Hemichannel-mediated volume regulation contributes to IPC-induced cardiomyocyte protection

Abstract

Cx43 has been documented to be involved in ischemic preconditioning (IPC). However, the participation of Cx43-formed hemichannels in IPC and the potential underlying mechanisms remain unclear. The present study focused on cardiomyocytes' volume regulation during IPC to investigate the role of hemichannels in the IPC-induced cardioprotection. In the study, mice cardiomyocytes were respectively treated with a hemichannel blocker, octanol or 18a-Glycyrrhizic acid (18a-GA), and a Cx43-silenced lentivirus. They were subsequently cultured in hypotonic solution to simulate ischemic reperfusion (SIR) and systemic ischemic preconditioning (SIP). Cell morphology and volumetric (area) change were detected by inverted microscopy at 30 min following the addition of hypotonic solution. Cardiomyocyte mortality was assessed by trypan blue stain assay. The analyses revealed that regardless of the treatments, hypotonic solution aggravated cell edema: Compared with the initial condition (the moment before the solution addition, 0 min), the volumetric area increased significantly 30 min later (for hypotonic+DMSO, 5,050±1,511 vs. 3,464±723 µm²; for hypotonic+scramble lentiviral vector, 5,517±1,128 vs. 2,331±536 µm²; P<0.05, respectively). Either treatment alleviated the edematous condition when a comparison was made between 30 min after the hypotonic addition and 0 min (for hypotonic+octanol, 2,990±765 vs. 2,821±773 µm²; for hypotonic+18a-GA, 4,817±1,306 vs. 4,762±1,271 µm²; for hypotonic+Cx43-silenced, 3,627±688 vs. 3,419±814 µm²; P>0.05 for all). Notably, results indicated that the SIP group had lower mortality rates compared with its SIR counterpart; the hypotonic+octanol, hypotonic+18a-GA, and hypotonic+Cx43-silenced group showed markedly-declined mortality when compared with their respective control groups (respectively, 35.70±1.02, 30.76±2.20 vs. 53.58±2.14%; 30.89±2.37 vs. 54.12±2.55%; P<0.05 for all). The results suggest that ischemic preconditioning may provide cardioprotection by blocking the opening of the hemichannels and further mediating the volume regulation of cardiomyocytes.

Keywords: capacity regulation; hemichannels; ischemic preconditioning; myocardial protection.

Figure 1.

Fluorescence outcomes of four lentiviral vectors at 72 h after the transfection (in pairs). (Aa and Ab) Scramble lentiviral vector (CON077), (Ba and Bb) lentiviral vector LV-Gja1-RNAi (51661–2), (Ca and Cb) LV-Gja1-RNAi (51660–1) and (Da and Db) LV-Gja1-RNAi (51662–1). (a) observed by inverted microscopy (magnification, ×200) and (b) stimulated by blue light under the fluorescence microscope (magnification, ×200). Cells with green fluorescence indicate successful transfection.

Figure 2.

Protein expression of Cx43 and phosphorylated Cx43 by western blotting. Representative western blot images manifested these protein expression levels in the five groups: The normal group, Scramble lentiviral vector (CON077) group, lentiviral vectors LV-Gja1-RNAi (51661–2) group, LV-Gja1-RNAi (51660–1) and LV-Gja1-RNAi (51662–1) group. Expression of proteins (both total and activated protein of Cx43) was markedly alleviated in the LV-Gja1-RNAi (51662–1) group when compared with that of the scramble lentiviral vector group.

Figure 3.

Gene expression of Cx43 in the four lentiviral vectors in comparison with normal cells by RT-qPCR. n=3 in each group. All results were presented as means ± SD. Gene expression in LV-Gja1-RNAi (51662–1) was lower compared with that of scramble lentiviral vector (P<0.05). Scramble lentiviral vector (CON077), lentiviral vector LV-Gja1-RNAi (51660–1), LV-Gja1-RNAi (51661–2) and LV-Gja1-RNAi (51662–1) were indicated.

Figure 4.

Volume changes of cardiomyocytes in the hypotonic solution. Areas are presented as means ± SD. The cell areas of the groups before the addition of hypotonic solution (baseline) and 30 min after the addition were shown in the first and second column, respectively. *P<0.05 vs. their corresponding baseline groups (0 min). 18a-GA, 18a-Glycyrrhizic acid; DMSO, dimethylsulphoxide.

Figure 5.

Mortality of cardiomyocytes in SIR groups. The mortality rates were expressed by means ± SD. *P<0.05 as compared with the SIR+DMSO group. ^#P<0.05 as compared with the SIR+scramble lentiviral group. SIR, simulate ischemic reperfusion; 18a-GA, 18a-Glycyrrhizic acid; DMSO, dimethylsulphoxide.

Figure 6.

Mortality of cardiomyocytes in SIP groups. The mortality rates were expressed by means ± SD. *P<0.05 as compared with the SIP+DMSO group. ^#P<0.05 as compared with the SIP+scramble lentivirus group. SIP, systemic ischemic preconditioning; 18a-GA, 18a-Glycyrrhizic acid; DMSO, dimethylsulphoxide.