygen species; HDACs, histone deacetylases; UPR, unfolded protein response; COX-IV, prolonged hypertrophy of the heart ultimately impairs the cardiac Cytochrome C oxidase IV function and results in heart failure [3]. The incidence of cardiac n Correspondence to: Department of Pharmacology and Toxicology, School of hypertrophy is dramatically increased with age [4], implying that Pharmaceutical Sciences, Sun Yat-sen University (Higher Education Mega Center), aging-associated mechanisms are involved in the pathogenesis of 132# East, Wai-huan Road, Guangzhou 510006, Guangdong, People's Republic of China. cardiac hypertrophy. nn Corresponding author at: Department of Pharmacology and Toxicology, School Sirtuins, a family of evolutionarily conserved histone deacety- of Pharmaceutical Sciences, Sun Yat-sen University (Higher Education Mega Cen- lases (HDACs), play pivotal roles in the regulation of aging-related ter), 132# East, Wai-huan Road, Guangzhou 510006, Guangdong, People's Republic pathophysiology. Distinguished from other HDAC classes, sirtuins of China. require nicotinamide adenine dinucleotide (NAD) for their acti- E-mail addresses: [email protected] (S. Chen), fi [email protected] (P. Liu). vation, and thus are de ned as class III HDACs [5,6]. Collective 1 These authors contributed equally to this work. evidence has indicated that sirtuins are critical regulators of many http://dx.doi.org/10.1016/j.yexcr.2016.07.006 0014-4827/& 2016 Published by Elsevier Inc. 262 Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273 cellular cascades, including stress responses, cell growth, energy (Shanghai, China). The adult male mice (8 weeks old) were used metabolism and apoptosis [7–9]. Seven subtypes of sirtuins, in the study. Wild type and knockout mice were infused with named as SIRT1–7, have been identified in mammals. SIRT3, lo- Ang II (Sigma, 3 mg/kg/d dissolved in 0.9% NaCl), with saline- cated in the mitochondria, is considered as the major mitochon- infusedmiceservingasvehiclecontrols.Allmicewereinfused drial deacetylase [10]. SIRT3 is involved in the regulation of mul- with Ang II by implanted osmotic minipumps (Alzet model tiple mitochondrial metabolic events, such as fatty acid oxidation, 1002, Alza Corp.) for 2 weeks [29].After14days,micewere tricarboxylic acid (TCA) cycle, the electron transport chain, and the sacrificed and subjected to hypertrophic analysis. urea cycle [11–13]. SIRT3 is abundantly expressed in organs with Additional materials and methods are listed in Supplementary highly metabolic turnover, such as the heart [14]. SIRT3 depletion Materials. Data were presented as means 7 standard error of the impairs mitochondrial and contractile function in the heart, due to mean (SEM). Statistical analysis was performed by SPSS statistic increased acetylation of various energy metabolic proteins and software13.0. Mean difference between two groups was tested by subsequent myocardial energy depletion [15]. Coincidently, SIRT3 Student’s t-test. Statistical analysis among the various groups was preserves heart function and capillary density in the setting of performed by One-way analysis of variance (ANOVA) with Bon- obesity [16]. SIRT3 protects cardiomyocytes from stress-mediated ferroni post hoc test. In all cases, difference between groups was cell death and preserves contractile function in response to a considered statistically significant at Po0.05. chronic increase in workload via FOXO3a-mediated suppression of oxidative stress [17,18]. Furthermore, lack of SIRT3 facilitates the opening of the mitochondrial permeability transition pore by in- 3. Results creasing acetylation and spatial dislocation of cyclophilin D [19]. Considering there are many downstream substrates of SIRT3 in 3.1. SIRT3 was downregulated in Ang II-induced cardiac hypertrophy mitochondria, the mechanisms by which SIRT3 regulates cardiac function and cardiac hypertrophy still need to be further We first determined the cellular localization of SIRT3 in cardi- elucidated. omyocytes. Our data showed that SIRT3 was present in the mi- NAD, a classic coenzyme for cellular redox reactions, has been tochondria (Fig. 1), in line with previous reports [10]. In response fi identi ed as a substrate for sirtuins [20,21]. In mammalians, NAD to Ang II (100 nM), a neurohumoral factor that stimulates cardiac can be synthesized through both salvage and de novo pathways hypertrophy, the mRNA and protein levels of SIRT3 in neonatal rat [22]. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate- cardiomyocytes (NRCMs) were decreased in a time-dependent limiting enzyme for NAD salvage from nicotinamide. Previous manner (Figs. 1 and 2A and B). Moreover, the enzyme activity of studies demonstrate that overexpression of NAMPT significantly SIRT3 was downregulated by Ang II treatment (Fig. 2C). In vivo, we increases NAD biosynthesis in primary cultured cardiomyocytes also observed that the protein level and enzyme activity of SIRT3 [23]. Nicotinamide mononucleotide adenylyltransferase (NMNAT) were also decreased in the mouse myocardium treated with Ang II is another central enzyme for NAD biosynthesis, which catalyzes (3 mg/kg/d) for 2 weeks (Fig. 2D and E). both de novo and salvage pathways [24]. Currently, three NMNAT isoforms have been identified, which varied in subcellular localization and tissue distribution [25]. Our previous study showed 3.2. SIRT3 protected cardiomyocytes against Ang II-induced that NMNAT2 attenuates Ang II (angiotensin II) induced cardio- hypertrophy myocyte hypertrophy via increasing NAD biosynthesis [26]. NMNAT3 is localized in the mitochondrial matrix and is the only To investigate the potential effect of SIRT3 on cardiomyocyte known enzyme of NAD synthesis residing within the organelles hypertrophy, NRCMs were transfected with plasmids encoding [27]. A recent study has reported that exogenous NAD could sup- Flag-tagged wild-type SIRT3, or the catalytically inactive mutant press the agonist-induced cardiac hypertrophy through activating H248Y-HA. Expression of SIRT3 was augmented after SIRT3-Flag or SIRT3, rather than SIRT1 [28]. Thus, it is plausible that SIRT3 in- H248Y-HA transfection (Fig. 3A), whereas, the enzyme activity of teracts withNMNAT3 in the mitochondria of cardiomyocytes. The SIRT3 was increased by SIRT3-Flag, rather than H248Y-HA mutant present study was designed to investigate this potential interac- (Fig. 3B). After treatment with 100 nM Ang II for 24 h, the ex- tion and the co-regulations of these enzymes in cardiac pression of hypertrophic biomarkers (atrial natriuretic factor (ANF) hypertrophy. and brain natriuretic peptide (BNP)), as well as the cell surface area, were significantly elevated (Fig. 3C and D). However, transfection with SIRT3-Flag, rather than H248Y-HA, reversed Ang II- 2. Materials and methods induced increase of hypertrophic biomarkers and cell surface area (Fig. 3C and D). Dulbecco's modified Eagle's medium (DMEM), fetal bovine By contrast, endogenous SIRT3 in cardiomyocytes was depleted serum (FBS), 4′, 6-diamidino-2-phenylindole (DAPI), penicillin/ by RNA interference. Quantitative RT-PCR was performed to eval- streptomycin,andLipofectamine2000werepurchasedfrom uate the efficiency of three independent siRNAs, marked as si001, Life technologies/ Invitrogen (Carlsbad, CA, USA). Anti-NMNAT3 si002, and si003, respectively. The si003 significantly decreased antibody was obtained from Santa Cruz Biotechnology (CA, the mRNA and protein level of SIRT3 (Fig. S1A), but had no sig- USA). Anti-acetyl-lysine (anti-AC-K), anti-SIRT3, anti-COX-IV, nificant effects on SIRT4 and SIRT5 (Fig. 4A), suggesting the spe- anti-Flag antibodies, and normal rabbit immunoglobulin G were cificity of siRNA. Therefore, si003 was renamed as siSIRT3 here- all bought from Cell Signaling Technology (Boston, MA, USA). after and used in the following experiments. Transfection with Anti-α-tubulin antibody, Anti-NAMPT antibody, and Angio- siSIRT3 significantly increased the mRNA levels of ANF and BNP, as tensin II (Ang II) were provided by Sigma-Aldrich (St. Louis, MO, well as the cell surface area (Fig. 4B and C). Additionally, silencing USA), Proteintech (Wuhan, China), and Merck Millipore (Bill- of endogenous SIRT3 further augmented Ang II-induced hyper- erica, MA, USA), respectively. SIRT3 genetic knockout mice trophic response (Fig. 4B and C). In line with the observations in in (strain#129-Sirt3tm1.1Fwa/J) were purchased from The Jackson vitro studies, genetic knockout of SIRT3 in mice significantly in- Laboratory(BarHarbor,ME,USA).Age-,sex-,andbackground- creased the cardiac expression of hypertrophic biomarkers. Ad- matched wild type mice were purchased from Shanghai Bio- ditionally, SIRT3 depletion could further exacerbate Ang II-induced model Organism Science & Technology Development Company cardiac hypertrophy (Fig. 4D). Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273 263

Fig. 1. The intracellular distribution of SIRT3 (green) and mitochondria (red) were detected by confocal immunoﬂuorescence microscopy. DAPI was used to counterstain the nuclei. The time-dependent effect of Ang II on SIRT3 protein expression was detected. Scale bar¼20 mm.

3.3. SIRT3 increased NMNAT3 activity by interacting with and dea- three-dimensional forms (Fig. 5B and Fig. S3). Co-im- cetylating NMNAT3 munoprecipitation experiments were conducted to test whether SIRT3 interacted with NMNAT3. As shown in Fig. 5C and D, SIRT3 To further investigate the molecular mechanism underlying was detected by Western blotting after NMNAT3 was im- SIRT3-mediated anti-hypertrophic effects, we explored the po- munopurified from both cultured cell extracts and mouse myo- tential interaction between SIRT3 and NMNAT3 in the mitochon- cardium, supporting the interaction between SIRT3 and NMNAT3. dria of cardiomyocytes. As shown in Fig. 5A, distribution of To determine whether NMNAT3 is a potential deacetylation NMNAT3 was similar to that of the mitochondrial mitotracker red substrate of SIRT3, we evaluated the acetylation level of NMNAT3 fluorescence, implying the mitochondrial localization of NMNAT3 with an acetylated lysine antibody. As shown in Fig. 6A, acetyla- [27]. Additionally, NMNAT3 co-localized with SIRT3 in cardio- tion level of NMNAT3 was remarkably increased in NRCMs treated myocytes, as shown by the immunofluorescence in both two- and with Ang II. Transfection with SIRT3-Flag, but not H248Y mutant, 264 Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273

Fig. 2. The mRNA, protein expression and enzyme activity of SIRT3 were decreased in cardiac hypertrophy induced by Ang II. Time-dependent effect of Ang II on SIRT3 mRNA and protein expression was detected by qRT-PCR (A) and Western blot (B), respectively. (C) The SIRT3 enzyme activity was determined in cardiomyocytes treated with 100 nM Ang II for indicated times. The protein expression (D) and enzyme activity (E) of SIRT3 were decreased in mouse myocardium treated with Ang II (3 mg/kg/d for 2 weeks). *P o0.05 vs. control (A–C) or vs. sham (sham-operated group) (D and E). reduced the acetylation level of NMNAT3 (Fig. 6A). In addition, the Moreover, the enzyme activity of NMNAT3 was elevated in acetylation level of NMNAT3 was augmented both in Ang II-in- cardiomyocytes overexpressed wild type SIRT3, rather than H248Y duced cardiac hypertrophic mice and SIRT3 knockout mice, and mutant, but decreased in SIRT3-depleted cardiomyocytes (Fig. 6C). was further increased in SIRT3-deﬁcient mice treated with Ang II Contrary to total acetylation level, the enzyme activity of NMNAT3 (Fig. 6F). was decreased by Ang II, but was rescued after SIRT3 Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273 265

Fig. 3. SIRT3 overexpression attenuated Ang II-induced cardiomyocyte hypertrophy. The neonatal rat cardiomyocytes were transfected with wild type SIRT3-Flag plasmid or catalytically inactive H248Y-HA SIRT3 mutant. The mitochondrial protein expression (A) and enzyme activity (B) of SIRT3 were determined. The cells were further treated with 100 nM Ang II for 24 h. Then, mRNA levels of ANF and BNP (C) and cell surface area (D) were measured, respectively. *P o0.05 vs. CON (control group), #P o0.05 vs. Ang II treatment. overexpression (Fig. 6B). Additionally, in vivo experiments de- almost abolished the anti-hypertrophic effect of SIRT3, evidenced monstrated that the enzyme activity of NMNAT3 was attenuated by the increased expression of cardiac fetal genes and cell surface in Ang II-induced cardiac hypertrophic mice and SIRT3 knockout area in response to Ang II stimulation (Fig. 7B and C). Moreover, mice, and was further decreased in mice combined the genetic overexpression of NMNAT3 attenuated the hypertrophic effect modification and Ang II stimulation. (Fig. 6G). induced by siSIRT3 (Fig. 7D and E). Taken together, these ob- To investigate the potential effect of SIRT3 or Ang II on mi- servations indicate that NMNAT3 was at least partially responsible tochondrial NAD level, NRCMs were transfected with plasmids for the anti-hypertrophic effect of SIRT3. SIRT3-Flag or the catalytically inactive mutant H248Y. However, the NAD content was unchanged by either SIRT3-Flag or H248Y 3.5. NMNAT3 depletion decreased mitochondrial NAD level and transfection (Fig. 6E). Additionally, the mitochondrial NAD levels in SIRT3 enzyme activity NRCMs were not significantly altered in response to Ang II (100 nM) from 6 to 48 h (Fig. 6D). Moreover, in the animal studies, Since NMNAT3 is the rate-limiting enzyme in NAD biosynthesis Ang II treatment, but not SIRT3 genetic depletion, decreased mi- in the mitochondria, NMNAT3 might probably affect the activation tochondrial NAD content (Fig. 6H). of SIRT3 by altering NAD synthesis. To test this hypothesis, NMNAT3 was knockdown by siRNA, then the mitochondrial NAD 3.4. The anti-hypertrophic effect of SIRT3 was partially dependent on level and SIRT3 enzyme activity were investigated in cultured activation of NMNAT3 cardiomyocytes. As shown in Fig. 8A and B, knockdown of NMNAT3 decreased mitochondrial NAD level, as well as SIRT3 To investigate whether the anti-hypertrophic effect of SIRT3 enzyme activity. In contrast, NAD level in cytoplasm was not was dependent on the activation of NMNAT3, RNA interference changed (Fig. 8C). Additionally, the mRNA levels of ANF and BNP was used to knockdown endogenous NMNAT3 in NRCMs. Quan- were significantly increased by NMNAT3 depletion (Fig. 8D). titative RT-PCR was performed to evaluate the silencing efficiency of three independent siRNAs, marked as si001, si002, and si003, 3.6. SIRT3 did not interact with or deacetylate NAMPT respectively. As shown in Fig. S1B, si001 remarkably reduced the mRNA expression and protein level of NMNAT3 without affecting NAMPT is an important NAD biosynthesis rate-limiting en- NMNAT1 and NMNAT2 isoforms (Fig. 7A), thus si003 was used for zyme, however, its presence and functions within the mitochon- NMNAT3 silencing and named as siNMNAT3. NMNAT3 depletion dria is controversial [30,31]. To investigate the subcellular 266 Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273

Fig. 4. SIRT3 depletion aggravated the hypertrophic responses in neonatal rat cardiomyocytes. Cells were transfected with siSIRT3 and negative control (Neg). (A) The mRNA expression of mitochondrial SIRT isoforms (SIRT3-SIRT5) after transfection with Neg or siSIRT3. (B and C) The mRNA levels of ANF and BNP and cell surface area were measured in SIRT3 depleted cardiomyocytes treated with or without 100 nM Ang II for 24 h. Cells in Neg group were transfected with negative control siRNA which was the negative control for siRNA transfection. (D) The mRNA levels of ANF and BNP were measured in myocardium of SIRT3 knockout mice treated with or without Ang II (3 mg/kg/ d for 2 weeks). *P o0.05 vs. Neg (A–C) or vs. sham (D); #P o0.05 vs. Ang II þNeg (B and C) or vs. shamþ Ang II (D). distribution of NAMPT in NRCMs, western blot was used to analyze energy production, which is supplied mainly by mitochondrial the protein expression of NAMPT in the mitochondria. As shown in respiration [32]. Regulation of mitochondrial function is crucial to Fig. 9A, NAMPT was predominantly expressed in the cytosol, but maintain the cardiac contractility. SIRT3, a mitochondria-localized not in the mitochondria. Co-immunoprecipitation experiments sirtuin, has been reported to regulate almost every major aspect of were conducted to test whether SIRT3 interacts with NAMPT. As mitochondrial biology, including detoxification of reactive oxygen shown in Fig. 9B, SIRT3 was detected by western blot after NAMPT species (ROS), nutrient oxidation, ATP generation, mitochondrial was immunopurified from whole cell extracts or mitochondria dynamics, and the mitochondrial UPR (unfolded protein response) extracts, suggesting that there was no direct interaction between [33–35]. Recent reports have shown that SIRT3 has multiple pro- SIRT3 and NAMPT. The acetylation levels of NAMPT were assessed tective effects in cardiomyocytes. Downregulation of SIRT3 has using an acetylated lysine antibody. As shown in Fig. 9C, acetyla- been observed in animal models of cardiac dysfunction [18,36]. tion level of NAMPT remain unchanged in NRCMs treated with Ang Deficiency of SIRT3 leads to spontaneous cardiac hypertrophy in II. Transfection with either SIRT3-Flag or H248Y mutant, did not non-stressed hearts and accelerates heart failure in response to affect the acetylation level of NAMPT (Fig. 9C). These data sug- pressure overload [15,16]. The present study has demonstrated gested that SIRT3 specifically interacted with and deacetylated that the mRNA level, the protein expression and the enzyme ac- NMNAT3, but not NAMPT, in cultured cardiomyocytes. tivity of SIRT3 were reduced in hypertrophic cardiomyocytes sti- mulated by Ang II and in the hearts of Ang II-treated mice (Fig. 1B– 4. Discussion D and Fig. 2). These observations were in line with Benigni's observations that Ang II downregulated mRNA level of SIRT3 and Permanent contractility of the heart requires continuous candesartan, an Ang-II type I (AT1) receptor antagonist, prevented Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273 267

Fig. 5. SIRT3 co-localized and interacted with NMNAT3. (A) The intracellular distribution of NMNAT3 (green) and mitochondria (red) was detected by confocal immuno- ﬂuorescence microscopy. DAPI (blue) was used to counterstain the nuclei. (B) Cells transfected with SIRT3-Flag were used to detect the intracellular distribution of SIRT3 (red) and NMNAT3 (green) and DAPI (blue) was used to counterstain the nuclei. (C and D) SIRT3 physically interacted with NMNAT3. Endogenous NMNAT3 was im- munopuriﬁed from cardiomyocytes (C) or mouse myocardium (D) with anti-NMNAT3 antibody, followed by western blotting with an anti-SIRT3 antibody. 268 Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273

Fig. 6. SIRT3 deacetylated and activated NMNAT3. (A and B) Cardiomyocytes transfected with SIRT3 or H248Y mutant were treated with 100 nM Ang II for 24 h. (F–H). Sham and SIRT3 genetic knockout mice were treated with 3 mg/kg/d Ang II for 2 weeks. Then, endogenous NMNAT3 was immunopurified with an anti-NMNAT3 antibody. Its acetylation status was detected by western blotting with an anti-acetyl-lysine antibody (A and F), and its enzymatic activity was quantified as described (B and G) then the mitochondrial NAD were measured (H). Cardiomyocytes were transfected with SIRT3, H248Y or siSIRT3 and its enzymatic activity was quantified as described (C) then the mitochondrial NAD was measured (E). (D) Cardiomyocytes were treated with 100 nM Ang II for indicated times, then the mitochondrial NAD was measured. *P o0.05 vs. vector group (B and C) or sham group (G and H). #Po0.05 vs. Ang II treated group (B and G) or vs. vector group (C). Cells (vector group) were transfected with control vector plasmid which was the negative control for plasmid transfection. this downregulation [37]. Our observations are also consistent administration of losartan could normalize SIRT3 protein level in with the findings observed by Mohsen et al. who showed that the ischemic heart [38]. However, the present findings do not SIRT3 protein expression in left ventricular ischemic tissue were support the observations in a previous study showing that Ang II reduced during acute myocardial ischemia reperfusion and that induced SIRT3 expression in human endothelial cells [39].Itis Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273 269

Fig. 7. NMNAT3 was involved in the protective effect of SIRT3 on Ang II-induced cardiomyocyte hypertrophy. Cells were transfected with siNMNAT3, or negative control siRNA (Neg) or plasmid NMNAT3, or empty vector. (A) The mRNA expression of NMNAT isoforms (NMNAT1-NMNAT3) was detected by qRT-PCR. After transfection with siNMNAT3, cardiomyocytes were overexpressed with SIRT3 and then treated with 100 nM Ang II for 24 h. After overexpression with NMNAT3 plasmid, cardiomyocytes were transfected with siSIRT3. The mRNA level of hypertrophic biomarkers (B and D) and cell surface area (C and E) were measured. *P o0.05 vs. neg (A, B and C) or vector (D and E), #P o0.05 vs. Ang II treatment group (B and C) or siSIRT3 group (D and E), $P o0.05 vs. SIRT3 overexpression plus Ang II treatment group. 270 Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273

Fig. 8. NMNAT3 depletion decreased mitochondrial NAD level and SIRT3 enzyme activity. Cardiomyocytes were transfected with siNMNAT3 and negative control for 48 h. The mitochondrial NAD (A), cytoplasmic NAD (C), SIRT3 enzyme activity (B) and mRNA levels of ANF and BNP (D) were measured. *Po0.05 vs. Neg group.

Fig. 9. SIRT3 did not interact with or deacetylate NAMPT. (A) Western blot analysis of NAMPT in whole cell extract, or cytoplasmic and mitochondrial fractions. Cytochrome C oxidase IV (COX-IV) is shown as a mitochondrial marker. (B) Endogenous NAMPT was immunopurified with anti-NAMPT antibody from total protein and mitochondrial protein in cardiomyocytes, followed by western blotting detection with an anti-SIRT3 antibody. (C) Endogenous NAMPT was immunopurified with anti-NAMPT antibody from cardiomyocytes treated with Ang II for 24 h or transfected with SIRT3 or H248Y mutant. NAMPT acetylation status was detected by western blotting with an anti-acetyl- lysine antibody. possible that this discrepancy arises from the differences of cell Several mechanisms are proposed to mediate the anti-hyper- types and experimental settings. Moreover, overexpression of trophic effect of SIRT3, including the activation of FOXO3a and LKB1, SIRT3 attenuated Ang II-induced cardiomyocyte hypertrophy, as and prevention of aberrant ROS production [18]. However, the evidenced by the decreased expression of hypertrophic markers downstream targets of SIRT3, especially those associated with cardi- (ANF and BNP), as well as the decrease of cell surface area (Fig. 3C ovascular diseases, are not yet fully understood. Since SIRT3 is a NAD- and D). On the contrary, SIRT3 depletion or deficiency aggravated dependent mitochondrial deacetylase, intracellular level of NAD, the hypertrophic response induced by Ang II (Fig. 4B–D). especially mitochondrial level of NAD, is essential for the activation of Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273 271

SIRT3. Recently, it has been reported that pathological cardiac hypertrophy was associated with depletion of cellular NAD, and NAD sup- plementation blocked Ang II-induced cardiac hypertrophy via activation of SIRT3 [28]. Thus, enzymes involved in NAD biosynthetic pathways may potentially affect the activation of SIRT3. NAMPT catalyzes the first reversible step in NAD biosynthesis and nicotinamide (NAM) salvage. It is considered as an important enzyme to regulate NAD consumption, notably, the aging-associated histone deacetylase SIRT1 [40]. However, whether NAMPT is present in the mitochondria remains controversial [30,41,42]. The present study aimed to investigate the subcellular distribution of NAMPT in NRCMs. As shown in Fig. 9A, the enzyme was mainly present in the cytosol, but was ab- sent in the mitochondria. Moreover, NAMPT was not involved in the anti-hypertrophic effect of SIRT3 in Ang II-induced cardiomyocyte hypertrophy, as supported by the following observations: (1) no physical interaction between SIRT3 and NAMPT was observed in NRCMs (Fig. 9B); (2) the acetylation level of NAMPT was not changed in Ang II- induced cardiomyocyte hypertrophy (Fig. 9C); (3) overexpression of SIRT3 did not affect the acetylation level of NAMPT (Fig. 9C). Fig. 10. A working model. In Ang II-induced cardiac hypertrophy, SIRT3 is down- NMNAT, is another important enzyme which catalyzes the last step regulated and loses the capability to deacetylate NMNAT3. The resulting hyper- in the biosynthesis of NAD. Three human NMNAT isoforms, with dif- acetylation status of NMNAT3 leads to a decrease in its enzyme activity and reduces ferent subcellular localization and catalytic properties, have been the synthesis of mitochondrial NAD. Deficiency of NAD further reduce SIRT3 ac- identified and characterized. NMNAT1 is a nuclear protein composed tivity and aggravates pathological cardiomyocyte hypertrophy. of 279 residues; NMNAT2 is enriched at the surface of the Golgi ap- paratus; and NMNAT3 is a mitochondrial protein with an N-terminal targeting sequence [25]. The rigorous subcellular localization of in a vast majority of metabolic redox reactions and is thus essential for mammalian NMNATs suggests a predominant role for this family of energy transduction; on the other hand, it is used as a substrate for enzymes in determining the subcellular NAD pool distribution. In this covalent protein modification in multiple signaling pathways [44].Mi- study, the subcellular distribution of NMNAT3 was observed in the tochondrial NAD can directly enhance the deacetylase activity of SIRT3, mitochondria, and NMNAT3 co-localized with SIRT3 in NRCMs (Fig. 5B which induces the concomitant deacetylation of its mitochondrial tar- and Fig. S3). Our co-immunoprecipitation results clearly indicated a gets. The present study suggested that NMNAT3 regulated the activity physical interaction between SIRT3 and NMNAT3 (Fig. 5CandD). of SIRT3 by altering mitochondrial NAD level. In NMNAT3-knockout Additionally, through gain- and loss-of-function approaches, we found NRCMs, the mitochondrial NAD level was decreased, and the deacety- that SIRT3 could positively regulate the enzyme activity of NMNAT3 lase activity of SIRT3 was subsequently reduced, but expression of (Fig. 6C). In Ang II-induced cardiac hypertrophy, increased acetylation cardiac hypertrophic biomarkers ANF and BNP were eventually aug- level of NMNAT3 was associated with decreased enzyme activity mented (Fig. 8A–D). Interestingly, our observations that NMNAT3 (Fig. 6). Overexpression of SIRT3, rather than H248Y mutant, deace- maintains mitochondrial NAD level seem contradictory to a recent re- tylated NMNAT3 and restored its enzyme activity (Fig. 6AandB). port showing that NAD level in the heart is not altered in NMNAT3 Moreover, SIRT3 genetic deficiency in mice could further increase the knockout mice [45]. However, NAD level was assessed in the whole acetylation level of NMNAT3 and decrease its enzyme activity (Fig. 6F tissue of the heart in that study, in which cardiomyocytes were not and G). Interestingly, Ang II infusion could further decrease NMNAT3 separated from other cells like the fibroblasts, and the mitochondrial enzyme activity in SIRT3-deficient mice (Fig. 6G). The possible reasons fraction was not extracted for testing NAD levels [45].Itispossiblethat are that the mechanisms of Ang II's effect on NMNAT3 activity were these contrary results are due to different experimental settings. In line complicated and that there were additional effects besides its reg- with our observations, the role of NMNAT3 in mitochondrial NAD sal- ulation in SIRT3, which needs to be further elucidated. It seems logical vage pathway was supported by the findings that downregulation of that the regulation of NMNAT3 by SIRT3 could influence NAD levels, NMNAT3 abolished the protective effect of NAMPT against methyl- since NMNAT3 is critical for NAD biosynthesis. However, over- methane sulfonate (MMS)-induced cell death [30].Moreover,ithas expression of SIRT3 or SIRT3 depletion did not affect mitochondrial been proved that the positive-charged cytoplasmic NAD cannot be NAD content (Fig. 6E and H). This could be explained by the possibility transported across the mitochondrial membrane in mammalian cells that depletion of SIRT3 could decrease mitochondrial NAD level via [31,46]. It seems impossible that cytosolic NAD is imported to the mi- attenuating NMNAT3 enzyme activity, meanwhile would increase NAD tochondria. The mitochondrial NAD is most likely generated by the level by less NAD consumption since it is a NAD-dependent deacety- mitochondrial NMNAT3 isoform, using NMN which is transported from lase. The balance between synthesis and consuming of NAD is the cytoplasm to the mitochondria as the substrate, and ATP which is probable reason why SIRT3 did not affect mitochondrial NAD content. readily available in mitochondria as energy source [27,47]. Overexpression of SIRT3 attenuated Ang II-induced hypertrophic Taken together, the present observations propose a feedback responses as indicated by the decrease in cell surface area and fetal loop in the interaction between SIRT3 and NMNAT3. SIRT3 regulates gene expression. However, the effect of SIRT3 was abolished when the enzyme activity of NMNAT3 by interacting and deacetylating NMNAT3 was simultaneously knockdown by RNA interference NMNAT3 protein; meanwhile, NMNAT3 could change the bio- (Fig. 7B and C). Additionally, overexpression of NMNAT3 also atte- synthesis of mitochondrial NAD to alter the activation of SIRT3. This nuated siSIRT3-induced pro-hypertrophic responses (Fig. 7D and E). feedback loop between SIRT3 and NMNAT3 might participate in the Taken together, these findings suggest that NMNAT3 is a substrate progression of cardiac hypertrophy. In Ang II-induced cardiac hy- of SIRT3, and the protective effect of SIRT3 against cardiac hyper- pertrophy, SIRT3 is downregulated and loses the capability to dea- trophy may be at least partially attributed to NMNAT3 activation. cetylate NMNAT3. The resulting hyper-acetylation of NMNAT3 leads A substantial fraction of the cellular NAD pool is compartmentalized to decrease in its enzyme activity and reduces the synthesis of within the mitochondria (about 75% in heart muscle) [43]. Mitochon- mitochondrial NAD. Deficiency of NAD might further reduce SIRT3 drial NAD has two important functions: On one hand, it is a coenzyme activity and aggravates cardiomyocyte hypertrophy (Fig. 10). 272 Z. Yue et al. / Experimental Cell Research 347 (2016) 261–273

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