Effects of Gap 26, a Connexin 43 Inhibitor, on Cirrhotic Cardiomyopathy in Rats

Abstract

Introduction Cirrhotic cardiomyopathy (CCM) is recognized by impaired cardiac responsiveness to stress, prolonged QT interval, and systolic and diastolic dysfunctions. Connexins are a family of transmembrane proteins that play a key role in cardiac physiology. Connexin 43 (Cx43) inhibition showed cardio-protective effects. Peptide drug Cx43 inhibitor, Gap 26, could inhibit gap junction 43. This study was designed to evaluate the effects of a connexin mimetic peptide, Gap 26, in the CCM model in rats. Methods The cirrhosis was induced through carbon tetrachloride (CCl4). On day 56, electrocardiography (ECG) was recorded, spleen weight was measured, and tissue and serum samples were collected. Further, Cx43 mRNA expression in heart tissue was checked. Results The chronotropic responses decreased in the CCl4/saline and increased in the CCl4/Gap. The spleen weight, QTc interval, and brain natriuretic peptide (BNP), tumor necrosis factor-alpha (TNF-α), aspartate aminotransferase (AST), alanine transaminase (ALT), and malondialdehyde (MDA) levels elevated in the CCl4/saline, and the spleen weight, QTc interval, and MDA and ALT levels were reduced by Gap 26 treatment. The level of nuclear factor (erythroid-derived 2) factor 2 (Nrf2) decreased in the CCl4/saline. The Cx43 expression was downregulated in the CCl4/saline and upregulated with the Gap 26 treatment. Conclusion Gap 26 not only alleviated the chronotropic hyporesponsiveness and the severity of liver damage and upregulated the atrial Cx43 expression, but it also had an antioxidant effect on the heart.

Keywords: cirrhotic cardiomyopathy; connexin 43; gap 26; inflammation; oxidative stress.

Figure 1. The spleen weight/body weight changes of sham (control) or CCl4-treated rats following saline or Gap 26 (1 µg/kg, PO) treatment. One-way ANOVA followed with post hoc Tukey's test, and the outcome was considered significant at P<0.05. The data are presented in mean±SEM. Six to eight rats were included in each experimental group. ***P<0.001 compared to the sham/saline group. #P<0.05 compared to the cir/saline group.

Cir: cirrhotic; sol: solvent

Figure 2. The heart sections' histopathology photomicrographs of sham (control) (Figure 2a) and CCl4-treated (cirrhotic) rats (Figure 2b) after saline treatment (Figure 2c) or Gap 26 (1 µg/kg, PO) (Figure 2d). The photomicrographs (100×) are shown in 56 days of treatment. The H&E was used for staining heart sections. Six rats were included in each experimental group.

Cir: cirrhotic; sol: solvent

Figure 3. The QTc intervals of sham (control) or CCl4-treated rats (0.4 g/kg, IP) after treatment with saline or Gap 26 (1 µg/kg, PO). The data for respective groups are mentioned after 56 days of treatment. One-way ANOVA was applied to analyze data with post hoc Tukey's test, and the significance level was P<0.05. The outcome is shown as mean±SEM, n=6. **P<0.01, ***P<0.001 compared to the sham/saline group. &&P<0.01 compared to the cir/saline group. ##P<0.01.

Cir: cirrhotic; sol: solvent

Figure 4. Average atrial beating rates of sham (control) and CCl4-treated (0.4 g/kg, IP) (cirrhotic) rats after treatment with saline or Gap 26 (1 µg/kg). The atrial beating rates were calculated pre- (4B) and post-stimulation with 10-10 to 10-5 M cumulative isoproterenol (4A). The data for respective groups are mentioned after 56 days of treatment. Two-way ANOVA was applied to analyze data with post hoc Bonferroni's test, and the results are significant at P<0.05. The data are presented as mean±SEM and n=6 rats. **P<0.01 compared to the sham/saline group. ####P<0.0001 compared to the cir/saline group.

Cir: cirrhotic; sol: solvent

Figure 5. The serum BNP, ALT, and AST levels of sham (control) and CCl4-treated rats (0.4 g/kg, IP), after treatment with saline or Gap 26 (1 µg/kg). The data are mentioned 56 days post-treatment. One-way ANOVA with post hoc Tukey's test analyzed the data, and the significant difference is at P<0.05. The data are presented as mean±SEM. Six rats were included per experimental group. **P<0.01, ***P<0.001, ****P<0.0001 compared to the sham/saline group. &P<0.05 compared to the sham/saline group. #P<0.05 compared to the cir/saline group.

Cir: cirrhotic; sol: solvent; BNP: brain natriuretic peptide; ALT: alanine transaminase; AST: aspartate aminotransferase

Figure 6. The tissue levels of TNF-α, Nrf2, and MDA in sham (control) and CCl4-treated (0.4 g/kg, IP) rats after treatment with saline or Gap 26 (1 µg/kg, PO). The data for the respective groups are mentioned after 56 days of treatment. One-way ANOVA was applied to analyze data with post hoc Tukey's test, and the significant level was at P<0.05. The data are presented as mean±SEM. Six rats were included per experimental group. *P<0.05, **P<0.01, ****P<0.0001 compared to the sham/saline group. #P<0.05 compared to the cir/saline group. &P<0.05 compared to the cir/sol group.

Cir: cirrhotic; sol: solvent; TNF-α: tumor necrosis factor-alpha; Nrf2: nuclear factor (erythroid-derived 2) factor 2; MDA: malondialdehyde

Figure 7. The tissue levels of TNF-α, Nrf2, and MDA in sham (control) and CCl4-treated (0.4 g/kg, IP) rats after treatment with saline or Gap 26 (1 µg/kg, PO). The data for the respective groups are mentioned after 56 days of treatment. One-way ANOVA was applied to analyze data with post hoc Tukey's test, and the significant level was at P<0.05. The data are presented as mean±SEM. Six rats were included per experimental group. *P<0.05, **P<0.01, ****P<0.0001 compared to the sham/saline group. #P<0.05 compared to the cir/saline group. &P<0.05 compared to the cir/sol group.

Cir: cirrhotic; sol: solvent; TNF-α: tumor necrosis factor-alpha; Nrf2: nuclear factor (erythroid-derived 2) factor 2; MDA: malondialdehyde