Effect Of SFRP5 On Heart Failure With Preserved Ejection Fraction And Its Mechanism

Background and objective: Heart failure with preserved ejection fraction(HFp EF)is a global public concern with its prevalence increasing at an alarming rate and its 5-year mortality was comparable to heart failure with a reduced ejection fraction.However,there is a lack of understanding on the pathophysiological mechanism of HFp EF and few effective treatments on HFp EF.Therefore,it is important to identify the key proteins which can affect the pathogenesis and development of HFp EF.Secreted frizzled related protein 5(SFRP5)is an emerging adipokine which is closely associated with obesity,diabetes mellitus and other metabolic diseases.Meanwhile,a number of studies have shown the protective role of SFRP5 in the cardiovascular diseases.However,there has been no report on whether SFRP5 is related to the occurrence and development of HFp EF which is often accompanied by metabolic syndrome.In the present study,we investigated the relationship between SFRP5 and HFp EF in a human cohort and animal experiments,and further studied the mechanism of SFRP5 affecting HFp EF,aiming at providing theoretical basis and therapeutic targets for the prevention and treatment of HFp EF.Methods:(1)Firstly,we enrolled 1607 individuals over 65 years old from 10 communities in Northern Shanghai Study(Registration Number: NCT02368938).Cardiovascular risk factors,organ damage indicators,diseases and treatments were investigated in all participants.The inclusion and exclusion criteria were showed in the study protocol which was published previously(BMJ Open,2017.7(3): e013880).Then we studied the basic characteristics and risk factors of HFp EF in the population.The relationship between serum SFRP5 level and HFp EF was also investigated in the cohort.(2)In the animal study,we generated a HFp EF mouse model by a combination of high-fat diet and L-NAME and evaluate the expression of SFRP5 in the myocardium of HFp EF mice.Then wild-type C57(WT)and SFRP5 gene knockout mice(KO)were used to construct HFp EF models,and their body weight,fasting blood glucose,blood pressure were measured.Cardiac systolic and diastolic function of mice was analyzed by echocardiography.Then we harvested and weighted heart tissues of mice and observed the changes of heart size,myocardial hypertrophy and myocardial fibrosis.The wet weight and dry weight of lung tissue were also analyzed to show the pulmonary congestion.Further,SFRP5 recombinant protein was administrated to KO-HFp EF mouse to confirm whether treating with SFRP5 can alleviate HFp EF phenotype.(3)We went through a transcriptome sequencing to compare the differential expressed genes in WT-HFp EF and KO-HFp EF mice and also analyzed the enrichment of KEGG pathway in two groups.Heart lipid deposition in WT-HFp EF and KO-HFp EF mice was investigated.The effect of SFRP5 on lipid deposition in cardiomyocytes was studied after inducing lipid accumulation in H9C2 cells by palmitic acid(PA).Western Blot was used to detect the PPARα/CD36 pathway protein in myocardial tissue of HFp EF mice and in lipid-accumulated H9C2 cell.Results:(1)In the Northern Shanghai Study(n=1607),the mean age of the participants was 71.3±6.0 years and 56.4% was women.There were 96(5.97%)HFp EF patients in the cohort and the prevalence of HFp EF increased with age(3.7% for <70years,5.9%for 70～79 years,and 15.5% for ≥80 years).Multivariate logistic regression showed that HFp EF was significantly associated with body mass index(OR 1.07,95%CI 1.01-1.15,P<0.05),fasting blood glucose(OR1.21,95%CI 1.06-1.38,P<0.01)and total cholesterol(OR 2.24,95% CI 1.12-4.51,P<0.05).Further,HFp EF was more prevalent in individuals with diabetes mellitus(DM)and obese obese people(DM vs non-DM 9.40% vs 5.20%,P<0.01;obese vs non-obese10.27% vs 5.28%,P<0.01).In the cohort,serum SFRP5 was positively correlated with NT-pro BNP after adjusting for age and sex(r=0.17,P<0.0001).Serum SFRP5 levels were higher in HFp EF patients than in individuals with normal heart function(5.7±3.6 ng/m L vs4.6±2.8 ng/m L,P<0.01).(2)In animal study,we firstly constructed HFp EF mouse model and evaluated the HFp EF phenotypes.There were no significant differences in ejection fraction(EF)and fractional shortening(FS)between HFp EF group and control,while E/A,E/E’ and left ventricular mass(LVM)were significantly increased in HFp EF mice.Compared to control,significantly increased levels of body weight,fasting blood glucose and systolic blood pressure(SBP)were observed in HFp EF mice.Meanwhile the heart weight/body weight ratio and lung wet weight/dry weight ratio were significantly increased in HFp EF group.Immunohistochemistry and Western blot results showed that the SFRP5 expression in myocardium of HFp EF mice was higher than that of control group.Then HFp EF models were constructed in wild-type(WT-HFp EF)and SFRP5knockout(KO-HFp EF)mice.KO-HFp EF mice had significantly increased body weight,fasting blood glucose and higher E/E’ compared to WT-HFp EF,but there was no difference in EF.Meanwhile the heart/body weight ratio was markedly increased and myocardial fibrosis was more severe in KO-HFp EF mice compared to WT-HFp EF group.After treating the KO-HFp EF mice with recombinant SFRP5,E/E’ was significantly decreased compared to the mice without treatment,while EF did not differ in two groups.Treatment with SFRP5 also decreased fasting blood glucose in HFp EF mice.Cross-sectional area of myocyte and myocardial fibrosis level tended to decrease in KO-HFp EF mice treated with SFRP5 compared to that without treatment,but there was no statistical difference.Meanwhile there was no significant difference in myocyte cross-sectional area and myocardial fibrosis between SFRP5-treated HFp EF mice and KO-Chow mice.(3)Transcriptome sequencing was performed in the heart tissues of WT-HFp EF and KO-HFp EF mice.KEGG enrichment analysis showed that the number of differentially expressed genes related to PPAR signaling pathway was the largest and most significant.In KO-HFp EF mice,triglyceride content in heart tissue was significantly higher than that of WT-HFp EF mice.And in vitro,treatment with recombinant SFRP5 protein could reduce the PA-induced lipid deposition in cardiomyocytes.Western blot analysis showed that PPARα/CD36 signaling pathway was activated in the heart of HFp EF mice,and this activation was more marked in KOHFp EF group.SFRP5 inhibited the activation of PPARα/CD36 signaling pathway induced by PA in vitro.Conclusion: SFRP5 plays a protective role in the development of HFp EF through inhibiting the activation of PPARα/CD36 pathway and reducing myocardial lipid deposition,thus alleviates the symptoms of HFp EF.