Sunitinib and Imatinib Display Differential Cardiotoxicity in Adult Rat Cardiac Fibroblasts That Involves a Role for Calcium/Calmodulin Dependent Protein Kinase II

Figures

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  Figure 1. Assessment of Cardiac Fibroblast phenotype. (A) CF phenotype was confirmed via immunohistochemistry by staining for vimentin, smooth muscle cell alpha actin (SMCa) and von Willebrand Factor (vWF). Staining was compared between CFs and myofibroblasts (MF). Smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVECs) were used as positive controls. Cells treated with DAPI alone and secondary antibodies alone were used as negative controls. (B) Brightfield images showing CF treated with sunitinib and imatinib (0.1–10 μM) for 18 h in the presence of serum. Scale bar 100 μm. Images are from one experiment, representative of two others.

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  Figure 2. Sunitinib treatment reduces cardiac fibroblast viability. Cells were treated with sunitinib or imatinib (0.1–10 μM) in the presence of serum for 18 h. Cell proliferation was assessed via MTT assays. (A) Sunitinib treated CF (B) imatinib treated CF. Results are expressed as mean ± S.E.M and are normalized to control (n = 3, *p < 0.05).

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  Figure 3. Sunitinib treatment causes increased necrosis in cardiac fibroblasts. Cells were treated with sunitinib (0.1–10 μM) in the presence of serum for 18 h. Cell viability was determined by PI and AnV staining. (A) Representative flow cytometry dot plots with double Annexin V-APC/PI staining for cells treated with sunitinib. (B) Histograms detailing the percentage of (i) healthy (ii) early apoptotic (iii) late apoptotic and (iv) necrotic cells following sunitinib treatment. Results are expressed as mean ± S.E.M (n = 3, *p < 0.05).

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  Figure 4. Imatinib treatment does not cause apoptosis or necrosis in cardiac fibroblasts. Cells were treated with imatinib (0.1–10 μM) in the presence of serum for 18 h. Cell viability was then determined by PI and AnV staining. (A) Representative flow cytometry dot plots with double Annexin V-APC/PI staining for cells treated with sunitinib. (B) Histograms detailing the percentage of (i) healthy (ii) early apoptotic (iii) late apoptotic and (iv) necrotic cells following imatinib treatment. Results are expressed as mean ± S.E.M (n = 3).

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  Figure 5. Sunitinib increases Angiotensin II—induced Ca2+ mobilization in cardiac fibroblasts. (A) Effect of increased AngII concentrations (0.01–10 μM) on intracellular Ca2+ mobilization (B) Representative trace of Ca2+ mobilization in response to 0.3 μM AngII stimulation (C) Effects of increasing concentrations of sunitinib (0.001–1 μM) on AngII induced Ca2+ mobilization. Sunitinib in the absence of Ang II is also shown. Results shown are mean data from one experiment, representative of five other experiments. (D) Comparison of maximal (1 μM) sunitinib Ang II-induced Ca2+ mobilization vs. control. (E) Effects of increasing concentrations of imatinib (1–10 μM) on AngII induced Ca2+ mobilization. (F) Effects of TKI-treatment on peak agonist-induced Ca2+ mobilization. Calcium mobilization was determined by measuring fluorescence at Ex:494/Em:525 nm. Results are expressed as mean ± S.E.M of 6 biological replicates, *p < 0.05.

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  Figure 6. Sunitinib treatment increases ox-CaMKII expression in cardiac fibroblasts. Following the indicated treatments with either sunitinib or imatinib, CaMKIIδ expression or CaMKII activation via phosphorylation or oxidation was determined via quantitative immunoblotting (A) CaMKIIδ expression, normalized to GAPDH (B) Phos-CaMKII expression, normalized to CaMKIIδ (C) Ox-CaMKII expression, normalized to CaMKIIδ (D) Representative immunoblot showing CaMKIIδ expression and CaMKII activation following TKI treatment. Results are expressed as mean ratios protein: GAPDH ± S.E.M and are normalized to control (n = 3, *p < 0.05).

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  Figure 7. Sunitinib and imatinib treatments both cause increased mitochondrial superoxide production in cardiac fibroblasts. (Ai) Histogram showing superoxide production assessed via fluorescence intensity in antimycin A and vehicle treated CFs (Aii) Representative images of MitoSOX Red analysis of antimycin A and vehicle treated samples. (Bi) Superoxide production assessed via fluorescence intensity in sunitinib treated CFs (Bii) Representative images of MitoSOX Red analysis of sunitinib treated samples. (Ci) Superoxide production assessed via fluorescence intensity in imatinib treated CFs (Cii) Representative images of MitoSOX Red analysis of imatinib treated samples. Scale bar 100 μm. Results shown are mean data from one experiment (n = 10), representative of two other experiments (*p < 0.05).

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  Figure 8. KN-93 reduces mitochondrial superoxide production in both sunitinib and imatinib treated cardiac fibroblasts. (Ai) Superoxide production assessed via fluorescence intensity in sunitinib treated CF in the presence (+) or absence (−) of 5 μM KN-93. (Aii) Representative images of MitoSOX Red analysis of sunitinib treated samples in the presence (+) or absence (−) of KN-93. (Bi) Superoxide production assessed via fluorescence intensity in imatinib treated CF in the presence (+) or absence (−) of KN-93. (Bii) Representative images of MitoSOX Red analysis of imatinib treated samples in the presence (+) or absence (−) of KN-93. Scale bar 100 μm. Results shown are mean data and images from one experiment where the same control (−) presented previously for non-KN 93 treatment has been used as a reference comparison for control (+) (n = 10). Imatinib and sunitinib treatments from the same experiment have been presented separately for clarity and are representative of two other experiments (*p < 0.05).

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  Figure 9. A proposed mechanism for sunitinib-induced mitochondrial dysfunction in adult cardiac fibroblasts. Schematic showing proposed sunitinib-induced events at the level of the mitochondria in CFs. Sunitinib treatment may exert direct effects on mitochondrial superoxide production that can then result in CaMKII oxidation. Sunitinib-induced increases in CaMKII oxidation and activation can then go on to cause elevated superoxide production so a positive feedback loop may exist exacerbating the cardiotoxic effects. The combination of elevated CaMKII oxidation and increased superoxide production ultimately causes toxicity at the level of the mitochondria and can contribute to CF cell death.