Effects And Mechanisms Of Mitigating The Mitochondrial Permeability Transition Through Dynlt1 Up-regulation By MAP4 In Early Hypoxia

The injury of cardiomyocyte cytoskeleton in the early burn stage is presumably one of the main mechanisms concerning cellular energy dysmetabolism after burn. A series of initial investigations have shown that during the pathophysiologic procedure of cellular energy dysmetabolism, the normal cytoskeleton structure and function lay great effect on stabilization of intracellular mitochondria. Hence, we may start from the research of regulation and control of cytoskeleton, aiming to find the bridge molecule between microtubule and mitochondria, which may mitigate the mitochondrial permeability transition after hypoxia and improve cardiomyocyte energy metabolism. The initial study indicated that up-regulation of MAP4 expression had the certain ability of keeping the cardiomyocyte energy metabolism and physiologic activity by maintaining the stabilization of cardiomyocyte microtubule during hypoxia, improving evidently the level of ATP and energy load of cardiomyocyte in early hypoxia (<3h) and preserving cellular activity.We have screened out a new protein molecule, dynein light chain 1 (Dynlt1) by yeast two-hybrid method. It possibly interacted with main component VDAC, consisted in mitochondrial outer membrane. Thus, we may consider that Dynlt1 is the bridge molecule, performing the regulation and control when microtubule acting on MPTP. The exact structure is MitoVDAC - Dynlt1 / Dynein - MT.The present study was undertaken to test the hypothesis that Dynlt1 stabilizes VDAC thus to mitigate mitochondrial permeability transition in early hypoxia. The study focuses on proving the scientific presumption of"Cytoskeleton plays important role in cardiomyocyte energy dysmetabolism in early hypoxia".Material and methods:1. Observing the expression and location of Dynlt1 and VDAC in cardiomyocytes of new born rats and HeLa cells, which were cultured in vitro by techniques of immunofluorescence and co-IP. Verifying if there is an interaction between Dynlt1-VDAC and Dynlt1-MT in mammalian cells. Further observation is planed to study the mapping rule of Dynlt1 and the relationship with microtubule.2. Using the re-combined adenovirus carrier comprising MAP4 gene to transfect HeLa cells. Observing the stabilization of up-regulation of MAP4 expression on microtubule through LCM, and the change of Dynlt1 expression during this procedure. Detect the cellular activity by method of MTT, the mitochondrial membrane potential(ΔΨ) by TMRE and the opening degree of MPTP by Calcein-AM. Accordingly, try to confirm that up-regulation of MAP4 may stabilize microtubule, and the increase of Dynlt1 content may mitigate the mitochondrial permeability transition in early hypoxia as well.3. Transfecting HeLa cells with Dynlt1 shRNA Plasmid to form the steady cell clone and build up cell line HeLa-dD of continuous low expression of Dynlt1. Using the methods of MTT, TMRE, Calcein-AM etc. to observe the cellular activity,ΔΨand the degree of MPTP opening in Dynlt1 low expression patterns. and we further test the cellular activity in cells induced to overexpress MAP4 under this condition.Results and Conclusion:1. Cytoplasmic Dynlt1 has the obvious mapping character of co-location with MT and with VDAC, representing that a large amount of Dynlt1 distributes along with microtubule. VDAC has the close co-location with Dynlt1 almost under normoxia. Dynlt1 molecule connects closely with cellular microtubule structure in space. Meanwhile, we have identified the protein interaction between Dynlt1 and VDAC. These findings reveal that Dynlt1 and VDAC have mutual effects on function aspect.2. The destruction of microtubule not only arouses the location of Cytoplasmic Dynlt1 change, it also makes the Dynlt1 molecule fell off from the microtubule, which would not be alleviated as hypoxia persists. This implys the close relationship between microtubule and Dynlt1 in the location. Hypoxia injures mitochondria, accompanied with content decrease of VDAC in mitochondrial outer membrane. This finding shows that Dynlt1 and VDAC have been widely combined in cytoplasm.3. This study involves the cellular activiy,ΔΨ, MPTP opening and Dynlt1 content after MAP4 up-regulation. We demonstrate that MAP4 up-regulation can definitely maintain the cellular activity in early hypoxia and mitigate the destruction of microtubule structure caused by hypoxia. It also can maintain the mitochondrial membrane potential and mitigate the mitochondrial permeability transition. Over expression of MAP4 may up-regulate the expression of Dynlt1.4. After establishing the continuous low expression cell line HeLa-dD through steady transfection of Dynlt1 shRNA lentiviral vector plasmids, researches have been done to clarify if there is causal relationship between Dynlt1 expression reinforcement and improvement of cellular energy and activity by MAP4 in hypoxia. Our results show that when down-regulation Dynlt1 expression is performed, hypoxia for 15 min may cause severe cellular injury, activity reduction, destruction of mitochondrial membrane potential and more MPTP opening. However, up-regulated the expression of MAP4 is irrelevant to these destructive changes.