Effects And Molecular Mechanisms Of Simvastatin And Metoprolol On Transient Outward Potassium Current And Calcium Currents In Rat Hypertrophic Cardiomyocytes

Objective:To investigate the effects and molecular mechanisms of Simvastatin and Metoprolol on transient outward potassium current(Ito) and calcium currents in rat hypertrophic cardiomyocytes.Methods:(1)Ventricular myocytes of Neonatal Spraque-Dawley (SD) rats (born from 1 to 5 days), were isolated and cultured.(2) The groups in the experiment are as follows: the control group without drugs, the induced group with 10-6mol/L Angiotensin II, and the interventional groups of Simvastatin and Metoprolol, further graded into five subgroups(①～⑤) by different concentrations of 10-8mol/L, 10-7mol/L, 10-6mol/L, 10-5mol/L, 10-4mol/L respectively, which were added 10-6mol/L Ang II for another 48 hours after 60-minute cultivations of Simvastatin or Metoprolol of these concentrations; (3) The proliferations and protein concentrations of ventricular myocytes were detected by MTT analysis and Coomassie analysis respectively.(4) Using patch-clamp technique, we recorded the following items: membrane capacitance, action potential, the current densities of transient outward current and calcium current;(5) the mRNA levels of Ito channels of Kv1.4, KV4.2 and Kv4.3 as well as calcium channels of Cav1.2, Cav3.1 and Cav3.2 were measured by Reverse Transcription- Polymerase Chain Reaction(RT-PCR);(6)α1-G protein expression levels of T-type calcium channels in hypertrophic ventricular myocytes in rats were detected with Western Blot.Results(1) Action potential(AP), Ito and ICa in hypertrophic cardiac myocytes were affected by Simvastatin. Rest potential(RP), maximum velocity of action potential(Vmax), action potential amplitude(APA), the overshort (OS) did not change significantly among the control group, the induced group and interventional groups of Simvastatin. Compared with the control group, the action potential duration(APD), APD25, APD50 and APD90 of the induced group have a significant delay respectivlely(p<0.05). However, no obvious change was observed between the induced group and the intervention group①or the intervention group②(p>0.05). APD25, APD50 and APD90 in the intervention group③, the intervention group④, and the intervention group⑤,were significantly shorted than that in the induced group(p <0.05).Compared with control group, the densities and the peak values of T and L-type calcium of hypertrophic ventricular myocyte were higher, but the density and the peak value of Ito were lower. There are no significant change in T and L type Ica and Ito among the induced group, the intervention group①and the intervention group②(p>0.05). However, compared with the induced group, the densities and the peak values of ICa-L, ICa-T increased in the intervention group③, the intervention group④and the intervention group⑤. The activated voltage, reversal potential, steady state-activation, steady state-inactivation, and recover time from inactivation, have no changes among the control group, the induced group and the interventional groups(p>0.05).(2) The mRNA expression levels of Ito and ICa in hypertrophic ventricular myocytes were affected by Simvastatin.Compared with the control group, the mRNA level of Kv4.2, Kv4.3 in the induced group decreased significantly (p<0.05); while Kv1.4, Cav1.2, Cav3.1, and Cav3.2 increased obviously (p<0.05); and so did theα1-G protein level of T type calcium channels(p<0.05). However, compared with the induced group, Kv4.2, Kv4.3, Kv1.4, Cav1.2, Cav3.1, and Cav3.2 have no significant change in the intervention group①and the intervention group②( p>0.05), the same as theα1-G protein level of T type calcium channel; As for the mRNA expression levels of Kv4.2, Kv4.3 in the inter- ventional group③, interventional group④and the interventional group,all increased significantly (p<0.05); and Kv1.4, Cav1.2, Cav3.1, and Cav3.2 decreased obviously (p<0.05), the same as theα1-G protein level of T type calcium channel(p<0.05).(3) Action potential(AP), Ito and ICa in hypertrophic cardiac myocytes were affected by Metoprolol.Rest potential(RP), maximum velocity of action potential(Vmax), action potential amplitude(APA), the overshort (OS) did not change significantly among the control group, the induced group and interventional groups of Metoprolol. Compared with the control group, action potential duration(APD), APD25, APD50 and APD90 of the induced group delayed significantly (p<0.05). However, no obvious change was observed between the induced group and the intervention group①or the intervention group②(p>0.05). APD25, APD50 and APD90 in the intervention group③, the intervention group④, and the intervention group⑤, shorted significantly than that in the induced group(p <0.05). Compared with control group, the densities and the peak current value of the T and L type calcium of hypertrophic ventricular myocytes were higher, but the density and the peak value of Ito were lower. There were no significant change in T and L type calcium current and Ito among the induced group, the intervention group①and the intervention group②(p>0.05). However, compared with the induced group, the densities and the peak value of ICa-L, ICa-T increased in the intervention group③, the intervention group④and the intervention group⑤. The activation voltage, reversal potential, steady stated activation, steady stated inactivation, and the recover time from inactivated state, had no change among the control group, the induced group and the interventional groups(p>0.05).(4) The mRNA expression levels of Ito and ICa in hypertrophic ventricular myocytes were affected by Metoprolol.Compared with the control group, the mRNA expression levels of Kv4.2, Kv4.3 in the induced group decreased significantly (p<0.05); of Kv1.4, Cav1.2, Cav3.1, and Cav3.2 have obviously increased(p<0.05); and so do theα1-G protein level of T type calcium channel(p<0.05). However, compared with the induced group, Kv4.3, Kv1.4, Cav1.2, Cav3.1, and Cav3.2 have no significant change in the intervention group①and the intervention group②(p>0.05), and so does theα1-G protein level of T type calcium channel. Compared with the induced group, the mRNA express level of Kv4.2, Kv4.3 in the intervention③,the intervention group④and the intervention group⑤increased significantly (p<0.05); while Kv1.4, Cav1.2, Cav3.1, and Cav3.2 decreased obviously (p<0.05); and so did theα1-G protein level of T type calcium channel(p<0.05). Conclusions:(1) Compared with the control group, the action potential duration of hypertropic myocardial delayed significantly, the densities of T type and L type calcium current increased obviously and Ito declined, which suggests an abnormal repolarization in myocardial hypertrophy underlying the main electrical remodeling of myocardial hypertrophic and leading to the prolongation of the action potential duration in hypertrophic myocardium. The mechanism of electrical remodeling is attribute to down regulation of the mRNA level of Kv4.2 and Kv4.3, while up regulation of Kv1.4, Cav1.2, Cav3.1, and Cav3.2 along with the increased regulation of theα1-G protein expression level.(2) With the treatment of Simvastatin, the current densities of T and L type calcium decreased, the density of transient outward current increased and the action potential duration was shortened. At mRNA levels, Cav3.1 and Cav3.2, Cav1.2 decreased as well as theα1-G protein expression level while Kv4.2, Kv4.3 encoding transient outward current channels increased along with Kv1.4 decreased, all of which indicated that Simvastatin rectifies the abnomal repolarization, counteracting and/or reversing ventricular remodeling.(3) With the application of Metoprolol, the densities of T and L type calcium current could be decreased with transient outward current increased and action potential duration shortened, then the gene expression levels of Cav3.1 and Cav3.2, Cav1.2 encoding T and L type calcium channels decreased as well as theα1-G protein expression level while Kv4.2, Kv4.3 encoding transient outward current channels increased along with Kv1.4 decreased, all of which indicated that Metoprolol rectifies the abnomal repolarization, counteracting and/or reversing ventricular remodeling.