Neonatal ketone body elevation regulates postnatal heart development by promoting cardiomyocyte mitochondrial maturation and metabolic reprogramming

Neonatal heart undergoes metabolic conversion and cell cycle arrest preparing for the increased workload during adulthood. Herein, we report that neonatal ketone body elevation is a critical regulatory factor for postnatal heart development. Through multiomics screening, we found that the expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), the rate-limiting enzyme of ketogenesis, was transiently induced by colostrum in the neonatal heart. Hmgcs2 knockout caused mitochondrial maturation defects. Meanwhile, postnatal heart development was compromised and cardiomyocytes reacquired proliferation capacity in Hmgcs2 knockout mice. Consequently, over 40% of newborn Hmgcs2 knockout mice died before weaning. The heart function of surviving Hmgcs2 knockout mice was also impaired, which could be rescued by ketone body supplementation during the suckling stage. Mechanistically, ketone body deficiency inhibited β-hydroxybutyrylation but enhanced acetylation of mitochondrial proteins, which might be responsible for the inhibition of the enzyme activity in mitochondria. These observations suggest that ketone body is critical for postnatal heart development through regulating mitochondrial maturation and metabolic reprogramming.


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For liquid chromatography-tandem mass spectrometry (LC-MS) analyses, TMT-labeled peptides were 35 resuspended in 0.1% formic acid and analyzed using an LTQ Orbitrap Fusion Lumos mass spectrometer 36 (Thermo Finnigan, USA) coupled to the Easy-nLC 1200. The trap column (75μm×2cm, Acclaim® 37 PepMap100 C18 column, 3μm, 100 Å; DIONEX, Sunnyvale, CA) effluent was transferred to a reverse- modifications, and carbamidomethylation of cysteine residues was set as a fixed modification. The 51 minimum peptide length was 6 amino acids, and the maximum missed cleavage for each peptide was 2.

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False discovery rates (FDR) cut-offs were set to 0.01 for both proteins and peptides. All other parameters 53 were the default settings of the MaxQuant software.

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For metabolome analysis, the heart samples were mixed and freeze-dried as required. Then the 56 sample was weighted to an EP tube, and extract solution (acetonitrile: methanol: water = 2: 2: 1)

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MS raw data (.wiff) files were converted to the mzXML format by ProteoWizard, and processed by 81 R package XCMS (version 3.2). The process includes peak deconvolution, alignment and integration.

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Minfrac and cut off were set as 0.5 and 0.3 respectively. An in-house MS2 database was applied for 83 metabolite identification.

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f Immunohistochemical analysis of HMGCS2 expression in the mouse hearts. Scale bar, 2 mm. g qPCR 105 detection of Oxct1 mRNA levels in mouse hearts from the embryonic period to adulthood (n = 5-13 for 106 each group). h Quantitative analysis of the OXCT1 protein levels in Fig. 2h (n = 3 for each group). Data 107 are presented as mean ± SEM. and the P values were determined by one-way ANOVA. *, P < 0.05; **, 108 P < 0.01; ***, P < 0.001.

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(such as IDH2 and SDHA) in mitochondria, which is essential for the activity of these enzymes and 235 mitochondrial function (left). After Hmgcs2 is deleted, the K-bhb of metabolic enzymes in mitochondria 236 is decreased. In addition, due to the lack of ketogenesis, acetyl-CoA is accumulated and participates in 237 the K-ac of metabolic enzymes in mitochondria. The decrease of K-bhb and the increase of K-ac together 238 lead to the decrease of enzyme activity and ultimately cause the abnormality of mitochondrial function 239 (right).