The Molecular Mechanism Of NaHSO₃ Induced Mitochondrial Damage And Apoptosis In H9C2 Cells

Sulfur dioxide is one of the main pollutants in the atmosphere.More and more people are concerned about cardiovascular diseases caused by inhalation of S02.Epidemiological studies have demonstrated an association between sulfur dioxide?SO₂?and an increase of morbidity and mortality of cardiovascular diseases.In our previous study,we found that the cytochrome c oxidase?COX?activity,mitochondrial membrane potential???m?,ATP contents,mitochondrial DNA?mtDNA?contents,and mRNA expression of complexes IV and V subunits encoded by mtDNA were decreased after NaHS03 treatment in vitro or S02 inhalation in vivo.The mitochondrial dysfunctions were accompanied by depressions of co-activator of peroxisome proliferator activated receptor gamma?PGC-1??,nuclear respiratory factor 1?NRF1?,and mitochondrial transcription factor A?TFAM?mRNA and protein.However,it is unclear that whether S02 and its derivatives induce mitochondrial dysfunction by PGC-1?-NRF1-TFAM-mtDNA-mtRNA-ATP signaling pathway.To investigate whether TFAM and NRF1 is a critical determinant of mitochondria dysfunction in response to NaHS03,we constructed TFAM-pcDNA3.1 and NRF1-pcDNA3.1 plasmids and transfected them in H9C2 cells.H9C2 cells were treated with 100?M NaHS03 for 24 h after TFAM-pcDNA3.1 or NRF1-pcDNA3.1 transfection.Protein levels of NRF 1 and TFAM were measured by Western blot.The mtDNA-encoded complex IV subunit CO3 mRNA levels were determined by real time RT-PCR analysis.The amount of ATP was measured by the luciferinluciferase method.Our results showed that NRF1 and TFAM protein expressions were increased after NRF1 overexpression,and the TFAM protein expression was increased after TFAM overexpression.At the same time,the mtDNA-encoded complex IV subunit C03 mRNA levels were also increased compare with NaHS03 treatment groups,we detected an amelioration of mitochondrial dysfunction marked by an increase of ATP contents after transient transfected TFAM and NRF1 in NaHS03 exposure H9C2 cells.In addition,H9C2 cells were treated with 100?M NaHSO₃ for different time?0,3,6,12,24 h?in vitro exposure.Flow cytometry was used to detect ROS production in cells.We found that NaHSO₃ can induce a time-dependent increase in ROS production.Cells were pretreated with or without 5 mM NALC?free radical scavenger?for 1 h.This was followed by treatment with 100?M NaHS03 for 24 h.The cells were harvested and ROS was analyzed by flow cytometry.The nuclear transcription factor TFAM mRNA levels were determined by real time RT-PCR.The ATP content was measured by a bioluminescence technique method.We found that NALC abolished NaHS03-induced increase of ROS.And NaHSO₃-induced TFAM mRNA expressions and ATP content reduction was restored.Finally,H9C2 cells were exposed to 100?M NaHSO₃ for different time?0,3,6,12,24 h?in vitro.Flow cytometry was used for the analysis of apoptosis.Immunofluorescence was used to analyze the expressions of apoptosis-related proteins?cytc,bax and p53?.The activity of caspase-3 was detected by spectrophotometry.And apoptosis-related proteins?cleaved caspase-3,cleaved caspase-9,p53,p53-serl5,bcl-2,and bax?were tested by Western blot.The results showed that NaHSO₃ induced apoptosis in H9C2 cells.The activity of caspase 3 and protein levels of cytc?cleaved caspase-3?cleaved caspase-9?p53?p53-ser15 and bax were increased.In contrast,the protein expressions ratio of bcl-2/bax was reduced.These results suggest that SO₂ could stimulate the production of ROS,and downregulate nuclear transcription factors NRF1 and TFAM,and thus affect the replication and transcription of mitochondrial DNA,and contribute to the mitochondrial dysfunction eventually result in cardiac myocyte apoptosis.It might lead to cardiovascular diseases such as heart failure and arrhythmias.