Abstract 3439: Nampt is a Critical Regulator of Nad+ Synthesis in Cardiomyocytes
Cellular NAD+ generated by the de novo and the salvage pathways. Despite the presence of the de novo pathway, the salvage pathway is essential in humans. Nicotinamide phosphoribosyl-transferase (Nampt) is a rate-limiting enzyme in the salvage pathway. NAD+ is not only required for cellular respiration and oxidative phosphorylation, but is also used as a substrate for NAD+-dependent enzymes, such as Sirt1, a longevity factor. Little is known about the function of Nampt in the heart. Protein and mRNA expression of Nampt in the heart was decreased in pathological conditions, including ischemia (44% p < 0.01, 22%, p < 0.001), ischemia/ reperfusion (40%, p<0.001, 30%, p < 0.01), pressure overload (58%, p<0.01, 46%, p < 0.02 ) and heart failure (63%, p<0.002, 61%, p < 0.01). Transduction of adenovirus (Ad) harboring Nampt increased both NAD+ and ATP concentrations in neonatal cardiac myocytes (CMs) whereas that of Ad harboring shRNA-Nampt decreased them, suggesting that Nampt critically regulates NAD+ and ATP levels in CMs. Overexpression of Nampt increased survival of CMs against stress imposed by methylmethane sulfonate (MMS, 1.2 mM), an agent known to hyperactivate PARP-1 and reduce the cellular NAD+ content, as well as glucose deprivation. Knockdown of Nampt alone induced death of CMs and made CMs more sensitive to cell death induced by MMS (0.6 mM). Death of CMs due to Nampt knockdown was accompanied by increases in caspase 3 cleavage and TUNEL positive nuclei (8.19 ± 0.25 to 14.87 ± 1.08%, p = 0.004) , suggesting that the death may be apoptotic. Nampt knockdown also increased LC3-II and accumulation of autophagosomes, which was, however, accompanied by accumulation of p62 and impaired autophagic flux. Sirt1 knockdown mimicked the effect of Nampt knockdown on autophagy. In summary, Nampt critically regulates the NAD+ and ATP content in CMs. Expression of Nampt is decreased in many pathological conditions in the heart. Nampt is protective against NAD+/ATP deprivation, whereas reduced expression of Nampt causes apoptotic cell death. Furthermore, downregulation of Nampt causes impairment of autophagic flux in CMs directly and/or through the effects of Sirt1. These results suggest that Nampt is an essential gatekeeper of energy status, autophagic flux and survival in CMs.