Cardiac Metabolic Deregulation Induced by the Tyrosine Kinase Receptor Inhibitor Sunitinib is rescued by Endothelin Receptor Antagonism

Figure 1. Study design: (A) represents investigation for short-term cardiotoxic effects on immunodeficient tumor-bearing mice (nude). Sunitinib-treated mice were studied at baseline and week 1 using a cancer PET protocol compared to vehicle. (B) represents investigation for short-term cardiotoxic effects on immunocompetent mice (C57BL/6). Sunitinib-treated mice were studied at baseline and week 1 using a cancer PET protocol and echocardiography compared to vehicle. (C) represents study design for long-term treatment on immunocompetent mice (C57BL/6). Sunitinib-treated mice were followed at baseline, week 1, week 2 and week 3 using a cardiac PET protocol and echocardiography compared to vehicle and sunitinib+macitentan groups. FDG: 2'-deoxy-2'-[18F]fluoro-D-glucose.

Figure 2. Sunitinib increases myocardial FDG uptake in fasted mice: (A) Example of PET scan images representing cardiac views of FDG-SUV at baseline (left) and post-treatment (right) in vehicle-treated mice and sunitinib-treated mice. (B) Difference post-treatment - baseline of myocardial FDG-SUV for sunitinib and vehicle groups in nude mice (n=6 for vehicle, n=10 for sunitinib) and C57BL/6 (n=6 for each). (C) Difference post-treatment - baseline of myocardial metabolic flux for sunitinib and vehicle groups in nude mice (n=5 for vehicle, n=10 for sunitinib) and C57BL/6 (n=2 for each). (D) Difference in cardiac output (CO, Heart Rate times stroke volume) for sunitinib and vehicle in C57BL/6 mice (n=8 for each groups). Data expressed as mean±SEM; *p <0.05 compared to baseline, **p <0.01 and ***p <0.001 compared to baseline, $p <0.05 compared to vehicle. CO: cardiac output; SUV: standard uptake values

Figure 3. Sunitinib-induced increased FDG uptake in fasted mice is associated with increased fibrosis: (A) Representative sections of myocardium stained for blood vessels (lsolectine B4 Griffonia Simplicifolia-FITC, green) and nuclei (DAPI, blue). (B) Quantification of microvascular density normalized by cell number in nude and C57Bl/6 mice treated with vehicle (open circles) or sunitinib (black circles). (C) Representative sections of myocardium stained for fibrosis (Picrosirius red) from hearts of mice treated with vehicle (left) or sunitinib (right). (D) Quantification of fibrosis (normalized by tissue area) in nude and C57Bl/6 mice treated with vehicle (open circles) or sunitinib (filled circles). (E) Representative blots and their associated quantification for GLUT1, HK2 and PGC1α (normalized by cyclophilin B) mice treated with vehicle (open circles) or sunitinib (filled circles). Data expressed as mean ± SEM; $$p <0.01 $$$p <0.001 compared to vehicle. HK2: Hexokinase 2; GLUT1: glucose transporter 1; PGC1α; Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; S: sunitinib; V: vehicle.

Figure 4. Macitentan prevents sunitinib-induced diastolic dysfunction. (A) Difference treatment - baseline of myocardial FDG-SUV for vehicle (n=9), sunitinib (n=10) and sunitinib+macitentan treated mice (n=8) at week 1 and week 3. (B) Difference treatment - baseline values of CO is represented for vehicle (n=7), sunitinib (n=10) and sunitinib+macitentan (n=8) groups at week 1 and 3. (C) Difference treatment - baseline of left ventricular internal diameter at diastole for the three groups. (D) Difference treatment - baseline of aortic velocity time integral for both groups. Data are expressed as mean ± SEM, *p <0.05 compared to baseline, ***p <0.001 compared to baseline, $p <0.05 compared to other at week 3. AoVTI: Aortic velocity tracking integral; CO: cardiac output; LVID: left ventricular internal diameter; SUV: standard uptake value.

Figure 5. Macitentan prevents myocardial flux dysfunction induced by sunitinib. (A) Difference treatment - baseline of myocardial metabolic flux and metabolic rate of glucose (MRglu) for vehicle (n=5), sunitinib (n=5) and sunitinib+macitentan (n=6) groups. Data are expressed as mean ± SEM, *p <0.05 compared to baseline, $p <0.05 compared to other at week 3. #p <0.05 compared to sunitinib group at week 3. (B) Micrographs of cardiac sections of microvascular staining (lectin in green and nuclei in blue) at D22 in non-fasted C57Bl/6 mice. (C) Quantification of vessels area reported on number of cells for vehicle (n=9), sunitinib (n=9) and sunitinib+macitentan (n=7) groups at D22 in non-fasted C57Bl/6 mice. MRGlu: metabolic rate of glucose.

Figure 6. Protective effects of macitentan involve ET receptors: (A) ng of ET-1, ETA and ETB receptor mRNA reported on ng of 18s RNA in the myocardium (n=6 for each). (B) Micrographs of cardiac sections of fibrosis marker (Picrosirius red) at D22 non-fasted C57Bl/6 mice. (C) Quantification of fibrosis for vehicle (n=9), sunitinib (n=9) and sunitinib+ macitentan (n=7) groups at D22 in non-fasted C57Bl/6 mice. Data are expressed as mean ± SEM, $p <0.05; $$$p <0.001 compared to other. EDN1: Preproendothelin-1; EDNRA: endothelin receptor type A; EDNRB: endothelin receptor type B.

Figure 7. Cardioprotective effects of macitentan. (A) Pathway studio representations of proteins involved in myocardial infarction and endothelial cell apoptosis showing the changes in protein expression levels in the myocardium of the sunitinib group and the (vehicle and sunitinib+macitentan) groups. Red indicates significantly (p<0.05) upregulated proteins, blue significantly (p<0.05) down-regulated proteins. (B) Ingenuity representation of the changes in Pyruvate dehydrogenase complex expression in the myocardium of the sunitinib group in comparison to the (vehicle and sunitinib+macitentan) groups. Green shows significantly (p<0.05) downregulated proteins in the sunitinib group. (C) Table of the diabetes-related pathways most significantly perturbed by sunitinib in mouse myocardium and correction by macitentan. n=6 for each group. n.s: non-significant. Proteins described in Table 2.

Figure 8. Schematic representation of the mechanism of sunitinib-induced cardiac side effects: (A) Sunitinib upregulates glycolysis and downregulates oxidative metabolism in cardiac mitochondria. (B) Sunitinib induces resistance to insulin stimulation of cardiac glucose uptake. The metabolic switch is an immediate early response to sunitinib while insulin resistance either appears later or is masked by the metabolic switch during the early stages of sunitinib treatment. Both mechanisms depend on signaling by the endothelin pathway, and lead to myocardial fibrosis and impaired cardiac function, and are reversed by the endothelin receptors antagonist macitentan. Red indicates upregulated proteins and pathways, blue indicates downregulated protein and pathways. ATP: Adenosine triphosphate; ET-1: endothelin 1; ETA: endothelin receptor type A; FA: fatty acid; GLUT: glucose transporter protein; O2: oxygen; OXPHOS: oxidative phosphorylation; TCA: tricarboxylic acid.