Application Of Liquid Chromatography-tandem Mass Spectrometry Analysis The Key Proteins And Signaling Pathways Regulating The Occurrence Of Heart Failure With Preserved Ejection Fraction

BackgroundHeart failure with preserved ejection fraction（HFpEF）is a heterogeneous clinical disease with increasing prevalence and unclear pathogenesis.With the change of people’s eating habits,the number of HFpEF phenotype patients with hypertension and metabolic diseases such as obesity（glucose and lipid metabolism disorders）and diabetes is increasing,and the research on energy metabolism of HFpEF has become a hot spot.Previous proteomics studies have found that changes in multiple proteins are associated with HFpEF.However,the pathogenesis of heart failure in HFpEF induced by high fat and high blood pressure,especially the regulatory mechanism at the protein level and signaling pathway,is still unclear.AimsLiquid chromatography-tandem mass spectrometry analysis and biological analysis were used to screen differential expressed proteins in the HFpEF induced by hyperlipidemia and hypertension,as well as the biological processes and signaling pathways involved,at the same time,found the key proteins in the model.It provides experimental basis for further study of the molecular mechanism of HFpEF.Methods1.Establishment of animal modelIn this experiment,Sprague-Dawley（SD）rats（8 weeks old）were used for the experiment.The rats were divided into control and“two hit”treatment for 3,6 and12 weeks（n=12）.We used a high-fat diet and N^[ω]-nitro-L-arginine methyl ester（L-NAME）to induce hyperlipidemia and hypertension.The treatment of“two hit”was used to construct the HFpEF model.The success of the model was determined by monitoring blood pressure,blood glucose,and cardiac function.2.Monitoring of blood pressure,blood glucose,and cardiac functionThe blood pressure changes of rats were evaluated by rat tail blood pressure measuring instrument,the blood glucose changes of rats were evaluated by blood glucose measuring instrument,and the cardiac function indexes of rats were evaluated by small animal ultrasound high-resolution imaging system.3.Cardiac histopathological analysisHematoxylin-eosin（H&E）,MASSON,α-SMA immunohistochemistry,WGA,dihydroethidium（DHE）,and TUNNEL staining were used to analyze the pathological changes of ventricular tissue.4.Liquid chromatography-tandem mass spectrometry analysis of heart tissueTime series proteomic analysis of ventricular tissue was performed by liquid chromatography combined with tandem mass spectrometry.Combined with biological analysis methods to clarify the biological processes and signaling pathways involved in related proteins,integrated co-expression relationships,constructed a network of significant signaling pathways involved in differential proteins,and screened proteins that play a core regulatory role in the network.5.Statistical analysis methodAll data were expressed as mean±standard deviation.The significant difference between the two groups was determined by T-test.Import Graph Pad Prism9.0 for statistical analysis and plotting.When P<0.05,there was a statistically significant difference.Results1.The treatment of“two-hit”induced cardiac dysfunction in ratsCompared with the control,the systolic blood pressure and fasting blood glucose of the model group increased with time,and echocardiography showed that there was no significant change in the EF%and FS%of the rats in the“two hit”treatment for 3-6 weeks,and the EF%and FS%rate of the rats decreased significantly in the“two hit”treatment for 12 weeks.In addition,E/A value decreased（E/A<1）at 6-12 weeks of“two hit”treatment.2.The treatment of“two-hit”induced rat ventricular cardiomyocyte hypertrophy,inflammatory response,oxidative stress,fibrosis,and cardiomyocyte apoptosisCompared with the control group,there were obvious pathological changes in the myocardial tissue of the model group,which were manifested as cardiomyocyte hypertrophy,increased infiltration of inflammatory cells,increased oxidative stress level,increased degree of myocardial fibrosis,increased apoptosis of cardiomyocytes,and the degree of lesions increased with time（p<0.05）.3.The treatment of“two-hit”activated inflammatory response,fibrosis and apoptosis signaling pathways in rat cardiomyocytesWestern Blot was used to verify that the expression of P65/pP65,Bcl-2,Bax,Caspase 3 and TGF-β1 was significantly changed under the treatment of“two-hit”.It indicates that the signaling pathways related to inflammation,fibrosis and apoptosis of rat cardiomyocytes are activated.4.The treatment of“two-hit”induced differential expression of proteins in myocardial tissueA total of 407 differentially expressed proteins（DEPs）were identified in the“two hit”model.Among them,198,176 and 237 proteins were significantly up-regulated and208,231 and 170 proteins were significantly down-regulated at 3,6 and 12 weeks after“two hit”.5.Application of biological analysis of differentially expressed proteins caused by changes in biology and signaling pathwaysWe performed GO enrichment and KEGG analysis on DEPs.The results showed that DEPs were mainly concentrated in mitochondria,and DEPs were mainly involved in molecular functions such as protein binding and biological processes such as glycolysis and redox.There are 50 signaling pathways that interact with each other.The significant signaling pathways are mainly related to energy metabolism,including citrate cycle,pyruvate metabolism and glycolysis.6.Trend significance analysis and verification of differential proteinsThe 406 DEPs identified in the ventricular tissue of the“two hit”model can be divided into 50 expression patterns.Among them,10 expression patterns（#40,9,11,37,26,14,1,18,15 and 23）had significant differences.We detected the expression levels of six cardiovascular disease-related proteins by Western blotting,including Profile 1（NACA）,Profile 9（OXCT1）,Profile 11（PSMB7）,Profile 18（ICAM-1,ITGAV）,and Profile 37（FBN1）.It was found that their expression was consistent with the trend of expression patterns.7.Identification and verification of key proteinsThe analysis of the protein co-expression network showed that Bsg played a core regulatory role in the co-expression network,directly regulating 69 adjacent proteins,and the expression level was verified by Western blotting.ConclusionUnder the treatment of“two hit”,the HFpEF model was successfully induced.Proteomic analysis showed that a total of 407 proteins were significantly different in expression,mainly located in mitochondria,involved in biological functions such as glycolysis,redox and fatty acids,and corresponding signaling pathways.Bsg plays a central regulatory role in the network,which may be involved in the occurrence and development of HFpEF.This study provides a direction for subsequent research.