The Circadian Variation Of β-adrenergic Receptor Modulation On The IKr Current Of Normal Guinea Pig Ventricular Cardiomyocytes

Background:The circadian variations of ventricular arrhythmia and sudden cardiac death(SCD) have been repeatedly reported. The ventricular arrhythmias and SCD occurred in the early morning. Moreover, the circadian variations of ventricular arrhythmias and SCD are not related to many known risk factors like age, sex, myocardial ischemia, chronic heart failure, myocardial hypertrophy and basic heart rhythm, suggesting that circadian variations have independent mechanisms.The QTc interval and ventricular refractories are significantly shorter during the daytime. The QTc interval of the nocturnal mice reached the trough during the nighttime, which was the active period corresponding to the daytime for the human kind.QTc interval and ventricular refractories reflect the myocardial repolarization and are crucial for arrhthmogenesis. We have tried to investigate the circadian variation of cardiac repolarization by the measurements of cardiomyocytes APD and IKr current. The rapid component of delayed rectifier potassium channel(IKr) current is the main outward current during the phase3repolarization of ventricular cardiomyocytes. The inhibition of IKr causes signigicant APD prolongation. IKr is modulated by β adrenergic receptors, mainly through the classical AC-cAMP-PKA pathway. Activation of β adrenergic receptors can inhibit IKr currents through the PKA phosphorylation of the specific sites on the hERG channel proteins.In the recent years, the cardiac circadian clock system has attracted more and more attentions. Numerous reports have been published on the cardiac circadian clock modulation on various aspects of cardiac physiological functions like cardiac metabolism, cardiac rhythm, and signal transduction.To investigate the circadian variation of cardiomyocyte repolarization and the possible role of cardiac circadian clock in it, healthy guinea pigs were preadapted under standard environmentally controlled rooms (12-hour light/dark cycle) for at least two weeks, then were randomly assigned into two groups:the DAY group (all the experiments were done at noon), and the NIGHT group (all the experiments were done at midnight). The surface ECQ the cardiomyocyte APD and IKr, as well as the β adrenergic modulation on the IKr current were measured respectively in the two groups.Part Ⅰ Isolation of adult guinea pig cardiomyocytesAim:To establish an effctive and stable way to isolate guinea pig ventricular cardiomyocytes.Methods:The guinea pig ventricular cardiomyocytes were isolated using traditional enzymatic methods, with a few improvements. We used the modified Langendorff to perfuse the heart with type Ⅱ collagenase. The free ventricular cardiomyocytes were obtained after the perfusion procedure was done.Results:The enzymatic method was effective in isolating ventricular cardiomyocytes. The majority (80%-95%) of the isolated cardiomyoctes were rod-shaped with clear striations, with the ability to endure the patch clamping experiments. The isolation procedure can be routinely repeated to obtain viable ventricular cardiomyocytes.Conclusion:The enzymatic method is effective in isolating viable ventricular cardiomyocytes for the experiments.Part Ⅱ The circadian rhythm of the APD and IKr currents in healthy guinea pig ventricular cardiomyocytesAim:To explore the molecular mechanism of the circadian variation in cardiac repolarization.Methods:After two weeks of preadaptation under controlled light cycles(12-hour light/dark cycle), healthy guinea pigs were randomly assigned into two groups:DAY group and NIGHT group. We recorded the surface ECG, and measured APD and IKr current of ventricular myocytes in both groups.Results:The QTc interval, cardiomyocytes APD and IKr tail current density showed a feature of circadian variations. The QTc intervals in the DAY group were significantly longer than those in the NIGHT group(312.40±2.30ms vs.294.10±6.77ms, P<0.05). Correspondingly, the cardiomyocytes APD90in the DAY group were a little bit longer than those in the NIGHT group(229.10±24.70ms vs.184.8±4.19ms, p>0.05), but the difference did not reach the statistical significance. At40mV, the IKr tail current densities in the DAY group were significantly larger (DAY1.07±0.07vs. NIGHT0.82±0.06, p<0.01). Correspondingly, the difference between APD90and APD50was shorter in the DAY group than those in the NIGHT group(DAY17.06±1.72ms vs.NIGHT53.28±10.62, p<0.01), which suggested that the phase3repolarization was faster in the DAY group. The IKr gating parameters (vhalf, slope, activation time constants and deactivation time constant) showed no significant difference between the two groups. The hERG mRNA level showed the fold change (night to day) of1.30±0.20, however, the difference did not reach the statistical significance.Conclusions:Healthy guinea pigs show circadian variations in the QTc interval, cardiomyocytes APD and IKr tail current density.Part Ⅲ The circadian rhythm of the β adrenergic modulation on the IKr current in healthy guinea pig ventricular cardiomyocytes Background:Ventricular arrhythmia(VT) and sudden cardiac death(SCD) show diurnal variations.β adrenergic modulation on the IKr current is one of the mechanisms underlying VT&SCD.Objective:To investigate the circadian variations in the ventricular cardiomyocytes βadrenergic modulation on the IKr current in healthy guinea pigs.Methods:Healthy guinea pigs were randomly assigned into two groups:DAY and NIGHT group. The ventricular cardiomyocytes were isolated at11:00(Day group) or23:00(NIGHT group) and the β adrenergic modulations on the IKr tail were measured and compared.Results:The non-selective β adrenergic agonist ISO (10uM) inhibited IKr tail current with significant diurnal fluctuations(45.79±3.38%in the DAY group vs.12.65±6.74%in the NIGHT group, p<0.01). Selective β14R agonist Xamoterol(10uM) showed similar circadian variations, with the inhibition percentage of47.60±7.67%in the Day group vs.23.06±4.20%in the NIGHT group(p<0.05). Selective activation of β2AR&β3AR were not significantly different between the two groups. When we directly activated the intracellular adenyly cyclase(AC) with forskolin(100uM), the circadian variations were largely abrogated(IKr inhibition percentage52.89±4.30%in the DAY group vs.54.36±2.71%in the NIGHT group, p>0.05). The RT-PCR results also showed circadian variations in the β1AR mRNA level (NIGHT to DAY fold change1.66±0.20, p<0.05).Conclusion:Our experiment showed that there was a circadian rhythm in the β adrenergic inhibition on the IKr current, possibly caused by the circadian variation of the β1AR expression.