Anti-PD-1 immune checkpoint inhibitor-induced cardiotoxicity is associated with dysfunctional metabolism, muscle wasting and autophagy

Figures

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  Fig. 1. Tumor characteristics of control and ICI-treated tumor bearing mice (Fig. 1a, b), and body mass and heart mass of control and ICI-treated mice (Fig. 1c-e). Relative tumor mass (a) was calculated by normalizing wet tumor mass in grams to body mass in grams. Estimated tumor volume (b) was calculated by using the following formula: tumor volume (mm3) = (a * b)2/2, with a being the longest diameter and b being the shortest diameter of the tumor. Body mass (c) was weighed in grams at baseline and endpoint. Absolute heart mass (d) was weighed in grams and then normalized to body mass in grams (e). CON + T, control tumor bearing group (n = 5); ICI + T, ICI treated tumor bearing group (n = 4 in Fig. 1A excluding one animal due to difficulties during tumor excision, n = 5 in Fig. 1B); N = 9/10. CON, control group (n = 5); ICI, ICI treated group (n = 5); N = 10. Values are reported as mean ± SD. *Significant difference, P < 0.05; **Significant difference, P < 0.01.

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  Fig. 2. In Vivo Echocardiography at Study Endpoint. Wall thicknesses (a, b) and left ventricular diameter (c, d) at end-diastole and end-systole represent an average of three consecutive cardiac cycles per mouse. Fractional shortening as a measure of cardiac function (e-g) represents an average of three consecutive cardiac cycles per mouse. Change scores (f) were calculated by subtracting average baseline from average endpoint scores and dividing results by baseline scores. CON, control group (n = 5); ICI, ICI treated group (n = 5); N = 10. Values are reported as mean ± SD. *Significant difference, P < 0.05; ***Significant difference, P < 0.001; ****Significant difference, P < 0.0001.

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  Fig. 3. Effects of ICI treatment on protein levels of P-AKT (b), P-FoxO1 (c), and P-FoxO3a (d) in cardiac muscle. Cropped, representative western blot images (a); original blots are presented in supplementary files. CON, control group (n = 5); ICI, ICI treated group (n = 5). N = 10. Values are reported as mean ± SD. *Significant difference, P < 0.05.

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  Fig. 4. Effects of ICI treatment on protein levels of MuRF1 (b) and Atrogin1 (c) in cardiac muscle. Cropped, representative western blot images (a); original blots are presented in supplementary files. CON, control group (n = 5); ICI, ICI treated group (n = 5). N = 10. Values are reported as mean ± SD. *Significant difference, P < 0.05.

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  Fig. 5. Autophagic flux analysis in cardiac muscle via Fluorescent Microscopy. Yellow puncta (b) represent early-phase autophagosomes. Red puncta (c) represent late-phase autolysosomes. Effects of ICI treatment on protein levels of p62 (d) in cardiac muscle. Representative fluorescent images have been taken at 40x magnification (a). Yellow arrows point towards yellow puncta/early-phase autophagosomes, red arrows point towards red puncta/late-phase autolysosomes (a). Cropped, representative western blot images (d); original blots are presented in supplementary files. CON, control group (n = 5); ICI, ICI treated group (n = 5); N = 10. Values are reported as mean ± SD. *Significant difference, P < 0.05. **Significant difference, P < 0.01.