Inhibition of gap junction intercellular communication is involved in silica nanoparticles-induced H9c2 cardiomyocytes apoptosis via the mitochondrial pathway

Abstract

Gap junction intercellular communication (GJIC) between cardiomyocytes is essential for synchronous heart contraction and relies on connexin-containing channels. Connexin 43 (Cx43) is a major component involved in GJIC in heart tissue, and its abnormal expression is closely associated with various cardiac diseases. Silica nanoparticles (SNPs) are known to induce cardiovascular toxicity. However, the mechanisms through which GJIC plays a role in cardiomyocytes apoptosis induced by SNPs remain unknown. The aim of the present study is to determine whether SNPs-decreased GJIC promotes apoptosis in rat cardiomyocytes cell line (H9c2 cells) via the mitochondrial pathway using CCK-8 Kit, scrape-loading dye transfer technique, Annexin V/PI double-staining assays, and Western blot analysis. The results showed that SNPs elicited cytotoxicity in H9c2 cells in a time- and concentration-dependent manner. SNPs also reduced GJIC in H9c2 cells in a concentration-dependent manner through downregulation of Cx43 and upregulation of P-Cx43. Inhibition of gap junctions by gap junction blocker carbenoxolone disodium resulted in decreased survival and increased apoptosis, whereas enhancement of the gap junctions by retinoic acid led to enhanced survival but decreased apoptosis. Furthermore, SNPs-induced apoptosis through the disrupted functional gap junction was correlated with abnormal expressions of the proteins involved in the mitochondrial pathway-related apoptosis such as Bcl-2/Bax, cytochrome C, Caspase-9, and Caspase-3. Taken together, our results provide the first evidence that SNPs-decreased GJIC promotes apoptosis in cardiomyocytes via the mitochondrial pathway. In addition, downregulation of GJIC by SNPs in cardiomyocytes is mediated through downregulation of Cx43 and upregulation of P-Cx43. These results suggest that in rat cardiomyocytes cell line, GJIC plays a protective role in SNPs-induced apoptosis and that GJIC may be one of the targets for SNPs-induced biological effects.

Keywords: cardiotoxicity; cell death; connexin 43; gap junction; silica nanoparticles.

Figure 1

Transmission electron microscopy images of silica nanoparticles in serum-free Dulbecco’s Modified Eagle’s Medium (×100,000).

Figure 2

Viability of H9c2 cells after exposure to silica nanoparticles at different concentrations and times. The results indicated that viability of H9c2 cells reduced in both concentration- and time-dependent manner (mean, n=6).

Figure 3

Silica nanoparticles (SNPs)-induced gap junction intercellular communication (GJIC) inhibition in H9c2 cells. (A) SNPs-induced downregulation of GJIC in H9c2 cells. Lucifer yellow (LY, green) transferred to adjacent cells via open gap junctions. (B) The average distance of LY spread from the side of the scraped edge from six different sites in each sample was obtained and quantified. (C and D) Reduced expression of connexin 43 (Cx43) and P-Cx43 after SNPs treatment was detected using Western blot. Values represent the mean ± standard deviation (n=6). ^aP<0.05, vs control group; ^bP<0.05, vs 25 μg/mL group; ^cP<0.05, vs 50 μg/mL group.

Figure 4

Effect of gap junction activator retinoic acid (RA) and inhibitor carbenoxolone disodium (CBX) on the survival of cells treated with silica nanoparticles (SNPs) (mean, n=6).

Figure 5

Effect of gap junctions modulator (activator retinoic acid [RA] and inhibitor carbenoxolone disodium [CBX]) on gap junction intercellular communication (GJIC) of H9c2 cells. (A) Modulation of GJIC in H9c2 cells. Lucifer yellow (LY, green) transferred to adjacent cells via open gap junctions. (B) The average distance of LY spread from the side of the scraped edge from six different sites in each sample was obtained for quantification. Values represent the mean ± standard deviation (n=6). ^aP<0.05, vs control group; ^bP<0.05, vs silica nanoparticles (SNPs) group.

Figure 6

Effect of gap junction intercellular communication on silica nanoparticles (SNPs)-induced apoptosis in H9c2 cells determined by flow cytometry. (A) The apoptotic rate after treatment with 100 μg/mL SNPs for 24 h was determined by flow cytometry using Annexin V/PI double staining. (B) Effect of gap junction modulators (activator retinoic acid [RA] and inhibitor carbenoxolone disodium [CBX]) on the apoptotic rates. Values represent the mean ± standard deviation (n=6). ^aP<0.05, vs control group; ^bP<0.05, vs SNPs group. Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 7

Effect of gap junction intercellular communication on the expression of apoptosis-related proteins following treatment with silica nanoparticles (SNPs) as determined by Western blot analysis. (A) Expression of apoptosis-related proteins in H9c2 cells following treatment with SNPs, retinoic adid (RA) combined with SNPs, and carbenoxolone disodium (CBX) combined with SNPs. (B and C) Columns are the means from densitometric scanning from blots, and bars are the standard deviation (SD). Values represent the mean ± SD (n=6). ^aP<0.05, vs control group; ^bP<0.05, vs SNPs group.