Function And Mechanism Of Adipsin In Diabetic Cardiomyopathy

Background:Diabetic cardiomyopathy(DCM)is the most serious cardiac complication of diabetes with complex etiology and unclear mechanism.According to statistics,DCM is the leading cause of heart failure and death.The pathological basis of DCM is reduced compliance and impaired diastolic function due to ventricular hypertrophy.The main pathogenesis is abnormal myocardial lipid metabolism,insulin resistance,endoplasmic reticulum stress,inflammation,etc.As an important endocrine organ,adipose tissue interacts with other organs through adipokines when the metabolic environment changes.Adipose tissue dysfunction will lead to a series of changes in the secretion spectrum of adipokines,which is a sign of metabolic dysfunction.Recent studies have shown that Adipsin has physiological effects such as protecting islet cells and improving insulin resistance.Aims:This study focuses on the role and molecular mechanism of Adipsin in DCM,and is expected to provide a new target and theoretical basis for the treatment of DCM.Methods:1.The animal model was based on C57BL/6J mice,and the cardiac function of the mice was observed after 60% kcal high-fat diet was fed for 16 weeks;Cell model treatment of primary cardiomyocytes with palmitic acid(300 μM)for 48 hours to simulate the lipotoxic injury of cardiomyocytes in a high-fat environment;2.Transvalvular flow velocity across the mitral valve was collected by mouse cardiac ultrasound,and the diastolic function was assessed by peak early diastolic blood flow/peak late diastolic blood flow(E/A);3.Diabetic status of mice was assessed by body weight,glucose and insulin tolerance tests;4.Western blot was used to detect the content of Adipsin in serum;5.Detection of Adipsin mRNA levels in various tissues and organs by RT-qPCR;6.Expression of Adipsin in Adipose Tissue by Immunohistochemistry;7.Detection of primary cardiomyocyte injury by flow cytometry and Tunel staining;8.Detection of Adipsin protein enrichment in various tissues and organs in vivo by near-infrared tracer technology;9.Detection of myocardial fiber hypertrophy by HE and WGA staining;10.Detection of myocardial tissue triglyceride and malondialdehyde content by microplate reader and commercial kits to evaluate cardiac lipid metabolism status;11.Construction of Adipsin overexpression and knockout mice using CRISPR/Cas 9technology;12.Detection of Adipsin enrichment in myocardial tissue of Adipsin overexpressing mice by tissue immunofluorescence;13.Analysis of downstream gene expression in myocardial tissue of DCM mice using myocardial tissue transcriptome gene sequencing and bioinformatics methods;14.Using protein immunoprecipitation combined with mass spectrometry to explore molecules that may interact with Adipsin;15.Adipsin-interacting molecules were knocked down by intramyocardial injection of adeno-associated virus vector,and the differences in downstream gene expression,pathological tissue changes and lipid metabolism-related indicators of the heart were further observed.Results:Firstly,significant reduction of Adipsin was found in the high-fat-induced DCM model serum,and Adipsin deficiency further aggravated the damage to the heart caused by high-fat diet.The reason is that high-fat diet will lead to a series of disorders such as obesity and insulin resistance,which will eventually increase the accumulation of lipids in the heart,further leading to heart failure and even life-threatening;We found that Adipsin overexpression alleviated the high-fat diet-induced DCM phenotype and significantly reduced lipid accumulation in myocardial tissue.Secondly,we used whole-genome sequencing to analyze the myocardial tissue of DCM model mice.We found that Adipsin overexpression significantly decreased the expression of genes related to fatty acid metabolism compared with controls.This result suggests that Adipsin may play a protective role against myocardial lipotoxicity by reducing the expression of genes related to fatty acid metabolism.Next,we performed a Co-IP combined mass spectrometry experiment on the myocardium of mice treated with high-fat after Adipsin overexpression,and the results suggested that Thrap3 directly acts on Adipsin.It has been reported that Thrap3,as a transcriptional cofactor,can regulate gene expression of fatty acid metabolism.Therefore,we hypothesized that Adipsin may limit the function of transcriptional cofactors,downregulate the expression of genes related to fatty acid metabolism,and further regulate myocardial lipid metabolism by directly binding to Thrap3.Finally,We down-regulated Thrap3 expression in mouse myocardium using AAV9 carrying sh RNA-Thrap3,followed by high-fat feeding to induce DCM.The results suggest that Thrap3 downregulation and Adipsin overexpression can both alleviate the DCM phenotype when they work alone,but when the two sides are combined,no more significant therapeutic effect can be produced.This indicates that Thrap3 downregulation and Adipsin overexpression are not two independent factors,and Thrap3 is involved in the cardioprotective mechanism of Adipsin.Conclusions:In conclusion,The adipokine Adipsin exerts cardioprotective effects in DCM;Adipsin can improve myocardial lipid accumulation by regulating myocardial fatty acid metabolism;This study revealed that Adipsin regulates downstream related gene expression through Thrap3,thereby improving the phenotype of DCM;The mechanism study of Adipsin in DCM provides a new direction for the prevention and treatment of the disease.