| BackgroundHeart failure is the clinical end stage of heart organic disease caused by hypertension,coronary artery disease and other major diseases,and remodeling runs through the whole pathological process.Research on genes and regulatory networks involved in remodeling is beneficial to the prevention and treatment of heart failure.We found that the expression of Adtrp decreased in the process of myocardial remodeling,and molecular flow regulation also confirmed that it is significantly related to cardiovascular diseases,which all reveal its importance to myocardial remodeling.However,at present,the role of Adtrp in the development of the heart,especially in the pathological processes closely related to myocardial remodeling such as myocardial hypertrophy and heart failure,is still unclear.Therefore,we first established a rat model of myocardial tissue-specific Adtrp overexpression and knockout,and compared it,and explored the role of Adtrp in the pathological process of myocardial hypertrophy and heart failure and its molecular mechanism from the three levels of the overall function and structure of the heart,histopathological changes and molecular signals,clarified the biological role of Adtrp,and provided ideas and basis for the prevention and treatment of cardiovascular diseases such as cardiac hypertrophy.MethodsI.Use immunofluorescence technique to observe the localization and expression of ADTRP in rat cardiomyocytes.Western blot was used to detect the time course expression characteristics of ADTRP in the myocardium of wild-type SD rats at 1,2,3,5,7,12,and 18 months of age,and changes in protein expression levels in the myocardial tissues of ADR-induced heart failure mice models,TAC-induced hypertrophic cardiomyopathy mice and rat models.2.An overexpression vector of Adtrp gene driven by α-MHC promoter was constructed in vitro,and a rat model of myocardial tissue-specific Adtrp overexpression was constructed using microinjection technology.PCR technology to identify genotypes.WB detects the expression efficiency of ADTRP in myocardial tissue.3.Using the principle of the recombination system(Cre/loxP),the established Adtrp cKO(Conditional knockout)rat and the myocardial tissue-specific Cre overexpression(αMHC-Cre)rat were subjected to two rounds of cross-breeding,namely The myocardial tissue specific Adtrp knockout(Adtrpflox/flox/MHC-Cre,KO)rats can be obtained.PCR technology to identify genotypes.WB detects the knockout efficiency of ADTRP in myocardial tissue.4.Ultrasonic imaging was used to detect the changes of heart function and structure in the three groups of WT,OV and KO rats at the age of 1,2,3,5,7,12,and 18 months;at the age of 7 months,after the rats were euthanized,their hearts were removed and recorded Wet weight data,calculate the ratio of HW/BW(heart weight/body weight).The heart was fixed and long-axis paraffin sections were prepared,and the histopathological changes of the myocardium at the microscopic level were observed by H&E and Masson staining.The heart was fixed to prepare electron microscope samples,and the histopathological changes of myocardium at the ultramicroscopic level were observed by transmission electron microscope.5.Three groups of rats of WT,OV and KO at the age of 2 months were selected,and an osmotic pump containing ISO was implanted subcutaneously for 28 days of continuous administration.During the period,the death data were recorded,the heart function and structural changes were detected by ultrasound imaging at the end of the administration,and the HW/BW index and histopathological changes were detected after euthanasia.6.Screening and verification of ADTRP interacting proteins was carried out by immunoprecipitation and immunoblotting technology.Results1.Analysis of the expression characteristics of ADTRP in physiological and pathological conditions1)The expression level of ADTRP in myocardial tissue is higher from birth to about 5 months of age,and the expression level decreases with age.2)In cardiomyocytes,ADTRP is mainly located on the cell membrane.3)The expression of ADTRP in the myocardial tissues of ADR-induced heart failure mouse models and TAC-induced hypertrophic cardiomyopathy rats and mouse models was significantly decreased.The expression of ADTRP in the myocardial tissues of clinical samples of hypertrophic cardiomyopathy was significantly decreased.2.Establishment and phenotype analysis of myocardial tissue-specific Adtrp over expression and knockout rat model1)The myocardial tissue-specific Adtrp overexpression rat was established,and the first established mouse line with high expression and stable inheritance was screened for subsequent analysis.2)Adtrp-cKO rats and aMHC-Cre rats constructed by using CRISPR/Cas9 technology,after two rounds of cross-breeding,the myocardial tissue-specific Adtrp knockout rats were obtained.3)Under physiological conditions,myocardial tissue-specific Adtrp overexpression rats have a slightly thicker ventricular wall at 1 month of age,and then the ventricular cavity gradually expands and the ventricular wall becomes thinner.3 months of age is the turning point,and significant differences are present at 5 months of age.,The thickness of the ventricular wall decreases,the ventricle dilates,and the function decreases.In myocardial tissue-specific Adtrp knockout rats,the ventricular wall is thickened and the ventricular cavity diameter becomes smaller.This difference accumulates to the maximum at 7 months of age.4)After treatment with ISO,myocardial tissue-specific Adtrp overexpression rats significantly reduced the ventricular wall thickness,ventricular dilatation,and decreased heart function.The myocardial tissue-specific Adtrp knockout rats can improve the above indicators.5)After ISO treatment,In the WT-ISO group,myocardial fibers appeared heterogeneous hypertrophy,irregular order,and myocardial mitochondria swelled.In the OV-ISO group,myocardial fibers were obviously broken and dissolved,arranged disorderly,sarcomeres were blurred,mitochondria were obviously swollen,and cristae rupture occurred.The myocardial fibers in the KO-ISO group showed local heterogeneous hypertrophy and swelling of myocardial mitochondria,which were alleviated compared with the histological changes in the WT-ISO group.3.Preliminary analysis of the molecular mechanism of ADTRP involved in the process of myocardial hypertrophy and decompensation regulation1)H9c2 cells were transfected with Flag-tagged Adtrp expression plasmid,the protein was extracted,and mass spectrometry was performed.The interaction proteins of ADTRP were RHOQ,Caveolin-1 and PRKAR2A.2)H9c2 cells were co-transfected with the Caveolin-1 expression plasmid with the Flag tag and the Adtrp expression plasmid,and the protein was extracted,and the reverse verification was performed.The results showed that Caveolin-1 is the interaction protein molecule of ADTRP.Conclusion1.ADTRP is mainly located on the myocardial cell membrane.The expression level is higher from birth to about 5 months of age in rats,and then decreases with age.Under the pathological conditions of myocardial hypertrophy and heart failure,its expression is significantly down-regulated.2.The myocardial tissue-specific Adtrp overexpression rat began to thicken the ventricular wall at 1 month of age,and then the ventricular cavity gradually expanded and the ventricular wall became thinner.The turning point was at 3 months of age.There was a significant difference at 5 months of age,and then gradually lost.Compensation,the thickness of the ventricular wall decreases,the ventricle expands,and the function decreases.Myocardial tissue-specific Adtrp knockout rats have thickened ventricular walls and reduced ventricular cavity diameter.The difference between the rats and the control group is the largest at 7 months of age.3.The changes in the overall function and structure of the heart,as well as histopathological microscopic and ultramicroscopic morphological changes,all show that Adtrp overexpression can accelerate,and Adtrp knockout can inhibit the pathological development of ISO-induced cardiac hypertrophy to heart failure.4.Through immunoprecipitation and immunoblotting technology,it was screened and confirmed that Caveolin-1 is an interacting molecule of ADTRP,which provided possible direction and target clues for subsequent signal mechanism analysis.This study revealed the role of ADTRP in the pathological process of myocardial hypertrophy decompensation,clarified the biological role of ADTRP in the development of heart and the pathological process of myocardial hypertrophy decompensation,and finally provided ideas,method and direction for the prevention and treatment of myocardial hypertrophy and heart failure.BackgroundSpliceosomes play an important role in the processing of nascent RNA transcripts in eukaryotic cells.At present,many studies have reported that splicing complex-related gene mutations are associated with a variety of clinical diseases,and PRPF40B is one of the genes that have been discovered to encode spliceosome compounds.PRPF40B belongs to the U2 ribonucleoprotein body-dependent splicing complex related genes.Studies have shown that PRPF40B gene mutations are related to myelodysplastic syndrome,chronic lymphocytic leukemia,acute myeloid leukemia,and a variety of solid tumors.Therefore,in order to further study the biological effects of PRPF40B,our laboratory used CRISPR/Cas9 technology to construct a PRPF40B gene knockout rat model for the first time,providing in vivo animal model tools.And in preliminary research and analysis,it was found that this gene is highly expressed in myocardial tissues.It is speculated that this gene may have an impact on heart development.Therefore,we use the gene knockout rat model to systematically observe the overall function and structure of the heart,as well as histopathological changes,and provide ideas and clues for the in-depth study of the biological function of the gene.Methods1.The Prpf40b knockout rats were constructed by CRISPR/cas9 technology.The genotype of the founders and offspring rats were identified by sequencing and PCR.2.Ultrasound imaging technology analyzes the changes in the morphology and function of the heart of knockout rats.3.After the rat was euthanized,the heart was removed,the wet weight data was recorded,and HW/BW(heart weight and body weight)was calculated.The heart was fixed and long-axis paraffin sections were prepared,and the myocardial histopathological changes were observed by H&E staining.Results1.Establishment and breeding of Prpf40b gene knockout rats PCR identification and sequencing comparison confirmed that the Prpf40b gene knockout rats was successfully constructed.2.Analysis of cardiac structure and function in Prpf40b knockout rats According to ultrasound imaging analysis,compared with the wt rats,the cardiac structure and morphology of 2-month-old knockout rats were not significantly abnormal,while the ventricular cavity diameter and volume of 12-month-old knockout rats decreased significantly in systole and diastole,and the stroke volume decreased significantly.3.Histological observation of myocardium in Prpf40b knockout rats Histopathological analysis showed that the myocardium of 12-month-old knockout rats was irregularly arranged,the thickness of myocardial fibers was uneven,and the sarcoplasmic reticulum was dilated.ConclusionThe deletion of Prpf40b gene can induce changes in the overall structure and morphology of the rat heart and abnormal myocardial histology. |
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