A Novel Positron Emission Tomography (PET) Approach to Monitor Cardiac Metabolic Pathway Remodeling in Response to Sunitinib Malate

Abstract

Sunitinib is a tyrosine kinase inhibitor approved for the treatment of multiple solid tumors. However, cardiotoxicity is of increasing concern, with a need to develop rational mechanism driven approaches for the early detection of cardiac dysfunction. We sought to interrogate changes in cardiac energy substrate usage during sunitinib treatment, hypothesising that these changes could represent a strategy for the early detection of cardiotoxicity. Balb/CJ mice or Sprague-Dawley rats were treated orally for 4 weeks with 40 or 20 mg/kg/day sunitinib. Cardiac positron emission tomography (PET) was implemented to investigate alterations in myocardial glucose and oxidative metabolism. Following treatment, blood pressure increased, and left ventricular ejection fraction decreased. Cardiac [18F]-fluorodeoxyglucose (FDG)-PET revealed increased glucose uptake after 48 hours. [11C]Acetate-PET showed decreased myocardial perfusion following treatment. Electron microscopy revealed significant lipid accumulation in the myocardium. Proteomic analyses indicated that oxidative metabolism, fatty acid β-oxidation and mitochondrial dysfunction were among the top myocardial signalling pathways perturbed. Sunitinib treatment results in an increased reliance on glycolysis, increased myocardial lipid deposition and perturbed mitochondrial function, indicative of a fundamental energy crisis resulting in compromised myocardial energy metabolism and function. Our findings suggest that a cardiac PET strategy may represent a rational approach to non-invasively monitor metabolic pathway remodeling following sunitinib treatment.

Fig 1. Physiological effects of sunitinib in two rodent models.

Effects of sunitinib treatment on MAP (A, n = 9/group) and LVEF (B, n = 13/group) in female Balb/CJ mice. D-E) Effect of sunitinib treatment on systolic blood pressure (C), LVEF (D) and heart rate (E) in female Sprague-Dawley rats (n = 6/group, Significant difference between groups (unpaired t-test, *p<0.05 **p<0.01 ***p<0.001), Significant change from pre-treatment values (paired t-test, †p<0.05 ††p<0.01 †††p<0.001). Error bars = SEM)

Fig 2. Effect of sunitinib treatment on myocardial metabolism and perfusion measured by PET.

A) Myocardial metabolic rate of glucose (MMRG) (mice, n = 10/group), B) K_mono (rats, n = 6/group). C-E) The effects of sunitinib treatment on overall perfusion of the myocardium in the rat model: (C) Representative images of the myocardium as seen in Carimas software (version 2.7) which is used to select region of interest (ROI), subsequently used to construct 17 segment heat maps for the 11C-Acetate data (rats) (D) which are used to determine global perfusion values. D) Representative pre- and 5 days post-treatment in one treated and one vehicle rat E) The effects of sunitinib treatment on overall perfusion of the myocardium (rats, n = 6/group, * = significant difference between groups (unpaired t-test, p<0.05), Significant change from pre-treatment (paired t-test, †p<0.05 ††p<0.01), bar* indicates significantly different overall on-treatment values (two-way ANOVA p < 0.05) Error bars = SEM).

Fig 3. Histopathologic assessment of rodent myocardium following sunitinib treatment.

All error bars represent SEM unless otherwise indicated. H&E staining in mice (A) and rats (B) Magnification as indicated, scale bars (black) 100 μM and 50 μM respectively. C) Masson’s trichrome staining to assess fibrosis in the myocardium of vehicle versus sunitinib treated mice (scale bars (black) represent 100 μm (left) and 50 μm (right), magnification as indicated). D) TUNEL staining for the assessment of apoptosis in mice (40 × magnification; scale bar (black): 50μm. 1000 nuclei were counted per organ using ImageJ software. % TUNEL positive nuclei shown). E) Microvessel density in mice (CD31 immuno-staining). Representative images shown; scale bar (black) 50 μm, x 40 magnification. Vascular density is expressed as % CD31 positive pixels to total pixels captured in each image. F and G) EM analysis of cardiac tissue (mice) to determine the presence of mitochondrial damage [high magnification; x 29000] (F) and the presence of lipid droplets (white arrows) [lower magnification; ×11500] (G), scale bars as indicated ** p = <0.01, n = 4/group. H) ORO analysis in rat heart cryosections to determine lipid droplet accumulation (black arrows indicate representative red lipid droplets, scale bars represent 500 μm; magnification is ×5; n = 3/group. Quantification a.u, arbitrary units **p < 0.01, compared with vehicle. Values are means ± SD).

Fig 4. Proteomic analyses of sunitinib effects in myocardial tissue.

The most significantly perturbed canonical pathways from (A) crude membrane and (B) cytosolic fractions from sunitinib treated and control mouse myocardial tissue (n = 4/group) C) Proteins perturbed in the three most significantly enriched canonical pathways. D) Ingenuity Pathway Analysis (IPA) to determine the most significantly enriched network which is centred on Mitochondrial complex 1. Colour intensity indicates the degree of up- (red) or down- (green) regulation. IPA predictions are shown in blue (predicted inhibition) and orange (predicted activation). Black indicates no predicted effect. Continuous lines indicate a direct relationship between two proteins; a discontinuous line indicates indirect association.

Fig 5. Confirmation of proteomic findings in mouse myocardial tissue.

A) ELISA to measure Mitochondrial complex 1 levels in myocardial tissue lysates. B) Immunoblot analysis of lysates from the cytosolic fraction of myocardial tissue from vehicle or sunitinib treated animals (n = 4/group) as described. C-G) Densitometry assessment of the presented western blots as described (* p < 0.05, error bars represent SEM).