The Expression Of Bitter Taste Receptors And The Mechanism Underlying Their Regulation Of Rat Heart Function

Background:G protein-coupled receptors（GPCRs）,the seven-transmembrane domain receptors in the cell membrane,play crucial roles in regulating of intracellular signal pathways and physiological processes.GPCR convert extracellular stimuli into intracellular signals and are important drug targets.Bitter taste receptors（TAS2Rs）referring to a large family of GPCR,which mediate bitter taste in taste buds cells of the tongue.However,ongoing research has yielded new evidence that the function of TAS2Rs may beyond the oral cavity and they play significant physiological roles in systems other than gustatory.Recent research shows that TAS2Rs are expressed in airway,gastrointestinal,myometrial,and urinary systems.However,the physiology and pharmacology of TAS2Rs in cardiovascular system have been hardly reported.Recent studies have shown that TAS2Rs are expressed in cultured human cardiomyocytes and neonatal rat whole heart.TAS2Rs is associated with the nutrient-sensing function of the heart and mediates the negative inotropy effects in a G-protein dependent manner.The characteristics of TAS2Rs expression in the left ventricle of rat heart,their function and signal pathways mediated by TAS2Rs in ventricular myocytes have not been clearly elucidated.Aim:This study investigates the expression of TAS2Rs in the left ventricle of rat heart,their regulation of rat heart function,and the underlying mechanism of bitter compounds regulating the contractility of cardiac muscle.Methods:The cardiac muscle and freshly isolated cardiomyocytes of rat left ventricle were processed to detect the expression levels of TAS2Rs mRNA by real time quantitative PCR（qPCR）.Langendorff isolated heart models were used to record the heart rate,electrocardiogram（ECG）and left ventricle pressure.Isometric tension recordings were used to measure the contractility of left ventricular muscle strips induced by electrical field stimulation（EFS,1 Hz-5 V）or epinephrine.We studied the mechanisms underlying the regulation of cardiac muscle contractility by bitter compounds,denatonium,quinine and chloroquine.Ca²⁺sparks,the basic units of Ca²⁺signals,are essential in cardiac excitation-contraction coupling process.Ca²⁺sparks are evoked by the entrance of Ca²⁺through L-type Ca²⁺channel and the induced Ca²⁺release from the sarcoplasmic reticulum（SR）Ca²⁺release channel.By using confocal microscopy with isolated myocytes,we studied the effects of bitter compounds on Ca²⁺sparks in freshly isolated myocytes.Results:We detected the mRNA of six TAS2Rs,including TAS2R108,TAS2R120,TAS2R121,TAS2R126,TAS2R135,and TAS2R143 in the rat left ventricle tissue and cardiomyocytes.The mRNA level of TAS2R120 is the highest among the six detected TAS2Rs,while TAS2R108 is the lowest.In the isolated rat perfused heart,the addition to the perfusate of denatonium,quinine and chloroquine lead to the decreasing of heart rate（P<0.05）.We also showed that the activation of TAS2Rs may prolong the transduction of electrical currents of the whole heart（P<0.05）.Our data demonstrated that the bitter compounds denatonium,quinine and chloroquine significantly prolonged systolic duration and diastolic duration（P<0.05）.The perfusion of denatonium and chloroquine significantly decreased+dp/dtmax and-dp/dtmax,whileas quinine did not show significant effect on the+dp/dtmax and-dp/dtmax（P>0.05）.Quinine increased LVDP at 50μM（P<0.05）and then decreased LVDP at 100μM（P>0.05）,while chloroquine reduced the LVDP of isolated rat heart（P<0.05）.Denatonium did not show significant effect on the LVDP（P>0.05）.Results from isometric tension recording shows that quinine,chloroquine,and denatonium were effective to inhibit the EFS-induced phasic contractions of cardiac muscle strip（P<0.05）.Denatonium（20μM,50μM,100μM,200μM）and quinine（20μM,50μM,100μM,200μM）mediated a complete inhibition of epinephrine-elicited phasic contractions.At concentrations of 100μM or 200μM,both denatonium and quinine exerted quick and sustained inhibition of the epinephrine-elicited phasic contractions,whereas the phasic contractions recovered in 1 min after the inhibition by chloroquine.In order to reveal the mechanisms underlying the inhibition of EFS-and epinephrine-induced phasic contractions in left ventricle muscle strips,we utilized inhibitors for ryanodine receptors（tetracaine）,L-type Ca²⁺channel（nifedipine）,β-adrenergic receptors（propranolol）,as well as Ca²⁺-free buffer.Our data shows that EFS increases the opening probability of sarcolemmal voltage-sensitive Ca²⁺channels and the influx of Ca²⁺.The influx of Ca²⁺induces the release of Ca²⁺from the sarcoplasmic reticulum（SR）through ryanodine receptors and further increases the cytosolic Ca²⁺,leading to heart muscle contraction.The L-type Ca²⁺channel blocker,nifidepine,attenuates the EFS-induced contractions in freshly-isolated left ventricle strips,and the subsequent addition of bitter compounds inhibited the remaining phasic contractions.In the presence of its competitive inhibitor,GABA,quinine lost its ability to inhibit the phasic contractions of ventricular muscle strips（P<0.05）.In freshly isolated ventricular myocytes,three bitter compounds,denatonium,quinine,and chloroquine,inhibited the localized intracellular Ca²⁺release,the Ca²⁺sparks,measured using laser scanning confocal microscopy（P<0.05）.Conclusions:This study was the first to provid evidence for the expression of TAS2Rs in rat left ventricle of rat,and demonstrated that the activation of TAS2Rs will lead to the decrease of heart rate,the transduction function of cardiac tissue,and the electricaly and chemically induced phasic contractions of the left ventricular muscle.The activation of TAS2Rs inhibits the intracellular Ca²⁺release through ryanodine receptors from sarcoplasmic reticulum（SR）in the cardiomyocytes,which contributes to the reduction of cardiac activity.This study suggests that TAS2Rs agonists could be useful pharmacological tools for the treatment of ventricular tachycardia and heart failure.