Integrated systems biology identifies disruptions in mitochondrial function and metabolism as key contributors to heart failure with preserved ejection fraction (HFpEF)

Figures

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  Fig. 1:. Clinical manifestations of HFpEF are observed in the ZSF1-Obese rat. Physiological characterization of HFpEF. (A) Schematic of study design. (B) Body weights of WKY (control), Lean (Hypertensive; HTN), and ZSF1-Obese (Metabolic Syndrome + HTN; HFpEF) rats. (C) Assessment of systolic (SBP) and diastolic (DBP) blood pressure obtained during invasive hemodynamics. (D) Distance run during a treadmill exercise capacity test. (E) Indices of cardiac left ventricular diastolic function assessed by echocardiography for the E/e’ ratio and invasive hemodynamics for the left ventricular end diastolic pressure (LVEDP). (F) Determination of cardiac systolic function assessed by the echocardiography for the left ventricular ejection fraction (LVEF%). (G) Gravimetric assessment of left ventricle and left atria normalized to tibia length (TL). n = 4-7 male rats per group, mean ± SEM. One-way ANOVA with Holm-Sidak’s post-hoc test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

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  Fig. 2:. Hypertension significantly impacts the cardiac metabolome with minimal impact on the transcriptome. RNAseq and metabolomic comparisons of hearts from Lean vs WKY control rats. (A) Summary of the upregulated and downregulated transcriptional and metabolic changes. Gene ontology analysis revealing the top upregulated (B) biological processes and (C) KEGG pathways of those genes found to be differentially expressed. Gene ontology analysis revealing the top downregulated (D) biological processes and (E) KEGG pathways of those genes found to be differentially expressed. (F) Pathway enrichment analysis of the cardiac metabolome indicated those pathways found to be most significantly affected in ZSF1 rats due to hypertension. Fold change cutoffs of ≥ 2.0 (RNAseq) and ≥ 1.25 (metabolomics) were employed with an FDR ≤ 0.05. n = 6 male rats per group for RNAseq and n = 7 male rats per group for metabolomics. FDR = false discovery rate.

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  Fig. 3:. Transcriptional cardiac remodeling is dependent upon the two hits of obesity and hypertension in ZSF1-Obese HFpEF rats. RNAseq and metabolomic comparisons of hearts from ZSF1-Obese vs Lean rats. (A) Summary of the upregulated and downregulated transcriptional and metabolic changes. Gene ontology analysis revealing the top upregulated (B) biological processes and (C) KEGG pathways of those genes found to be differentially expressed. Gene ontology analysis revealing the top downregulated (D) biological processes and (E) KEGG pathways of those genes found to be differentially expressed. (F) Pathway enrichment analysis of the cardiac metabolome indicated those pathways found to be most significantly affected in ZSF1 rats due to hypertension. Fold change cutoffs of ≥ 2.0 (RNAseq) and ≥ 1.25 (metabolomics) were employed with an FDR ≤ 0.05. n = 6 male rats per group for RNAseq and n = 7 male Lean and 8 male ZSF1-Obese rats for metabolomics. FDR = false discovery rate.

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  Fig. 4:. Integrated network analysis of RNAseq and metabolomic dataset reveals unique metabolic pathways impacted in HFpEF. Integrated analysis of cardiac omics datasets to identify those pathways most impacted by transcriptional and metabolic remodeling due to (A) hypertensive phenotype (Lean vs WKY) or (B) the observed HFpEF phenotype (ZSF1-Obese vs Lean). Fold change cutoffs of ≥ 2.0 (RNAseq) and ≥ 1.25 (metabolomics) were employed with an FDR ≤ 0.05. A greater pathway impact indicates a greater influence at the transcriptional and metabolic level to a given pathway. Labeled pathways had a p-value ≤ 0.05. n = 6 male rats per group for RNAseq and n = 7 male Lean and 8 male ZSF1-Obese rats for metabolomics. FDR = false discovery rate.

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  Fig. 5:. HFpEF results in the transcriptional and metabolic downregulation of pathways central to energy metabolism. Pathway maps of the transcriptional and metabolic alterations in Lean vs WKY and ZSF1-Obese vs Lean rats in energy generating pathways, specifically (A) aerobic glycolysis, (B) fatty acid oxidiation, (C) and the Krebs cycle. Additional pathways indicated in boxes provide an objective summary of the transcriptional and metabolic increase or decrease observed. Genes and metabolites significantly increased or decreased in expression or abundance (Fold change ≥ 2.0 (RNAseq) and ≥ 1.25 (metabolomics); FDR ≤ 0.05) are as indicated.

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  Fig. 6:. Mitochondrial ultrastructural remodeling in the HFpEF heart is characterized by a decrease in mitochondrial content, cristae disorganization, and lipid droplet association. Transmission electron micrographs of cardiomyocyte mitochondrial and lipid droplet (LD) ultrastructure. (A) Representative images of WKY, Lean, and ZSF1-Obese cardiomyocyte ultrastructure indicating cristae disorganization (yellow arrows) and lipid droplet accumulation and interaction with mitochondria (red arrows). (B) mitochondrial area (i.e., content) quantified by the number of mitochondria per image area and the percent area of mitochondria to total area. (C) Mitochondrial shape quantified by the Feret’s diameter and circularity index. (D) Quantification of LD area (i.e., content) quantified by the number of LDs per image area and the percent area of LDs to total area. (E) LD shape quantified by the Feret’s diameter and circularity index. (F) Analysis of mitochondrial-LD interaction quantified by the number of mito-LD contacts per image area, the outer mitochondrial membrane (OMM) perimeter in contact with an LD, and the LD perimeter in association with the OMM. (G) Determination of sarcomeric length…

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  Fig. 7:. Mitochondrial dysfunction characterized by impaired respiratory activity and disrupted calcium handling is a key feature of the HFpEF heart. Functional assessment of the mitochondrial function in the HFpEF heart. (A) Determination of mitochondrial content assessed by citrate synthase activity. Interrogation of mitochondrial respiratory function by assessing oxygen consumption rates (OCR) of (B,C) complex I (pyruvate + malate)-specific substrates, (D,E) complex II (succinate)-specific substrate + rotenone (complex I inhibitor), and (F,G) fatty acid oxidation capacity (palmitoyl-L-carnitine): state 3 (substrate-mediated) oxygen consumption and the respiratory control ratio (RCR) providing an index of oxygen consumption to ATP-production coupling. (H,I) Mitochondrial calcium uptake in response to repeated 2.5 μM boluses. Mitochondrial swelling in response to a 500 μM bolus, displayed as both (J) uncorrected and (K) normalized prior to calcium addition. Quantification of mitochondrial swelling indicated by (L) percent change to WKY baseline and (M) area above the curve. Immunoblotting of (N) VDAC1/3 and components of the mitochondrial calcium uniporter and (O) subunits of ETC…