Effects Of Aldosterone On Delayed Rectifier Potassium Current In Adult Guinea Pig Cardiomyocytes

Pathological electrophysiological remodelling in cardiac hypertrophy, heart failure makes electrophysiological instability, which leads to arrhythmias. More than 50% of patients with heart failure die of sudden cardiac death(SCD) due to malignant arrhythmias. The systemic and local renin-angiotensin-aldosterone system(RAAS) plays a very important role in regulating cardiovascular function but may contribute to the progression of various cardiovascular diseases, including cardiac hypertrophy and heart failure. In addition to the well-known effector of RAAS, angiotensin II, more and more evidences show that aldosterone not only involves in regulating cardiac ion channel protein expression and participating electrophysiological remodelling besides regulating water electrolyte balance, blood volume and pressure by modulating the reabsorption of water and sodium in kidney. Two clinical trials have indicated that aldosterone antagonists, in combination with standard therapy, significantly reduce the occurrence of sudden cardiac death among patients with severe heart failure or acute myocardial infarction. The results suggest that aldosterone may contribute to ventricular arrhythmogeneity by modulation of cardiac ionic channels through mineralocorticoid receptor(MR)-mediated effects.Action potential duration(APD) prolongation of ventricular cardiomyocytes is a characteristic electrophysiological remodelling in heart failure(HF) and hypertrophy. Rapid ventricular arrhythmias most likely result from the combination of triggered activity such as early after depolarization(EAD) and reentry induced by enhanced dispersion of repolarization across the ventricular wall. A lot of experiments have shown that reduced depolarization K+ current is the important reason of APD prolongation. Delayed-rectifier K+ current(IK)plays a key role in cardiac repolarization, which includes rapid delayed-rectifier K+ current(IKr) and slow delayed-rectifier K+ current(IKs). Human ether-a-go-go-related gene(h ERG or KCNH2) encodes the pore forming subunit of the channel underlying IKr. The channel carrying IKs is a heteromultimer consisting of α-subunit encoded by KCNQ1 and β-subunit encoded by KCNE1. It has been proven that mutation of h ERG, KCNQ1 or KCNE1 is the molecule base of cardiac channelopathies. Dowregulation of IKr or IKs plays an important role for APD prolongation in hypertrophy and HF. Our previous research has shown that effector of RAAS, angiotensin II, downregulates IKr via angiotensin II receptor 1(AT1). Several studies have demonstrated that aldosterone upregulates L-type and T-type Ca2+ channels and downregulates transient outward K+ current(Ito) in ventricular myocytes. However, up to now, available information is limited regarding the effect of aldosterone on delayed-rectifier K+ current in cardiac ventricular myocytes. Guinea pig hearts are considered to lack endogenous Ito and thus delayed-rectifier potassium currents, IKr and IKs constitute major repolarizing outward currents in ventricular myocytes. Our previous experimental data have demonstrated that aldosterone prolongs APD in adult guinea pig papillary muscle, which suggests aldosterone may modulate IKr and IKs. Therefore, the present study was designed to investigate the modulation of aldosterone on IKr and IKs in adult guinea pig ventricular myocytes in hyperaldosteronemia animals and in in vitro cultured isolated cardiomyotes using electrophysiological technique and analyse the mechanism of action.Part 1 Effects of hyperaldosteronemia on delayed rectifier potassium currents in adult guinea pig cardiomyocytesOblective: To investigate whether hyperaldosteronemia regulates delayed rectifier potassium currents in adult guinea pig cardiomyocytesMethods: Male adult guinea pigs weighing 200–250 g were acclimated to their new environment for one week. Animals were treated with aldosterone over a period of 28 days via in implanted osmotic minipump. Aldosterone was dissolved in polyethylene glycol 400(PEG-400, aldosterone release 1μg·h–1). Pumps were implanted subcutaneously under anesthesia with tribromoethanol (225 mg·kg–1) by intraperitoneal injection. Control animals were treated with vehicle. The animals(10 per group) had free access to food containing 0.5% sodium chloride and water and were maintained in a constant environment with a conventional 12 h/12 h light–dark cycle starting at 6 am.After 4 weeks of drug treatment(day 28), 1.5-2ml of guinea pig blood was taken to prepare plasma. Plasma sodium and potassium levels were measured. Then hearts were rapidly excised and left ventricular myocytes were freshly isolated by enzymatic method. The conventional whole-cell patch-clamp technique was used to record APD, IKr and IKs respectively in fresh isolated ventricular cardiomyocytes. Borosilicate glass electrodes had tip resistances of 1–3 M? when filled with the pipette solution. We used V-clamp to record IKr and IKs respectively in fresh isolated ventricular cardiomyocytes. The pipette solution contained(in m M) KCl 140, Mg-ATP 4, Mg Cl2 1, EGTA 5, and HEPES 10, with p H adjusted to 7.2 with KOH. The external solution contained(in m M) Na Cl 132, KCl 4, Ca Cl2 1.8, Mg Cl2 1.2, glucose 5 and HEPES 10; with p H adjusted to 7.4 with Na OH. Nimodipine(Nim, 1 μM) was added to the external solution to block the L-type Ca2+ current. Na+ and T-type Ca2+ currents were inactivated by holding a potential of-40 m V. To record IKr, chromanol 293B(20 mM) was added to the external solution to block IKs. To record IKs, E4031(2 mM) was added to the external solution to block IKr. Action potential recordings were performed in isolated guinea pig ventricular myocytes using the I-clamp patch technique at 0.25 Hz, 0.5Hz, 1Hz respectively. The pipette solution contained(in m M):glutamate potassium 120, KCl 25, Mg Cl2 1, Ca Cl2 1, HEPES 10(p H 7.4 with KOH);The external solution contained(in m M):Na Cl 138, KCl 4, Mg Cl2 1, Ca Cl2 2, Na H2PO4 0.33, glucose 10 and HEPES 10(p H 7.4 with Na OH).All experiments were performed at room temperature(24–25° C) using an Axopatch 700 B amplifier(Molecular Devices, USA). The electrical signals were sampled at 2.5–10 k Hz, filtered at 1 k Hz using a low-pass filter, and digitized with an A/D converter(Digidata 1322; Axon Instruments). The p Clamp software(Version 10.2; Molecular Devices) was used to generate voltage-pulse protocols and to acquire and analyse the data.Data are expressed as means ± SEM. SPSS 19.0 software was used for data analysis. Group comparisons were performed with unpaired Student’s t tests(for single 2-group comparisons) and ANOVA with Dunnett’s post hoc tests(for multiple-group comparisons). The Chi-square test was used for incidence rate comparisons. Differences were considered significant if P<0.05.Results:1 There were no significant differences in the levels of serum potassium and sodium among different groups(P>0.05). Our previous experimental data have demonstrated that this experimental hyperaldosteronemia model does no effects on blood pressure.2 Hyperaldosteronemia significantly prolonged APD90 in adult guinea pig cardiomyocytes.3 Hyperaldosteronemia downregulated IKs in adult guinea pig cardiomyocytes.However, aldosterone treatment did not alter the voltage-dependent activation of the channel and the half-maximal activation potentials were 37.4 ± 2.8 m V in control and 39.3±2.0 m V in aldosterone-treated animals(P>0.05), respectively.4 Aldosterone treatment did not alter IKr density and voltage-dependent activation of the channel. These results show that regulation of aldosterone on potassium channels are selective.Conclusion: Hyperaldosteronemia downregulates IKs in adult guinea pig cardiomyocytes and thus delays the ventricular repolarization, independent of blood pressure and electrolyte disorder. Aldosterone treatment has no action on IKr, which suggests regulation of aldosterone on potassium channels are selective.Part 2 Effects of adosterone on delayed rectifier potassium currents in cultured cardiomyocytes of adult guinea pigsObjective: To recapitulate the in vivo experiments, we further tested the effect of aldosterone on delayed rectifier potassium currents in in vitro cultured cardiomyocytes.Methods: Guinea pig ventricular cardiomyocytes were isolated aseptically. Cells were reintroduced to calcium by a stepwise. Fresh medium(Hyclone M199+Earle’s salts and L-glutamine) was then added containing aldosterone(1μmol·L–1) or vehicle in dimethyl sulphoxide(DMSO). Cells were kept in culture for an additional 24 hours in 37° C, 5% CO2 cell incubation box. The medium M199 was supplemented with 10% fetal calf serum, 2m M L-carnitine, 5m M sarcosine, and 100 IU·ml–1 penicillin and 100 μg·ml–1 streptomycin.The conventional whole-cell patch-clamp technique was used to record IKs and IKr respectively in in vitro cultured cardiomyocytes and to test the action of hyperaldosteronemia on IK in in vitro cultured cardiomyocytes.Results:1 Aldosterone downregulated IKs in in vitro cultured cardiomyocytes, which is in concentration-dependent manner. However, aldosterone did not affect the voltage-dependent activation of the channel.2 Sustained aldosterone exposure had no significant effect on IKr density and voltage-dependent activation of the channel.Conclusion: Aldosterone selectively downregulates IKs in in vitro cultured cardiomyocytes, which is consistent with the result in in vivo experiments.Part 3 Mechanism underlying the downregulation of IKs by aldosterone in ventricular myocytesObjective: To address potential mechanism for the downregulation of IKs by aldosterone in hyperaldosteronemia animal models or cultured cardiomyocytes were used to show how aldosterone downregulates IKs in ventricular myocytes.Methods: The method for treating the whole animals with aldosterone was same as the first part. To assess the involvement of MR activation, some aldosterone-treated animals concomitantly received the MR antagonist spironolactone(100 mg·kg–1·day–1)by gavage. After 28 days treatment,the IKs current was recorded and the current density was compared between ldosterone-treated animals and those treated with aldosterone in combination of spironolactone.The method of culturing cardiomyotes was same as the first part. To test whether mineralocorticoid receptor, angiotensin Ⅱ /angiotensin Ⅱ receptor1(ANGⅡ/AT1R)and calcium were involved in the regulation of IKs, some tool drugs were added to cuture cardiomyocytes for 24 h along with aldosterone, which included MR antagonist spironolactone(10μmol·L–1), hot shock protein 90(HSP90) inhibitor geldanamycin(1μmol·L–1), angiotensin coverting enzyme inhibitor(ACEI) enalapril(1μmol·L–1), angiotensin receptor 1(AT1) blocker losartan(1μmol·L–1), Ca2+ channel blocker nimodipine(1μmol·L–1), permeant Ca2+ chelator BAPTA-AM(2.5μmol·L–1). Cells from the same batch were in parallel exposed to aldosterone as an internal control.The conventional whole-cell patch-clamp technique was used to record IKs in in vitro cultured cardiomyocytes and to test whether these tool drugs were involved in the regulation of IKs.Total m RNA was extracted from cells isolated from animals after 28 days treatment or cells after 24-hr culture in intervention- or vehicle- medium. Real-time PCR was performed to test whether aldosterone acts on the KCNQ1,KCNE1 and ERG m RNA level of IK channel. At the same time the membrane protein or total protein was extracted from cells. Westernblot was used to test whether aldosterone acts on the KCNQ1,KCNE1 and ERG protein level of IK channel.Results:1 MR antagonist Spironolactone, HSP90 inhibitor geldanamycin, ACEI enalapril, AT1 blocker losartan reversed action of aldosterone on IKs.2 Ca2+ channel blocker nimodipine and Ca2+ chelator BAPTA-AM did not affect the effect of aldosterone on IKs in in vitro cultured cardiomyocytes. However, they, per se, downregulated IKs.3 m RNA and protein expression of both KCNQ1 and KCNE1 were significantly decreased after a 28 days aldosterone treatment. However, sustained aldosterone exposure had no effect on ERG expression, which is the upposed core subunit of the IKr channel.4 mRNA and protein expression of both KCNQ1 and KCNE1 were significantly decreased after 24-hour aldosterone exposure(1μM). However, sustained aldosterone exposure had no effect on ERG expression, which was the supposed pore subunit of IKr channel.Conclusion: The present study demonstrates that MR stimulation downregulates IKs by inhibiting expression of KCNQ1 and KCNE1 and thus delays the repolarization of guinea pig hearts. Otherwise, the interactions between MR and angiotensin Ⅱ or AT1 receptor may involve in the downregulation of IKs by aldosterone. Downregulation of IKs by aldosterone was not secondary to the modulation of intracellular Ca2+ signalling, which probably arises from the aldosterone-induced increase in ICa,L.