Na(+)/Ca(2+) exchanger 1 (NCX-1) mediates the anti-apoptotic effect of Akt1 in neonatal rat cardiomyocytes during ischemia/reperfusion

Abstract

The purpose of this study was to investigate the anti-apoptotic role of Akt1 gene in neonatal rat cardiomyocytes and the relationship with Na(+)/Ca(2+) exchanger 1 (NCX1) during ischemia/reperfusion (IR). The cultured original rat cardiomyocytes were randomly divided into five groups: normal control group (C group), hypoxia/reoxygenation group (HR group), the control vector pLVX-EGFP-3FLAG group (CV group), the gene pLVX-EGFP-3FLAG-Akt1 transfection group (A group), and Akt1 inhibitor LY294002 group (LY group). Cardiomyocyte vitality was determined using MTT, and the apoptosis was determined by TUNEL to verify the anti-apoptotic role of Akt1. The mRNA levels of Akt1 and NCX1 were determined by RT-PCR, the protein expression of Akt1, p-Akt1, NCX1 and the apoptotic proteins of mitochondrial pathway cytochrome C (Cyto C) and caspase-9 were measured by Western blot. As a result, transfected Akt1 (A group) showed increased myocardial cell viability and reduced apoptosis, with increase in Akt1 expression and decrease in NCX1 expression. The levels of apoptotic proteins Cyto C and caspase-9 also declined. This study demonstrated that lentivirus-mediated transfection of Akt1 played an anti-apoptotic role during IR of rat cardiomyocytes, via inhibition of NCX1 and other mitochondrial proteins.

Keywords: Akt1; NCX1; apoptosis; cardiomyocytes; ischemia/reperfusion.

Figure 1

Identification of myocardial cells. Myocardial cells showed the red fluorescence, the nuclei of all cells showed the blue fluorescence.

Figure 2

Tested the optimal MOI value. Because of the eGFP, the myocardial cells showed the green fluorescence. When the MOI was 25, the transfection rate reached 83%, as the MOI was 30, the transfection rate reached 88%, when the MOI was 35, 40 and 45, there was no obvious difference for the transfection rate.

Figure 3

The changes in neonate rat myocardial cells vitality. Data are presented as mean ± SEM. C normal control group, HR hypoxia-reoxygenation group, CV the control vector group, A the gene transfection group, LY Akt inhibitor LY294002 group. *P value < 0.05 as compared with C group; #P value < 0.05 as compared with HR group; $P value < 0.05 as compared with CV group; &P value < 0.05 as compared with A group. Compared with C group, the other groups of cell vitality decreased significantly (P < 0.05), compared with HR group, the myocardial cells of the A group vitality increased significantly (P < 0.05).

Figure 4

TUNEL detected apoptosis of cardiomyocytes. The nuclei of apoptotic myocardial cells were dyed brown. Data are presented as mean ± SEM. C normal control group, HR hypoxia-reoxygenation group, CV the control vector group; A the gene transfection group, LY Akt inhibitor LY294002 group. *P value < 0.05 as compared with C group; #P value < 0.05 as compared with HR group, $P value < 0.05 as compared with CV group; &P value < 0.05 as compared with A group. Statistical results showed that compared with C group, the other groups of myocardial cell apoptosis rate increased significantly (P < 0.05). Compared with the HR group, A group apoptosis rate decreased (P < 0.05), LY group and CV group all had no significant difference Compared with the HR group (P > 0.05).

Figure 5

RT-PCR detected the mRNA level of Akt1 and NCX1. Data are presented as mean ± SEM. C normal control group, HR hypoxia-reoxygenation group, CV the control vector group, A the gene transfection group, LY Akt inhibitor LY294002 group. *P value < 0.05 as compared with C group; #P value < 0.05 as compared with HR group, $P value < 0.05 as compared with CV group, &P value < 0.05 as compared with A group. Compared with C group, Akt1 and NCX1 mRNA levels were no significant change in HR, CV and LY groups (P < 0.05); compared with HR group, A group Akt1 mRNA levels increased significantly (P < 0.05), however, the level of NCX1 mRNA decreased (P < 0.05).

Figure 6

Expression of Akt1 and p-Akt1 determined by Western blotting. Data are presented as mean ± SEM. C normal control group, HR hypoxia-reoxygenation group, CV the control vector group, A the gene transfection group, LY Akt inhibitor LY294002 group. *P value < 0.05 as compared with C group, #P value < 0.05 as compared with HR group, $P value < 0.05 as compared with CV group, &P value < 0.05 as compared with A group. Compared with C group, HR group Akt1 protein expression had no significant change (P > 0.05), but the background of p-Akt1 expression increased (P < 0.05), Compared with HR group, A group, Akt1 and p-Akt1 protein expression increased significantly (P < 0.05).

Figure 7

Expression of NCX1 determined by Western blotting. Data are presented as mean ± SEM. C normal control group; HR hypoxia-reoxygenation group, CV the control vector group; A the gene transfection group, LY Akt inhibitor LY294002 group. *P value < 0.05 as compared with C group; #P value < 0.05 as compared with HR group, $P value < 0.05 as compared with CV group; &P value < 0.05 as compared with A group. Compared with C group, hypoxia 2 h and reoxygenation 24 h could not make NCX1 protein take place obvious change (P > 0.05), but when transfected Akt1 gene, the NCX1 protein level was upregulated (P < 0.05).

Figure 8

Expression of Cyto C and caspase-9 determined by Western blotting. Data are presented as mean ± SEM. C normal control group; HR hypoxia-reoxygenation group; CV the control vector group, A the gene transfection group, LY Akt inhibitor LY294002 group. *P value < 0.05 as compared with C group; #P value < 0.05 as compared with HR group, $P value < 0.05 as compared with CV group, &P value < 0.05 as compared with A group. Compared with C group, hypoxia 2 h and reoxygenation 24 h could make the apoptotic related proteins of caspase-9, Cyto C increased obviously (P < 0.05), but transfected Akt1 gene could downregulate the Cyto C and caspase-9 protein levels (P < 0.05).