Connexin 43 and Connexin 26 Involvement in the Ponatinib-Induced Cardiomyopathy: Sex-Related Differences in a Murine Model

Abstract

Cardiac connexins (Cxs) are proteins responsible for proper heart function. They form gap junctions that mediate electrical and chemical signalling throughout the cardiac system, and thus enable a synchronized contraction. Connexins can also individually participate in many signal transduction pathways, interacting with intracellular proteins at various cellular compartments. Altered connexin expression and localization have been described in diseased myocardium and the aim of this study is to assess the involvement of Cx43, Cx26, and some related molecules in ponatinib-induced cardiac toxicity. Ponatinib is a new multi-tyrosine kinase inhibitor that has been successfully used against human malignancies, but its cardiotoxicity remains worrisome. Therefore, understanding its signaling mechanism is important to adopt potential anti cardiac damage strategies. Our experiments were performed on hearts from male and female mice treated with ponatinib and with ponatinib plus siRNA-Notch1 by using immunofluorescence, Western blotting, and proteomic analyses. The altered cardiac function and the change in Cxs expression observed in mice after ponatinib treatment, were results dependent on the Notch1 pathway and sex. Females showed a lower susceptibility to ponatinib than males. The downmodulation of cardiac Cx43, Cx26 and miR-122, high pS368-Cx43 phosphorylation, cell viability and survival activation could represent some of the female adaptative/compensatory reactions to ponatinib cardiotoxicity.

Keywords: Notch1; cardiotoxicity; connexin; heart; mouse model; ponatinib; sex-dependent differences.

Figure 1

Heart Cx43 protein modulation. (A) Differential Proteomics Analysis demonstrated the downregulation of Cx43 only in female ponatinib-treated mice compared to control and siRNA-Notch1 ones. Histogram representing the proteomics data related to Cx43 abundance in heart mice samples. Data were reported as mean ± standard deviation of LFQ Intensity parameter analytical replicates (n = 3). &: p < 0.05 at ANOVA test and validated with post hoc-Tukey’s HSD test. Proteomics data were confirmed by Western Blot analysis as shown in Panel (B) of the figure. This shows a representative blot of one group of the three groups analysed. (C) Histogram representing the Cx43 protein expression in heart mice samples. Data are expressed as mean ± standard deviation, n = 3. ** p < 0.001 vs. CNTRL; * p < 0.05 vs. PON + siRNA-Notch1; ^§: p < 0.01 vs. CNTRL; Φ: p < 0.05 vs. males; Φ Φ: p < 0.01 vs. males.

Figure 2

Female proteomic analysis. Upstream network effects of a quantified protein in the Ingenuity Pathway Analysis (IPA) revealed the activation of ERK (A) and Akt (B) as well as the inhibition of miR-122 (C) in female mice cardiac tissue treated with ponatinib compared to control ones. Red and green shapes represent increased or decreased measurements of quantified proteins, respectively. Color intensity is directly proportional to the fold change value of each protein reported in the figure. Circles show proteins related to Cx43. Color key and symbols are reported in the legend.

Figure 3

Male proteomic analysis. Upstream network effects of a quantified protein in the Ingenuity Pathway Analysis (IPA) revealed the activation of IL6 in male mice cardiac tissue treated with ponatinib compared to control ones. Red and green shapes represent increased or decreased measurements of identified proteins, respectively. Color intensity is directly proportional to the fold change value of each protein reported in the figure. Circles show target molecules of Cx43. Color key and symbols are reported in the legend.

Figure 4

Cx43 protein expression at cardiomyocyte membrane level. (A) Confocal laser scanning microscopy: representative three-dimensional images of the maximum intensity projection of mice cardiomyocyte longitudinal sections. Cx43 (red) is confined at the intercalated discs in CNTRL and in siRNA-Notch1 groups whereas it lies also on the lateral border (arrows) of cardiomyocytes (grey) in PON groups. Nuclei of cardiomyocytes and connective cells are marked with DAPI in blue. Magnification: 600×. Bars: 10 µm. (B) Immunofluorescence staining: representative images of Cx43 immunoreaction (red). Magnification: 200×. Bars: 50 µm. (C) Image analysis of Cx43-immunofluorescence on cardiomyocytes. Data are expressed as mean ± standard deviation, n = 4. * p < 0.001 vs. CNTRL; ** p < 0.05 vs. PON + siRNA-Notch1; ^§: p < 0.001 vs. CNTRL.

Figure 5

pS368-Cx43 protein expression. (A) Representative of a Western blot analysis of one group of the three analysed groups. (B) Histogram obtained by Western blot analysis, representing the total cardiac pS368-Cx43 protein expression of heart mice samples. (C) Immunofluorescence staining: representative images of cardiomyocyte pS368-Cx43 immunoreaction (red). Magnification: 200×. Bars: 50 µm. (D) Image analysis of cardiomyocyte pS368Cx43-immunofluorescence. Data are expressed as mean ± standard deviation, n = 4. * p < 0.001 vs. CNTRL; ** p ≤ 0.001 vs. CNTRL and PON + siRNA-Notch1; ^§: p ≤ 0.001 vs. CNTRL; Φ: p < 0.01 vs. females; Φ Φ: p < 0.05 vs. females.

Figure 6

PKCps immunofluorescence staining in cardiomyocytes. Representative images of PKCps immunopositivity (red) at intercalated discs (arrows) and at lateral borders (arrowheads). Magnification: 200×. Bars: 10 µm.

Figure 7

Cardiac Cx26 protein expression. (A) Representative of a Western blot analysis of one group of the three analysed groups. (B) Histogram obtained by Western blot analysis, representing the total cardiac Cx26 protein expression of heart mice samples. (C) Immunofluorescence staining: representative images of cardiomyocyte Cx26 immunoreaction (red). Magnification: 200×. Bars: 50 µm. (D) Image analysis of cardiomyocyte Cx26 immunostaining * p ≤ 0.05 vs. CNTRL and PON + siRNA-Notch1; ** p < 0.01 vs. CNTRL and PON + siRNA-Notch1.