| Background It is known that there are many kinds of ion channels scattered in the membrane of cardiomyocytes, including some voltage-dependent potassium channels. The human ether-a-go-go related gene (hERG) encodes the pore-for ming subunit of a voltage-dependent potassium channel, which conductsthe rapidly activating delayed rectifying potassium current(IKr current). The IKr current presents slow activation and deactivation kinetics, coupled with rapid voltage-dependent inactivation and recovery from inactivation. These kinetics make IKrcurrent critical in controlling the orderly repolarization at the end of each cardiac action potential (AP), particularly near the threshold potential of early afterdepolarizations. When hERG gene mutates or hERG channels are inhibited by some drugs, namely the congenital long QT syndrome type 2 (LQTS-2) and the acquired long QT syndrome, the IKr current will decrease, which causes the delay in cardiac reploarization, the prolongation in action potential duration (APD), and the prolongation in QT interval in the ECG. All these changes are liable to trigger lethal tachyarrhythmias, such as torsade de pointes (TdP), and even lead to sudden cardiac death (SCD).Stimulation of the sympathetic nervous system in response to physical or emotional stress activate the alpha-adrenergic receptor (a-AR) and beta-adrenergic receptor (β-AR), including a-AR and β-AR in cardiomyocytes. Recently, mountains of studies have revealed that IKr current can be modulated by acute activation of cardiac α1-AR and β1-AR. Furthurmore, some researchers put forward that there exists interactive influences betweenα1-AR and β1-AR stimulation on IKr current, which is necessarilya protective feedback regulatory mechanismfor humans who are under stress conditions. Nevertheless, the direct evidence and the exact mechanism remained blank and unclear.Therefore, this study is to investigate the effects of acute stimulation of different subtype of adrenergic receptors (α1-ARand β1-AR) respectively on IKr current from health guinea pig ventricular myocytes, and the effects of acute stimulation of both of ai-AR and β1-AR on IKr current, which may provide some new evidence and details of the complex and finely reticular regulatory mechanism. More importantly, we anticipate this study to be a real start in exploring this cross-talk regulation in IKr current, which may provide a novel target for preventing and treating malignant arrhythmia events in clinic.Part I Isolation of Ventricular Cardiomyocytes from Adult Guinea Pigs and Patch Clamp Recording of IKrObjective To establish an effctive and stable method to isolate single guinea pig ventricular cardiomyocyte, and the patch clamp recording of IKrcurrent.Methods Using a modified Langendorff equipment, the guinea pig ventricular cardiomyocytes were isolated by enzymatic methods. The viably dissociative ventricular cardiomyocytes were put in the perfusion chamber for whole cell patch clamp recording of the current of IKr.Results The enzymatic method was proved to be effective in obtaining viable ventricular cardiomyocytes from guinea pig. The majority (about 70-80%) of the dissociative cardiomyoctes were rod-shaped with clear striations, with the ability to endure the patch clamping experiments for about fourty minutes. The isolation procedure had a good repeatability and was relatively easy to grasp. And in the instrument of patch clamp,IKr of guinea pig ventricular cardiomyocyte was steadily induced by a certain protocol by using the whole cell voltage clamp method.Conclusion The enzymatic method is uncomplicated, reliable, and effective in isolating viable ventricular cardiomyocytes from guinea pigs, and the protocol in this study is stable to induce IKr by whole cell voltage clamp.Part Ⅱ Effects of Acute Stimulation of α1-AR and β1-AR Separately or Simultaneously on IKrin Guinea Pig Left Ventricular MyocytesObjective To investigate the effects of acute stimulation of different subtype of adrenergic receptors (α1-AR and β1-AR) on IKr-from guinea pig ventricular myocytes, and the effects of acute stimulation of both α1-AR and β1-ARon IKr current.Methods Single ventricular myocyte was obtained from guinea pig by using enzymatic dissociation technique. The cardiomyocytes were randomly divided into three groups, given α1-adrenergic agonist (phenylephrine, PE) and β1-adrenergic agonist (xamoterol, Xamo) separately, or α1-plus β1-adrenergic agonists (PE+Xamo). The IKrcurrent was recorded by the wholecell patch clamp technique and observed during certain time. The amplitudes of IKr current before and after stimulation of adrenergic receptors were measured to reflect the effects of acute stimulation of different ARs. The expression levels of hERG channel protein which conducted IKr after acute stimulation of different subtype of adrenergic receptors in three groups were detected by western-blot.Results Phenylephrine(PE)and xamoterol(Xamo)inhibited IKr current amplitude by a content dependent way, the IC50 was 0.93 μmol/L and 6.40 μmol/L respectively. In our study,1 μmol/L PE reduced IKr current to 0.79±0.02, and shifted the voltage-dependent activating curve to the negative voltage, where the half-maximal activation voltage (V0.5) changed from-2.99 mV±1.44mV to-9.10 mV±1.74 mV, and the slope factor(k) changed a little.10 μmol/LXamo reduced IKr current to 0.72±0.01, and shifted the voltage-dependent activating curve to the negative voltage, where V0.5changed from-4.54 mV±1.48 mV to-7.24 mV±1.93 mV, and k changed a little. While simultaneously ad ministration of 1 μmol/L PE and 10 μmol/L Xamo only reduced Ikr current to 0.69±0.02, and it also shifted the voltage-dependent activating curve to the negative voltage, where V0.5changed from-2.71 mV±1.95 mV to-8.45 mV±1.97 mV, and k changed a little. In PE group, XAMO group and PE+XAMO group, Ikr current decreased by (20.73±2.46)%, (27.99±0.68)% and (30.56±1.80)%, separately.Nevertheless, the expression levels of hERG channel protein which conducted Ikr current in three different groups, by western-blot, did not show any statistical differences(P>0.05).Conclusion Acute stimulation of α1-ARand β1-ARseparately inhibited IKr current in different degrees, while simultaneously acute stimulation of α1-AR and β1-AR did not generate more dramatic inhibitory effects. During acute adrenergic stimulation, hERG channel protein which conducted IKrcurrent did not change at transcription and translation level, but at post-translational modification level. This kind of phenomenon suggests that there exists a certain degree of overlap in regulation, namely "cross-talk", between α1-and β1-adrenergic signaling cascades. Most importantly, this kind of "cross-talk" regulation is necessarily a protective mechanism for humans who are under stress conditions.Part Ⅲ Functional Cross-talk between α1-AR and β1-ARModulates IKr Current in Guinea-pig Left Ventricular MyocytesObjective To elucidate the inhibition effect of α1-AR and β1-AR stimulation on IKr current, especially in the presence of the other subtype of adrenergic receptor stimulation, and to explore at which level the cross function happens between α1-and β1-adrenergic signaling pathways.Methods To address the hypothesis, two experimental groups were designed in this part, which are referred to as Ppre+X and Xpre+P. In the Ppre+X group, PEwas applied for a certain time (the changes of PE on IKrcurrent had stabilized), followed by co-application of Xamo and PE for another certain time. Conversely, in the Xpre+P group,Xamo was pre-applied for a certain time, followed by simultaneous application of both PE and Xamo for another certain time. Hence, we were able to compare the reductions and shifts in activation curves of IKrcurrent caused by exposure to PE alone, and Xamo alone. More importantly, we compared PE-induced IKrcurrent reduction in the presence of Xamo, and Xamo-induced IKrcurrent reduction in the presence of PE, as well as shifts in the activation curve of IKrcurrent under varying conditions. Results In two groups,1 μmol/L PE reducedIKrcurrent by (21.67 ±3.19)%, while 10 μmol/L Xamo reducedIKrcurrent by (27.57 ±0.64)%. And the voltage-dependent activating curves shifted to the negative direction in different degrees, in which V0.5changed (-6.35±1.53) mV and (-1.95±2.22) mV, while k changed a little in both groups. In the presence of PE,10 μmol/L Xamo only reduced Ikrcurrent by (12.85 ±1.05)%, at the same time,1 μmol/LPE only reduced IKrcurrent by (14.44 ±2.94)% in the presence of Xamo. Moreover the voltage-dependent activating curves only shifted a little, with the changes in V0.5and k not obvious. As stated, α1-adrenergic activation-induced inhibition of IKr current was less than β1-adrenergic activation-induced inhibition ((21.67 ±3.19)% & (27.57 ±0.64)%), but the difference between the two groups did not reach statistical significance (P>0.05). However, the α1-AR induced inhibition of IKr current significantly differed in cells that were pre-incubated with the β1-AR agonist compared to the control ((21.67 ±3.19)% & (14.44 ±2.94)%, P<0.05). Similarly, the β1-AR activation-induced inhibition of IKrcurrent significantly differed between unstimulated (control) versus α1-AR stimulated myocytes ((27.57±0.64)% & (12.85±1.05)%, P<0.001).Conclusion HERG channel activation is strongly modulated by acute stimulation of α1-AR and β1-AR, as well as theIKr currentconducted the channel. Remarkably, stimulation of either receptor class markedly attenuates the inhibitory effects of the other receptor class on IKr current. These results proved that there exists functional cross-talk effect between α1-AR and β1-AR in inhibitory modulation of IKr current in from another aspect. Further investigations are required to elucidate the molecular mechanisms that underlie the functional cross-talk, or to identify putative intermediate proteins in the signal transduction pathways involved in modulation of IKr current via adrenergic activation, providing new thoughts for clinical prevention and treatment of certain malignant arrhythmias. |
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