Mechanisms Of Regulating Mitochondrial Protein Import Machinery (PIM) Components Expression And Function In Response To Stress In Skeletal Muscle

Mitochondria are the "power house" within the cell and are the key organelle to cell metabolism. Mitochondrial proteins are encoded by nuclear and mitochondrial DNA. As we known,99%of mitochondrial proteins are encoded by nuclear DNA, those proteins have to be transcribed and translated, then formed as precursor proteins, carried specific signal sequences, in cytosol. With the help of cytosolic chaperone proteins, precursor proteins are imported to different compartments of mitochondria through mitochondrial protein import system on mitochondrial membrane. Once imported into mitochondria, mitochondrial enzyme will cut off the specific signal sequences of precursor proteins and form to mature proteins that participate in mitochondrial respiratory chain formation or other mitochondrial biogenesis process. Therefore, mitochondrial protein import machinery (PIM) is an essential step of regulating mitochondrial biogenesis. PIM concerns about hundreds of components of mitochondrial protein import and the function of different import pathways and it is composed of mitochondrial outer membrane translocases complex (TOM)、mitochondrial inner membrane translocases complex (TIM)、matrix chaperone proteins and cytosolic chaperone proteins.Purpose:PGC-1α and Bax/Bak were used as entry point in this research, based on their own features, we investigated the mechanisms of regulating mitochondrial protein import components expression and function in skeletal muscle in response to different stress through determining the role of PGC-1α and Bax/Bak in mitochondrial PIM components expression and PIM function, which is the first time to focus on whether PGC-1α or Bax/Bak play a role in mitochondrial PIM and reveal the regulation molecular mechanisms of mitochondrial PIM.Methods:1. The role of PGC-1α in mitochondrial PIM in skeletal muscle1) Cell culture research:C2C12myotubes were used to subject2hours acute contractile activity, with or without the treatment of specific inhibitors, such as NAC (ROS↓)、 BAPTA-AM (Ca2+↓)、Compound c (AMPK↓)、BIRB796(p38↓).Investigate the involvement of multiple signaling pathways in PGC-1αgene regulation in response to acute stimulation. Experiment Ⅰ:Group:Control, Stimulation and Stimulation+Recovery. Western Blot method was used to examine protein expression involved in PGC-1αrelated multiple signaling pathways and immunoflurencense method was used to determine the role of contractile activity on PGC-1α protein translocation within the myotube.Experiment Ⅱ:Group:Control, Control+Inhibitor, Stimulation, Stimulation+Inhibitor. Myoblasts were transfected with ⅰ) the full length of mouse PGC-1α promoter sequence fused with a luciferase vector, or ⅱ) the full length of PGC-1α protein DNA sequence fused with a luciferase vector. Luciferase assay was used to look at the role of multiple signaling pathways on the activity of PGC-1α transcription and coactivation.2) Animal research:PGC-la genetic mice were used in this study. Mitochondrial PIM components expression and function were examined in both PGC-la WT and PGC-la KO mice skeletal muscle. Group:PGC-1α WT and PGC-1α KO. Western Blot method was used to investigate mitochondrial PIM components expression, radiolabeled protein was used to look at mitochondrial PIM function.2. The role of Bax/Bak in mitochondrial PIM in skeletal muscleAnimal research:Bax/Bak double knock out mice were used in this study. Bax/Bak apoptotic role, Bax/Bak non-apoptotic roles, mitochondrial PIM components expression and PIM function were examined in both Bax/Bak WT and Bax/Bak DKO mice skeletal muscle. Group:Bax/Bak WT and Bax/Bak DKO. Western Blot method was used to determine the release of cytochrome c, mitochondrial dynamic proteins expression and mitochondrial PIM components expression. JC-1dye was used to look at mitochondrial membrane potential. Radiolabeled protein with35S was used to look at mitochondrial PIM function.3. Exercise adaptation of mitochondrial PIM in skeletal muscleAnimal research:Bax/Bak double knock out mice were used in this study, combined with6-week voluntary running wheel. Mitochondrial PIM components expression and function were investigated between Bax/Bak WT and Bax/Bak DKO mice in the absence or presence of exercise training. Group:Bax/Bak WT Control, Bax/Bak DKO Control, Bax/Bak WT training, Bax/Bak DKO training. Western Blot method was used to investigate mitochondrial PIM components expression. Synthesized test protein radiolablled with35S was used to look at mitochondrial PIM function.Results:1. The activity of PGC-la transcription and coactivation were significantly increased by52%and35%respectively, in response to acute contractile activity through cellular ROS/Ca2+/AMPK/p38MAPK molecular signaling pathways.2. The absence of PGC-la has different effects on mitochondrial PIM components expression, and has no influence on PIM function in skeletal muscle.3. Cytosol with the absence of PGC-la cause44%to57%significantly decreased in mitochondrial OCT import in skeletal muscle.4. Cytochrome c release from SS and IMF mitochondria were both significantly reduced by64%and70%, respectively, in Bax/Bak DKO mice under stimuli treatment, which suggest Bax/Bak plays an important role in mitochondrial induced cell apoptosis.5. Basal and state IV mitochondrial membrane potential were both significantly reduced by20%and16%with the absence of Bax and Bak, which associated with changed mitochondrial dynamic proteins expression.6. Mitochondrial PIM components Tom40、mtHsp60and mtHsp70expression were all significantly reduced from38%to40%in Bax/Bak DKO mice, whereas cytosolic stress proteins Hsp90and Bip displayed abnormal high level, this resulted in the import of OCT and Tom40protein were both significantly decreased by37%and41%, mitochondrial matrix and outer membrane protein import pathway were both impaired.7. Exercise training up-regulated the key mitochondrial PIM components expression, Tom40and optimized cytosolic stress proteins expression in skeletal muscle, which recovery the defect mitochondrial PIM function.Conclusions1. Multiple cellular signaling pathways:Ca2+、ROS、AMPK、p38were involved in the regulation of acute contractile activity-induced PGC-1αgene expression, both in transcriptional and post-translation level.2. The role of PGC-la in mitochondrial PIM in skeletal muscle rather through regulating gene expression that involved in ER stress and UPR, but not related to regulating TOM、TIM components expression.3. Bax/Bak has both apoptotic and non-apoptotic functions, the non-apoptotic functions manifested in maintaining mitochondrial membrane potential, regulating mitochondrial dynamic process and support mitochondrial PIM in skeletal muscle.4. Bax/Bak plays a vital role in the function of mitochondrial matrix and outer membrane protein import pathways, via regulating PIM components expression, mitochondrial membrane potential and PIM related chaperone proteins.5. Impaired function of mitochondrial protein import in Bax/Bak DKO mice was recovery by exercise training, which was associated with up-regulation of mitochondrial PIM components expression and optimized cytosolic stress proteins expression. This suggested exercise adaptation of mitochondrial PIM may relate to ER stress, UPR or mitochondrial stress.