Discovery The Endogenous Bitter Taste Receptor Agonists In Bile Acids And The Study Of Bitter Compounds Regulation Of Heart Function

Background:Bitter taste receptors(Tas2rs)are G protein-coupled receptors(GPCR)and express on taste cells membrane.They are the biological basis for organisms to have a bitter sensation.GPCRs are the essential cell membrane receptors that regulate the physiological functions of the cardiovascular system.Neurotransmitters,hormones,and cytokines regulate cardiovascular function by activating corresponding GPCRs.The discovery of endogenous GPCR agonists is vital for cardiac physiology.There are twenty-five human Tas2rs in non-gustatory tissues,including the respiratory tract,gastrointestinal tract,brain,bladder,uterus,and cardiovascular system.Tas2rs have specific physiological functions in various organ systems.Tas2rs agonists can reduce heart rate,regulate heart metabolism and nutrition sense.The extensive-expression and physiological function of Tas2rs suggest that Tas2rs endogenous agonists may exist and affect cardiac function.Bile is endogenous bitter substance in humans.The bile acids in the saliva and blood of patients with liver disease induce a bitter sensation,and it might be mediated by the activation of Tas2rs on the tongue.At present,there are no confirmed endogenous Tas2rs agonists.In this project,we studied the Tas2rs endogenous agonists using the functional study of bile acids on left ventricular musle.We further studied the candidate compounds'effect on rat cardiac function and determined the underlying mechanisms.Both in vivo and in vitro experiments have showed that Tas2rs signals interacted with adrenergic recepters signals in hearts.The computer-aided modeling were also used to theoretically calculate and analyze the binding ability of different bile acids with Tas2rs.Then use cell biology experiments to screen out bile acids with higher activating Tas2rs effects.We found that the ability of different bile acids to activate Tas2rs is consistent with their ability to affect heart function.Therefore,our project explored the effects and mechanism of bile acid on heart function with the ventricular muscle function experiment,organ,tissue or cell physiology and pharmacology experiments.We preliminarily judged the Tas2rs endogenous agonists from both theoretical and experimental aspects.Our results provided the foundation for studying Tas2rs as the potential target for treating specific heart disease.Research objectives:1.Screening a potential endogenous agonist of Tas2rs in bile acids;2.Finding evidence for Tas2rs as a therapeutic target of the treatment of heart disease.Method and results:1.The effects and mechanism of CDCA and DCA on rat heart function(1)Using organbath experimeriment to determine the effect of bile acids on the left ventricular muscle contractions and the data revealed that chenodeoxycholic acid(CDCA)and deoxycholic acid(DCA)increased the ventricular muscle contractility dependent on their concentrations.Our immunocytochemistry experiments showed that Tas2r108,120,and their coupled G?gustdu_cin protein expressed in rat left ventricular myocytes.The immunofluorescence intensity of G?gustducin and the Pearsons's colocalization ratio of G?_gustducin anda-actinin significantly decreased after the treatment with quinine,CDCA,and DCA(100?M).The results indicated that Tas2rs activation led to the change of G?gustducin spatial localization.(2)The CDCA and DCA effects on rat heart function and the underlying mechanismWe used the Langendorff perfused isolated rat heart to determine the effects of cumulative addition of CDCA and DCA on the heart rate,heart contractility,and ECG.The results showed that in the concentration range of 10?100?M,CDCA and DCA gradually increased myocardial contractility and decrease heart rate.In the concentration range of 50-100 m M,both CDCA and DCA significantly increased the left ventricular developed pressure(LVDP)and the maximum rate of positive and negative change in LV pressure(±d P/dtmax)compared with the control group(P<0.05),and decreased the heart rate(HR)(P<0.05).Both CDCA and DCA(100?M)significantly prolonged the RR and QT interval,and at 50?M,they significantly prolonged the QRS interval.When their concentration reached 200?M,both CDCA and DCA arrested the isolated heart,indicating that the two bile acids may be toxic to the isolated heart at high concentrations.To determine whether CDCA and DCA directly exert a positive inotropic effect on the left ventricular muscle,we used an isolated tissue bath system to test their effects on left ventricular muscle(LVM)isometric contractions paced by 1 Hz electrical stimulation.The results show that the LVM developed-tension significantly increased30 minutes after CDCA and DCA(100?M).CDCA and DCA(200?M)also significantly increased the developed tension and TTI of the left ventricular muscle.However,when the concentration increased to 300 or 500?M,CDCA and DCA inhibited the EFS-paced myocardial contractions.We used laser scanning confocal fluorescence imaging technology to determine the bile acid effects on calcium signal and[Ca²⁺]_i of rat cardiomyocytes.The results show that both CDCA and DCA(100?M)significantly increased the[Ca²⁺]_i of resting cardiomyocytes,the frequency of calcium sparks,and amplitude of spontaneous calcium waves(P<0.05).The correlation analysis of CDCA and DCA effects on the heart function,LVM,and cardiomyocytes show that LVM development tension increase positively correlated with the isolated-heart LVDP increase and the myocyte[Ca²⁺]_iincrease,respectively.To determine the mechanism of CDCA and DCA,reducing heart rate,we tested CDCA and DCA on the beating frequency of the isolated sinoatrial node tissue.The sinoatrial node's average beating frequency in the K-H solution saturated with oxygen at37?was 334.2±10.7 beats/min.CDCA and DCA(10-200?M)concentration-dependently reduced the beating frequency.CDCA(150?M)and DCA(100?M)reduced the beating frequency to 80.6±3.0%and 79.7±0.9%,respectively.In summary,Tas2r108 and 120 proteins express in cardiomyocytes and regulate rat hear functions.CDCA and DCA increased the developmental tension of left ventricular muscle by increasing cardiomyocytes[Ca²⁺]_i,and ultimately led to the positive muscle strength of the isolated heart.CDCA and DCA also reduced the rat heart rate by suppressing the beating frequency of the sinoatrial node.2.Bitter compounds inhibit epinephrine-induced heart rateA previous study found that multiple Tas2rs agonists could suppress the beating rate of isolated rat hearts.The Tas2r agonists,quinine,denatonium,and chloroquine,inhibited the 10?M epinephrine-induced phasic contractions in LVM.Our current study shows that CDCA and DCA(200?M)also completely inhibited the epinephrine-induced LVM phasic contractions.We speculate that Tas2rs might inhibit the?-adrenergic signaling in the heart.(1)In vivo heart function experiment:(1)SD rats were selected and randomly divided into four groups according to the heart rate measured by the tail vein.We administered 100 mg/kg,150 mg/kg,200 mg/kg quinine,and 0.01 m L/g 0.9%normal saline to the four groups via intraperitoneal injection.The in vivo ECG showed that quinine could dose-dependently reduce rats'heart rate and prolong PR,RR,QRS,QT intervals,and P wave duration(P<0.05).(2)The SD rats were divided into three groups:epinephrine group,epinephrine and 100 mg/kg quinine group,epinephrine and 200mg/kg quinine group.Intraperitoneal injection of 0.8 mg/kg epinephrine caused acute tachycardia in rats.The average heart rate increased from 376.6±32.3 beats per minute to 478.1±32.2 beats per minute.After 30 minutes of epinephrine injection,the heart rate reached a stable level,and then intraperitoneal injection with quinine(100,200mg/kg)followed.Quinine(100,200 mg/kg)significantly reduced the heart rate of rats(P<0.05)30 min after injection.(2)Isolated heart function experiment:The addition of 0.1?M epinephrine increase the beating rate of the Langendorff-perfused isolated rat hearts,and the subsequent cumulative addition of quinine,denatonium benzonate,or chloroquine attenuated the epinephrine-induced tachycardia(P<0.05)and prolonged the systolic duration and diastolic duration(P<0.05).However,quinine and denatonium benzoate did not affect the LVDP and±d P/dtmax of the perfused heart.(3)Acutely-isolated left ventricular myocytes experiment:Epinephrine(10?M)could significantly increase the frequency of spontaneous calcium waves in isolated cardiomyocytes(P<0.05)and maintained a stable level for 4-10 minutes.The subsequent perfusion with quinine(50-100?M)4 min after epinephrine significantly reduced epinephrine-induced calcium wave frequency.3.Theoretical prediction and cell biology test of Tas2rs endogenous agonist(1)Use a computer simulation to model Tas2rs proteins,and then use molecular docking with bile acids to predict their binding ability.In this study,we used SWISS-MODEL,GPCR-I-TASSER,and DS 3.0 homology modeling to establish the protein model of Tas2rs.According to the Model Appraising and the ROC curve of molecular docking score with Tas2rs agonists and decoy molecules,we selected a proper method to model Tas2rs.The area under the ROC curve of the GPCR-I-TASSER-constructed Tas2r46 model for identifying agonists and decoy molecules was 0.77±0.03,and it was the highest close to one.This result indicated that the GPCR-I-TASSER method was the best option for Tas2rs molecular modeling.We used this method to construct the model 25 subtypes of human Tas2rs and evaluate the molecular docking with 15 bile acids.The average C-score was higher than 7.0 and was similar to that of the positive control,the Tas2r4 agonist taurocholic acid(7.4).These results indicated bile acids could be the candidate agonists of Tas2rs.(2)Use HEK293T cells to determine bile acids be the potential Tas2r agonists.RT-PCR experiments detected 23 subtypes of Tas2rs m RNA in HEK293T cells.The immunocytochemical staining using the specific antibodies showed the protein expression of Tas2r3,4,14,21,46 in HEK293T cells.The bitter compounds quinine,and difenidol,increased the intracellular Ca²⁺levels in HEK293T cells in a concentration-dependent manner with p EC50 of 1.62 and 1.25.Both?-aminobutyric acid and(500?M)and N?,N?-Bis(carboxymethyl)-L-lysine hydrate(50?M),the competitive inhibitor of quinine binding to Tas2r4,significantly inhibited the calcium increase induced by quinine.These results further support the expression of the functional Tas2rs in HEK293T cells.We used HEK293T cells as a screening tool for potential Tas2rs agonists in the bile acids.The Ca²⁺imaging exprimetns with HEK293T cells show that chenodeoxycholic acid(CDCA)and deoxycholic acid(DCA)(200,500?M)increased the intracellular calcium level more effectively than the positive control compounds,quinine and diphenidol at the same concentration.CDCA and DCA increased intracellular Ca²⁺concentration with p EC50 of 3.51 and 3.56,respectively.Conclusion:This study preliminarily screened the effects of different bile acids on ventricular muscle function and found that CDCA and DCA can effectively increase ventricular muscle developed tension.CDCA and DCA can cause positive inotropic and negative chronotrophic effects,and their influence on cardiac function is dose dependent.In order to study the regulation of Tas2rs on cardiac function,the protein expression of Tas2r108 and 120 was detected in adult rat cardiomyocytes and co-localized with the?-actinin.When Tas2rs were actived,the relative position of G?_gustducin and?-actinin has changed significantly.This study also found that the Tas2rs agonists quinine,benzodiazepine and chloroquine can significantly inhibit the heart rate increasing caused by the epinephrine,suggesting that Tas2rs signaling may inhibit the adrenetic receptor signaling in the heart.The results suggest that Tas2rs in the heart are related to heart function,reveal the influence of bile acids on heart function,and provide part of the experimental basis for studying the relationship between bile acid elevation and heart disease in the body.In order to explore the possibility of binding between bile acids and Tas2rs,this project also used the method of computer-aided drug design.Through the comparison of three protein model construction methods,the GPCR-I-TASSER method is selected to construct the Tas2rs protein model,and the Tas2rs protein is used.The protein models were docked with 15 kinds of bile acid,and it was preliminarily judged that bile acids could be candidate compounds for the endogenous agonist of Tas2rs.This subject discovered for the first time that HEK293T cells expressed m RNAs of 23 endogenous Tas2rs subtypes and at least 5 subtypes of proteins.Tas2rs mediated the increase of calcium ions in HEK293T cells,and based on this experiment,it preliminarily judged that chenodeoxycholic acid(CDCA)and deoxycholic acid(DCA)are potential endogenous agonists of Tas2rs.