Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Oct 16;7(20):e010378.
doi: 10.1161/JAHA.118.010378.

Molecular Atlas of Postnatal Mouse Heart Development

Virpi Talman  1 , Jaakko Teppo  2   3 , Päivi Pöhö  2 , Parisa Movahedi  4 , Anu Vaikkinen  2 , S Tuuli Karhu  1 , Kajetan Trošt  5 , Tommi Suvitaival  5 , Jukka Heikkonen  4 , Tapio Pahikkala  4 , Tapio Kotiaho  2   6 , Risto Kostiainen  2 , Markku Varjosalo  3 , Heikki Ruskoaho  1
Affiliations

Molecular Atlas of Postnatal Mouse Heart Development

Virpi Talman et al. J Am Heart Assoc. .

Abstract

Background The molecular mechanisms mediating postnatal loss of cardiac regeneration in mammals are not fully understood. We aimed to provide an integrated resource of mRNA , protein, and metabolite changes in the neonatal heart for identification of metabolism-related mechanisms associated with cardiac regeneration. Methods and Results Mouse ventricular tissue samples taken on postnatal day 1 (P01), P04, P09, and P23 were analyzed with RNA sequencing and global proteomics and metabolomics. Gene ontology analysis, KEGG pathway analysis, and fuzzy c-means clustering were used to identify up- or downregulated biological processes and metabolic pathways on all 3 levels, and Ingenuity pathway analysis (Qiagen) was used to identify upstream regulators. Differential expression was observed for 8547 mRNA s and for 1199 of 2285 quantified proteins. Furthermore, 151 metabolites with significant changes were identified. Differentially regulated metabolic pathways include branched chain amino acid degradation (upregulated at P23), fatty acid metabolism (upregulated at P04 and P09; downregulated at P23) as well as the HMGCS ( HMG -CoA [hydroxymethylglutaryl-coenzyme A] synthase)-mediated mevalonate pathway and ketogenesis (transiently activated). Pharmacological inhibition of HMGCS in primary neonatal cardiomyocytes reduced the percentage of BrdU-positive cardiomyocytes, providing evidence that the mevalonate and ketogenesis routes may participate in regulating the cardiomyocyte cell cycle. Conclusions This study is the first systems-level resource combining data from genomewide transcriptomics with global quantitative proteomics and untargeted metabolomics analyses in the mouse heart throughout the early postnatal period. These integrated data of molecular changes associated with the loss of cardiac regeneration may open up new possibilities for the development of regenerative therapies.

Keywords: heart development; heart regeneration; metabolomics; neonatal mouse cardiomyocyte; proteomics; transcriptomics.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Experimental design for the multiomics analysis of postnatal mouse hearts. A, Two separate sets of mouse ventricular tissue samples collected on postnatal day 1 (P01), P04, P09, and P23 were used. The postnatal loss of cardiac regenerative capacity is illustrated for comparison, and numbers of animals in each sample group are presented in the table. *Total sample sizes are indicated for metabolomics (5) and proteomics (14). B, Analysis techniques and bioinformatics analyses used in the study. GCxGC‐MS, 2‐dimensional gas chromatography–mass spectrometry; GO, gene ontology; LC‐MS, liquid chromatography–mass spectrometry; LC‐MS/MS, liquid chromatography–tandem mass spectrometry; RNAseq, RNA sequencing.
Figure 2
Figure 2
Gene expression changes in the neonatal mouse heart. A, Heat map of the top 1000 genes with the smallest q values between postnatal day 1 (P01) and P04. B, The numbers of up‐ and downregulated genes (q<0.01, fold change >1.5). C, Expression patterns of selected cardiomyocyte‐specific structural proteins. The data were normalized to P01 and are expressed as mean±SEM (n=3 pooled samples, each from 3 hearts). D, Selected significantly enriched (q<0.01) biological process gene ontology (GO) terms for each time point comparison from gene set enrichment analysis. Blue indicates downregulation, and red indicates upregulation. All gene symbol explanations are available in Appendix S1.
Figure 3
Figure 3
Proteomic changes in the neonatal mouse heart. A, The number of proteins quantified in each sample group, expressed as mean±SEM (identification false discovery rate <0.01 on both peptide and protein levels). B, The numbers and fold changes of differentially expressed (q<0.01) proteins in sample group comparisons. C, Hierarchical clustering of proteins and samples. All proteins detected in more than 67% of samples of at least 1 sample group are included in the heat map. Gray indicates missing values (protein not quantified). B and C, Data presented are from set 2; corresponding images for set 1 are in Figure S4. P indicates postnatal day.
Figure 4
Figure 4
Metabolite changes in the postnatal mouse heart. A, Heat map of linear mixed effect (LME) model estimates for lipids. B, Heat map of LME model estimates for polar metabolites with Glass's Δ effect sizes from postnatal day 1 (P01). *q<0.01 compared with P01. Cer indicates ceramide; FA, fatty acid; LysoPC, lysophosphatidylcholine; LysoPE, lysophosphatidylethanolamine; MG, monoacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin.
Figure 5
Figure 5
Multiomics integration with fuzzy c‐means clustering. A, Median abundance patterns of mRNAs, proteins, and metabolites in each cluster. Detailed images of clusters and the scales for the y‐axis are in Figure S6. B, The percentage of proteomics clusters covered by the RNA sequencing (RNAseq) clusters at the level of transcripts/proteins (left) and enriched biological process gene ontology (GO) terms (q<0.01) in the clusters (right). C, A heat map of −log10 false discovery rate (FDR) values of selected GO terms and KEGG pathways in RNAseq and proteomics clusters. D, Metabolite set enrichment analysis performed on the union of metabolites in all clusters indicating the biological processes associated with metabolite changes. CoA indicates coenzyme A; NA, not assessed.
Figure 6
Figure 6
Postnatal changes in branched chain amino acid degradation in the mouse heart. A, Concentrations of branched chain amino acids valine, leucine, and isoleucine in mouse ventricular tissue. B, The KEGG pathway map of valine, leucine, and isoleucine degradation indicating up‐ and downregulated transcripts and proteins. *Multiple enzymes may catalyze the same reaction. All gene symbol explanations are available in Appendix S1. The KEGG pathway image is modified and reprinted with permission from the Kyoto Encyclopedia of Genes and Genomes.65 P indicates postnatal day; RNAseq, RNA sequencing.
Figure 7
Figure 7
Postnatal changes in fatty acid metabolism in the mouse heart. A, The levels of free fatty acids (FAs) are regulated by ACOTs (acyl‐CoA [acyl‐coenzyme A] thioesterases) that hydrolyze acyl‐CoAs to CoASH (free coenzyme A) and free FAs, ACSLs (long‐chain FA–CoA ligases) that activate free FAs by ligation of CoA, and FASN (FA synthase). B, Relative mRNA expression of enzymes regulating the concentrations of free FAs, shown as mean±SEM (n=3 pooled samples, each from 3 hearts). C, Normalized label‐free quantification intensities of FA‐regulating enzymes detected in proteomics. D, The abundances or concentrations of selected free FAs. MS1 indicates precursor ion mass spectrum; P, postnatal day.
Figure 8
Figure 8
Ketogenesis and mevalonate pathways in the early postnatal heart. A, ACATs (acetyl‐CoA [acetyl‐coenzyme A] acetyltransferases) and HMGCSs (hydroxymethylglutaryl‐CoA synthases) catalyze HMG‐CoA (hydroxymethylglutaryl‐CoA) synthesis. HMG‐CoA serves as a substrate for the ketogenesis route producing 3‐hydroxybutyrate and the mevalonate pathway producing mevalonate, which can be further used for cholesterol synthesis. B, Relative mRNA expression of selected ketogenesis and mevalonate pathway components, shown as mean±SEM (n=3 pooled samples, each from 3 hearts). C, Normalized label‐free quantification intensities of proteins detected in the ketogenesis and mevalonate pathways. D, Concentrations of 3‐hydroxybutyrate and cholesterol over the early postnatal period. E and F, Effect of HMGCS inhibition with hymeglusin and HMGCR (HMG‐CoA reductase) inhibition with simvastatin on neonatal rat ventricular cardiomyocyte viability (E) and proliferation (F). Cell viability was assessed using the MTT assay, and cell proliferation was quantified as the percentage of BrdU‐positive cells after 24‐hour exposure. The data are expressed as mean±SEM from 3 independent experiments. *P<0.05, **P<0.01 compared with control (Ctrl); Welch ANOVA followed by Games‐Howell. All gene symbol explanations are available in Appendix S1. BDH1 indicates 3‐hydroxybutyrate dehydrogenase 1; FBS, fetal bovine serum; HMGCL, hydroxymethylglutaryl–coenzyme A lyase; IDI1, isopentenyl‐diphosphate δ‐isomerase 1; MVD, mevalonate diphosphate decarboxylase; MS1, precursor ion mass spectrum; MVK, mevalonate kinase; OXCT1, 3‐oxoacid CoA‐transferase 1; PMVK, phosphomevalonate kinase.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation. 2000;101:2981–2988. - PubMed
    1. Talman V, Ruskoaho H. Cardiac fibrosis in myocardial infarction‐from repair and remodeling to regeneration. Cell Tissue Res. 2016;365:563–581. - PMC - PubMed
    1. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabe‐Heider F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H, Jovinge S, Frisen J. Evidence for cardiomyocyte renewal in humans. Science. 2009;324:98–102. - PMC - PubMed
    1. Senyo SE, Steinhauser ML, Pizzimenti CL, Yang VK, Cai L, Wang M, Wu TD, Guerquin‐Kern JL, Lechene CP, Lee RT. Mammalian heart renewal by pre‐existing cardiomyocytes. Nature. 2013;493:433–436. - PMC - PubMed
    1. Laflamme MA, Murry CE. Heart regeneration. Nature. 2011;473:326–335. - PMC - PubMed

Publication types

LinkOut - more resources

-