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

Abstract

The use of Doxorubicin (DOXO), a potent antineoplastic agent, is limited by the development of cardiotoxicity. DOXO-induced cardiotoxicity is multifactorial, although alterations in calcium homeostasis, seem to be involved. Since even the Connexin43 (Cx43) plays a pivotal role in these two phenomena, in this study we have analyzed the effects of DOXO on Cx43 expression and localization. Damage caused by anthracyclines on cardiomyocytes is immediate after each injection, in the present study we used a short-term model of DOXO-induced cardiomyopathy. C57BL/6j female mice were randomly divided in groups and injected with DOXO (2 or 10 mg/kg i.p.) for 1-3 or 7 days once every other day. Cardiac function was assessed by Echocardiography. Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCAII) and phospholamban (PLB) expression were assessed by Western blot analysis, intracellular [Ca²⁺] were detected spectrofluorometrically by means of Fura-2 pentakis (acetoxymethyl) ester (FURA-2AM), and Cx43 and pCx43 expression and localization was analyzed by Western blot and confirmed by immunofluorescence analysis. DOXO induces impairment in Ca²⁺ homeostasis, already evident after a single administration, and affects Cx43 expression and localization. Our data suggest that DOXO-induced alterations in Ca²⁺ homeostasis causes in the cells the induction of compensatory mechanisms until a certain threshold, above which cardiac injury is triggered.

Keywords: calcium homeostasis; cardiotoxicity; connexin 43; doxorubicin; mitochondria.

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. from at least three independent experiments each performed in duplicate. Data were analyzed by Student’s t-test. *p < 0.05 vs. control.

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).

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.