Cardiotoxic Effects of Short-Term Doxorubicin Administration: Involvement of Connexin 43 in Calcium Impairment

Figures

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  Figure 1. Effect of DOXO on calcium homeostasis. Mice received a single administration (1th group), two administrations (2nd group) or three administrations (3rd group) of DOXO (2 or 10 mg/kg; i.p.) and primary cardiomyocytes were isolated by enzymatic digestion. Intracellular calcium content in cells suspension was evaluated by using ionomycin (1 μM) (A); reticulum calcium content was evaluated by means of thapsygargin (100 nM) (B) and mitochondrial calcium content was evaluated by using FCCP (50 nM) (C). Results were expressed as mean ± S.E.M. of delta (δ) increase of FURA-2 AM ratio fluorescence (340/380 nm) from at least three independent experiments each performed in duplicate. Data were analyzed by Student’s t-test. * p < 0.05, ** p < 0.005 and *** p < 0.001 vs. control. Effect of DOXO on SERCA II (D) and PLB (E) expression. Mice received a single administration (first group), two administrations (second group), or three administrations (third group) of DOXO (2 mg/kg or 10 mg/kg; i.p.) and SERCA II and PLB expressions were detected by Western blot analysis into tissue homogenates from mice; GAPDH protein expression was used as loading control. Values were expressed as mean ± S.E.M.…

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  Figure 2. Mice received a single administration (first group), two administrations (second group), or three administrations (third group) of DOXO (2 mg/kg and 10 mg/kg; i.p.) and Cx43 and pCx43 expressions were detected by Western blot analysis into tissue homogenates from mice; GAPDH protein expression was used as loading control (A,B); Effects of DOXO on mCx43 (C) and mpCx43 (D) expression were detected by Western blot analysis on mitochondrial protein lysate from mice; TOM20 protein expression was used as loading control. Values were expressed as mean ± S.E.M. from at least three independent experiments each performed in duplicate. Values are expressed as mean ± S.E.M. from at least three independent experiments each performed in duplicate. Data were analyzed by Student’s t-test. * p < 0.05, ** p < 0.005 and *** p < 0.001 versus control. Representative Western blots of Na+/K+ ATPase and Ox-Phos Complex II were used as markers of sarcolemma (SF) and mitochondria (MF), respectively, to demonstrate the purity of the mitochondrial extracts (E).

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  Figure 3. Effect of DOXO (2 and 10 mg/kg; i.p.) on Cx43 localization in heart of C57BL/6j mice which received a single administration (first group), two administrations (second group), or three administrations (third group). Frozen myocardial tissue sections were stained with Anti-Cx43 (green), TOM20 (red) and nucleus with DAPI (blue) and were determined by Immunofluorescence assay at confocal microscopy for mitochondrial Cx43 localization. Scale bar 10 µm.