Nicotinamide riboside cardioprotection is mediated through glycolysis activation
Authors: Xiao Y , Wang Q , Preckel B , Hollmann M W , Weber N C , Schomakers B V , Van Weeghel M , Zuurbier C 
Published: November 2023

Abstract

Purpose
Low NAD+ levels are observed in pathologies such as ageing, diabetes, heart failure and ischemia-reperfusion (IR) injury. Nicotinamide riboside (NR) has emerged as promising therapeutic NAD+ precursor [1,2]. NR was recently reported to protect against in vivo cardiac IR injury [3], but underlying mechanisms of protection were unexplored. Here we used the isolated mouse heart allowing metabolic control to examine NR’s metabolic mechanisms of cardiac protection.

Methods
Isolated C57BL/6N hearts were Langendorff-perfused with 5.5 mM glucose and 0.2 mM fatty acids (FA), and subjected to 35 min global ischemia and 90 min reperfusion. NR (50 mg/L) or vehicle were administered for 25 min before ischemia, and during the first 25 min of reperfusion. Cardiac functional recovery and cell death parameters (lactate dehydrogenase (LDH) release, % infarct size) were determined. To examine NR’s effects on cardiac metabolism, hearts were perfused with 5.5 mM 13C-glucose and 0.2 mM FA and analyzed by metabolomics and fluxomics using LC-MS techniques before ischemia. The dependency of NR’s protection on glycolysis was tested in hearts perfused with glucose replaced by pyruvate and lactate ("no glycolysis" hearts). Finally, NR’s protection and effects on cardiac metabolism were also examined in "high glycolysis" hearts, by adding insulin to the perfusate.

Results
NR reduced infarct size, LDH release and improved cardiac functional recovery for glucose/FA perfused hearts, demonstrating NR’s cardioprotection. In these glucose/FA hearts, metabolomics analysis showed NR increased NAD+, glycolytic intermediate phosphoenolpyruvate (PEP), Krebs cycle intermediate succinate and pentose phosphate pathway (PPP) intermediates ribose-5P (R5P) and sedoheptulose-7P (S7P) (Fig. 1). Fluxomics analysis demonstrated that NR’s protection was associated with activated glycolysis, without changes in tricarboxylic acid (TCA) cycle or PPP activities. No 13C-labeling of S7P was observed (Fig. 1). NR protection was lost in the "no glycolysis" hearts. In the "high glycolysis" hearts, NR was unable to protect the heart and increase glycolysis and reduced anaerobic glycolysis and ATP with increases in AMP, indicative of NR-induced energetic stress (Fig. 2). Remarkably, in these insulin perfused hearts, NR still increased PPP intermediates, with high 13C-labeling of S7P (Fig. 2).

Conclusions
NR’s protection against IR injury is only present in hearts with low glycolysis, associated with activation of pre-ischemic glycolysis. When activation of glycolysis was prevented, through either examining "no glycolysis" hearts or "high glycolysis" hearts, NR protection was abolished. NR treatment increased pre-ischemic PPP intermediates in either low or high glycolysis hearts, whereby insulin was necessary to incorporate 13C-glucose into S7P. The data suggest that NR’s acute cardioprotective effects are mediated through the activation of glycolysis.NR effects in low-glycolysis heartsNR effects in high-glycolysis hearts