Nicotinamide phosphoribosyltransferase regulates cell survival through NAD+ synthesis in cardiac myocytes

Abstract

Rationale: NAD+ acts not only as a cofactor for cellular respiration but also as a substrate for NAD(+)-dependent enzymes, such as Sirt1. The cellular NAD+ synthesis is regulated by both the de novo and the salvage pathways. Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the salvage pathway.

Objective: Here we investigated the role of Nampt in mediating NAD+ synthesis in cardiac myocytes and the function of Nampt in the heart in vivo.

Methods and results: Expression of Nampt in the heart was significantly decreased by ischemia, ischemia/reperfusion and pressure overload. Upregulation of Nampt significantly increased NAD+ and ATP concentrations, whereas downregulation of Nampt significantly decreased them. Downregulation of Nampt increased caspase 3 cleavage, cytochrome c release, and TUNEL-positive cells, which were inhibited in the presence of Bcl-xL, but did not increase hairpin 2-positive cells, suggesting that endogenous Nampt negatively regulates apoptosis but not necrosis. Downregulation of Nampt also impaired autophagic flux, suggesting that endogenous Nampt positively regulates autophagy. Cardiac-specific overexpression of Nampt in transgenic mice increased NAD+ content in the heart, prevented downregulation of Nampt, and reduced the size of myocardial infarction and apoptosis in response to prolonged ischemia and ischemia/reperfusion.

Conclusions: Nampt critically regulates NAD+ and ATP contents, thereby playing an essential role in mediating cell survival by inhibiting apoptosis and stimulating autophagic flux in cardiac myocytes. Preventing downregulation of Nampt inhibits myocardial injury in response to myocardial ischemia and reperfusion. These results suggest that Nampt is an essential gatekeeper of energy status and survival in cardiac myocytes.

Figure 1. Nampt is downregulated by pathological stimuli in vivo

(A) Heart homogenates were prepared from mice subjected to ischemia for 24 hours, ischemia for 45 minutes and reperfusion for 24 hours, and transverse aortic constriction (TAC) for 2 or 4 weeks. Expression of Nampt, GAPDH and actin was evaluated by immunoblots. (B) The effect of pathological stress on the expression level of Nampt mRNA in the heart. Nampt mRNA level was determined by RT-qPCR and normalized by S15RNA. The expression level of sham operated mice is expressed as 1. (C) Representative Nampt stainings in heart sections from sham operation and ischemia/reperfusion are shown. (D) Heart homogenates were prepared from mice with indicated ages. Expression of Nampt and tubulin was evaluated by immunoblots.

Figure 1. Nampt is downregulated by pathological stimuli in vivo

(A) Heart homogenates were prepared from mice subjected to ischemia for 24 hours, ischemia for 45 minutes and reperfusion for 24 hours, and transverse aortic constriction (TAC) for 2 or 4 weeks. Expression of Nampt, GAPDH and actin was evaluated by immunoblots. (B) The effect of pathological stress on the expression level of Nampt mRNA in the heart. Nampt mRNA level was determined by RT-qPCR and normalized by S15RNA. The expression level of sham operated mice is expressed as 1. (C) Representative Nampt stainings in heart sections from sham operation and ischemia/reperfusion are shown. (D) Heart homogenates were prepared from mice with indicated ages. Expression of Nampt and tubulin was evaluated by immunoblots.

Figure 2. Nampt regulates NAD⁺ and ATP levels in cardiac myocytes

Cardiac myocytes were transduced with Ad-LacZ, Ad-shRNA-scramble, Ad-Nampt or Ad-shRNA-Nampt at indicated MOIs. In A and D, cell lysates were subjected to immunoblot analyses with anti-Nampt, anti-GAPDH and anti-tubulin antibodies. In B, C, E and F, Cellular [NAD⁺] and [ATP] were measured using the EnzyChrom^TM NAD⁺/NADH Assay Kit and ATP Bioluminescent Assay Kit, respectively. In B, cells were cultured with either normal culture media or those supplemented with 20 mM nicotinamide for 16 hours. In C-F, the level of Nampt, [NAD⁺] and [ATP] in control virus transduced myocytes is expressed as 1. In E and F, cardiac myocytes were cultured with or without 500 µM NAD⁺ or glucose free medium for 24 hours. Data represent the mean of four experiments ± SEM.

Figure 3. Knockdown of Nampt causes apoptotic cell death in cardiac myocytes

Cardiac myocytes were transduced with Ad-shRNA-scramble, Ad-shRNA-Nampt, or Ad-shRNA-Sirt1 alone or in combination. In some cases, Ad-LacZ or Ad-Bcl-xL was co-transduced. Myocytes were then cultured for 96 hours. (A) (upper) TUNEL staining of myocytes. Nuclei were counterstained with DAPI. (lower) Relative TUNEL-positive nuclei among different groups. The value in control myocytes was designated as 1. (B) Cell lysates were subjected to immunoblot analyses with anti-Nampt, anti-Bcl-xL, anti-cleaved caspase-3 (C. Caspase-3), and anti-tubulin antibodies. Myocytes treated with chelerythrine (CE, 10 µM for 2 hours) were used as a positive control for apoptosis. (C) Cardiac myocytes were transduced with Ad-shRNA-scramble (Scr) or Ad-snRNA-Nampt (Nampt) (3 MOI) and cultured for 5 days. The cytosolic fraction was subjected to immunoblot analyses with anti-cytochrome c (Cyto c), anti-cytochrome c oxidase I (Cox) and anti-tubulin antibodies. The mitochondrial fraction (Mito) was included as a positive control for Cox. (D) Nuclei were stained using the Texas Red labeled blunt end probe synthesized with pfu. Myocytes treated with 1.2 mM methylmethane sulfonate (MMS) for three hours were used as a positive control for necrotic cell death. (left) Representative images of DAPI and hairpin 2 staining and merged images. (right) Quantitative analyses of hairpin 2 positive cardiac myocytes. The value in control myocytes was designated as 1. * p<0.05 vs Ad-shRNA-scramble. The results are mean of 4 experiments.

Figure 4. Knockdown of Nampt affects autophagic flux, in a manner similar to chloroquine treatment

(A and B) Myocytes were transduced with Ad-shRNA-scramble (Ad-sh-Scr.) or Ad-shRNA-Nampt (Ad-sh-Nampt) at indicated MOIs and cultured for 96 hours. Cell lysates were subjected to immunoblot analyses with anti-LC3 (A), anti-p62 (B), and anti-tubulin (B) antibodies. The values of LC3-II/LC3-I and p62 expression in myocytes transduced with Ad-shRNA-scramble are designated as 1. Experiments were conducted 3 times. (C) Myocytes were transduced with Ad-GFP-LC3 (10 MOI) and either Ad-shRNA-scramble or Ad-shRNA-Nampt at 3 MOI and cultured for 96 hours. Some myocytes were treated with chloroquine (Cq, 6 µM) for 2 hours or NAD⁺ (500 µM) for 24 hours. (left) Representative images of GFP-LC3 staining are shown. (right) The percentage of cells with punctate GFP-LC3 staining is shown. N=4. (D) Myocytes were transduced with Ad-shRNA-scramble, Ad-shRNA-Nampt, or Ad-shRNA-Sirt1 alone or in combination and cultured for 96 hours. Cell lysates were subjected to immunoblot analyses with anti-LC3, anti-p62, anti-Sirt1, anti-Nampt, and anti-tubulin antibodies. The results shown are representative of three experiments.

Figure 5. Generation of Tg-Nampt mice

(A) Representative immunoblots of heart homogenates obtained from Tg-Nampt and NTg mice with anti-Nampt and anti-tubulin antibodies. (B) Representative immunostaining of Nampt in left ventricular myocardial sections. (C) The level of [NAD⁺], [NADH] and [NAD⁺]/[NADH] in NTg and Tg-Nampt (line #9) was evaluated. Data represent the mean of 5–6 experiments ± SEM. (D) Tg-Nampt (line 8) or NTg control mice were subjected to ischemia or sham operation for 24 hours. Expression of Nampt and tubulin was determined by immunoblot analyses. The results shown are representative of three experiments.

Figure 6. I/R injury is attenuated in Tg-Nampt mice

(A) Tg-Nampt and NTg mice were subjected to 45 minutes of ischemia and 24 hours of reperfusion. (upper) Gross appearance of LV myocardial sections after Alcian blue and triphenyltetrazolium chloride (TTC) staining. (lower left) The area at risk (AAR) (% of LV) was comparable between NTg and Tg-Nampt. (lower right) The infarction area/AAR was significantly smaller in Tg-Nampt than in NTg mice. (B) (left) LV myocardial sections were subjected to TUNEL and DAPI staining. Representative images of the staining in the border zone are shown. (right) The number of TUNEL-positive myocytes is expressed as a percentage of total nuclei detected by DAPI staining. (C and D) Tg-Nampt and NTg mice were subjected to 2 hours of ischemia. (C) (upper) Gross appearance of LV myocardial sections after Alcian blue and TTC staining. (lower left) The area at risk (AAR) (% of LV) was comparable between NTg and Tg-Nampt. (lower right) The infarction area/AAR was significantly smaller in Tg-Nampt than in NTg. (D) Expression of p62 and tubulin at the baseline and after 2 hours of ischemia was determined by immunoblot analyses. The results shown are representative of five experiments.

Figure 7. Current hypothesis

The proposed Nampt pathway in cardiac myocytes under stresses