Increased Afterload Augments Sunitinib-Induced Cardiotoxicity in an Engineered Cardiac Microtissue Model

Figure 1. Detecting Changes in Cell Viability With Sunitinib Treatment Using a Rat CMT Model (A) Activated caspase 3/7 levels in response to treatment with 0.1% dimethyl sulfoxide (vehicle), 1 μmol/l sunitinib, or 1 μmol/l staurosporine for 8 h (dimethyl sulfoxide and sunitinib) or 6 h (staurosporine). ****p < 0.0001 relative to vehicle (n = 3 experiments); ***p < 0.001 1 μmol/l sunitinib versus staurosporine (n = 3 experiments). (B) Time-dependent changes in caspase 3/7 activation. Fold changes (relative to vehicle) in caspase levels for cardiac microtissues (CMT) treated with 1 μmol/l or 10 μmol/l sunitinib for 4 h, 8 h, or 12 h. **p < 0.01 1 μmol/l sunitinib 4 h versus 8 h; *p < 0.05 1 μmol/l sunitinib 4-h versus 12-h time points (n = 2 experiments for all time points); ‡‡p < 0.01 10 μmol/l sunitinib 4 h versus 8 h; ‡p < 0.05 10 μmol/l sunitinib 4-h versus 12-h time points (n = 2 experiments for all time points). (C) Dose-dependent changes in caspase activation in CMT treated with 50 nmol/l, 200 nmol/l, 1 μmol/l, 10 μmol/l sunitinib for 8 h. *p < 0.05 50 nmol/l versus 200 nmol/l sunitinib (n = 2 experiments); *p < 0.05 200 nmol/l versus 1 μmol/l sunitinib (n = 2 experiments for 200…

Figure 2. Modeling Variations in Cardiac Function Following Administration of Sunitinib in Rat CMT (A) Dose-dependent decreases in spontaneous beating rates following 24 h of treatment with 0.1% dimethyl sulfoxide (vehicle n = 10 tissues), 1 μmol/l sunitinib (n = 11 tissues), or 10 μmol/l sunitinib (n = 11 tissues). ****p < 0.0001 versus vehicle; *p < 0.05 1 μmol/l sunitinib versus 10 μMmol/l sunitinib. (B) Dose-dependent decreases in static (diastolic) tension generated by CMT treated for 24 h with 0.1% dimethyl sulfoxide (n = 12 tissues), 1 μmol/l sunitinib (n = 20 tissues), or 10 μmol/l (n = 10 tissues) sunitinib. **p < 0.01 vehicle versus 1 μmol/l sunitinib, 1 μmol/l sunitinib versus 10 μmol/l sunitinib; ****p < 0.0001 vehicle versus 10 μmol/l sunitinib. Decrease in active (systolic) tension with 10 μmol/l sunitinib treatment. ****p < 0.0001 vehicle (n = 4 tissues) or 1 μmol/l sunitinib (n = 5 tissues) versus 10 μmol/l sunitinib (n = 9 tissues). We did not observe any changes in excitation threshold (C) or maximum capture rate (D) following 24 h treatment with 0.1% dimethyl sulfoxide or 10 μmol/l sunitinib. (A, C) Data are box-and-whisker plots; median-first quartile are plotted in…

Figure 3. Characterizing Changes in Mitochondrial Function and Cell Energetics With Sunitinib Treatment (A) Flow cytometry histogram showing levels of tetramethylrhodamine (TMRM). Neonatal rat ventricular myocytes grown in flat culture were treated with 0.1% dimethyl sulfoxide (vehicle), 1 μmol/l sunitinib, or 50 μmol/l carbonyl cyanide m-chlorophenyl hydrazine (CCCP) for 30 min (CCCP) or 4 h (dimethyl sulfoxide and sunitinib) and then were labeled with 10 nmol/l TMRM. (B) Time-dependent decreases in mitochondria membrane potential following treatment with 1 μmol/l sunitinib. Neonatal rat ventricular myocytes grown in flat culture were treated with 0.1% dimethyl sulfoxide or 1 μmol/l sunitinib for 30 min to 24 h and labeled with TMRM to assess mitochondria membrane potential. *p < 0.05 (n = 3 experiments). (C) Decreases in adenosine triphosphate (ATP) levels in neonatal rat ventricular myocytes following 24 h treatment with 1 μmol/l sunitinib. *p < 0.05 (n = 3 experiments). (D) Upstream activation of adenosine monophosphate-activated protein kinase with molecule 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) did not reverse sunitinib-induced caspase activation in rat cardiac…

Figure 4. Using CMT to Assess the Contribution of Afterload to Observed Cardiotoxic Effects of Sunitinib (A) Rat CMT cultured on stiff (5:1) or soft (15:1) pillars were treated with 0.1% dimethyl sulfoxide (vehicle) or 1 μmol/l sunitinib for 8 h. Differences in active caspase 3/7 levels were observed between soft and stiff vehicle-treated CMT. **p < 0.01 (n = 3 experiments) and sunitinib-treated CMT. ***p < 0.001 (n = 3 experiments). (B) Results in (A) plotted as fold changes in active caspases 3/7 relative to vehicle-treated CMT. **p < 0.01 (n = 3 experiments). Abbreviations as in Figure 1.

Figure 5. Sunitinib Cardiotoxicity in CMT Composed of Human iPS-CM Induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) were combined with 7% human mesenchymal stem cells to create human CMT. (A) Fold changes in activated caspase 3/7 levels of human CMT on stiff pillars treated 8 h with 0.1% dimethyl sulfoxide (vehicle) or 1 μmol/l, 10 μmol/l sunitinib for 8 h compared with neonatal rat CMT. **p = 0.0116 10 μmol/l sunitinib human CMT (n = 2 experiments) versus neonatal rat CMT (n = 2 experiments). (B) Human CMT were cultured on stiff (5:1) or soft pillars (15:1) and treated with 1 μmol/l sunitinib for 8 h. Plotted are fold changes (relative to vehicle) in caspase 3/7 levels in human and rat CMT. **p < 0.01 human CMT on stiff versus soft pillars (n = 2 experiments); **p < 0.001 human CMT (n = 2 experiments) versus neonatal rat CMT (n = 3 experiments) cultured on soft pillars. Abbreviation as in Figure 1.