Mechanisms Of Ca²⁺ Regulation By Berberine In Adult Rat Cardiomyocytes Under Physiological And Hypertrophic Conditions

Background:Nowadays,death from cardiovascular diseases has been the leading cause of the total deaths of urban(43.56%)and rural(45.91%)residents.These are far greater than those caused by cancer or respiratory disease.Heart failure is the end stage of multiple heart diseases,and ventricular remodeling is regarded as a key factor for its genesis.Berberine is an effective ingredient in Coptis Chinensis Franch,which is commonly used to treat gastrointestinal infections and bacillary dysentery in clinical.Recent decades,it has attracted great attention for its inhibitory effects on various cardiovascular diseases,such as hypertension,cardiac hypertrophy,heart failure and hyperlipidemia.Some clinical studies showed the beneficial effects of berberine on the heart function.Moreover,its anti-hypertrophy effects have been widely verified in multiple animal models.Therefore,it is meaningful to clarify the underlying mechanisms for its application from bench to bedside.Ca²⁺is an important second messenger in cells.It can regulate the contractility of cardiomyocytes through the excitation-contraction coupling(ECC)signaling pathway.Besides,Ca²⁺-dependent signaling pathways also play a pivotal role in the activation of hypertrophic gene transcription,which further promotes the development of cardiac hypertrophy.Berberine shows positive inotropic effects both in physiological and pathological states.However,the underlying mechanisms were unclear.So far,most studies merely reported the effects of berberine on intracellular Ca²⁺level and LTCCs in cardiomyocytes.These studies could not draw consistent conclusions because of variable experimental factors,such as species of experimental animals,physiological solutions and so on.Moreover,little is known about the potential relationship of inhibitory effect of berberine on cardiac hypertrophy and Ca²⁺regulation.Previous study of our team showed that berberine had positive inotropic effects on isolated hearts and left ventricular tissues of healthy rats,and inhibited the pressure overload-induced cardiac hypertrophy in rats.As a continuation of the previous study,this current study aimed to explore the mechanisms of Ca²⁺regulation by berberine in ARCs under the physiological condition,and mechanisms of its inhibitory effects on the cardiac hypertrophy from the perspective of Ca²⁺regulation.We expected this study would provide a sound theoretical basis for the prevention and treatment of heart failure with berberine in clinical.This would be profound for the treatment of cardiovascular disease.Objectives:1.To study the mechanisms of Ca²⁺regulation by berberine in ARCs under the physiological condition;2.To illuminate the underlying mechanisms of the inhibitory effect of berberine on cardiac hypertrophy from the perspective of Ca²⁺regulation.Methods and Results:1.The effect of berberine on the intracellular Ca²⁺level of ARCs under the physiological condition.Methods:To study the effect of berberine on the intracellular Ca²⁺level of ARCs,experiments were carried out by using laser confocal microscope with Ca²⁺probe Fluo4-AM.Results:30-300?M berberine significantly increased the intracellular fluorescence intensity in ARCs in the quiescent state.100-300?M berberine increased the intracellular fluorescence intensity at the peak and baseline of Ca²⁺transient in paced-ARCs(15 V,4 ms,1Hz).These suggested that berberine can increase the intracellular Ca²⁺in ARCs.2.The effect of berberine on the Ca²⁺influx in ARCs under the physiological condition.Methods:(1)To study the influence of berberine on the Ca²⁺influx in ARCs,Ca²⁺-free KH buffer was employed.(2)Then,nifedipine and FPL-64176,the antagonist and agonist of LTCCs,respectively,were used to investigate whether the LTCC-mediated Ca²⁺influx was involved in the action of berberine.(3)Inhibition of the reverse mode of NCX was accomplished with low Na⁺-KH buffer,Ni²⁺and KB-R7943.This was used to test the role of the Ca²⁺influx via the reverse mode of NCX in the action of berberine.(4)To detect the effect of berberine on the expression of associated ion channels in ARCs,RT-PCR and Western-blot experiments were performed.Results:(1)The 300?M berberine-induced increase in the fluorescence intensity in ARCs was significantly inhibited in the Ca²⁺-free KH buffer(p<0.001).(2)10?M nifedipine had no influence on the 300?M berberine-induced increase in the fluorescence intensity.10?M FPL-64176 could increase the fluorescence intensity in ARCs,and the subsequent addition of 300?M berberine caused a further increase in the fluorescence intensity(p<0.001).(3)The 300?M berberine-induced increase in the intracellular fluorescence in ARCs was significantly hindered in low Na⁺KH buffer or in the presence of 1 m M Ni Cl₂ and 5?M KB-R7943,respectively(p<0.001).(4)30-300?M berberine had no effect on the NCX1 expression at the m RNA and protein level.These suggested that berberine enhances Ca²⁺influx via activating the reverse mode of NCX.3.The effect of berberine on the Ca²⁺release in ARCs under the physiological condition.Methods:(1)To determine the effect of berberine on the sarcoplasmic reticulum(SR)Ca²⁺release,Ca²⁺sparks were observed by using laser confocal microscope with Ca²⁺indicator Fluo4-AM.Then,this was confirmed with the blocking of SR Ca²⁺release by1.5 m M tetracaine.(2)Inhibition of the PKA and Ca MK?mediated phosphorylation in Ry Rs by H-89 and KN-93,respectively,was conducted to verify the effect of berberine on the activity of Ry Rs.(3)Experiment with 10 m M caffeine was carried out to examine the effect of berberine on the SR Ca²⁺content.(4)To detect the effect of berberine on the expression of Ry R2 and SERCA2a,RT-PCR and Western-blot experiments were performed.Results:(1)The mean mass of Ca²⁺sparks significantly increased after incubation with berberine for 1 min(p<0.01),and their total events and frequency also increased,but without significance.The 300?M berberine-induced increase in the fluorescence intensity of ARCs was significantly blocked by 1.5 m M tetracaine(p<0.001).(2)Both3?M H-89 and 10?M KN-93 could significantly reduce the 300?M berberine-induced increase in the fluorescence intensity(p<0.001).(3)300?M berberine significantly increased the amplitude(p<0.001)and the fluorescence intensity-time integral of 10m M caffeine evoked-Ca²⁺transient(p<0.01).(4)30-300?M berberine had no effects on the expression of Ry R2 and SERCA2a.These suggested that berberine enhances SR Ca²⁺release by activating Ry Rs.This was correlated the PKA and Ca MK?mediated phosphorylation in Ry Rs and the increased SR Ca²⁺content.4.The effect of berberine on the expression of genes involved in the ECC signaling pathway under the hypertrophic condition.Methods:(1)Cardiac hypertrophy was induced in rats by abdominal artery banding(AAB).Blood pressure of right forelimb and right carotid artery of rats were detected by using non-invasive blood pressure meter and acquisition system of biological signal,respectively.Heart index,interstitial collagen deposition,cardiomyocyte cross sectional area,as well as Nppa and Nppb m RNA expression were used to evaluate the effects of berberine on the pressure overload-induced cardiac hypertrophy in rats.(2)Cardiac hypertrophy was constructed in ARCs with 1?M NE or 10?M PE.Cell surface ratio and the m RNA expression level of Nppa and Nppb were used to evaluate the effect of berberine on the cardiac hypertrophy in ARCs.(3)To estimate the effect of berberine on the expression of SERCA2a,Ry R2 and NCX1 under the hypertrophic condition,RT-PCR and Western-blot experiments were carried out with rat left ventricular tissues and ARCs.Results:(1)After the 24-week berberine treatment,systolic and mean blood pressure of the right forelimb artery and systolic blood pressure of the right carotid artery were significant lower than those of AAB group(p<0.05).(2)80 mg/kg berberine could significantly inhibit hypertrophic response,evaluated by reduced heart index,interstitial collagen deposition,cross sectional area,as well as Nppa and Nppb m RNA expression in left ventricular tissues of AAB rats.(3)1-3?M berberine and 3-6?M berberine could significantly reduce the cell surface ratio of ARCs stimulated with 1?M NE and 10?M PE,respectively.Moreover,the 10?M PE-induced increases in the expression of Nppa and Nppb m RNA in ARCs were diminished by 3?M berberine.(4)In vivo,the expression level of SERCA2a and Ry R2 m RNA and protein showed decrease and increase in rat left ventricular tissues,respectively;the 80 mg/kg berberine-treatment had no influences on those.No significant difference appeared both at the m RNA and protein level of NCX1 in sham,AAB and AAB with berberine group.In vitro,the SERCA2a m RNA expression level showed no significant difference in ARCs treated with vehicle,10?M PE,and 3?M berberine plus 10?M PE.10?M PE downregulated Ry R2 m RNA expression level in ARCs,but without significance.3?M berberine had no impact on this.NCX1 m RNA expression level increased with no significance,which could be inhibited by 3?M berberine(p<0.05).At the protein level,10?M PE upregulated the expression of SERCA2a and Ry R2 with significance,and berberine showed no effects on this.NCX1did not change among ARCs treated with vehicle,10?M PE,and 3?M berberine plus 10?M PE.These suggested that berberine can inhibit cardiac hypertrophy both in vivo and in vitro,which was not accomplished by regulating the expression of genes involved in the ECC signaling pathway.5.The effects of berberine on the expression of STIM1 and the STIM1-dependent SOCE under the hypertrophic condition.Methods:(1)To explore the effect of berberine on the expression of STIM1 under the hypertrophic conditions,RT-PCR and Western-blot experiments were carried out.(2)Laser confocal microscope was used with Ca²⁺indicator Fluo4-AM to evaluate the effect of berberine on the STIM1-dependent SOCE in ARCs under the hypertrophic condition,Results:(1)Berberine showed no effects on the STIM1 m RNA expression under the hypertrophic conditions both in vivo and in vitro.At the protein level,Berberine significantly inhibited the increase in the STIM1 expression in AAB rats(p<0.01),and the 10?M PE-stimulated ARCs(p<0.01).(2)The 10?M PE-induced increase in the SOCE in ARCs was significantly attenuated by 3?M berberine(p<0.001).Conclusions:1.Under the physiological condition,berberine enhances Ca²⁺influx and SR Ca²⁺release via activating the reverse mode of NCX and Ry R,respectively,which ultimately contributes to the intracellular Ca²⁺increase in ARCs.The activation of Ry R may be associated with its phosphorylation by PKA and Ca MK?and the increased SR Ca²⁺content.2.Berberine inhibits cardiac hypertrophy in rats by downregulating the expression of STIM1 at the protein level and consequently decreases the STIM1-dependent SOCE.However,the effect of berberine on cardiac hypertrophy is not correlated to its regulation of the expression of ECC related genes.