The Regulation Of Mitochondrial Calcium By SIRT1

The mitochondrion is a factory of energy in cells,it has an important role in the various activities of whole cells.Calcium ions work as intracellular second messengers to participate in the activities of body.Calcium ions in the mitochondria have an important regulation role of oxidation of respiratory chain.Mitochondrial calcium participate in maintaining oxidative reaction and producing ATP in normal condition,while under pathologic stimulus,mitochondrial calcium overload leads to increased cellular oxidation,and producing too much peroxide to activate the downstream apoptotic factors,finally leads to the cell apoptosis.Mitochondrial oxidative respiration is regulated by extracellular calcium waves that induced by different stimulus.In the process of cerebral ischemia reperfusion,there also accompanied with excessive superoxide caused by mitochondrial calcium overload.So the mitochondrial calcium homeostasis is particularly important for the activities of cells.Mitochondrial calcium intake is mainly controlled by MCU.MCU(mitochondrial calcium uniporter)is a protein(35Kda)regulated by a various of subunits,including MICU1(Mitochondrial calcium uptake 1)which play a important role in mitochondrial calcium uptake through directly combined with MCU.Loss-of-function mutations in MICU1 caused a brain and muscle disorder linked to altered mitochondrial calcium.Knockout of MICU1 leads to the death of newborn mice and the difficulty of liver tissue regeneration.Our previous work,yeast two-hybrid experiments,showed that MICU1 interact with SIRT1(Silent mating type information regulation 2 homolog-1)which plays an important role in lifespan.Studies have shown that SIRT1 can prolong the life of the yeast.What's more,it also has life prolonging effects in animals.Further researches suggest that SIRT1 exists in mitochondria and participate in the activities of the whole cell and body.So studying the relationship between SIRT1 and mitochondrion,the biologic function of MICU1 involved in mitochondrial calcium homeostasis is the focus of our research.At first,in order to explore the influence of SIRT1 on mitochondria,we did cell immunofluorescence,Western blot to detect SIRT1 protein and found it exists in mitochondria.SIRT1 specific inhibitor EX527 also influenced Hela mitochondrial morphology,leading to the fragmentation of the mitochondria in whole cell.Then,we treated Hela cells respectively with EX527 or interference plasmid to lower SIRT1 activity,then observed the effects on mitochondrial calcium intake with Rhod-2/AM fluorescent probe or CMV-mito-R-GECO1 plasmid as mitochondrial calcium indicator.We found that EX527 significantly enhanced mitochondrial calcium overload under the stimulus of histamine(10 ?M and 100 ?M)compared with DMSO group,while basal mitochondrial calcium level compared with DMSO group did not have significant difference.Under the stimulus of high concentrations of histamine(100 ?M),mitochondrial calcium intake of SIRT1 interference group increased significantly compared to shNC(control)group,and basal calcium signal was also significantly higher than shNC(control)group.Finally,combined with previous yeast two hybridization experiment and in order to verify whther the SIRT1 affect mitochondrial calcium intake through MICU1.We directly treated SD rat cortex neuron and Hela cells with EX527,WB detection with antibodies,and found EX527 did not affected MICU1 protein,mitochondrial calcium uniporter(MCU)protein.Inhibition of whole cell protein synthesis with cycloheximide,EX527 also did not affected the degradation of MICU1 protein,what's more,the experiment also found MICU1 degrade through proteasome pathway.After interfered SIRT1 with plasmid,the SIRT1 protein was decreased,MICU1 protein was also decreased,while MCU protein was still unchanged.From what has been done above,we may drawed the conclusions:First,the SIRT1 which mainly expressed in nuclear and cytoplasm also exsisted in mitochondria of Hela and neuron cells.Second,the overall SIRT1 protein in EX527 treated group did not changed,while the mitochondrial morphology was affected Third,SIRT1 involved in maintaining mitochondrial calcium homeostasis in normal condition;EX527 significantly promoted mitochondrial calcium intake under the stimulus of histamine compared to control,but it did not significantly influenced the protein level of MCU and MICU1.Four,knocking down SERT1 protein increased resting mitochondrial calcium level,increased mitochondrial calcium intake under the stimulus of histamine,while MICU1 protein was decreased,and MCU protein level was still unchanged.To sum up,SIRT1 involved in maintaining mitochondrial calcium homeostasis,two ways to lower the SIRT1 activity can enhance the strength of mitochondrial calcium overload.Our findings revealed a new mechanism that SIRT1 resides in mitochondria,it may affect mitochondrial calcium overload via MICU1.As the SIRT1 inhibitor treatment result isn't completely consistent with the result of interference of SIRT1,the specific mechanism needs further exploration,which may provide a new direction for looking for new drug targets.