Assessment of left ventricular tissue mitochondrial bioenergetics in patients with stable coronary artery disease

Figures

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  Fig. 1. Study overview, workflow and CMR results. a, Diagram outlining the experimental workflow of the AMBITION study. Consented patients underwent a full clinical evaluation, including quantitative stress perfusion CMR. Guided by the CMR results, paired LV biopsies from areas with and without inducible ischemia were acquired on the beating heart during CABG. All samples were rapidly clamp-frozen in liquid nitrogen within theater. The myocardial biopsies were analyzed using up to three methods: (1) HEP quantification using a luciferin/luciferase-based bioluminescence assay; (2) metabolomic analysis by LC–MS; and (3) transcriptomic analysis by snRNA-seq. b, Summary of the laboratory analyses undertaken on the myocardial biopsies. The a priori aim was to acquire paired LV biopsies from regions with (‘ischemic’ biopsy) and without (‘remote’ biopsy) inducible myocardial ischemia, permitting HEP quantification and LC–MS in all patients. Surgical discretion and insufficient sample resulted in a lack of HEP and LC–MS data in some patients. Where it was deemed safe, additional myocardium was acquired from the same sites for snRNA-seq. c, Violin box plots demonstrating a significant difference…

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  Fig. 2. Global alterations in LV HEP content and single-nucleus transcriptomic landscape between patients with CAD and control donor myocardium. a, Violin box plots demonstrating the global myocardial HEP content of 26 patients with stable CAD versus donor control myocardium from four individuals, analyzed and quantified using a luciferase-based bioluminescence assay. The results demonstrate a lower ATP:ADP ratio in CAD biopsies as compared to the donor heart samples. The violin box plots visualize the distribution of the data, alongside the median value, hinges corresponding to the first and third quartiles, and whiskers corresponding to the largest/smallest value no farther than 1.5× IQR from the hinge. Analyzed using the Wilcoxon–Mann–Whitney U-test. b, UMAP representation of 65,886 nuclei from five pairs of CABG samples and seven donor controls, color-coded by cell type. c, Gene Ontology analysis of the snRNA-seq data demonstrating upregulation of several biological processes associated with normal cellular bioenergetic function within vCMs in patients undergoing CABG compared to control donor myocardium. The x axis shows the significance level, represented as −log10(FDR), and the…

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  Fig. 3. Regional differences in LV metabolic profile in patients with stable CAD. a, Violin box plots demonstrating paired analysis of the myocardial HEP content in 23 patients with preoperative evidence of regional myocardial ischemia, quantified using a luciferase-based bioluminescence assay. Each color represents an individual patient. The results show that myocardial ATP levels and the ATP:ADP ratio are not different between regions with and without inducible ischemia from within the same LV. Analyzed using the Wilcoxon signed-rank test. b, PCA of the LC–MS data revealing no clusters based on ischemic (square) or remote (triangle) biopsies and no clear clusters of patients. Each color represents an individual patient, with the three analytical repeats from each patient included in the plot. c, log2FC of the LC–MS differential metabolite analysis (comparing ischemic versus remote samples) with hierarchical clustering. The metabolite correlation between patients within those clusters is relatively minor (predominantly 20–50%), suggesting that the patterns of regional metabolite changes within individuals are not significantly replicated across the cohort. d, Violin box plots detailing…

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  Fig. 4. Exploratory analysis comparing the bioenergetic profile of patients with CAD with preserved LVEF versus impaired LVEF. a, Exploratory univariate analysis of myocardial HEP content in 26 patients with CAD, stratifying by LV function, analyzed and quantified using a luciferase-based bioluminescence assay. The results demonstrate a significant reduction in ATP levels and the ATP:ADP ratio in patients with impaired LVEF compared to preserved LVEF. b, Volcano plot of the LC–MS data highlighting metabolites that were significantly altered between patients with and without reduced LVEF (P < 0.05 with log2FC > 0.5 or log2FC < −0.5). c, Exploratory univariate analysis of the LC–MS data in 29 patients, the results suggesting elevated succinate levels in patients with CAD with reduced LVEF compared to patients with preserved LVEF. The violin box plots visualize the distribution of the data, alongside the median value, hinges corresponding to the first and third quartiles and whiskers corresponding to the largest/smallest value no farther than 1.5× IQR from the hinge. In these exploratory experiments, biopsies from regions with and without preoperative inducible ischemia were individually…

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  Extended Data Fig. 1. Summary of the study findings. Patients undergoing CABG had LV biopsies acquired on the beating heart. LV samples from donor hearts were included as controls. First, global alterations between CAD patients and control donor myocardium were assessed. a: Violin box-plot demonstrating the lower ATP/ADP ratio in CAD biopsies as compared to donor samples. b: Gene ontology analysis demonstrating upregulation of several biological processes associated with normal cellular function within ventricular cardiomyocytes of CAD patients compared to donor myocardium. Second, regional differences in LV metabolic profile were assessed, using paired biopsies from segments with and without inducible ischaemia (as determined by CMR). c: Principal component analysis of the liquid chromatography–mass spectrometry data revealing no clusters based on ischaemic (square) or remote (triangle) biopsies and no clear clusters of patients. d: Gene Ontology analysis demonstrating no significant differential expression of oxidative phosphorylation genes in ventricular cardiomyocytes between segments with or without inducible ischaemia. ADP = adenosine diphosphate; ATP = adenosine triphosphate; CABG = coronary…

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  Extended Data Fig. 2. Overview of the snRNAseq data and additional analyses. a. Heatmap showing the specificity of marker genes used to annotate the cell type label for each cluster. b. Boxplot showing the abundance of each cell type per sample for each group, calculated from snRNAseq data. c. UMAP embedding of all nuclei highlighting those from CABG patients. d. UMAP embedding of all nuclei highlighting those from controls. e. Analysis of specific genes implicated in glucose transport, fatty acid metabolism and bioenergetic regulation between ischemic and remote samples (ventricular cardiomyocytes only). CABG versus control sample results included as a reference. The results demonstrate no significant differential expression in the selected genes between regions. AD = adipocytes; CABG = coronary artery bypass grafting; EC = endothelial cells; FB = fibroblasts; FA= fatty acid; FC = fold change; FDR = false discovery rate; NA = not applicable; ns = non-significant; SMC = smooth muscle cells; snRNAseq = single-nuclei ribonucleic acid sequencing; PC = pericytes; vCM = ventricular cardiomyocytes.

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  Extended Data Fig. 3. Volcano plot of snRNAseq data. Volcano plot detailing the differential gene expression within ventricular cardiomyocytes (a) and fibroblasts (b), CABG versus controls. FB = fibroblasts; FC = fold change; NS = non-significant; vCM = ventricular cardiomyocyte.

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  Extended Data Fig. 4. Volcano plot of snRNAseq data. Volcano plot detailing the differential gene expression within endothelial cells (a) and pericytes (b), CABG versus controls. EC = endothelial cells; FC = fold change; NS = non-significant; PC = pericytes.

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  Extended Data Fig. 5. Volcano plot of snRNAseq data. Volcano plot detailing the differential gene expression within smooth muscle cells, CABG versus controls. FC = fold change; NS = non-significant; SMC = smooth muscle cells.

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  Extended Data Fig. 6. Supplementary univariate metabolomic results. a. Violin box plots detailing paired univariate analysis of myocardial hypoxanthine and lactate content (assessed by LC-MS) in chronically ischemic versus remote LV regions from the same heart. The results demonstrating no significant difference in the levels of these metabolites between regions. b. Violin box plots detailing paired univariate analysis of myocardial glucose content and glycolytic intermediate levels (assessed by LC-MS) in chronically ischemic and remote LV regions in the same heart. The results demonstrating no significant difference in the levels of these metabolites between regions. c. Exploratory univariate analysis of the LC-MS data in 29 patients, the results suggesting elevated succinate and taurine levels in CAD patients with ≥3 dysfunctional viable segments (that is, hypocontractile region with ≤50% transmurality of late gadolinium enhancement) compared to patients with <3 dysfunctional viable segments. The boxplots visualise the median value, in addition to hinges corresponding to the first and third quartiles, and whiskers corresponding to the largest/smallest value no further than 1.5*IQR from the hinge.…

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  Extended Data Fig. 7. Supplementary volcano plots of the LC-MS data. a. Volcano plot of the LC-MS data highlighting metabolites that were significantly altered between patients with and without global MPR < 1.5; P-value of <0.05 with log2FC > 0.5 or log2FC < −0.5. b. Volcano plot of the LC-MS data highlighting metabolites that were significantly altered between patients with and without 3 or more dysfunctional viable LV segments (that is hypokinetic/akinetic segment with <50% LGE transmurality); P-value of <0.05 with log2FC > 0.5. FC = fold change; LC-MS = Liquid chromatography–mass spectrometry; LGE = late gadolinium enhancement; LV = left ventricular; MPR = myocardial perfusion reserve.