| About half of patients with heart failure(HF)are heart failure with preserved ejection fraction(HFpEF).Randomized clinical trials of patients with HFpEF using traditional HF medications have not been shown to significantly reduce their mortality.Multiple clinical studies have shown that the SGLT-2 inhibitors can significantly reduce the incidence of primary composite endpoint events(cardiovascular death and hospitalization due to heart failure)and can improve chronic HF(regardless of LVEF and with or without diabetes).This includes canagliflozin(CANA),one of the inhibitors of SGLT-2.However,the mechanism by which SGLT-2 inhibitors benefit patients with HF is not fully understood.SGLT-2 inhibitors have been shown to improve cardiac contractility in patients with reduced ejection fraction(HFrEF),and the mechanism may be related to normalizing the oxidative metabolism of myocardial glucose and fatty acids,increasing the ketogenic effect,and thus increasing cardiac ATP production.CANA increases fatty acid oxidation and ketogenic action by inducing transcription factor activation in the liver’s catabolic pathway.Similarly,caloric restriction activates the myocardial SIRT1/adenosine 5-monophosphate-activated protein kinase(AMPK)/PGC-1αpathway,but it remains unclear whether calorie restriction is caused by SGLT-2 inhibitors can activate the same pathway of the myocardium in HFpEF rats caused by hypertension.To further explore its possible mechanism,we fed Dahl salt-sensitive(DSS)rats 8%high salt to induce hypertensive left ventricular diastolic dysfunction.Then,DSS rats were treated with CANA intervention for 12 weeks,and we used a metabolic-proteomics combination to analyze the effect of CANA on myocardial metabolism and cardiac function in rats,and explored the possible mechanism of CANA.In addition,we collected chronic HF cases from the clinic to further analyze the expression level of pyruvate,a key metabolite of the myocardium,and its relationship with cardiac function,to provide new targets for cardiometabolic therapy.Part one Establishment and evaluation of the hypertensive animal model of heart failure with the preserved ejection fractionObjectives:By feeding DSS rats with the 8%high-salt diet for 12 weeks,we established a chronic HFpEF model,in the meanwhile the safety of the CANA medication intervention was also evaluated.Methods:1.Thirty-seven-week-old male DSS rats were randomly allocated into three groups.Ten rats were fed with the 8%high-salt diet(HSD)to establish a chronic HFpEF model group,10 rats were fed with the 8%HSD combined with CANA(20mg/kg/d)gavage intervention treatment group(HSD+CANA),another 10 rats were fed with 0.3%normal-salt diet(NSD)as the blank control group.2.The rats were fed for 12 weeks and their blood pressure,weight,food intake,mental state,appearance,and signs were monitored.3.Before the end of the trial,All the DSS rats were placed in the metabolic cage system to monitor 24h water consumption,food intake,activity distance,and 24h urine parallel oral glucose tolerance experiment.4.The serum of DSS rats was collected after the experiment.An automatic biochemical analyzer was used to detect serum albumin,triglyceride,total cholesterol,LDL-C,creatinine,serum sodium,3-hydroxybutyric acid,and random urine biochemistry in rats,including urine glucose,urine sodium,urine albumin,and urine creatinine.5.The serum BNP content of DSS rats was detected by ELISA.6.Cardiac echocardiography was performed in rats.Left ventricular anterior wall at end-diastole,left ventricular anterior wall at end-systole,left ventricular internal diameter at end-systole,left ventricular internal diameter at end-diastole,left ventricular posterior wall at end-systole,left ventricular posterior wall at end-diastole,and the E/A ratio were collected to evaluate.Results:1.After 12 weeks of observation,the body weight of rats in the HSD+CANA group was significantly lower than that in the HSD and NSD groups,meanwhile,the urine volume,water intake,and food intake were significantly increased.There was no significant statistical difference in the activity distance between the HSD+CANA group and the NSD group.Compared with the HSD group,the heart weight and heart weight ratio of the HSD+CANA group were significantly decreased after 12 weeks of treatment,and there was a statistical difference.2.After 12 weeks of the 8%HSD,the HSD group’s systolic and diastolic blood pressure was significantly increased.After 12 weeks of CANA treatment,compared with the HSD group,the systolic blood pressure(176.30±12.25 vs.211.38±12.89 mm Hg,P<0.01)and diastolic blood pressure(101.50±11.14 vs.134.25±18.08 mm Hg,P<0.01)were decreased.3.After 12 weeks of the HSD,comparing HSD and HSD+CANA with the NSD group,we found that urine glucose,urine sodium,and urine protein were significantly increased.After 12 weeks of CANA treatment,compared with the HSD group,the urine glucose in the HSD+CANA group increased,while the urine protein decreased significantly.After 12 weeks of HSD,serum creatinine and urinary creatinine in the HSD group were significantly increased.After 12 weeks of CANA treatment,serum and urinary creatinine decreased,but only urinary creatinine was statistically different.CANA significantly increased the concentration of 3-hydroxybutyric acid in the blood circulation of DSS rats.4.There was no significant statistical difference in the area under the oral glucose tolerance test curve and insulin levels among the three groups of DSS rats.5.According to echocardiographic parameters and serum BNP in three groups of DSS rats were showed that after 12 weeks of HSD in DSS rats,the average E/A<1,LVEF>60%,and serum BNP were significantly increased in HSD group.After 12 weeks of CANA treatment,the average E/A>1,BNP,left ventricular anterior wall end-diastole,left ventricular internal diameter end diastole,left ventricular mass were significantly lower than the HSD group,and there was a statistical difference.Conclusions:1.The HFpEF model was established by feeding DSS rats with the8%HSD for 12 weeks,which was consistent with the model characteristics of left ventricular diastolic dysfunction caused by hypertension.2.CANA can reduce both blood pressure and weight.At the same time,it could also have the effects such as diuresis,reducing urinary creatinine and urinary protein,but does not affect normal blood glucose.3.After 12 weeks of modeling,DSS rats fed with 8%HSD had left ventricular hypertrophy and diastolic dysfunction,while CANA could reduce left ventricular hypertrophy and improve diastolic function.Part two Effect of canagliflozin on ventricular remodeling and oxidative stress in Dahl salt-sensitive ratsObjective:To observe the data changes of ventricular structure and oxidative stress indexes in DSS rats fed with the 8%HSD after 12 weeks.And to further explore the effect of CANA on ventricular remodeling and oxidative stress in DSS rats.Methods:1.By using Masson and wheat germ agglutinin staining separately performed on the left ventricular pathological sections of rats in the three groups.To analyze the collagen fiber area and myocardial cell cross-sectional area of rat myocardial interstitial.2.The m RNA expression levels ofα-MHC andβ-MHC in myocardial tissue were detected by qRT-PCR.3.The protein expression levels of NOX4 in the myocardium were detected by Western Blotting.4.The content of AOPP in myocardial tissue was detected with the AOPP kit.Results:1.The results of wheat germ agglutinin and Masson staining showed that the cross-sectional area of left ventricular myocytes and myocardial interstitial collagen fibers were significantly increased in the HSD group,respectively.After 12 weeks of treatment with CANA,the cross-sectional area of myocardial cells and myocardial interstitial fibrosis were significantly reduced,and there were statistical differences.2.Myosin heavy chain subtypeβ-MHC/α-MHC is a marker of cardiac hypertrophy.The ratio ofβ-MHC/α-MHC in the HSD group was significantly increased,and the ratio ofβ-MHC/α-MHC was significantly decreased after CANA treatment,and there was a statistically significant difference.3.Compared with the NSD group,AOPP in the HSD group was significantly increased.AOPP content decreased significantly after 12 weeks of CANA treatment.The content of NOX4 protein in the myocardium was significantly increased in the HSD group and decreased in the HSD+CANA group,and these data show there were both statistically different.Conclusions:1.After 12 weeks of feeding with the 8%HSD,DSS rats showed ventricular reconstruction such as hypertrophy of left ventricular cardio-myocytes and increased myocardial interstitial fibrosis,and the CANA can alleviate the above pathological changes.2.After 8%HSD feeding 12 weeks DSS rat ventricular muscle oxidation stress symbol AOPP level and NOX4 protein expression was significantly increased.CANA can reduce the improvement of ventricular reconstruction by reducing oxidative reactions.Part three The effect of canagliflozin on myocardial metabolism and its mechanism in Dahl salt-sensitive ratsObjective:Through combined metabolome-proteomics analysis of myo-cardial metabolic pathways in 8%high-salt diet-fed DSS rats treated with CANA for 12 weeks,explore the possible mechanism of CANA to improve heart function and regulate myocardial metabolism.Methods:1.We used LC-MS/MS metabolomics to detect the changes of meta-bolites in the left ventricular myocardium of DSS rats.2.Changes in metabolic enzymes in left ventricular muscle tissue of DSS rats were detected by TMT isotope-labeled proteomics.3.By using ATP kit to setting-out concentration in myocardial tissue of DSS rats.4.Western Blotting was used to detect the protein of Pdk4,Ndufb4,Bdh1,Acox1,Ehhadh,Acadsb,AMPK,p-AMPK,SIRT1,PGC-1αexpression levels in myocardial tissue.5.The m RNA expression levels of SIRT1 and PGC-1αin myocardial tissue were detected by qRT-PCR.6.The protein expression levels of p-AMPK,SIRT1,and PGC-1αin heart tissues were further detected by immunohistochemical staining.Results:1.By analyzing the effects of CANA on myocardial metabolites in DSS rats from non-targeted metabolomics,we can conclude that after CANA treatment,compared with the HSD group,the glucose metabolites,including pyruvic acid,D-glucose,glyceraldehyde,gluconolactone,and D-Xylitol were significantly reduced.On the contrary,the oxidative degradation products of fatty acids increased,including 3-HTA,5Z-Dodecenoic acid,nervonic acid,cholesterol sulfate,ethyl oleate,ricinoleic acid,and 3-Hydroxycapric acid.Among them,5Z-Dodecenoic acid,ricinoleic acid,and ethyl oleate are monounsaturated fatty acids.DHA increased significantly after CANA treatment.And after CANA treatment,myocardial ketone 3-Hydroxybutyric acid increased,but there was no significant statistical difference compared with the HSD group.L-Valine and L-Alloisoleucine decreased compared with the HSD group,but there was no statistical difference.2.Proteomics analysis of cardiac non-targeted TMT isotope-labeling showed that after CANA treatment,compared with HSD,the detected glucose metabolism-related proteins Gpi,Dlat,G6pdx,Slc2a1,Pgm1,Pygb and Ugp2were down-regulated,but there was no significant statistical difference,and only Stt3b protein involved in glycosylation was down-regulated significantly.TCA cycle,mitochondrial electron transport chain-related proteins,Fatty acidβ-oxidation,and ketogenic protein expression were significantly up-regulated,including Pdk4,Ndufb4,COX2,Cox4i1,Cox5b,Cox6a1,Acox1,Acaa1a,Ehhadh,Plin2,Acadsb,Gpam,Hmgcs2.Although the key enzymes Bckdk and Bckdha in the branched-chain amino acid degradation pathway increased after CANA treatment,there was no significant statistical difference,and only the Pccb protein related to isoleucine metabolism was significantly up-regulated.3.The ATP level in the HSD group was significantly lower than that in the NSD group,and the ATP level showed an upward trend after 12 weeks of CANA treatment,but the difference did not reach statistical significance(P=0.27).4.Western Blotting and qRT-PCR data showed that compared with the HSD group,the protein expression levels of p-AMPK,SIRT1,PGC-1α,and the m RNA expression levels of SIRT1 and PGC-1αwere significantly increased after CANA intervention.Immunohistochemistry showed that compared with the HSD group,the protein levels of p-AMPK,SIRT1 and PGC-1αwere also significantly increased after CANA treatment.Conclusions:1.CANA increased fatty acid oxidation metabolism in the myocardium,promoted ketone formation,and up regulates the expression of tricarboxylic acid cycles and mitochondrial electron transport chain-related proteins in myocardial tissue of DSS rats with HFpEF.2.CANA could regulate myocardial metabolism,reduces oxidative stress,and improves cardiac diastolic function in DSS rats with HFpEF,possibly through activation of the AMPK/SIRT1/PGC-1αsignaling pathway.Part four Concentration of pyruvate in heart failure patients with different left ventricular ejection fractions and its corre-lation with cardiac functionObjective:To analyze the expression level of pyruvate in patients with different LVEF heart failure and to explore its correlation with cardiac function.Methods:1.Selected patients with heart failure diagnosed and treated in the Department of Cardiology,Hebei General Hospital from November 2020 to June 2021.The inclusion criteria were in line with the diagnostic criteria for HFpEF or HFrEF in the"Chinese Heart Failure Diagnosis and Treatment Guidelines 2018".A total of 60 patients were included.At the same time,31patients with non-heart failure due to other diseases were selected as the control group.The general clinical situation was collected for all included patients,including age,gender,height,weight,smoking,drinking history,past chronic disease history,clinical medication and blood pressure at admission,clinical-related laboratory indexes,and echocardiography.2.According to the inclusion criteria,they were divided into the HFpEF group,HFrEF group,and non-heart failure control group.All patients collected plasma samples,and the content of pyruvate was determined according to the instructions of the pyruvate kit.3.SPSS 25.0 software was used for data analysis.The measurement data of normal distribution were represented by(mean±SD),and a one-way analysis of variance was used for comparison among multiple groups.Non-normally distributed measurement data is represented by M(Q1,Q3),and the Kruskal-Wallis H rank-sum test is used for comparison among the groups.Enumeration data were expressed as frequencies and percentages,using theχ~2test and Fisher exact probability method.Spearman correlation was used to analyze the correlation of pyruvate and LVEF with each indicator.Multivariate logistic regression analysis was used to analyze the influencing factors related to HFrEF.P<0.05 was considered to indicate a statistically significant difference.Results:1.Comparison of general clinical data among three groups show that there was no significant difference in age,body mass index,diastolic blood pressure,smoking history,and statins history among the three groups.Patients in the HFrEF group were mostly males,and there was a statistically significant difference compared with the control group.Systolic blood pressure was significantly lower in the HFrEF group compared with the control group and it was statistically different.2.Comparison of laboratory indexes and LVEF among three groups shows that there was no significant difference in albumin,and it also conveys that ALT,triglyceride,and fasting blood glucose levels among the three groups have no significant difference.The level of NT-proBNP in the HFrEF group was significantly higher than that in the HFpEF group and control group,and the corresponding LVEF was significantly lower than that in the control group and HFpEF group.Pyruvate increased with the decrease of LVEF.The level of pyruvate in the HFrEF group was higher than that in the control group,and the difference was statistically significant(53.46±24.39 vs.40.22±14.93μg/m L,P<0.05).Serum creatinine in HFrEF and HFpEF groups was significantly higher than that in the control group.Compared with the blood lipid indexes of the three groups,the LDL-C and total cholesterol in the HFrEF and HFpEF groups were significantly lower than those in the control group,while the high-density lipoprotein cholesterol in the HFrEF group was significantly higher than that in the control and HFpEF groups,and there were statistical differences.3.Spearman correlation analysis showed that pyruvate was negatively correlated with LVEF(r=-0.271,P=0.009).LVEF was negatively correlated with male,creatinine,and NT-proBNP,and positively correlated with systolic blood pressure and HDL-C.4.By performing multivariate logistic regression analysis,we can tell HFrEF was taken as the dependent variable(HFpEF and the control group as0,HFrEF as 1),others such as gender,systolic blood pressure,albumin,total cholesterol,HDL-C,creatinine,NT-proBNP and pyruvate were taken as independent variables.The results showed that pyruvate[OR=1.040,95%CI(1.007,1.073),P<0.05]was the positive influencing factor for HFrEF.Conclusions:Plasma pyruvate level in patients with chronic HF was higher than that in non-heart failure subjects,which may be a positive influencing factor of chronic HFrEF. |
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