Expression And Function Of G Protein Gated Inwardly Rectifying K Channels In Mouse Ventricular Cardiomyocytes

G-protein regulated inward-rectifier potassium channels (GIRK) are part of a superfamily of inward-rectifier K+ channels which includes four main members: GIRK1-4. GIRK channels express in cardiac myocytes, central and peripheral neurons and other tissues. GIRK channels are G-protein-activated and mediate the effects of certain G-protein-coupled receptors (GPCRs) on electrical activity in cardiac, neuronal and neurosecretory cells. In mammalian heart, GIRK1 and GIRK4 are mainly localized in atrial myocytes and sinoatrial node cells, and play essential physiological roles in modulating excitability of heart, and is the molecular basis of IKACh in cardiac myocytes.Parasympathetic nerve and sympathetic nerves contribute in functional regulation of heart. Vagus nerve releases Acetylcholine (ACh), which can bind with muscarinic type 2 (M2) receptor. ACh could negatively regulate heart rate through activate pertussis toxin sensitive Gi protein family; during this process, Gβγreleased from G protein activate GIRK channels and open GIRK channels. This pathway is believed to be the main pathway activation of GIRK channels through PTX-sensitive G proteins. Activation of GIRK induces hyperpolarization of the membrane and reduces cell excitability, which is the molecular basis of vagus nerve effect on the excitability of heart. It has been debated whether GIRK channels express in ventricular cardiomyocytes, and if so what their functions are. Some studies show the expression of GIRK1 and GIRK4 in some animal ventricular cardiomyocytes. However, others found no evidences of existence of GIRK channels in ventricular cardiomyocytes. As for the functional significance of GIRK channel in ventricular cardiomyocytes, little is known.The basal inwardly rectifying potassium currents (IK1) play important role in maintaining of resting membrane potential of cardiomyocytes. It is generally accepted that the classical inwardly rectifying potassium channel (IRK) contribute to IK1 currents. As for GIRK, which has a similar property of inwardly rectification as IRK, their role in maintaining of resting membrane potential has not been reported. Therefore, this study will investigate the expression of GIRK in mouse cardiomyocytes and the roles in modulation of cardiac excitability.Based on the above studies, we will further study possible roles of GIRK in heart failure.Objective: To investigate expression and function of GIRK1/4 channels in mouse ventricular myocytes; to study changes of expression and function of GIRK during heart failure; to study the sensitivity of the inward rectifier potassium currents to Ba2+.Methods: Adenosine (Ado) was used to activate GIRK channels. Ado binds to A1 receptor and activates the corresponding G proteins. Gβγreleased from activated G protein directly activates GIRK channels. Tertiapin-Q, a specific blocker of GIRK current, was used to dissect GIRK current from other currents, and Ba2+ was used to block inward rectifier potassium currents. We recorded IKACh in cardiac myocytes using the whole-cell patch-clamp technique. In addition, we used western blot and immunofluorescence techniques to detect expression of GIRK1 and GIRK4 proteins in ventricular myocytes.Mouse heart failure model was made by ligation of aorta. After establishment of heart failure, we further used above methods to observe the changes of GIRK subjected to heart failure.Sensitivity of basal inwardly rectifying K+ channels (IK1) to Ba2+ was tested by constructing concentration-response relationship.Results: (1) Using Langendorff perfusion technique, we isolated single mouse ventricular myocytes. Currents from mouse epicardial and endocardial ventricular myocytes were recorded using the whole-cell patch-clamp technique. We first used oxo-M, a M type acetylcholine receptor agonist, to study the activation of GIRK. Oxo-M can activate GIRK through interaction with M2 receptor; however, the effect of oxo-M is not specific, due to a similar interaction with M1 receptor, which eventually leads to inhibition of GIRK currents. Adenosine, through activating A1 receptor, can also activate Gi protein thus GIRK channel. Thus, the present study mainly used adenosine as the activator of GIRK channels. An inwardly rectifying current could be recorded when the membrane potentials were changed from -40 mV to -120 mV; an outwardly rectifying current was seen when the membrane was depolarized above -40 mV. This outward current should be the outward K+ current which is not the subject of this study. We first ascertained that the inward current we recorded was the inward rectifier potassium currents. The basal inward current showed property of inward rectification, and could be completely inhibited by 500μM Ba2+. From this, we obtained the reversal potential of inward current which was around -80 mV, near the K+ equilibrium potential of -82.3 mV calculated form Nernst equation, suggesting that the inward current we recorded is the inward rectifier potassium current. Adenosine (10μM) induced an increase of the inwardly rectifying currents, which could be inhibited by 10 nM tertiapin-Q, the specific blocker of GIRK channels. In addition, 500μM Ba2+ can completely inhibit both the basal and adenosine-activated current. Taken together, the above results indicate that adenosine can activate GIRK currents in mouse ventricular myocytes. Our statistical results suggest that in all the mouse ventricular myocytes we measured, 10μM adenosine induced an increase of 22.3%±3.9 and 49.6%±23 in inward rectifier current (versus the basal current) in endocardial and epicardial ventricular myocytes, respectively. Based on the cell membrane area (measured using cell capacitance), we calculated the current densities in endocardial and epicardial ventricular myocytes. No significant difference was found between the current densities of basal and adenosine-activated inwardly rectifying currents, indicating a similar level of expression of GIRK in endocardial and epicardial ventricular myocytes. (2) We detected the expression of GIRK1 and GIRK4 in atrial, endocardial and epicardial ventricular myocytes using Western blot. From the statistical results of the protein expression densities, we observed no difference for expression of GIRK1/GIRK4 in normal mouse endocardial and epicardial ventricular myocytes. (3) Using laser confocal microscopy, we observed the immunofluorescence of GIRK1 and GIRK4 in mouse atrial, endocardial and epicardial ventricular myocytes. (4) In epicardial ventricular myocytes of heart failure mouse, Adenosine increased GIRK by 25.4%±8.8 (versus basal inward rectifying current). In addition,we detected the expression of GIRK1 and GIRK4 in atrial, endocardial and epicardial ventricular myocytes of failing heart myocytes using Western blot and immunofluorescence study. Because of the calcification of atrial myocytes in heart failure mouse, we could not obtain the atrial myocytes and experiments on atrial cells were therefore excluded for this part of the study. GIRK currents activated by 10μM adenosine in normal and heart failure epicardial ventricular myocytes were compared. The results show that in epicardial ventricular myocytes, the absolute GIRK amplitudes in heart failure mouse were larger than that in normal mouse. However, when the current densities were compared between normal and failing hearts, opposite results were found. These results indicate an increasement of the cell size in ventricular myocytes from failing hearts. There were no statistical difference for the basal current densities in epicardial ventricular myocytes of normal and heart failure mouse. (5) The effect of Ba2+ on the basal inwardly rectifying current. Concentration-response relationship for Ba2+-induced inhibition of inward rectifying potassium currents was constructed. Ba2+ inhibited endocardial and epicardial ventricular myocytes with IC50 of 0.32μM and 0.55μM, respectively. These results laid a fundation for further study of constitution of inwardly rectifying potassium currents in ventricular myocytes and for further study of cardiac excitability including rest membrane potential.Conclusions: Electrophysiological results show that endocardial and epicardial ventricular myocytes of normal and heart failure mouse express GIRK, and the current densities between these two cell types are not statistically different; Western blot and immunofluoresecence results verify the expression of GIRK1/4 in normal and heart failure mouse ventricular myocytes. The comparison of the current densities of GIRK between epicardial ventricular myocytes of normal and heart failure mouse indicate that current densities of GIRK are decreased in the cells of failing heart, which suggests that GIRK may be involved in the pathophysiological changes of heart failure myocytes. The IC50 for Ba2+ inhibition of the basal inwardly rectifying current in endocardial and epicardial ventricular myocytes are 0.32μM and 0.55μM, respectively.