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. 2022 Jul 5;11(13):e023868.
doi: 10.1161/JAHA.121.023868. Epub 2022 Jun 22.

Integrated Multilayer Omics Reveals the Genomic, Proteomic, and Metabolic Influences of Histidyl Dipeptides on the Heart

Keqiang Yan  1 , Zhanlong Mei  1 , Jingjing Zhao  2   3 , Md Aminul Islam Prodhan  4 , Detlef Obal  5 , Kartik Katragadda  2   3 , Benjamin Doelling  2   3 , David Hoetker  2   3 , Dheeraj Kumar Posa  2   3 , Liqing He  4 , Xinmin Yin  4 , Jasmit Shah  6 , Jianmin Pan  7 , Shesh Rai  7 , Pawel Konrad Lorkiewicz  2   3 , Xiang Zhang  4 , Siqi Liu  1 , Aruni Bhatnagar  2   3 , Shahid P Baba  2   3
Affiliations

Integrated Multilayer Omics Reveals the Genomic, Proteomic, and Metabolic Influences of Histidyl Dipeptides on the Heart

Keqiang Yan et al. J Am Heart Assoc. .

Abstract

Background Histidyl dipeptides such as carnosine are present in a micromolar to millimolar range in mammalian hearts. These dipeptides facilitate glycolysis by proton buffering. They form conjugates with reactive aldehydes, such as acrolein, and attenuate myocardial ischemia-reperfusion injury. Although these dipeptides exhibit multifunctional properties, a composite understanding of their role in the myocardium is lacking. Methods and Results To identify histidyl dipeptide-mediated responses in the heart, we used an integrated triomics approach, which involved genome-wide RNA sequencing, global proteomics, and unbiased metabolomics to identify the effects of cardiospecific transgenic overexpression of the carnosine synthesizing enzyme, carnosine synthase (Carns), in mice. Our result showed that higher myocardial levels of histidyl dipeptides were associated with extensive changes in the levels of several microRNAs, which target the expression of contractile proteins, β-fatty acid oxidation, and citric acid cycle (TCA) enzymes. Global proteomic analysis showed enrichment in the expression of contractile proteins, enzymes of β-fatty acid oxidation, and the TCA in the Carns transgenic heart. Under aerobic conditions, the Carns transgenic hearts had lower levels of short- and long-chain fatty acids as well as the TCA intermediate-succinic acid; whereas, under ischemic conditions, the accumulation of fatty acids and TCA intermediates was significantly attenuated. Integration of multiple data sets suggested that β-fatty acid oxidation and TCA pathways exhibit correlative changes in the Carns transgenic hearts at all 3 levels. Conclusions Taken together, these findings reveal a central role of histidyl dipeptides in coordinated regulation of myocardial structure, function, and energetics.

Keywords: genomics; heart; histidyl dipeptides; metabolomics; proteomics; transcriptomics; triomics.

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Figures

Figure 1
Figure 1. Transcriptomic analysis of the wild‐type (WT) and carnosine synthase transgenic (CarnsTg) hearts.
A, Volcano plot of the transcriptome between the CarnsTg and WT hearts (n=4 mice in each group). Statistical significance log10 of P value y‐axis was plotted against log2‐fold change (x‐axis). B, Heat map of the differentially expressed genes between the CarnsTg and WT hearts. C, Gene ontology analysis of the differentially regulated noncoding and coding genes between the CarnsTg and WT mice hearts, which were divided between 3 main categories: cellular component, biological component, and molecular function.
Figure 2
Figure 2. EuKaryotic Ortholog Groups (KOG) of proteins and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed proteins (DEPs) between the wild‐type (WT) and carnosine synthase transgenic (CarnsTg) hearts.
The Uniprot IDs of total proteins identified in the WT and CarnsTg hearts (n=3 in each group) via tandem mass spectrometry analysis were used to annotate the proteins with the corresponding KOG annotation. Functional enrichment analysis of (A) upregulated proteins showed that the greatest number of these proteins were allocated to metabolism and (B) downregulated proteins showed that the greatest number of these proteins were allocated to cellular processes and signaling. C, KEGG enrichment analysis: the vertical axis represents the pathway terms with high enrichment and the horizontal axis represents the Rich factor. The size of the q value is represented by the color of the dots. The smaller the q value, the closer the color is towards red. D, Enrichment of the specific KEGG pathway annotations for the upregulated (red) and downregulated (blue) proteins in the CarnsTg hearts. TCA indicates citric acid cycle.
Figure 3
Figure 3. Metabolomic analysis of the wild‐type (WT) and carnosine synthase (Carns) transgenic (CarnsTg) hearts under basal conditions and after short durations of ischemia.
Changes in the global cardiometabolomic profile by Carns overexpression were assessed using an unbiased metabolomic approach. A and B, Volcano plot represents the metabolites identified by N‐trimethylsilyl‐N‐methyl trifluroacetamide (MTBSTFA) and N‐trimethylsilyl‐N‐methyl trifluoroacetamide (MSTFA) derivatization in the WT and CarnsTg hearts under basal conditions. Isolated hearts from the WT and CarnsTg mice were subjected to 5 and 15 minutes of ischemia. The volcano plot represents the metabolites in the WT and CarnsTg hearts identified after (C) 5 minutes and (D) 15 minutes of ischemia (n=5–7 mice hearts in each group).
Figure 4
Figure 4. Pathway impact and enrichment of the differentially regulated metabolites in the wild‐type (WT) and carnosine synthase transgenic (CarnsTg) hearts under basal and ischemic conditions.
A and B, Pathway impact analysis, and (C and D) pathway enrichment of the differentially regulated metabolites identified by N‐trimethylsilyl‐N‐methyl trifluroacetamide and N‐trimethylsilyl‐N‐methyl trifluoroacetamide derivatizations, respectively, between the WT and CarnsTg mice hearts under basal conditions. Pathway impact analysis after (E) 5 minutes and (F) 15 minutes of ischemia, and pathway enrichments of the differentially regulated metabolites (G) after 5 minutes and (H) 15 minutes of ischemia, between the WT and CarnsTg mice hearts (n=5–7 hearts per group). TCA indicates citric acid cycle.
Figure 5
Figure 5. Transcriptomic, proteomic, and metabolic interactions of the fatty acid metabolism in the carnosine synthase transgenic (CarnsTg) hearts.
Schematic overview of the fatty acid metabolism, potential target of miRNA‐3100, and levels of detected metabolites and proteins in the wild‐type (WT) and CarnsTg hearts. Relative fold changes in the expression of (A) Carnitine palmitoyltransferase 2 (CPT2), (B) acyl‐CoA hydratase (ACoA‐DH), (C) 3‐hydroxyacyl‐cCoA dehydrogenase (3HCoA DH), (D) 2,3 enoyl‐CoA hydratase (2,3‐ECoAH), and (E) 3‐ketoacyl‐CoA thiolase (KCoAT) between the WT and CarnsTg hearts. Free fatty acid levels (F) decanoic acid and (G) stearic acid in the WT and CarnsTg hearts following 5 minutes of global ischemia. *P<0.05 vs WT (n=4–8 mice in each group). FABP indicates fatty acid–binding protein; and miRNA, microRNA.
Figure 6
Figure 6. Transcriptomic, proteomic, and genomic interactions in the carnosine synthase transgenic (CarnsTg) hearts.
Schematic overview of the citric acid cycle (TCA), potential target of microRNA (miRNA)‐6989, and levels of TCA intermediates. A, Levels of succinic acid under basal conditions and (B) fumaric acid following 15 minutes of ischemia. Relative fold changes in the expression of (C) isocitrate dehydrogenase (ICD) and (D) succinate dehydrogenase (SDH) between the WT and CarnsTg hearts. Schematic for glyoxylic acid formation and potential target of miRNA‐6913. Levels of (E) glycolic acid and (F) expression of aldehyde dehydrogenase (Aldh) in the WT and CarnsTg hearts. *P<0.05 vs WT (n=4–8 mice in each group). WT indicates wild type.
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