Oxaliplatin-induced cardiotoxicity in mice is connected to the changes in energy metabolism in the heart tissue

Figures

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  Figure 1. Weight loss of mice in the control and oxaliplatin treated mice. (Oxaliplatin, 10 mg/kg x 8 weekly injections, for control dextrose was used).

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  Figure 2. Effect of oxaliplatin on resting heart rates. By week 8, oxaliplatin-treated mice had a significantly lower resting heart rate. * p<0.05, Anova.

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  Figure 3. Histology of the heart of animal treated with oxaliplatin. A: Right atrial histology from a mouse treated with oxaliplatin for 8 weeks. Inset box is magnified in B. There is minimal interstitial infiltration by neutrophils and mononuclear cells; C: Ventricular histology near the apex of the mouse heart. Insert box is magnified in D. Focal myocardial necrosis is evident. This is a small focus of coagulative necrosis, with loss of cross-striations and loss of nuclei; E: Ventricular histology from near the right atrial appendage. Inset box is magnified in F. Focal myocardial necrosis is evident in the right atrial appendage that also shows infiltration of a small number of associated neutrophils.

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  Figure 4. Gross effect of oxaliplatin on the heart. A: Heart weight/body weight (HW/BW, mg/g) index for mice treated with oxaliplatin. B: Examples images of the enlarged heart measured at the atrio-ventricular sulcus. C: Correlation between the heart size and the Heart weight/body weight index. The Pearson correlation between the size and the HW/BW = 0.86

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  Figure 5. Changes in expression of markers of heart damage in mice by qPCR-RT in oxaliplatin (10 mg/kg, 8 dosages weekly) vs vehicle (control).

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  Figure 6. Effect of oxaliplatin on the RNA transcription in the heart. A: Volcano plot representation of genes from the heart tissue: Y threshold (FC) −1.25 to 1.25, X threshold FDR = 0.05. Total number of identified genes in the heart =13,755, upregulated =1,636, downregulated =972 (19.3% genes affected). B: Heatmap of all differentially expressed genes in the heart (total 2608): control dextrose-treated mice – top 4 rows, oxaliplatin -treated (bottom 5 rows). C: Top 20 processes affected by oxaliplatin by GO pathway enrichment analysis of all DEGs in the heart indicates metabolic pathway as one of the top affected pathways.

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  Figure 7. Effect of oxaliplatin on the RNA transcription of 75 metabolic DEGs the heart. A: Top 20 processes from the GO pathway enrichment analysis of 75 differentially expressed energy-related genes in the heart. B: Protein-protein interaction network and extracted 4 MCODE components/clusters. Network of enriched terms: (a) colored by cluster ID, where nodes that share the same cluster ID are typically close to each other; (b) colored by p-value, where terms containing more genes tend to have a more significant p-value. Cluster 1: Acaa2, Acadm, Acads, Acadvl, Cpt2, Decr1, Eci1, Ech1, Hadh, Hadhb, Hadha, Ivd; Cluster 2: Adpat 1, Agpat 2, Bcat1,Cth, Eno1, Pgam1, Pfkp; Cluster 3: Ndufs2, Nd6, Nd4l, Nd4, Cox2; Cluster 4: Bcat2, Pfkm, Hk1.

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  Figure 8. FA transport from capillary to the cytoplasm of cardiomyocytes affected by oxaliplatin. A: Lipoprotein lipase, LPL (Lpl) is transferred to the luminal side of the capillary endothelium where it binds to glycosylphosphatidylinositol-anchored protein 1 GPIHBP1 (Gpihbp1). LPL hydrolyzes TGs contained in the chylomicrons, VLDLs producing free FAs. The released FAs are transported across the lumen-faced endothelium membrane by CD36 FA transporter (Cd36), or a flip-flop mechanism controlled by flippases ABCA1 and ABCG1 (Abca1 and Abcg1). B: In the capillary endothelium, free FAs are picked up by FA binding proteins 4 and 5, FABP4,5 (Fabp4 and Fabp5) and delivered to the interstitial space via a flip-flop mechanism. C: Interstitial albumin binds FAs and transports them to the cardiomyocyte sarcolemma via the FA translocase transport proteins such as FATP1 (Slc27a1), or flip-flop transport. D: In the cardiomyocytes, the heart FA binding protein 3, H-Fabp (Fabp3) pick the free FAs and transports them and their esters from the cell membrane to mitochondria for oxidation. Blue – upregulated genes, grey – unaffected genes, red downregulated genes.

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  Figure 9.. ß-oxidation pathway for fatty acids is strongly downregulated by oxaliplatin. Abbreviations: ACAT1, acetyl-CoA acetyltransferase 1 (Acat1), ACSL1, acyl-CoA synthetase long chain family member 1 (Acsl1); CACT, Carnitine acylcarnitine translocase (Slc25a20); CPT1B, Carnitine O-palmitoyltransferase 1 (Cpt1b); CPT2, Carnitine O-palmitoyltransferase 2 (Cpt2); ECHS1, enoyl-CoA hydratase, short chain 1 (Echs1); HADH, hydroxyacyl-CoA dehydrogenase (Hadh); HADHA, hydroxyacyl-CoA dehydrogenase alpha and beta subunits of the mitochondrial trifunction protein complex (Hadha and Hadhb); H-FABP, heart fatty acid binding protein (Fabp3); LCAD, long chain acyl-CoA dehydrogenases (Acadl); MCAD, medium chain acyl-CoA dehydrogenase (Acadm); MTP, mitochondrial trifunction protein complex; SCAD, small chain acyl-CoA dehydrogenase (Acads); VLCAD, very long chain acyl-CoA dehydrogenases (Acadvl); VDAC, voltage dependent ion channels (Vdac1, Vdac2, or Vdac3). The pathway is derived from the KEGG database. Blue – upregulated genes, red downregulated genes, grey – not affected genes.

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  Figure 10. TCA cycle is affected by oxaliplatin. E1 component of 2-oxoglutarate dehydrogenase complex (OGDH) enzyme (Ogdh) exhibited statistically significant downregulation. The remaining two subunits of this complex encoded by Dlst and Dld and other genes controlling the TCA cycle were not affected. The other enzymes of the TCA cycle remained unaffected by oxaliplatin. That includes citrate synthase (Cs) that converts acetyl-CoA from β-oxidation of FAs or from glycolysis to form citrate, aconitase isomerases (Aco1 and Aco2) that convert citrate to isocitrate, isocitrate dehydrogenases (Idh3b and Idh3g) that form 2-oxoglutaric acid, succinate coenzyme A ligase (Sucla2, Suclg1, Suclg2), succinate dehydrogenases (Sdha, Sdhb and Sdhc) that forms fumarate, fumarate hydratase 1 (Fh1) that produce malate, and mitochondrial malate dehydrogenases (Mdh1 and Mdh2) that form oxaloacetate. The pathway is derived from the KEGG database. Blue – upregulated genes, red downregulated genes, grey – not affected genes.

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  Figure 11. Electron transport chain and oxidative phosphorylation in oxaliplatin treated mice. Oxaliplatin affects the oxidative phosphorylation by downregulation genes associated with the proteins for Complex I, Complex II and Complex IV, reducing the strength of the hydrogen ion gradient. Uncoupling Protein 2 (UCP2 encoded by Slc25a8) was the only upregulated. UCP2 generate heat by dissipating the hydrogen ion gradient of the cristae instead of allowing ATP formation. The pathway is derived from the KEGG database. Blue – upregulated complexes, red downregulated complexes, grey – not affected.

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  Figure 12. Glycolysis in cardiomyocytes is upregulated in the oxaliplatin treated mice. Abbreviations of enzymes and proteins with encoded genes is in parenthesis: ALDOA, aldolase A (Aldoa); ALDOB, aldolase B (Aldob); ENO1/2/3, enolase 1, 2 and 3 (Eno1, Eno2 and Eno3); GAPDH, glyceraldehyde-3-phosphate dehydrogenase (Gapdh); GCK, glucokinase (Gck); GLUT1, glucose transporter 1 (Slc2a1); GLUT3, glucose transporter 3 (Slc2a3); GLUT4, glucose transporter 4; HK2, hexokinase 2/3 HK2 and HK3 (Hk2 and Hk3); LDHB, lactose dehydrogenase B (Ldhb); PGAM2, phosphoglycerate mutase 2 (Pgam2); MCT1,3,6 monocarboxylic acid transporters 1, 3, 6 (Slc16a1, Slc16a3, Slc16a6); phosphofructokinases, PKFL, liver type (Pfkl); PKFM, muscle type (Pfkm); and PKFP, pallet type (Pfkp); PKM, pyruvate kinase, muscle type (Pkm); TPI1, topoisomerase 1 (Tpi1). The schematics is in part based on KEGG Pathways and ref (67). Blue – upregulated genes, grey – unaffected genes, red downregulated genes.