Myocardial Metabolomics of Human Heart Failure With Preserved Ejection Fraction

Abstract

Background: The human heart primarily metabolizes fatty acids, and this decreases as alternative fuel use rises in heart failure with reduced ejection fraction (HFrEF). Patients with severe obesity and diabetes are thought to have increased myocardial fatty acid metabolism, but whether this is found in those who also have heart failure with preserved ejection fraction (HFpEF) is unknown.

Methods: Plasma and endomyocardial biopsies were obtained from HFpEF (n=38), HFrEF (n=30), and nonfailing donor controls (n=20). Quantitative targeted metabolomics measured organic acids, amino acids, and acylcarnitines in myocardium (72 metabolites) and plasma (69 metabolites). The results were integrated with reported RNA sequencing data. Metabolomics were analyzed using agnostic clustering tools, Kruskal-Wallis test with Dunn test, and machine learning.

Results: Agnostic clustering of myocardial but not plasma metabolites separated disease groups. Despite more obesity and diabetes in HFpEF versus HFrEF (body mass index, 39.8 kg/m² versus 26.1 kg/m²; diabetes, 70% versus 30%; both P<0.0001), medium- and long-chain acylcarnitines (mostly metabolites of fatty acid oxidation) were markedly lower in myocardium from both heart failure groups versus control. In contrast, plasma levels were no different or higher than control. Gene expression linked to fatty acid metabolism was generally lower in HFpEF versus control. Myocardial pyruvate was higher in HFpEF whereas the tricarboxylic acid cycle intermediates succinate and fumarate were lower, as were several genes controlling glucose metabolism. Non-branched-chain and branched-chain amino acids (BCAA) were highest in HFpEF myocardium, yet downstream BCAA metabolites and genes controlling BCAA metabolism were lower. Ketone levels were higher in myocardium and plasma of patients with HFrEF but not HFpEF. HFpEF metabolomic-derived subgroups were differentiated by only a few differences in BCAA metabolites.

Conclusions: Despite marked obesity and diabetes, HFpEF myocardium exhibited lower fatty acid metabolites compared with HFrEF. Ketones and metabolites of the tricarboxylic acid cycle and BCAA were also lower in HFpEF, suggesting insufficient use of alternative fuels. These differences were not detectable in plasma and challenge conventional views of myocardial fuel use in HFpEF with marked diabetes and obesity and suggest substantial fuel inflexibility in this syndrome.

Keywords: branched-chain amino acid; heart failure, preserved ejection fraction; human; lipid metabolism; metabolic networks and pathways; metabolomics; obesity.

Figure 1.. Myocardial metabolomic signatures in HFpEF, HFrEF and control.

A) Principal component analysis of myocardial metabolomics shows fairly distinct clusters for each subject disease group. B) Principal component analysis of plasma metabolomics shows substantial overlap between disease groups. C) Hierarchical clustering of participants (columns) using the myocardial metabolome demonstrates similar separation of the disease groups. There were 3 main clusters of metabolites (rows): cluster 1 was lower in HFpEF uniquely, cluster 2 lowest in HFrEF and intermediate in HFpEF, cluster 3 was highest in HFpEF. D) Hierarchical clustering of participants using the plasma metabolome yielded 3 clusters with substantial overlap between disease groups. PC: principal component; 3-HBA: 3-hydroxybutyric acid, a-KG: alpha-ketoglutarate, HFpEF: heart failure with preserved ejection fraction; HFrEF: heart failure with reduced ejection fraction.

Figure 2.. Medium and long-chain acylcarnitines (MLAC) and fatty acid metabolism gene expression.

A) MLAC in HFpEF, HFrEF, and control in myocardial tissue are displayed in a polar (flower) plot. The mean (dark line) and ± standard error (shaded region) Z-scores for each metabolite are shown by their distance from the polor plot origin. Statistical differences between each set of group comparisons are denoted by colored circles surrounding the plot, as defined in the figure. B) Same comparison based on plasma MLAC. For both, a Kruskal-Wallis test with post-hoc Dunn’s test for multiple comparisons was used, and adjusted p values (Benjamini-Hochberg) annotated by colored dots corresponding to each comparison. C) Genes related to fatty acid uptake and metabolism are lower in myocardium from HFpEF vs. control. HFpEF: heart failure with preserved ejection fraction; HFrEF: heart failure with reduced ejection fraction.

Figure 3.. Tricarboxylic acid cycle intermediates and genes related to glucose metabolism.

A) Several metabolites (succinate, fumarate, malate) in the tricarboxylic acid cycle are lower in myocardium from HFpEF vs. control. Pyruvate is higher in HFpEF vs. control. Raw data were scaled for visualization purposes. Analyzed using Kruskal-Wallis test with post-hoc Dunn’s test for multiple comparisons, adjusted p-values (Benjamini-Hochberg) provided. B) Myocardial gene expression of genes related to glucose metabolism and its regulation/uptake in HFpEF vs. control. Gene expression adjusted p value determined as described in methods. HFpEF: heart failure with preserved ejection fraction; HFrEF: heart failure with reduced ejection fraction; alpha-KG: alpha-ketoglutarate.

Figure 4.. Branched-chain amino acid (BCAA) metabolites and amino acids are altered in HFpEF.

A) Polar – flower plot of myocardial BCAA and their downstream catabolites in all three groups. Data displayed as described for Figure 2A. BCAA were higher in HFpEF, but several corresponding mitochondrial carnitine catabolites were lower. BCKA were not measured. C05:1 includes an unresolved mixture of tiglyl and 3-methylcrontonyl. B) Similar display of these metabolites in plasma shows fewer significant differences and less asymmetry between BCAA and catabolites in HFpEF. P values calculated by Kruskal-Wallis test with post-hoc Dunn’s test for multiple comparisons and adjustment for multiple comparisons (Benjamini-Hochberg), and annotated by colored dots for respective comparisons. C) Box-plot for gene expression of key proteins in the BCAA metabolic pathway. Adjusted P-values from Benjamini-Hochberg multiple comparisons tests are shown for each. D) Summary diagram of differences in metabolites and gene expression of regulating proteins in the BCAA metabolic pathway comparing HFpEF to control. HFpEF: heart failure with preserved ejection fraction; HFrEF: heart failure with reduced ejection fraction. Other abbreviations in text.

Figure 5.. HFpEF subgroups identified by non-negative matrix factorization (NMF) finds substantial overlap in metabolome.

A) Patient-patient correlation plot shows classification into three groups based on 5 metabolites selected by the NMF algorithm (see text and panel C for details). B) Principal component analysis using all myocardial metabolites shows significant overlap between HFpEF NMF subgroups, with Groups 1 and 2 being the most different. C) NMF-metabolite, clinical, and tissue metabolomics differences for NMF-derived Groups 1 and 2.