Effects Of Aldosterone On Cardiac Electrophysiology In Guinea Pig

Arrhythmia is the leading cause of mortality in cardiovascular diseases. As an important factor of renin-angiotensin-aldosterone system, aldosterone participates in the regulations of physiological function and pathological process. Two famous experiments （RALES and EPHESUS） haveshown that aldosterone antagonist treatment based on conbination of diuretic and ACEI treatment can significantly reduce the incidence of sudden cardiac death in patients with heart failure,suggesting that aldosteronehas the potential trend to arrhythmia.Mineralocorticoid receptors exist in heart. Aldosterone not nolyparticipates in pathological cardiac hypertrophy、myocardial fibrosis, but alsomodulates electrical remodeling through regulating cardiac ion channelprotein expressions. Studies have shown that aldosterone up-regulated L-typeCa2+channels of ventricular myocardium, increased sodium channel proteindensity、enhanced INacurrent, markedly decreased transcription of mRNA andprotein expression of Kv4.2, Kv4.3(I_to), Kv1.5(I_Kur), Kir2.1and Kir2.3(I_K1),respectively.The up-regulation of depolarizing current and down-regulation ofrepolarized K+current prolong action potential duration （APD）, increaseoccurrences of early after-depolarization（EAD）. In additions, aldoseroneincrease the excitability of the myocardial cells by up-regulating I_fcurrent.Previous results suggested aldosterone could directly regulate thefunctions of cardiac ion channels, participate in the occurrence of arrhythmia.However, by now, studies mainly confined in mice, rats and other rodentsanimals. It is known that guinea pigs and other large mammals has obviousspecies differences in myocardial electrophysiology, compared with therodents animals. I_tois the main portion of depolarization current in smallanimals. Rodents animals lack of delayed rectifier potassium current in therepolarizing currents. But delayed rectifier potassium current is the important repolarization current in human heart, so our study mainly focus on theelectrophysiology effect in guinea pigs heart and its related mechanism.The animal model was made by planting osmotic pump in guinea pigsubcutaneous, which releases aldosterone （1μg/h） constantly for28days. Weinvestigated the effect of aldosterone with pathological concentrations onguinea pig heart, including the shape and parameters of ECG and actionpotential. By using IKrspecific blocker （dofetilide）, I_Ksspecific blocker （293B）and later sodium current blocker （ranolazine）, we investigated the possibleion channels involved in its cardiac electrophysiological effects. The resultswill provide evidences to explaining aldosterone related mechanism of cardiacremodeling.Objective: To observe the effect and relative mechanism of aldosteroneon cardiac electrophysiology in guinea pig.Methods:（1） Groups of Experiments:32male guinea pigs （200-250g） weredivided into four groups. Control group： feed for28days in normalenvironment; Aldosterone group: guinea pigs anesthetized by tribromoethanol（225mg/kg）, The osmotic pump filled with aldosterone solution （4mg/mL）was subcutaneously implanted in guinea pigs, release rate is1μg/h, feed for28days; Spironolactone group: mix spironolactone in food of guinea pigs,spironolactone was orally administrated for28days （100mg/kg/day）;Aldosterone and spironolactone group: implanted osmotic pump filled withaldosterone solution in guinea pigs subcutaneously, spironolactone was orallyadministrated for28days also through above way.（2） Electrophysiological parameters measurement: in the experiment, weneed to detect ECG between groups before and after making model, and tomeasure the change of QT interval.28day later, the conventional glasselectrode technique was used to record the action potential of papillary musclein guinea pig, to observe duration of90%depolarization (APD₉₀), restingpotential （RP）, absolute refractory period （ERP）, the frequency dependence（0.2Hz,1Hz,2Hz） and incidence of the early afterdepolarization （EAD）. Then perfuse IKrblocker dofetilide, I_Ksblocker293B, Na+blocker ranolazine,observe the change of APD₉₀for each group respectively.（3） Electrolyte measurement: FAAS method were used to detect the urinepotassium and urine sodium concentrations and total weight of the guinea pigurine collected in metabolic cage for24hours on the day of25.（4） Measurement of blood pressure and heart function:28day later,guinea pigs were anesthetized by intraperitoneally administration ofpentobarbital sodium （30-35mg/kg）. The blood pressure of guinea pigs wasmeasured through the carotid artery intubation, then weigh heart.（5） Measurement of plasma concentration of aldosterone: to measueplasma concentration of aldosterone by aldosterone radioimmunoassay kit.（6） Statistics: experimental data showed by mean±standard deviation,comparison between groups use two sample t test, comparison before and afterapplication use paired t test, p<0.05represent the difference was statisticallysignificant.Results:（1） Electrophysiological parameters measurement:RR intervals of control group, aldosterone （ALD） group,（spirolactone）SPI group and aldosterone+spirolactone （ALD+SPI） group were242.7±11.6ms,247.5±10.6ms,251.5±14.7ms, and251±11.3ms, respectively. Therewere no significant difference in the all other groups （p<0.05） compared tocontrol group. QT interval and QRS duration of control group were150.3±5.6ms,30.8±3.1ms respectively, ALD group were163.8±8.1ms,36.6±1.9ms. QTinterval and QRS duration of ALD group is longer than control group, thedifference is significant （p<0.05）, suggesting aldosterone can prolong QTinterval and QRS duration; QT interval and QRS duration of SPI group is146.4±6.5ms,28.8±1.8ms, which have no significant difference with thecontrol group （p>0.05）, suggesting spirolactone itself have little effect on QTinterval and QRS duration; QT interval and QRS duration of ALD+SPI groupis145.6±7.1ms,28.2±2.0ms, which all have significant difference with theALD group （p<0.05）, suggesting spirolactone can antagonize the effect of aldosterone significantly.APD₉₀of control group and ALD group was192.1±12.9ms,216.2±12.3ms respectively, APD₉₀of ALD group is longer than control group, thedifference is significant （p<0.05）, suggesting aldosterone can prolong APD;APD₉₀of SPI group is191.7±17.5ms, which have no significant differencewith the control group （p>0.05）, suggesting spirolactone itself have little effectwith APD₉₀; APD₉₀of ALD+SPI group is200.9±13.8ms, spirolactone canweaken the effect of aldosterone, which have significant difference with theALD group （p<0.05）, suggesting spirolactone can antagonize the effect ofaldosterone significantly.The frequency dependences （0.2Hz,1Hz,2Hz） for control group andALD group were （200.5±12.0ms,155.3±19.5ms,121.5±10.4ms）,（231.9±16.5ms,182.8±19.8ms,138.8±16.2ms） respectively, APD₉₀of ALD group aretotally prolonged significantly in every frequency （p<0.05）, but the shortenedtrend, along with the increase of frequency, was not changed, compared withcontrol group; The frequency dependences for SPI group and ALD+SPI groupwere （197.3±10.2ms,171.8±4.3ms,125.0±7.2ms）,（205.2±8.4ms,170.0±6.0ms,122.9±16.2ms） respectively, which have no significant difference with thecontrol group （p>0.05）, suggesting spirolactone itself have little effect onfrequency dependence and can antagonize the effects of aldosteronesignificantly.The absolute refractory period （ERP） of control group, aldosterone groupwas201.5±5.9ms,234.9±12.1ms respectively, ERP of ALD group is longerthan control group, the difference is significant （p<0.05）, suggestingaldosterone can prolong ERP; ERP of SPI group is198.3±15.0ms, whichhave no significant difference with the control group （p>0.05）, suggestingspirolactone itself have little effect with ERP; ERP of ALD+SPI group is200.6±12.4ms, demonstrating that spirolactone can weaken the effect ofaldosterone. There are significant difference between ALD+SPI group andALD group （p<0.05）, suggesting spirolactone can antagonize the effect ofaldosterone significantly. The occurrences of EAD for control group, ALD group were1/15,5/8respectively, the occurrences in ALD group were significantly higher thancontrol group, suggesting aldosterone can increase the incidence of arrhythmia;the occurrences of EAD in SPI group, ALD+SPI group were0/7,1/8respectively, which have no significant difference, compare to control group（p>0.05）, suggesting spirolactone itself have no effect on arrhythmia, but itcan antagonize the arrhythmia caused by aldosterone.After application of IKrantagonist dofetilide, APD₉₀was prolonged by21.8%,25.9%in control group and ALD group, respectively. There were nosignificant difference （p>0.05）, suggesting there was not different in IKrbetween ALD group and control group. Aldosterone has little effects on IKr.After application of I_Ksantagonist293B, APD₉₀was prolonged by26.6%,4.5%in control group and ALD group, respectively. There were significantdifference （p<0.05）, suggesting I_Ksin myocardial cell in guinea pig are morethan control group. Aldosterone reduces expression of I_Ks.After the application of late Na+channel antagonist ranolazine, APD₉₀was prolonged by8.0%and8.1%in control group and aldosterone group,respectively, there were no significant difference （p>0.05）, suggesting therewas not different in late Na+channel between ALD group and control group.Aldosterone has little effect on late Na+currents.（2） Electrolyte measurement:Urine potassium ion concentrations of the control group and ALD groupwere73.6±65.8mmol/L,85.9±42.9mmol/L, there were not significantdifference （p>0.05）; Urinary sodium ion concentrations were54.6±70.6mmol/L,80.2±14.4mmol/L, there were not significant difference （p<0.05）.Urine potassium ion weight excreted in24hour for control group and ALDgroup were2.13±0.87mmol,2.08±1.28mmol; Urine sodium ion weightexcreted in24hour for control group and ALD group were0.94±0.76mmol,1.74±1.18mmol, there were significant differences （p<0.05）. Thosedemonstrate there are not abnormal in Na+and K+metabolism, aldosteronehave little effect on electrolyte metabolism, suggesting the change of cardiac electrophysiology is not caused by abnormality of electrolyte.（3） Arterial blood pressure and heart weight/body weight measurement:Blood pressure of control group, ALD group, SPI group and ALD+SPIgroup was71.2±7.6mmHg,71.5±3.1mmHg,71.8±6.6mmHg,67.2±8.6mmHg, compared with control group, there was no significant difference inthe all other groups （p>0.05）. Relative heart weight （heart weight/body weight）was4.01±0.68×10-3,4.33±0.36×10-3,3.59±0.14×10-3,3.60±0.37×10-3,respectively, there was no significant difference in the other all groups（p>0.05）, compared with contol group.（4） Measurement of plasma concentration of aldosterone:Plasma concentrations of control group and ALD group were:190±44ng/L,554±84ng/L, plasma concentration of ALD group is higher than controlgroup （p<0.05）, suggesting aldosterone can be released into the body of guineapig at a steady speed; plasma concentrations of control group and ALD groupwere:4118±1568ng/L,7900±1262ng/L, also higher than the level of controlgroup （p<0.05）, suggesting spirolactone can promot the secretion and releaseof aldosterone in guinea pig by feedback regulation.Conclusion: The effects of aldosterone on cardiac electrophysiology inguinea pig include prolonging QT interval QRS duration in ECG and APD₉₀of ventricular papillary muscles, increasing spontaneous EADs occurrences.Apirolactone, aldoserone antagonist, can specifically inhibit above effectcaused by aldoserone, suggesting aldosterone exerts its effect throughactivating aldosterone receptor. Na+, K+excretion and blood pressure have nosignificant changes, suggesting electrical remodeling effects induced byaldosterone are not secondary to the change of Na+, K+and hypertension.Significant down-regulating response of aldosterone treated animals to I_Ksinhibitor, suggests aldosterone maybe prolong cardiac repolarization intervalby down-regulating I_Ks.