Title: Protective effects of Metoprolol on myocardial cell apoptosis of neonatal mouse after hypoxia/ reoxygenation
Abstract: Objective
To investigate the effect of metoprolol on hypoxia/ reoxygenation (H/ R) – induced cell apoptosis in primary neonatal mouse cardiomyocytes and to clarify the underlying mechanism.
Methods
Primary neonatal mouse cardiomyocytes from C57BL/ 6 are randomly (random number) divided into four groups: ①Control group (Control, Con) , in which cardiomyocytes were incubated with routine medium [a DMEM medium containing 10% fetal bovine serum (FBS) and 1% streptomycin/ penicillin] for 12 hours in a specific environment (a 37 ℃ and 5% CO2 humidified atmosphere) ; ②Metoprolol group (Meto) , in which cardiomyocytes were pretreated with 5 μmol/ L metoprolol for 24 hours, and then continuously cultured for another 12 hours in the routine medium and in a specific environment; ③H/ R group, in which cardiomyocytes were incubated with glucose-free and serum-free DMEM medium in hypoxia environment (95% N2 and 5% CO2) for 4 hours, and then were returned to the specific environment with the routine medium for 8 hours; ④Meto+ H/ R group, in which cardiomyocytes were pretreated with 5 μmol/ L metoprolol for 24 hours, and then exposed to H/ R treatment. The cell viability and apoptosis were separately detected by trypan blue and TUNEL staining. The concentration of cytochrome c (cyt c) was assayed by using cyt c immunocytochemistry. The caspase-3 and calpain activity were separately determined using caspase-3 and calpain activity detection kit.
Results
Compared to the control group, there was significant decrease in cell viability in the H/ R group [(91.67±4.38) %vs. (60.09±5.68)%, P<0.05] and there was remarkable increase in numbers of apoptotic cardiomyocytes [(6.60±0.53)%vs. (15.95±2.01) %, P <0.05] in the H/ R group. Meanwhile, after cardiomyocytes exposed to H/ R, there were significant increases in cyt c release [(2.55±0.28) ng/μg proteinvs. (5.60±0.56) ng/μg protein, P <0.05], in caspase-3 activity [(0.26±0.04) pmol/μg proteinvs. (0.83±0.08) pmol/μg protein, P <0.05] and in calpain activity (intensity) [(113.23±4.29) vs. (222.04±16.86) , P<0.05]. However, pretreatment with metoprolol significantly reduces H/ R-induced loss of cell viability (60.09±5.68)%vs. (71.82±6.25) %, P<0.05] and apoptosis [(15.95±2.01) %vs. (10.72± 1.93) %, P <0.05]. In addition, metoprolol pretreatment significantly suppresses cyt c release [(5.60± 0.56) ng/μg proteinvs. (3.59 ± 0.46) ng/μg protein, P<0.05] and markedly inhibits the caspase-3 [(0.83±0.08) pmol/μg proteinvs. (0.61±0.07) pmol/μg protein, P<0.05] and calpain activity (intensity) [(222.04±16.86) vs. (170.62±13.26) , P<0.05] compared with H/ R-treated cells.
Conclusions
Metoprolol can protect neonatal mouse cardiomyocytes against H/ R-induced apoptosis, which might be associated with inhibition of cyt c release and suppression of caspase-3 activity as well as calpain activity.
Key words:
Metoprolol; Cardiomyocytes; Hypoxia/ reoxygenation; Apoptosis
Publication Year: 2015
Publication Date: 2015-07-01
Language: en
Type: article
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