Cardiomyocyte Pdk4 response is associated with metabolic maladaptation in aging

Abstract

Ischemic heart disease (IHD) is the leading cause of death, with age range being the primary factor for development. The mechanisms by which aging increases vulnerability to ischemic insult are not well understood. We aim to use single-cell RNA sequencing to discover transcriptional differences in various cell types between aged and young mice, which may contribute to aged-related vulnerability to ischemic insult. Utilizing 10× Genomics Single-Cell RNA sequencing, we were able to complete bioinformatic analysis to identity novel differential gene expression. During the analysis of our collected samples, we detected Pyruvate Dehydrogenase Kinase 4 (Pdk4) expression to be remarkably differentially expressed. Particularly in cardiomyocyte cell populations, Pdk4 was found to be significantly upregulated in the young mouse population compared to the aged mice under ischemic/reperfusion conditions. Pdk4 is responsible for inhibiting the enzyme pyruvate dehydrogenase, resulting in the regulation of glucose metabolism. Due to decreased Pdk4 expression in aged cardiomyocytes, there may be an increased reliance on glucose oxidization for energy. Through biochemical metabolomics analysis, it was observed that there is a greater abundance of pyruvate in young hearts in contrast to their aged counterparts, indicating less glycolytic activity. We believe that Pdk4 response provides valuable insight towards mechanisms that allow for the young heart to handle ischemic insult stress more effectively than the aged heart.

Keywords: Pdk4; aging; glycolytic activity; transcriptional analysis.

FIGURE 1

Graphical synopsis of experimental design. We isolated left ventricle tissue from young (3–5 months) and aged (24–26 months) C57BL/6J mice under sham operations or 45 min of ischemia/24‐h of reperfusion (I/R) conditions. Once single‐cell suspensions were achieved, cells were prepared for sequencing using the 10× Chromium system.

FIGURE 2

(a) UMAP dimensional projection of each integrated dataset, clusters colored by cell‐type manually identified by marker genes. (b) Matching UMAP dimensional projections, clusters colored by sample age. (c–f) Corresponding dot plots for each integrated sample depicting key marker genes used for manual cell‐type identification. Displays cluster gene expression level compared to global expression in assay (color) and gene expression level within each specific cell‐type cluster (size). (g) Proportion of each cell‐type observed in each integrated dataset.

FIGURE 3

Differential Expression Testing. (a) Differentially expressed features in cardiomyocyte cell populations for all comparative datasets showing gene regulation based on foldchange (Positive is upregulated and negative is downregulated in testing variable). All genes presented received an adjusted p‐value of >0.05 from Seurat and plotted with −log₁₀ for visualization. Gene Ontology (GO) Biological Process Enrichment. (b) Enriched terms from Aged and Young I/R versus Sham cardiomyocyte integrated datasets. Testing variable is I/R condition (i.e., heart contraction under I/R conditions is downregulated in aged and young samples compared to sham operations). (c) Enriched terms from I/R and Sham Aged versus Young cardiomyocyte integrated datasets. Testing variable is Aged condition (i.e., cardiac muscle tissue morphogenesis in the aged sample is downregulated under I/R conditions and upregulated under Sham operations compared to young sample). All enriched terms received an adjusted p‐value of >0.05 from EnrichR and plotted with −log₁₀ for visualization.

FIGURE 4

Pdk4 expression in I/R versus Sham datasets. (a) Split feature plots displaying location and origin of Pdk4 expression in the UMAP dimensional plot. (b) Expression level plots displaying amount of cells expressing Pdk4 in each cell‐type. (c) Expression level plots displaying amount of cells expressing Pdk4 in each sample age and condition. (d) Log2FoldChange statistical expression values of Pdk4. Pdk4 expression values retained a 0 adjusted p‐value.

FIGURE 5

Pdk4 expression in Aged versus Young datasets. (a) Split feature plots displaying location and origin of Pdk4 expression in the uniform manifold approximation and projection (UMAP) dimensional plot. (b) Expression level plots displaying of cells expressing Pdk4 in each cell‐type. (c) Expression level plots displaying amount of cells expressing Pdk4 in each sample age and condition. (d) Log2FoldChange statistical expression values of Pdk4. Pdk4 expression values retained a 0 adjusted p‐value.

FIGURE 6

(a) Representative immunoblotting (left) and quantitative analysis (right) of Pdk4 levels in young and aged hearts under sham operations (shaded circles) or I/R conditions (open circles), n = 9. (b) Processed LC–MS/MS metabolomic analysis by XCMS and MetSign showing relative abundance of metabolites. All metabolites received a Grubs' two‐tail test p‐value of >0.05. Metabolite abundance in response to I/R stress compared to sham conditions in aged and young mice. (c) Metabolite abundance comparing aged to young mice under I/R and sham conditions. (d) The oxygen consumption rate (OCR) and (e) the glucose oxidation dependency measured by Seahorse XF analyzer in young/aged cardiomyocytes under sham operations or I/R conditions with or without Pdk4 inhibitor, PDK4‐IN‐1, n = 8.