The Preliminary Study On The Functional Regulation Of Vimentin By PKA-Anchored Protein Cypher In Cardiomyocytes

BackgroundDilated cardiomyopathy(DCM)is a multifactorial disease in clinical.This kind of cardiomyopathy often leads to severe heart failure and arrhythmia-induced sudden death.The causes of dilated cardiomyopathy are many,such as viral infections,autoimmune abnormalities,and poisoning.In the past few decades,studies on related pedigrees showed that genetic defects played an important role in the pathogenesis of DCM.Recently,some researchers have found that imbalance of function caused by mutation of cardiac cytoskeletal protein gene can bring about the development of DCM finally.Z line in the myocardial sarcomere is composed of multiple scaffold proteins,which is responsible for the force transfer between two sarcomeres.Z-line not only has a significant structural function,but also is an important signaling node that receives the signals generated by some outside mechanical stimulations in cardiomyocytes.ZASP(Z-band,alternatively Spliced PDZ-motif protein)is a vital cytoskeletal protein on the Z line of the sarcomere,and its corresponding gene is LDB3.Mutation of this gene resulted in dilated cardiomyopathy in clinical.Knockout of the Cypher(the corresponding gene in mice)in the myocardium leads to cardiomyocyte dysfunction and then causes DCM.Our previous study showed that Cypher not only plays a structural role in the Z line,but also is a novel anchor protein of PKA.However,Changes of myocardial global phosphoproteomic profiling resulting from Cypher absence and specific function of Cypher as an anchor protein in signal transduction are unknown.ObjectiveChanges of myocardial global phosphoproteomic profiling resulting from Cypher absence and specific function of Cypher as an anchor protein in signal transduction are unknown.Therefore,the aim of this study is to explore these changes in general and to identify the downstream unknown proteins of PKA-anchored proteins Cypher and their correlations.Methods1.Use Label free LC-MS/MS to detect a wide variety of differential myocardium proteins between Cypher-knockout and wild-type mouse hearts and screen significant differential proteins2.Through a series of bioinformatics analysis and comparison of differential proteins,observe the relevant changes in general and pick out some key proteins.3.Determine phosphorylation level and the protein expression level of the key protein in the tissue.4.Determine interactions at the tissue and cellular level and study phosphorylation levels and protein expression levels of key proteins after Cypher-overexpressed expression on cells.Results1.Mass spectrometry identified that a total of 225 phosphorylation sites in 145 phosphoproteins meaningfully changed(both upregulation and downregulation).2.In the phosphorylation profile of myocardium after Cypher knock-out,the most obvious changes in terms of molecular function,cellular components and biological processes are respectively ion binding protein(15.29%)cytoskeletal constitution(18.97%)and regulation of DNA transcription(17.65%).Interestingly,by pathway enriching,there was a significant change in cellular connectivity(8.89%).3.The level of phosphorylation of vimentin72 site in Cypher knockout mice was significantly lower than that in wild type mice,but the level of its expression was significantly higher than that of wild type mice.4.When Cypher overexpressed in HEK293 cells and H9c2cells,phosphorylation of the vimentin-72 site was significantly increased after adding the PKA agonist while overexpression of Cypher and vimentin in HEK293 cells made the half-life of vimentin degradation advanced compared with expression of vimentin alone when adding the cycloheximide.Conclusions1.The knock-out of Cypher in cardiac muscle may not only affect the stability of the cytoskeleton,but also play a role in the connection between cells.2.As a novel PKA anchoring protein,Cypher enhances the phosphorylation level of vimentin72 site,while Cypher might promote the degradation of vimentin.