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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 (nicotinamide adenine dinucleotide)-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 [mechanistic target of rapamycin complex 1]-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 [H]chloroquine (endosomal pH), and [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 VG1/Vd2) 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-knockout 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.

背景

长期过量摄入脂质，随后脂质在心脏中过度积累，导致心肌病。脂质诱导性心肌病的原因涉及质子泵液泡型 H+-ATP 酶（v-ATPase）的关键作用，v-ATPase使内体酸化，以及脂质转运蛋白 CD36 的作用，CD36 储存在酸化的内体中。在脂质过度暴露的情况下，v-ATPase感知到进入心肌细胞的脂质增加，然后解体，导致内体去酸化，并将储存的 CD36 从内体逐出到肌膜。一旦到达肌膜，CD36 不仅增加脂质摄取，还与炎症受体 TLR4（Toll 样受体 4）相互作用，共同导致脂质诱导的胰岛素抵抗、炎症、纤维化和心脏功能障碍。通过诱导 v-ATPase 重新组装的策略，即实现 CD36 的再内化，可能纠正这些适应性改变。为此，我们使用了 NAD（烟酰胺腺嘌呤二核苷酸）前体烟酰胺单核苷酸（NMN），通过刺激糖酵解酶与 v-ATPase 结合来诱导 v-ATPase 重新组装。

方法

用诱导心肌病的高脂肪饮食喂养大鼠/小鼠，并补充 NMN，并与赖氨酸/亮氨酸/精氨酸鸡尾酒（mTORC1[雷帕霉素复合物 1]介导的 v-ATPase 重新组装）进行比较。我们使用了以下方法：RNA 测序、mRNA/蛋白表达分析、免疫荧光显微镜、（共）免疫沉淀/邻近连接分析（v-ATPase 组装）、[H]氯喹的肌细胞摄取（内体 pH）和[C]棕榈酸、靶向脂质组学和超声心动图。为了确认 v-ATPase 在这两种补充剂的有益作用中的参与，我们给予了 mTORC1/v-ATPase 抑制剂（雷帕霉素/巴弗洛霉素 A1）。此外，还对 2 种心脏特异性 v-ATPase 敲除小鼠模型（亚基 VG1/Vd2）进行了这些测量。在脂质超负荷的药理学/遗传学操作的心肌细胞模型中证实了机制。

结果

NMN 通过维持 v-ATPase 活性，成功地在心肌脂质超负荷期间保持内体酸化，从而防止 CD36 介导的脂质积累、CD36-TLR4 相互作用引起的炎症、纤维化、心脏功能障碍和全身胰岛素抵抗。脂质组学显示，富含 C18:1 的二酰基甘油作为脂质类别在高脂肪饮食中显著增加，并随后被赖氨酸/亮氨酸/精氨酸/NMN 治疗逆转/保留。用 mTORC1/v-ATPase 抑制剂和心脏特异性 v-ATPase 敲除小鼠进行的研究进一步证实了 v-ATPase 在这些有益作用中的关键作用。