The Role Of TGF-BMP In Early Amyloidosis Of Rat Cardiomyocytes

Background:Amyloidosis is a systematic disease characterized by the pathological deposition of misfolded proteins known as immunoglobulin light chain(LC).Prognosis is closely related to the organs involved,and the most serious result is associated with cardiac involvement,which is up to 50% of patients with primary amyloidosis.Although this disease was identified early in the mid-19 th century and had lower mobility,the mortality rate was high resulting from difficult early diagnosis of the disease and little efficiency of current treatment.Patients with cardiac amyloidosis had elevation of serum TNT and BNP and finally died of arrhythmia and congestive heart failure with a median survival of less than 8 months and 5-year survival rate higher than 10%.The disease progressed rapidly and the prognosis was poor,thus new strategies of treatment are needed to improve survival rate.However,the integrated molecular mechanisms inducing cardiac amyloidosis remain unclear.Previous and recent researches have confirmed that increased oxygen species(ROS)production in cardiomyocytes was induced by LC resulting in impaired diastolic function and apoptosis.Jianru Shia's study confirmed that P38 MAPK pathway was an important mediator inducing cardiac dysfunction.Helen P'S research demonstrated that metabolism condition changed in cardiac amyloidosis.And other studies also explored other mechanisms of cardiac amyloidosis,including oxidative stress,mitochondrial dysfunction,impaired lysosomal and autophagy,altered contractility and calcium loading.The genetics of the pathogenesis of cardiac amyloidosis has not been defined.Over the past decade,microarray gene expression profile has provided lots of useful information for the diagnosis,treatment and prognosis of cardiovascular diseases.In this study,we investigated the DEGs of amyloid cardiomyopathy in cardiomyocytes model by microarray analysis.We further analyzed the roles of partial DEGs by using gene ontology and pathway analysis.TGF-? pathway plays an important role in the occurrence and development of this disease.BMP4 and BMP6 are two important functional proteins in this pathway.The increased expressions of BMP4 and BMP6 are closely related to the activation of this pathway,leading to the functional and structural changes of the heart.Objectives:The present study was designed to investigate the mechanism of cardiac amyloidosis in cardiomyocytes of neonatal Sprague Dawley(SD)male rats.Methods:1.Synthesis of amyloidogenic light chain protein AL-09 and collection of serum sampleThe AL-09 protein is an amyloidogenic light chain extracted by LA Sikkink et al.from the urine of patients with multiple myeloma who affected the heart.LA Sikkink and his colleagues not only validated the amino acid sequence of AL-09,but also synthesized and expressed the protein in E.coli BL21(DE3)cells.In this experiment,we used the same method as the LA Sikkink experimental group to reconstitute the AL-09 protein.To facilitate the successful expression of the protein,we added the His tag to the N-terminus.The protein was synthesized and expressed by Nanjing Jinsilui Company.The purified protein was stored at-80°C.The serum of patients with amyloidosis was collected from patients with myocardial amyloidosis in the Department of Cardiology of the First Affiliated Hospital of Nanjing Medical University;the serum of the control group was collected from normal subjects.Specimen collection was informed in writing by the patient.This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University(ratification number 2016SR169).2.extraction of primary cardiomyocyteSterile Dawley(SD)rat cardiomyocytes were harvested from 1-3 days after germination.Digestion,separation and extraction of the cardiomyocytes with the digestive fluid.More than 90% of high-concentration cardiomyocytes were cultured in cardiomyocyte culture medium.The cell counting plate counts,and adjusts the cell number to 3?105/ml,and finally plant the cells on the culture plate.3.Establishment of rat myocardial amyloidosis modelThe cells were divided into protein group and serum group.The protein group was cultured with AL-09 protein and His short peptide,respectively.The serum group was cultured with amyloidosis and normal serum,respectively.The cultured cells were placed at 37°C and incubated in a CO2 5% incubator for 48 h and then starved for 6-8 h in serum-free DMEM.The control group continued to be cultured in serum-free DMEM for 48 h.The serum group was 10-fold diluted with DMEM.Dilutions of human serum were cultured for 48 h,and the protein group was cultured for 48 h in serum-free DMEM cell culture medium at different concentrations.Each group has 6 complex holes.4.The inhibition of cell activity in each group by cck-8:Each group of cells was seeded into a 96-well plate at a density of 3?105 cells per well.After the end of the experimental intervention,CCK-8 assay was used to examine the inhibition of cell activity in each group.4.Quantitative detection of apoptosis by flow cytometryThe protein group cardiomyocytes were seeded in 24-well plates for 48 hours.The apoptosis rate was determined by Annexin V Alexa Fluor 647 / PI / apoptosis detection kit(Fcmrcs,Nanjing).The rate of apoptosis was assessed using a flow cytometer(BD FACSCalibur,USA).5.Gene chip and QRT-PCRThe cardiomyocytes of neonatal rats after intervention with protein group and serum group were extracted.Total RNA was extracted by Guangdong Ruibo Company.Differential genes were screened by gene chip.GO function analysis and Pathway signal pathway enrichment were used to analyze the biological functions and signal pathway of differential genes which related to cardiac amyloidosis.Focus on the TGF-? pathway-related m RNA,p53 signaling pathway,cell cycle and cell migration and proliferation-related genes.QRT-PCR was used to verify the above results.Results:1.AL-09 induces apoptosis in cardiomyocytesTo assess the cell viability of each group of cells,we analyzed the effect of AL-09 protein and patient serum on cardiomyocyte activity.Cell viability was assessed using the cck8 assay.Before the detection of cck8,each set of cytoscopic images was taken under a 400 magnification microscope by an ordinary optical microscope.As shown,myocardial cell debris at the highest concentration of AL-09 protein intervention was significantly greater than other groups.The difference between the other groups was not obvious.In the cck8 experiment,the viability of cultured cardiomyocytes at a concentration of 0.2 mg/ml of AL-09 protein was significantly lower than that of the control group(P < 0.05).The test also confirmed that the cell survival rate of the patient sera group was significantly lower than that of the normal serum control group(P < 0.05).Next we verified whether AL-09 induces cell death through apoptotic mechanisms.Annexin V / PI double labeling was used to detect PS externalization and showed early cell apoptosis.Consistent with the cck8 assay,the results showed that AL-09 protein caused apoptosis but not necrosis,and the proportion of early apoptotic cells accounted for the majority(P < 0.05)compared to the control group.2.Identification of differentially expressed genes(DEGs)The microarray results suggest that there are 256 identical DEGs in the proteome and genome.The 256 genes including 127 up-regulated genes and 88 down-regulated genes were analyzed by gene enrichment pathway analysis.In differential gene chip expression profiles,the expression of BMP4,BMP6,PTGS1,PTGS2,EREG,TGFA,PLOD2,and LBP in the two groups of commonly-expressed differentially expressed genes was significantly increased(P < 0.05).The expressions of CCNB1,RRM2,PTTG1 and CDKN2 C were down-regulated,the difference was statistically significant(P <0.05).Among the up-regulated DEGs,the differentially expressed genes with high functional enrichment were related to the irregular transport of calcium ions into cytoplasm,protein activation cascade,growth factor activity,catalase activity,and membrane anchoring component changes.These genes are involved in signal transduction,metabolism,apoptosis,adhesion and cardiac physiology.The results of KEGG pathway analysis included aminosugar and nucleotide sugar metabolism,lysine degradation,TGF-? signaling pathway and Erb B signaling pathway..In down-regulated DEGs,the major high-function enrichment analysis of differentially expressed genes correlates with cell cycle and cell contractile function.These genes are involved in cell proliferation and systolic and diastolic processes.The KEGG pathway analysis showed that differentially expressed genes were associated with p53 signaling pathway,cell cycle,meiosis of oocytes,and purine metabolism.3.Gene chip resultIn this study,DEGs related to the development of amyloid cardiomyopathy were screened by gene chip technology.The TGF-? pathway plays an important role in the occurrence and development of the disease.BMP4 and BMP6 are two important functional proteins in this pathway.Increased expression of BMP4 and BMP6 is closely related to the activation of TGF-? signaling pathways,leading to functional and structural changes in related tissues and the development of end-stage heart failure.Conclusion:1.Myocardial amyloidosis serum and protein AL-09 intervention experiment can induce the cardiomyocyte viability of neonatal rats significantly lower,compared with the control group while the early apoptosis rate of cells increased in the AL-09 group.2.Experimental group genes were down-regulated about p53 signaling pathway,cell cycle,oocyte meiosis and purine metabolic pathway,while other genes were up-regulated about aminosugar and nucleotide sugar metabolism,lysine degradation,TGF-? signaling pathway and Erb B signaling pathway.3.BMP4 and BMP6-mediated TGF-? signaling pathway may be involved in the pathological process of early disease in cardiomyocytes.