Effect And Mechanism Study Of Mitochondrial Fusion And Fission In Response For Cell Cold Stress

【Background】The process for cell in response for the stress of environmental changes is controlled by a complex biological system. Although there are many studies about this, but the molecule mechanism and physiological procedure remains largely unknown. As the most important organelle of the cell, mitochondria undergo frequent fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology, but their physiological means remain unclear. Mitochondrial fusion and fission is a conservative physiological process of cells. Plants, nemathelminth, drosophila, rat and human all remain this old dynamic process. It is supposed that this process has the close relation with the organism adaptation for the surrounding environmental changes in the evolutionary process.Cold, as the surrounding environmental change factor of organism, has always induced a complex changes and stress response in cells. Mitochondria are the center of energy metabolism, the function is important for the cell response for cold stress. Long time and strong cold stress will induce the cell injury occurred, it is necrosis or apoptosis of cell. The necrosis is observed when the temperature is lower than the freezing point, and it crystallized in the cell and cellular membrane breaken. But the apoptosis is frequently observed at 4℃environment. Previous studies have shown that ROS is the mean reason for cold induced cell apoptosis, and accompany with the mitochondrial impairment, but the mechanism is not clear. In the main model of mitochondrial participation in apoptosis induction in mammalian cells, the Bcl-2-family members regulate this process, and it hypothesis it participation in cold induced the cell apoptosis. Mitochondrial fusion and fission have the closed relation with cell apoptosis. Increased mitochondrial fission induced fragmentation of mitochondria, and this process of fragmentation similar with morphous ofmitochondria in the early apoptosis cell. The mitochondrial function impairment is also observed, such as the lost of mitochondrial membrane potential. It is hypothesis that mitochondrial fission participate in mitochondrial pathway cell apoptosis. Mitochondrial fussion can improve the mitochondrial function, and has the protect role for cell injury. To maintain the normal mitochondrial function is most important for cell response for cold stress, and we hypothesis that mitochondrial fusion and fission participate in cold induced cell stress.【Aims】To research the cold stress induced cell injury, investigate the molecular mechanism of cell response for cold stress and cell injury through mitochondrial fusion and fission view and elucidate the protective role of pyruvate on cells after cold stress. 【Methods】(1) HEK293 cells were incubated at 4℃for different time as the cell cold stress sample. (2) After cell cold stress for different time, the effect of cold stress on cellular morphology, viability and apoptosis were determined by inverted microscope observation, MTT assay, trypan blue assay, Hoechst stain and Flow cytometry assay after cell stained with Annexin V and PI. (3) Immunofluorescence analysis is used to investigate the cellular location of Bax, Mfn1, Drp1. (4) Cell stained with MitoTracker Deep Red 633 and imaged with confocal microscopy for examine the mitochondrial dynamic morphology changes. (5) RT-PCR, Western blot analysis are used to investigate the expression mitochondrial fusion protein and fission protein. (6) Flow cytometry assay for cellular ROS after cell stained with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). (7) Intracellular ATP level was measured by the luciferin–luciferase method. (8) Liposome is used to transfect the the small interference RNA vector of Mfn1 and the corresponding empty vector and into the HEK293 cells. The stable clones are screened by G418. Liposome is used to transfect pre-design chimera si-Mfn2 RNA into the HEK293 cells. (9) Flow cytometry assay for cellular mitochondrial transmembrane potential (?Ψm) after cell stained with rhodamine 123.【Results】1 Cell injury is found during cold stress.MTT assay showed a reduction in cell viability at 2h, 4h, 8h and 12h, and the lowest level is observed at 12h, while a higher cell viability at 4h than the viability at 2h. The morphologic alterations observed by inverted microscope showing a marked cellular retraction and cell floatation at 2h and 12h. The cellular nucleus is stained with Hoechst showing numerous thick and strong stained at 2h and 8h. Flow cytometry assay showing cell apoptosis ratio is 42.2% at 2h, 33.6% at 4h, and 40.7% at 8h. It indicates that cold stress induced the cell apoptosis, but the 4h's apoptosis ratio is lower than the 2h's.2 Cold-stress-induce cell injury is through the mitochondrial pathway apoptosis.Bcl-2 expression increased after the beginning of cold stress, 4h showing the peak level, and decreased with prolonged time. Bax expression has no marked change, but the immunofluorescence analysis showing that Bax translocate from cytoplasm to the mitochondrial outer membrane after cold stress at 2h and 8h in HEK293 cells, 2h's cold-stress-cell showing some cellular nucleus shrinkaged and broken to pieces, and the mitochondrial morphology fragmented. It is mean that mitochondria are important for cell injury during cold stress.3 Mitochondrial fusion and fission participate in cold induced cell stress, and have the closed relation with ATP production.The mitochondrial morphology is observed by confocal microscopy showing dynamic changes during cold stress. For the same mitochondria, the fragmentation is observed at 2h, then the fragmentation is repaired at 4h, and obviously fragmentation is observed at 10h. With electron microscopy observation, mitochondria show the continuous big and long peri-nuclear mitochondrion after 4h cold stress, and the mitochondria change to small structure and vacuolization. Western blot result showing Mfn1, Mfn2 protein expression increase in cells at 4h, but Drp1 expression marked decrease at 4h. It indicates that there is increased mitochondrial fusion at 4h. Drp1 expression marked increase at 2h, while Mfn1 and Mfn2 expression did not much increase, and Drp1 expression significantly increased at 8h, while Mfn1 and Mfn2 expression marked decrease at 8h. It is showing that there is increased mitochondrial fission at 2h and 8h. We also found that ATP level decreased after cold stress, resulted in 46.4% decrease at 2h, 37.1% decrease at 4h,64.0% decrease at 8h,and lowest level at12h, 76.7%. The ATP level at 4h is higher than the level at 2h. It indicates that increased mitochondrial fusion induced ATP production at 4h and increased mitochondrial fission with the decreased the cellular ATP level.4 Mfn2 play a key role in cell cold stress process.Silence Mfn1 has not much influence on cell survival after 4h cold stress; but Silence Mfn2 can cause a marked reduction in cell viability at 4h, and cell death was observed. After 4h cold stress, silence Mfn2 result in Mfn1, Mfn2 and Bcl-2 expression significantly decreased, Drp1 expression significantly increased, ?Ψm and ATP production significantly decreased, cells death are found. It indicates that Mfn2 play an important role in cell cold stress process.5 Drp1 participate in cold induced cell apoptosis and pyruvate has the protective role.After 2h cold stress, mitochondrial fission increased, Drp1 translocate from cytoplasm to the mitochondrial outer membrane, the cellular ATP production decreased, flow cytometry assay showing cell apoptosis. Treatment with pyruvate resulted in increase ATP level, inhibit Drp1 translocation, a marked increase in cell viability by MTT assay, and decrease in cell apoptosis ratio. It indicates pyruvate has the protective role in cold stress of HEK293 cells.【Conclusions】1. Cold stress induced 2 cell apoptosis peaks. One is at 2h, the other is at 8h. But the cell appeared to have the adaptive reaction at 4h. It indicates that cell has the ability of itself to response for cold stress.2. Cold stress induced cell apoptosis is through the mitochondrial pathway apoptosis. Mitochondrial impairment accompaniment with the cell injury process, showing the lower ATP production.3. Mitochondrial fusion and fission participate in cold induced cell stress, and have the closed relation with the mitochondrial function. Mitochondrial fusion can improve mitochondrial function and inhibit the cell apoptosis. It indicates that mitochondrial fusion can elevate the ability of cell response to cold stress. But increased mitochondrial fission promotes mitochondrial fragmentation and cell death. It indicates that the excess mitochondrial fission response for cold-induced cell death.4. It is tentative confirmation that Mfn2 play a key role in cell cold stress process.5. Treatment with pyruvate can increase cellular ATP level, and inhibit the mitochondrial translocation of Drp1 and mitochondrial fission. It suggested that pyruvate has the protective role for cell in cold stress.