Glycolysis-Mediated Activation of v-ATPase by Nicotinamide Mononucleotide Ameliorates Lipid-Induced Cardiomyopathy by Repressing the CD36-TLR4 Axis

Abstract

BACKGROUND: Chronic overconsumption of lipids followed by their excessive accumulation in the heart leads to cardiomyopathy. The cause of lipid-induced cardiomyopathy involves a pivotal role for the proton-pump vacuolar-type H + -ATPase (v-ATPase), which acidifies endosomes, and for lipid-transporter CD36, which is stored in acidified endosomes. During lipid overexposure, an increased influx of lipids into cardiomyocytes is sensed by v-ATPase, which then disassembles, causing endosomal de-acidification and expulsion of stored CD36 from the endosomes toward the sarcolemma. Once at the sarcolemma, CD36 not only increases lipid uptake but also interacts with inflammatory receptor TLR4 (Toll-like receptor 4), together resulting in lipid-induced insulin resistance, inflammation, fibrosis, and cardiac dysfunction. Strategies inducing v-ATPase reassembly, that is, to achieve CD36 reinternalization, may correct these maladaptive alterations. For this, we used NAD + -precursor nicotinamide mononucleotide (NMN), inducing v-ATPase reassembly by stimulating glycolytic enzymes to bind to v-ATPase. METHODS: Rats/mice on cardiomyopathy-inducing high-fat diets were supplemented with NMN and for comparison with a cocktail of lysine/leucine/arginine (mTORC1-mediated v-ATPase reassembly). We used the following methods: RNA sequencing, mRNA/protein expression analysis, immunofluorescence microscopy, (co)immunoprecipitation/proximity ligation assay (v-ATPase assembly), myocellular uptake of [ 3 H]chloroquine (endosomal pH), and [ 14 C]palmitate, targeted lipidomics, and echocardiography. To confirm the involvement of v-ATPase in the beneficial effects of both supplementations, mTORC1/v-ATPase inhibitors (rapamycin/bafilomycin A1) were administered. Additionally, 2 heart-specific v-ATPase-knockout mouse models (subunits V 1 G1/V 0 d2) were subjected to these measurements. Mechanisms were confirmed in pharmacologically/genetically manipulated cardiomyocyte models of lipid overload. RESULTS: NMN successfully preserved endosomal acidification during myocardial lipid overload by maintaining v-ATPase activity and subsequently prevented CD36-mediated lipid accumulation, CD36-TLR4 interaction toward inflammation, fibrosis, cardiac dysfunction, and whole-body insulin resistance. Lipidomics revealed C18:1-enriched diacylglycerols as lipid class prominently increased by high-fat diet and subsequently reversed/preserved by lysine/leucine/arginine/NMN treatment. Studies with mTORC1/v-ATPase inhibitors and heart-specific v-ATPase-KO mice further confirmed the pivotal roles of v-ATPase in these beneficial actions. CONCLUSION: NMN preserves heart function during lipid overload by preventing v-ATPase disassembly.

Preventing the Fatty Acid-Transporter CD36 From Taking its Toll on the Heart

“…Alternatively, NMN has been shown to enter muscle cells after a dephosphorylation step by the CD73/NT5E (cluster of differentiation/ ecto-5'-nucleotidase) at the membrane, which converts NMN into nicotinamide riboside that in turn enters cells through the nucleoside equilibrative transporters and is converted to NMN by the NMRK2 (nicotinamide riboside kinase 2). 10,11 The study by Wang et al 4 shows that NMN supplementation restores NAD + levels and the association of the glycolytic enzyme aldolase with the v-ATPase V1 subcomplex, accompanied by a reassembly with V0 subcomplex. The test of this hypothesis was actually driven by previous research from other teams working in yeast who had shown that glycolytic complexes assembly with v-ATPase subunits can lead to v-ATPase reassembly on glucose restoration in the medium after a period of starvation, allowing the localized production of ATP for the use of the v-ATPase proton pump and resulting in the reacidification of endosomes.…”

“…In the current issue of Circulation Research, Wang et al 4 specifically address the question of reducing the impact of cardiac lipotoxicity by assessing the benefit of specific nutrients supplementation to inhibit the fatty acid-induced increase in recycling of the fatty acid transporter CD36 (cluster of differentiation 36) at the membrane. Indeed this mechanism traps the cardiomyocytes in a vicious circle of further enhancement of lipid uptake reinforcing lipotoxicity.…”