The Role of Autophagy and Death Pathways in Dose-dependent Isoproterenolinduced Cardiotoxicity

Fig. (1). Effect of ISO administration at different doses on the survival rate of animals. (n=24-41/group). The significance of differences among groups was evaluated with Chi-square and Fisher’s exact tests. p values of 0.05 or less were considered significant in each graph. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively.

Fig. (2). Effect of ISO administration at different doses on serum biomarkers. (A) Cardial Troponin T, (B) creatine-kinase iso-enzyme MB (CK-MB), (C) lactate-dehydrogenase levels were obtained from blood samples taken from the left jugular vein. Results are expressed as the average magnitude of each value within a group of animals ± SEM (n=17-22/groups). The significance of differences among groups was evaluated with one-way analysis of variance (ANOVA) followed by the Dunnett multiple comparison tests. p values of 0.05 or less were considered significant in each graph. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively.

Fig. (3). Effect of ISO administration at different doses on necrosis. (A) TTC staining was performed to measure the level of necrosis. Results are expressed as the average magnitude of each value within a group of animals ± SEM (n=4-6/group). The significance of differences among groups was evaluated with one-way analysis of variance (ANOVA) followed by the Dunnett comparison test. p values of 0.05 or less were considered significant in each graph. * and *** represent p<0.05 and p<0.001, respectively. (B) Representative pictures of infarct size by TTC staining.

Fig. (4). Effect of ISO administration at different doses on the apoptotic marker. (A) TUNEL assay was carried out to study the expression level of DNA fragmentation. Results are presented as a percent of the ratio of TdT+ to total nuclei (n = 4/ groups). (B) Representative immunofluorescence pictures obtained in heart tissue samples from all groups. Blue: total nuclei by DAPI staining, Red: TdT+ nuclei. Scale bar: 20 µm. (C) Caspase-3 was evaluated using Western blot analysis. Results are expressed as the average magnitude of each value within a group of animals ± SEM (n=7). The significance of differences among groups was evaluated with one-way analysis of variance (ANOVA) followed by the Dunnett comparison test. p values of 0.05 or less were considered significant in each graph. *, and ** represent p<0.05 and p<0.01, respectively. (The color version of the figure is available in the electronic copy of the article).

Fig. (5). Effect of ISO administration at different doses on autophagic markers. The expression levels of (A) Beclin-1, (B) LC3B-II and (C) p62 in left ventricular tissues were evaluated using Western blot analysis. Results are expressed as the average magnitude of each value within a group of animals ± SEM (n=7-11). The significance of differences among groups was evaluated with one‐way analysis of variance (ANOVA) followed by the Dunnett comparison test. p values of 0.05 or less were considered significant in each graph. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively.

Fig. (6). Effect of ISO administration at different doses on the investigated pathways. Based on the experiments and results, theoretically, isoproterenol treatment initiates first autophagy, and then apoptosis, and finally necrosis-induced cell death, suggesting that necrosis masks the processes of autophagy and apoptosis.