Importance Of Mitochondrial Calcium Uniporter In High Glucose-induced Cardiac Myocytes Apoptosis

AIM:To investigate the importance of mitochondrial calcium uniporter（MCU）in high glucose-induced cardiac myocytes apoptosis.METHODS:1.Cardiac myocytes（CM）were separately exposed for 72 h to normal glucose[5.5 mM glucose+19.5 mM mannitol（control）],high glucose[25 mM glucose（HG）],or[HG+5 uM spermine（HG+Sp）].2.The levels of mRNA expression of MCU were detected by method of Realtime-PCR.3.The levels of protein expression of MCU,caspase-3 and caspase-9 were detected by method of Western blot.4.Rhod-2 AM probes were used to detect Ca²⁺fluorescence in mitochondria of H9c2cells.5.Detection of pyruvate dehydrogenase（PDH）activity by method of absorbance.6.The method of firefly luciferase was used to detect ATP concentration in cell lysates.7.JC-1 staining method was used to detect mitochondrial membrane potential（Δψm）.8.MitoSOX?method was used to detect reactive oxygen species（ROS）.RESULTS:1.Influence of high-glucose（HG）treatment on expression of proteins and mRNA of MCU were decreased compared with control group（n=6 per group.P<0.05）,and 5 uM spermine has no influence on expression of MCU proteins.2.Decreased MCU expression made mitochondrial calcium homeostasis and tricarboxylic acid cycle disorders.From comparisons of[Ca²⁺]m,pyruvate dehydrogenase（PDH）activity and ATP levels between control group,HG group,and HG+Sp group,the results show that influence of HG on levels of[Ca²⁺]m,pyruvate dehydrogenase（PDH）activity and ATP levels were decreased compared with control group（n=8 per group.P<0.05）.Adding 5 uM spermine in HG group returned these parameters toward control levels（n=8 per group.P<0.05）.3.Decreased MCU expression made mitochondrial dysfunction.From comparisons of mitochondrial membrane potential（Δψm）and ROS levels between control group,HG group and HG+Sp group,the results show that influence of HG on levels ofΔψm（JC-1aggregates/monomer）and ROS were increased compared with control group（n=8 per group.P<0.05）.Adding 5 uM spermine in HG group returned these parameters toward control levels（n=8 per group.P<0.05）.4.Decreased MCU expression made mitochondrial apoptosis increased.Caspase-3protein and caspase-9 protein levels were increased after 72 h in HG group compared with control group（P<0.05）.Moreover,apoptosis was reduced in HG group by adding spermine（P<0.05）.CONCLUSION:Thus we conclude that HG-induced cardiac myocytes apoptosis may be associated with the decreased MCU levels,abnormal mitochondrial Ca²⁺handling,deviant mitochondrial respiratory chain,and mitochondrial dysfunction.