Cardiomyocyte tetrahydrobiopterin synthesis regulates fatty acid metabolism and susceptibility to ischaemia–reperfusion injury

Figures

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  FIGURE 1. Generation and characterisation of cardiomyocyte‐specific deletion of Gch1. (a) Schematic diagram of LoxP site insertion and the resultant excised allele after crossing with α‐MHC Cre line. (b) A representative gel image of genomic DNA PCR products showing the knockout allele and floxed allele in tissue from mice with (+) or without (−) α‐MHC Cre across different tissue types. (c) Gch1 mRNA expression in different tissues and isolated cardiomyocytes (CM). (d,e) BH4 levels in the heart (d) and in cardiomyocytes (e). Data are presented as means ± SD and analysed using multiple t‐tests (c) or an unpaired Student's t‐test (d,e). Each data point represents an individual animal.

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  FIGURE 2. RNA sequencing of hearts from WT and cmGCh1 KO mice reveals downregulation of fatty acid metabolism genes. (a) Principal component (PC) analysis plot of RNA‐seq data of WT and cmGch1 KO hearts. (b) Cytoscape layout of pathway analysis using ClueGo plug‐in; each node represents a GO term and they are connected if 4% of genes overlapped. (c) Enrichment pathway analysis of the RNA‐seq data using IPA software. Percentages of downregulated and upregulated genes over the total known genes associated with the pathways are shown in green and red, respectively. P‐value of the pathway significance is shown by yellow squares. n = 6, genes with FDR < 0.05 are considered significantly differentially expressed and used in pathway analysis. TCA, tricarboxylic acid.

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  FIGURE 3. Downregulation of enzymes involved in fatty acid oxidation in cmGch1 KO hearts. (a–c) Protein levels of three key fatty acid oxidation enzymes (ACSL, acyl‐coenzyme A synthase (long‐chain); ACAD, acyl‐coenzyme A dehydrogenase; HADH, hydroxylacyl‐coenzyme A dehydrogenase). Brain and liver tissues were used as negative (−ve) and positive (+ve) controls, respectively. (d) Intracellular triacylglycerol (TAG) levels are increased in cmGch1 KO hearts compared to WT. (e) A schematic diagram of fatty acid catabolism pathway. Fatty acid can either undergo oxidation for ATP production or be incorporated into TAG. Fold changes (log) of mRNA expression by RNA sequencing are shown in dotted boxes. Data are presented as means ± SD and analysed using unpaired Student's t‐test. Each data point represents an animal used.

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  FIGURE 4. Genetic deletion of Gch1 in cardiomyocyte protects against ischaemia–reperfusion injury. Recovery of left ventricular development pressure (LVDP) and final infarct size in female (a,b) and male (c,d) from WT and cmGch1 KO mice subjected to 35 min global ischaemia (zero flow) followed by 60 min reperfusion. Representative scanned images of infarcted heart sections are shown. Data are presented as means ± SD and analysed using two‐way ANOVA with Bonferroni post hoc test (a,c) or unpaired Student's t‐test (b,d). Each data point represents an individual animal (n = 5–7 per group).

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  FIGURE 5. Metabolomic analysis of long‐chain fatty acid and its oxidation intermediates from WT and cmGch1 KO hearts in response to ex vivo ischaemia–reperfusion (IR) injury. (a) A diagram showing the sample collection protocol. Langendorff isolated hearts were subjected to sham, 35 min global ischaemia (I), or 35 min global ischaemia followed by either 5 min reperfusion (I + R5) or 30 min reperfusion (I + R30). Snap‐frozen samples were subject to metabolomic analysis. (b) Principal component analysis plot of the metabolomic data from the IR injury hearts. Shapes are drawn around the data points for visualisation. (c) Heatmaps showing levels of long‐chain fatty acids and their oxidation intermediates at each time point of IR. Data are presented as the mean scaled intensity, normalised from the original raw detection intensity. n = 7–8 in each group.

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  FIGURE A1. Measurements of biopterin species using HPLC in isolated adult cardiac endothelial cells and cardiomyocytes. (a) Levels of biopterin species in isolated adult cardiac endothelial cells. (b) Levels of biopterin species in isolated adult cardiomyocytes. (c) nNOS protein expression in the heart. Data are presented as means ± SD and analysed with multiple unpaired t‐tests (a, b) and single unpaired t‐test (c).

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  FIGURE A2. Hierarchical clustering heatmap. Hierarchical clustering was performed on full transcriptomic data using Bioconductor R. Sample A1–6 were from WT mice and sample B1–6 were from cmGch1 KO mice.

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  FIGURE A3. Mitochondrial DNA copy number and cardiac mitochondrial oxidative phosphorylation rate. (a) Cardiac mitochondria (mt) DNA copy number averaged from 2Δ C T of Actb versus Cytb, and sdha versus mtCo2. (b–d) Oxidative phosphorylation in isolated mitochondria from WT and cmGch1 KO hearts using Clark electrode. Data are presented as means ± SD and analysed using Student's t‐test (a) and two‐way ANOVA (b–d). n = 5.

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  FIGURE A4. Blood pressure measurement at 16 weeks old for WT and cmGch1 KO mice. Data are presented as means ± SD and analysed with Student's t‐test.

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  FIGURE A5. Coronary perfusion pressure (CPP) and heart rate of the isolated Langendorff heart model in male and female mice. The hearts were subjected to 35 min global ischaemia (zero flow) followed by 60 min reperfusion (2 ml/min constant flow). Data are presented as means ± SD and analysed using two‐way ANOVA.

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  FIGURE A6. Functional recovery of the left ventricular and infarct size does not affect by α‐MHC Cre activity. (a) Recovery of left ventricular development pressure (LVDP) and (b) the final infarct size in isolated Langendorff hearts from female WT and Gch1+/+ α‐MHC Cre+/− mice subjected to 35 min global ischaemia (zero flow) followed by 60 min reperfusion (2 ml/min constant flow). Data are presented as means ± SD and analysed using two‐way ANOVA (a) or Student's t‐test (b).

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  FIGURE A7. PCA plots of the metabolomic data from the different time points of ischaemia reperfusion injury. n = 7–8.