| Background and Specific AimsHeart disease was first identified in human muscular dystrophy one hundred and fifty years ago,there has been no cure so far.70-90%of muscular dystrophy patients, that have genetic defects in the dystrophin glycoprotein complex(DGC),have cardiovascular disease,and about 20%succumb to heart failure.The DGC is a critical complex for the maintenance of the structure and the function of normal cardiac and skeletal muscle,as a mechanosignaling complex with dual mechanical and nonmechanical membrane stabilizing functions,playing a pathogenic role in the cardiomyopathy associated with muscular dystrophy.DGC is a multimeric transmembrane protein complex,containing dystrophin,dystroglycan(DG) and a sarcoglycan(SG) / sarcospan complex.DG is the central protein in DGC.DG is generated from a propeptide,which is cleaved into a heavily glycosylated extracellularαDG protein(α-DG) and a transmembraneβDG protein(β-DG).α-DG is heavily glocosylated and completes the link from the cytoskeleton to the basal lamina by binding with high affinity to extracellular matrix proteins,such as laminin.β-DG binds intracellularly to dystrophin,which binds the actin cytoskeleton,and binds extracellularly toα-DG.Mutations in any one of the above components result in variable phenotypes of muscular dystrophy.For example,absolute or relative deficiency in dystrophin leads to Duchenne Muscular Dystrophy(DMD) or Becker Muscular Dystrophy(BMD).DMD usually complicates with cardiac muscle problem and mental retardation,BDM has a slower down progression.The mutations of the genes encodingγ-,α-,β-,andδ-SG subunits generate Limb Girdle Muscular Dystrophy(LGDM) -2C,2D,2E and 2F, respectively.DG is the molecular target for mutations in known and putative glycosylation enzymes that lead to abnormal glycosylation of DG,and its dysfunction as an extracellular matrix receptor,and the loss of binding with the ligand in skeleton muscle,which is responsible for severe congenital muscular dystrophies.The common and severe complication of these disorders is the associated cardiomyopathy.Therefore, the defects in glycosylation and function of DG are a cause of several human muscular dystrophies and associated cardiomyopathies.Because the glycosylation pathways are often promiscuous,several groups have hypothesized that other proteins may be targets for this enzymatic pathway and responsible for disease phenotypes.One logistic question,does genetic ablation of DG gene recapitulate many or all of the disease phenotypes in mice? The DG knockout mouse is embryonic lethal due to a developmental defect specific to rodents.Therefore, currently,there's no description about mutations ofα- orβ-DG itself involved in genetic disorders.Using Cre-LoxP technology to specifically delete the DG gene in heart to obtain cardiac muscle tissue-specific DG knockout mice will allow us to study the structural and functional roles of DG in cardiovascular system.First,it will be identified that whether the loss of DG is sufficient and necessary to explain the occurrence of the cardiomyopathy associated with muscular dystrophy due to abnormal glycosylation. Second,it will be explored that what role the mutation of DG plays in associated cardiomyopathy,structural or functional,and what the mechanism is.Third,it will be investigated that whether DG is involved in cell survival signaling,and what the phosphorylated levels of the associated enzymes are,and is there any other possible proteolysis system involved in,so that the novel potential strategy for clinical treatment will be sought out.As alluded to above,patients with DMD,BMD,and SG associated LGMD,mdx mice and knockout mouse models ofβ-,δ- andγ-SG associated LGMD often develop cardiomyopathy.The mechanisms of cardiomyopathy in muscular dystrophies have not been fully understood.Currently,the mechanisms by which DG and DGC cause cardiomyopathies associated with muscular dystrophy still remain elusive.The possible reasons are,first,the expression distribution and function of DGC in cardiovascular system is complicated,the disruption of DGC may render cardiac myocytes sensitive to membrane damage.Second,whether each protein primarily plays a structural or a functional role within DGC is far from defined.Third,although patients with abnormal glycosylation in skeleton muscle can develop cardimyopathy,the mechanic role of DG in cardiomyopathy has not been established.Using combinations of mutant and genetarget mouse models,and in vivo and in vitro approaches,the research is uniquely suited to address the questions:what the roles DG play in cardiovascular system are, structural or functional or both,what the mechanism by which DG defects cause the cardiomyopathy associated with muscular dystrophy is,providing a means by which the mechanism and target for treatment of cardiomyopathy associated muscular dystrophy can be identified.Materials and Methods1.Generation and analysis of Cradiac tissue-specific DG knockout mice(Mlc2vCre) and calpastatin transgene(CAST Tg) mice and strains' maintenance.Generate cardiac tissue-specific DG knockout mice Mlc2vCre using Cre-LoxP technology.The mutant mouse is Cre+/floxed DG,the control wild type is Cre-/floxed DG.Generate calpastatin transgene mice using transgene technology.Then cross the CAST Tg mice with Mlc2vCre mice to generate target mouse model whose genotype is CAST Tg;Cre/floxed DG.Genotyping and screening by PCR.2.Lysate preparation from cultured cells or cardiac muscle tissue and SDS-PAGE and Western blotting.Cells were washed with PBS and scraped in equal amounts of a lysis buffer. Cardiac muscle tissue was homogenized in 10 volumes of sample size's lysisi buffer. The samples were sonicated for 10 sec and centrifuged at 14000 rpm for 2 min to pellet cell or tissue debris.All of the resulting cell lysates was loaded or volumes were normalized to the total protein content determined using Dc Protein Assay.Proteins were separated by a 3-15%gradient SDS-PAGE and then transferred to a polyvinylidene fluoridine membrane.The membranes were blocked with 5%nonfat milk in TBS(150 mM Nacl,20 mM Tris pH 7.5) followed by incubation with primary antibody and secondary antibody conjugated to horseradish peroxidase,respectively. The membranes were developed using an enhanced chemiluminiscence assay.3.DG protein purification and laminin binding assay.To obtain purified DG protein using agarose wheat germ agglutinin(agarose-WGA) and N-acetylglucosaminyl glycoprotein(NAG).Western Blot and laminin Biniding assay(both membrane phase and solid phase) were performed on the purified DG protein,the rational and procedure of the memebrane phase assay are similar to Western Blot's.The solid phase was performed in 96-well microplate coated with gradient concentrations of laminin,0,0.02,0.1,1,2,10,20 nM,incubating for 1.5 hours,triplicate for each sample,incubated with anti-laminin and responding secondary antibody incubation,then washed,finally added developing reagent and 2M H2SO4 to stop the color reaction,plating the microplate to the reader to read data.4.Frozen tissue specimen preparation and slicing and histochemical staining and immunosatining.Heart tissue was placed onto the cork,dipped into -120℃isopentane for 30 seconds,then stayed in -196℃liquid nitrogen to be frozen,stored in -80℃freezer. Using cryostat did the slicing with transection for later histochemical staining and immunostaining.Follow the protocol to work on the histochemical staining,such as HE,SR staining and FG counterstaining.Immunostaining was performed using primary antibody and responding secondary antibody with immunofluroresence label.5.Assessment of PI3k-ARK signaling in adult cardiac myocytes.The activation of PI3 kinase signaling pathway in hearts in vivo will be assessed in whole heart homogenates by Western blotting using anti-phospho-AKT and antiphospho -GSK3βas well as the overall levels by anti-AKT and anti-GSK3β.To determine if the interaction of DG with ligands is necessary for the activation of AKT,adult cardiac myocytes will be isolated from the same group of animals and phosphorylations of AKT and GSK3βwill be assessed prior to and after the addition of exogenous laminin as the above procedures.6.Mouse cardiac myocytes primary Culture in vitro study.(1) Adult mouse cardiac myocytes primary culture. Adult mouse cardiac myocytes primary culture was performed by protocol worked out based on our previous studies.The heart was cannulated via the ascending aorta and mounted on a modified Langendorff perfusion apparatus.The heart was perfused with Myocyte Buffer and Enzyme Solution.The atria and aorta were removed;the ventricles were cut in two pieces,disaggregated gently with Dumont #5/45 forceps.Cells were pelleted and resuspended for a couple of times.Finally,cells were resuspended in Plating Media and plated into glass coverslips pre-coated with 56 nM laminin for over 30min.The plating density was 20,000 cells/coverslip.After 2 h,serum-containing Plating Media was switched to Culture Media.Culture Media was changed daily.Recombinant adenovirus preparation and transduction.The calpastatin adenoviruses were generated with the AdEasy XL adenoviral vector system using a 2637-bp mouse calpastatin cDNA.Recombinant virus was amplified in HEK293 cells and purified using a cesium chloride gradient as described.The titer of the virus was assessed by a plaque assay and was 1-2.4×109 plaque forming units(pfu) per 1 ml. Following 2 h of attachment,cardiac myocytes were transduced with 200-480 MOI AdCAST in 200μl culture Media for 1h.After 24-48 hours,collect cells which were lysed by lysis buffer.The SDS-PAGE and Western Blot were performed on Lysate.(2) Newborn mouse cardiac myocytes primary culture.Newborn pups at age of 1 day old was decapitated and the heart was removed from chest cavity and dissociated by 0.25%Trysin-EDTA and the cells were centrifuged at speed of 1000g for 4min and resuspended in media and plated into glass coverslips pre-coated with 7 nM purified laminin for about 120min,and placed in 37℃, 5%CO2.After 24 hour,the coverslips were removed for immunostaining,watching cells attachment and matrix assembly and picture-taking under the immunofluorescence microscope.7.The establishment of Acute and Chronic heart mechanic strain overloads mouse model In vivo study.In order to assess the effects of acute and chronic mechanic strain on adult DG deficient myocardium,treadmill exercise stress will be performed on mutant Mlc2vCre-DGnull mice compared to littermate controls.After 5 hours,the subjected mice were killed and the heart tissue was partly frozen for slicing and immunostaining thereafter,and was partly lysed for later Wetsern blotting.Chronic increases in ventricular pressure can be accomplishment by suprarenal abdominal aortic banding (AAB).Mutants and controls were both divided into operation and sham-operation groups.After 4 weeks,the mice were killed and the heart tissue was frozen and slicing for later HE,SR staining and FG counterstaining.8.Protein analysis and statistics software and method.Quantified protein acquired by western blot using software provided by Alpha Innotech.Statistics software-Prism 5.0 was used for data analysis such as t-test,Anova, linear regression and nonlinear regression,data were shown as mean±se.Results1.Mlc2vCre is the heart tissue-specific DG knockout mouse.Frozen sections were stained with an antibody againstα-DG(ⅡH6).The mutants displayed the indistinct, discontinuous,patched borders of the cardiac myocytes compared to controls with distinct,continuous borders.2.Western blotting and laminin binding assay(solid phase) for purified DG showed that there is almost no DG expression or loss of ability ofα-DG to bind laminin. Furthermore,the additional study on the relationship between DG and aging showed that the activity ofα-DG binding to laminin decreased,but no significant difference.3.Neonatal mice cardiac myocytes primary culture showed reduced cells attachment and lack of matrix assembly in Mlc2vCre lacking DG compared to controls but no defect in assembly in adult cardiac myocytes.4.Assessment of PI3k-ARK signaling in adult myocytes showed that lower levels of phosphorylated AKT and GSK3β(down stream of active AKT) was obtained in Mlc2vCre-DGnull mice whole heart homogenates compared to littermate controls, which indicates DG is involved in this cell survival signaling.Higher levels of phospho-AKT and phospho-GSK3βwere found after laminin addition to control myocytes,but had no effect on DG null cardiac myocytes,which indicates that laminin confers a cell survival signal to cardiac myocytes through DG and the interaction of DG with ligands is necessary for the activation of AKT,DG is indispensable to mediate this critical signaling transduction. 5.Assessment of the effects of acute and chronic mechanic strain on adult DG deficient myocardium showed that cardiac myocytes lacking DG was more sensitive to membrane damage due to overload.In Mlc2vCre mice 4 weeks after AAB,HE staining showed the disorders of cardiac myofibers,part of which discontinued,presenting uneven cytoplasmic staining,varied in intercellular space,infiltration of inflammatory corpuscle.SR staining and FG counterstaining showed that focal areas of collagen deposition suggesting focal regions of damage and remodeling in mutants,was performed on mutant Mlc2vCre-DGnull mice compared to littermate controls.In Mlc2vCre mice 5 hours after treadmill exercise stress,immunostainings showed that membranes of the cardiac myocytes were damaged severely and some cytoplasmic proteins such as cTnI and cTnT that leaked out through the disrupted membranes.6.Western blotting after treadmill exercising showed that the common substrates of Calpain(CAPN) such as cTnI,cTnT,and talin were degraded to some degrees.The mutant's degradation percentages compared to the control's are cTnI 13.6%vs 6.81% with p＜0.01,cTnT 21.2%vs 10.55%with p＜0.01,talin 3.01%vs 2.28%without significant defference.7.Assement of the effects of AdCAST transfection on proteins expression in cardiac myocytes primary culture showed that the expression of the CAPN substrates such as cTnI,cTnT,and talin were significantly higher in AdCAST transfection group compared to no transfection group,p＜0.05,on the contrary,the expression of CAPN significantly lower in transfection group,p＜0.05.8.The Western blotting and Zymogram analysis showed that Tg mice have the much higher expression of hCAST by which CAPN activity was highly inhibited, whereas there is almost no detection of intact hCAST but CAPN was normally activated.Repeating treadmill exercising showed that CAPN expression in CAST nontransgene(NTg) group the mutant subgroup increased significantly.In CAST Tg group either mutant or control mice CAPN was inhibited signicantly,there is no significant difference between these two subgroups.Futhermore,there are no differences in CAPN substrates such as cTnI,cTnT,and talin between these two subgroups.Conclusions1.The establishment of cardiac tissue-specific DG knockout mouse model renders it possible to directly study the structural and functional roles that DG,the central protein in DGC associated with muscular dystrophy,plays in cardiomyopathy associated with muscular dystrophy,and the associated cellular and molecular mechanism.2.DG at least plays critical role in matrix assembly in neonatal mice.3.DG is involved in the PI3K/ART cell survival signaling,and is indispensable to mediate this critical signaling transduction.4.Cardiac tissue-specific DG deficient leading to the disruption of DGC renders cardiac myocytes sensitive to membrane damage due to heart overload.5.Calpain protease system is involved in the development of the injury responding to mechanical strain overload on DG deficient heart.6.Overexpression of calpastatin can effectively decrease calpain activity and expression,especially in DG deficient heart with highly inhibition of abnormally activated Calpain,to some degree,playing a role in cardiac protection and providing a beneficial clue and a novel strategy for gene therapy and clinic medication of muscular dystrophy and associated cardiomyopathy due to DG deficient.
|
PDF Full Text Request