Abstract:Objective To explore the effects of nicotine on cardiomyocyte apoptosis and the roles of Cytochrome P450 Family 1 Subfamily A Member 1 (Cyp1a1) and Cholinergic Receptor Nicotinic Beta 4 Subunit (Chrnb4) genes in nicotine-induced cardiomyocyte apoptosis.Methods The cells were divided into control group, nicotine group, high-glucose and high-fat model group, nicotine + high-glucose and high-fat model group, shRNA-NC group, shRNA-Cyp1a1 group, AMPK inhibitor group, shRNA-Cyp1a1 + AMPK inhibitor group, shRNA-Chrnb4 group, and shRNA-Chrnb4 + AMPK inhibitor group. The high-glucose and high-fat model of H9C2 cells was constructed, transfected with Cyp1a1 or Chrnb4 interference plasmids, and treated with nicotine or AMPK inhibitor. Apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were detected by flow cytometry. The intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected by ELISA. Besides, Western blotting was used to detect the expressions of Cyp1a1, Chrnb4, Caspase-2, Caspase-3, Caspase-9, and p-AMPK in cells.Results Compared with the control group, the SOD activity was lower in nicotine group, high-glucose and high-fat model group, and nicotine + high-glucose and high-fat model group (P < 0.05). The SOD activity was even lower in the nicotine + high-glucose and high-fat model group than that in the high-glucose and high-fat model group (P < 0.05). The MDA content in the nicotine + high-glucose and high-fat model group was higher than that in the high-glucose and high-fat model group (P < 0.05). The ROS level in the high-glucose and high-fat model group and nicotine + high-glucose and high-fat model group was higher than that in the control group (P < 0.05), and that in the nicotine + high-glucose and high-fat model group was even higher compared with the high-glucose and high-fat model group (P < 0.05). The mitochondrial membrane potential was lower in the nicotine group, high-glucose and high-fat model group, and nicotine + high-glucose and high-fat model group than that in the control group (P < 0.05), while that was even lower in the nicotine + high-glucose and high-fat model group compared with the high-glucose and high-fat model group (P < 0.05). The mRNA levels of Cyp1a1 and Chrnb4 were higher in the nicotine group, high-glucose and high-fat model group, and nicotine + high-glucose and high-fat model group than those in the control group (P < 0.05), and those were even higher in the nicotine + high-glucose and high-fat model group compared with the high-glucose and high-fat model group (P <0.05). Compared with the control group, the protein levels of Caspase-2, Caspase-3, Caspase-9, Cyp1a1, and Chrnb4 were higher but the protein level of p-AMPK was lower in the nicotine group and high-glucose and high-fat model group (P < 0.05). The protein level of p-AMPK was lower but the protein levels of Caspase-9, Cyp1a1, and Chrnb4 were higher in the high-glucose and high-fat model group than in the control group (P < 0.05). The protein levels of Caspase-2, Caspase-3, Caspase-9, Cyp1a1, and Chrnb4 were higher but the protein level of p-AMPK was lower in the nicotine + high-glucose and high-fat model group than in the high-glucose and high-fat model group (P < 0.05). Compared with the shRNA-NC group, the cardiomyocyte apoptosis rate in the shRNA-Cyp1a1 group was lower (P < 0.05), but that in the AMPK inhibitor group and shRNA-Cyp1a1 + AMPK inhibitor group was higher (P < 0.05). Besides, the cardiomyocyte apoptosis rate in the shRNA-Cyp1a1 + AMPK inhibitor group was lower than that in the AMPK inhibitor group (P < 0.05). Compared with the control group, the protein levels of Caspase-2, Caspase-3, Caspase-9, and Cyp1a1 were lower (P < 0.05), but the protein level of p-AMPK was higher in the shRNA-Cyp1a1 group (P < 0.05). Compared with the shRNA-Cyp1a1 + AMPK inhibitor group, the protein levels of Caspase-2, Caspase-3, Caspase-9, and Cyp1a1 were higher (P < 0.05), but the protein level of p-AMPK was lower in the AMPK inhibitor group (P < 0.05). There was no difference in the protein level of Cyp1a1 between the shRNA-Cyp1a1 + AMPK inhibitor group and the AMPK inhibitor group (P > 0.05). The cardiomyocyte apoptosis rate in the shRNA-Chrnb4 group was lower than that in the shRNA-NC group (P < 0.05), while that in the AMPK inhibitor group and the shRNA-Chrnb4 + AMPK inhibitor group was higher compared with the control group (P < 0.05). In addition, the cardiomyocyte apoptosis rate in the shRNA-Chrnb4 + AMPK inhibitor group was lower than that in the AMPK inhibitor group (P < 0.05). The protein levels of Caspase-2, Caspase-3, Caspase-9 and Chrnb4 in the shRNA-Chrnb4 group were lower than those in the control group (P < 0.05), while the protein level of p-AMPK in the shRNA-Chrnb4 group was higher than that in the control group (P < 0.05). The protein levels of Caspase-2, Caspase-3 and Caspase-9 in the AMPK inhibitor group and shRNA-Chrnb4 + AMPK inhibitor group were higher than those in the control group (P < 0.05), whereas the protein level of p-AMPK in the AMPK inhibitor group and shRNA-Chrnb4 + AMPK inhibitor group was lower than that in the shRNA-NC group (P < 0.05). The protein level of Chrnb4 in the shRNA-Chrnb4 + AMPK inhibitor group was lower than that in the shRNA-NC group (P < 0.05). Compared with the AMPK inhibitor group, the protein levels of Caspase-2, Caspase-3, Caspase-9 and Chrnb4 in the shRNA-Chrnb4 + AMPK inhibitor group were lower (P < 0.05), but the protein level of p-AMPK was higher (P < 0.05). There was no difference in the protein level of Chrnb4 between the shRNA-Chrnb4 + AMPK inhibitor group and the shRNA-Chrnb4 group (P > 0.05).Conclusions Cyp1a1 and Chrnb4 are involved in nicotine-induced cardiomyocyte apoptosis. The mechanism may be associated with the inhibition of AMPK phosphorylation that leads to mitochondrial dysfunction and increased oxidative stress, ultimately activating the Caspase-2 apoptosis pathway.
