[3-¹¹C]Pyruvate PET detects alterations in cardiac pyruvate metabolism induced by doxorubicin chemotherapy.

Figures

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  Fig. 1. DOX exposure leads to decreased cardiac pyruvate metabolism in mice. a Selected significantly downregulated KEGG pathways at 4 weeks after DOX exposure. b Selected significantly downregulated GO:BPs at 4 weeks after DOX exposure. The number of genes in each pathway is indicated in parenthesis. c EnhancedVolcano plot from the bulk RNA sequencing performed on cardiac tissue samples collected at 4 weeks (Log2 fold change (FC) > | 1 | , p < 0.0001). Selected genes associated with pyruvate transport and metabolism, Mpc1, Mpc2, Pdhb, Pdha1, Dlat, Dld, Vdac1, Pdhx, Slc16a1 (monocarboxylate transporter 1), and Slc16a3 (monocarboxylate transporter 4) are highlighted. d Western blot analysis of cardiac MPC1 and MPC2 expression. HSP60 was used as a reference. e ROI quantification of each protein expression level was performed using ImageJ. f Map of TCA cycle intermediates assessed during [3-13C]pyruvate stable isotope tracing metabolomics experiments. The illustration was created with BioRender. g–j Comparison of total pool size and M + 0 and M + 1 fractional enrichments of lactate (g), alanine (h), citrate (i), and glutamate (j) between the control and DOX groups in hearts collected 10 min…

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  Fig. 2. DOX exposure affects pyruvate uptake by reducing MPC1/2 in HCM. a [3-14C]Pyruvate flux in HCMs was evaluated in a time-dependent manner (0, 2, 4, 6, and 8 min). Cells were treated with vehicle (control; CTRL) or 0.1 µM DOX for 2 × 48 h (n = 6 per condition). b Western blot analysis of MPC1, MPC2, and MCT1 expression from HCM treated with vehicle (CTRL), UK5099 (100 µM for 72 h), or DOX (0.1 µM for 2 × 48 h). Vinculin was used as a reference. c [3-14C]Pyruvate uptake was assessed in HCMs treated with vehicle (CTRL), UK5099 (100 µM for 72 h), or DOX (0.1 µM for 2 × 48 h) for 8 min. d [3-14C]Pyruvate uptake in HCM treated with scramble siRNA (100 nM for 24 h) or MPC1/2 siRNA (50 nM of each MPC siRNA—total concentration of 100 nM—for 24 h) for 8 min. e Hypothesized effect of DOX treatment on the cellular fate of [3-14C]pyruvate, which can be retained within the cell as [3-14C]pyruvate or another labeled metabolite, released into the media as [3-14C]lactate, [3-14C]pyruvate, or another species, or released to the air as [14C]CO2. The illustration was created with BioRender. f [14C]CO₂ capture assay was performed following an 8-min incubation of HCM with [3-14C]pyruvate. Data are…

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  Fig. 3. [3-11C]Pyruvate PET reveals DOX-induced alterations in cardiac pyruvate flux at 4 weeks. a Description of [3-11C]pyruvate dynamic PET/CT imaging. The lateral tail vein was cannulated for intravenous (i.v.) administration before imaging. b Representative summed sagittal PET images from the first 10 min post-injection in mice at the 4-week time point. c TACs over the initial 10-min PET acquisition period. Individual activity measurements were normalized to maximum uptake. d Representative multi-shot dynamic curves of HP [1-13C]spectroscopy in a control mouse heart. Intensities are scaled by the indicated factor to facilitate comparison. e Cardiac lactate/pyruvate ratio derived from HP [1-13C]pyruvate MR spectra. Mice (n = 3 in the CTRL group and n = 2 in the DOX group) were injected multiple times to provide a total of 10–12 independent measurements. f Cardiac bicarbonate/lactate ratios from the control (n = 2) and DOX (n = 2) groups. Data are presented as the mean ± s.d. *p < 0.05; **p < 0.01. Statistical analysis was performed using the Mann–Whitney test between CTRL and DOX (c) or an unpaired t-test (d, e). PET positron emission tomography, TAC Time-activity curve, HP…

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  Fig. 4. MPC1/2 expression rebounds 16 weeks after exposure in concert with the recovery of cardiac mass. a HW/TL ratio at 16 weeks after DOX exposure. b EnhancedVolcano plot from the bulk RNA sequencing at 16 weeks (Log2 fold change (FC) > | 0.5 | , FDR < 0.05). The Mpc1 and Mpc2 genes are highlighted. c Western blot analysis of MPC1 and MPC2 expression in cardiac tissue collected at 16 weeks. HSP60 was used as a reference. d ROI quantification of each protein expression level was performed using ImageJ. Data are presented as the mean ± s.d. **p < 0.01. Statistical analysis was performed using an unpaired t-test (a) or two-way ANOVA (d). HW/TL heart weight-to-tibia length, FDR false discovery rate.

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  Fig. 5. [3-11C]Pyruvate PET imaging captures changes in cardiac pyruvate metabolism. Comparison of total pool size and M + 0 and M + 1 fractional enrichments of a lactate, b alanine, c glutamate, and d citrate assessed during [3-13C]pyruvate stable isotope tracing metabolomics experiments. The experiment was performed 16 weeks after the first DOX exposure. Heart samples were collected 10 min post-injection of [3-13C]pyruvate. e Representative summed sagittal PET images from the first 10 min post-injection of [3-11C]pyruvate in mice at the 16-week time point. f TACs constructed using an ROI over the whole heart over the initial 10-min acquisition period. Activities at each measurement were normalized to maximum uptake. Data are presented as the mean ± s.d. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Statistical analysis was performed using a two-way ANOVA (a–d) or the Mann–Whitney test (c). TCA Tricarboxylic acid, PET positron emission tomography, TAC time-activity curve, ROI Region-of-interest.