Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): DZHK Deutsche Forschungsgemeinschaft (DFG-SFB_1470_A02) Cardiac metabolic remodelling in heart failure with preserved ejection fraction (HFpEF) underlies the pathogenesis of this syndrome. Yet, our understanding of metabolic alterations occurring in HFpEF hearts is still limited. Ketone bodies (KBs) – β-hydroxybutyrate (β-OHB) in particular – can become a major, alternative source of fuel for failing hearts. However, the biological relevance of KBs goes beyond their role as energy carrier. We used a ‘two-hit’ mouse model of HFpEF induced by high-fat-diet feeding and L-Name, aiming to evaluate metabolic alteration among various energy substrates (e.g., glucose, fatty acids), in particular the role of β-OHB in HFpEF hearts. Here, we showed that HFpEF heart exhibited impaired glucose oxidation and increased fatty acid oxidation. Impaired glucose oxidation was attributed to the reduced pyruvate dehydrogenase (PDH) activity and increased phospho-PDH expression via the activation of PDH kinase 4 (PDK4). The oxidation of β-OHB was reduced as a result of decreased β-OHB dehydrogenase (BDH1) and succinyl-coA-3-oxaloacid CoA transferase (SCOT) expression. Furthermore, we showed that β-OHB governs mitochondrial function and myocardial bioenergetics in HFpEF, acting as protein modifiers through β-hydroxybutyrylation (Kbhb) – a lysine-based post-translational modification (PTM). Immunoblot analysis for pan- β-hydroxybutyryllysine revealed a significant increase in Kbhb levels in the heart of ‘two-hit’ HFpEF mice compared to control mice. To identify the specific proteins and sites of Kbhb in the heart, Kbhb peptides from control and HFpEF mouse hearts were isolated by immunoaffinity enrichment using a pan-Kbhb antibody-conjugated beads. Volcano plot and gene ontology analysis showed that the most Kbhb-modified proteins in HFpEF hearts are mitochondrial enzymes. Glutamate oxaloacetate transaminase (GOT2) is one of the top hits, participating in the TCA cycle and malate-aspartate shuttle (MAS) in the mitochondria. We further demonstrated that GOT2 activity and MAS were enhacned in the mitochondrial of HFpEF hearts. To conclude, in the heart of ‘two-hit’ model of HFpEF, the oxidation of pyruvate was impaired resulting from decreased PDH activity. The oxidation of β-OHB was also reduced due to the down-regulated BDH1 activity. Instead of oxidation, Kbhb was highlighted as the primary mode of KB utilisation in HFpEF, affecting the GOT2 enzyme activity in MAS pathways. Our results shed light on identifying a novel role of KBs, which can facilitate the understanding of pathogenesis in HFpEF.Ketone-based metabolism and signalling

中文翻译：

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射血分数保留的心力衰竭 (HFpEF) 中基于酮的代谢和信号传导


资金致谢 资金来源类型：公共拨款 - 欧盟资金。主要资金来源：DZHK Deutsche Forschungsgemeinschaft (DFG-SFB_1470_A02) 射血分数保留的心力衰竭 (HFpEF) 的心脏代谢重塑是该综合征发病机制的基础。然而，我们对 HFpEF 心脏中发生的代谢改变的了解仍然有限。酮体（KB）——尤其是β-羟基丁酸（β-OHB）——可以成为衰竭心脏的主要替代燃料来源。然而，KB 的生物学意义超出了其作为能量载体的作用。我们使用高脂肪饮食喂养和 L-Name 诱导的 HFpEF 的“二次打击”小鼠模型，旨在评估各种能量底物（例如葡萄糖、脂肪酸）之间的代谢变化，特别是 β-的作用HFpEF 心脏中的 OHB。在这里，我们发现 HFpEF 心脏表现出葡萄糖氧化受损和脂肪酸氧化增加。葡萄糖氧化受损归因于丙酮酸脱氢酶 (PDH) 活性降低以及通过激活 PDH 激酶 4 (PDK4) 增加磷酸化 PDH 表达。由于β-OHB脱氢酶（BDH1）和琥珀酰辅酶A-3-草酰酸辅酶A转移酶（SCOT）表达减少，β-OHB的氧化减少。此外，我们发现 β-OHB 在 HFpEF 中控制线粒体功能和心肌生物能，通过 β-羟基丁酰化 (Kbhb)（一种基于赖氨酸的翻译后修饰 (PTM)）充当蛋白质修饰剂。泛β-羟基丁酰赖氨酸的免疫印迹分析显示，与对照小鼠相比，“两次打击”HFpEF 小鼠心脏中的 Kbhb 水平显着增加。 为了鉴定心脏中 Kbhb 的特定蛋白质和位点，使用泛 Kbhb 抗体缀合珠通过免疫亲和富集分离来自对照和 HFpEF 小鼠心脏的 Kbhb 肽。火山图和基因本体分析表明，HFpEF 心脏中 Kbhb 修饰最多的蛋白质是线粒体酶。谷氨酸草酰乙酸转氨酶 (GOT2) 是最热门的酶之一，参与线粒体中的 TCA 循环和苹果酸-天冬氨酸穿梭 (MAS)。我们进一步证明 HFpEF 心脏线粒体中 GOT2 活性和 MAS 增强。总之，在 HFpEF“二次打击”模型的核心中，丙酮酸的氧化因 PDH 活性降低而受损。由于 BDH1 活性下调，β-OHB 的氧化也减少。 Kbhb 不是氧化，而是 HFpEF 中 KB 利用的主要模式，影响 MAS 途径中的 GOT2 酶活性。我们的结果揭示了 KB 的新作用，有助于理解 HFpEF 的发病机制。基于酮的代谢和信号传导