The Effect Of Rgs5on Cardiac Electrophysiology And Arrhythmogenesis

Objective1. To elucidate the effects of Rgs5on atrial repolarization and tachyarrhythmia in mice.2.To elucidate the effect of Rgs5on acetylcholine-related atrial tachyarrhythmia in mice.3.To investigate the effect of Rgs5on cardiac repolarizing K+currents in mice.4.To investigate the effect of Rgs5on spatial and temporal fluctuation of cardiac repolarization in mice.MethodsMale wild-type (WT) and Rgs5knockout (Rgs5-/-) mice of C57BL/6background (8～10weeks) were provided from the Animal Model Centre of Cardiovascular Disease Institute of Wuhan University. All protocols were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University.1. ECG was recorded by Telemetry ECG and the P-wave duration and amplitude (Pdur and Pamp) and PR interval were measured in Rgs5-/-and WT mice; In Langendorff-perfused heart, atrial MAP and ERP were measured, and atrial tachyarrhythmia (ATA) was induced by PES and Burst pacing protocol,; TAP were recorded with borosilicate glass microelectrodes during regular pacing frequency in samples of atrial myocardium; repolarizing K+currents and kinetics were measured in atrial myocytes of both groups by patch clamp; application of real-time PCR and PSR staining to assay the atrial fibrosis; Echocardiography was performed to assess left atrial diameter.2. Telemetry ECG analyses the heart rates of conscious Rgs5-/-and WT mice during administration of carbachol (0.1mg/kg, IP); ERP and function of sinus node were measured in Langendorff-perfused heart; the incidence of ATA was analyzed between two groups by PES and Burst pacing during perfusion of1μM Ach, and the power spectrum analysis was applied to quantitate the dominant frequency (DF) of ATA before and after administration of Ach; acetylcholine sensitive K+current (Ik,Ach) were recorded by patch clamp in Rgs5-/-and WT atrial myocytes, and the mRNA of Kir3.1and Kir3.4were assayed by real-time PCR. 3. Surface and Telemetry ECG were applied to measured the heart rate and QT interval in Rgs5-/-and WT mice; In Langendorff-perfused heart, ventricular MAP and ERP were measured; TAP were recorded with borosilicate glass microelectrodes during regular pacing frequency in samples of ventricular myocardium; repolarizing K+currents and kinetics were measured in ventricular myocytes of both groups by patch clamp; application of real-time PCR and western blot to assay the mRNA and protein level of Kv1.5、Kv2.1、Kv4.2. Kv4.3and Kir2.1.4. Application of telemetry ECG to analyze the QT variability (QTV) and heart rate variability (HRV) in Rgs5-/-and WT mice; MAP and ERP were recorded in different sites of ventricular chamber, and the dispersion of epicardial and transmural MAP were analyzed; APD restitution curves were constructed by PES pacing and dispersion of maximal slope was calculated; application of PES and Burst pacing to induce ventricular tachyarrhythmia (VA), and incidence and duration of it were compared between Rgs5-/-and WT group; ventricualr fibrosis was assayed by real-time PCR and PSR staining; Echocardiography was performed to assess the diameter of ventricular chamber and cardiac function.Results1.The incidence of ATA were increased in Rgs5-/-Langendorff-perfused mouse hearts during program electrical stimulation (PES)(46.7%,7of15) and burst pacing (26.7%,4of15) compared with wild-type (WT) mice (PES:7.1%,1of14; burst:7.1%,1of14)(P<0.05). And the duration of ATA also shown longer in Rgs5-/-heart than that in WT,2out of15hearts exhibited sustained ATA (>30s) but none of them observed in WT mice. Atrial prolonged repolarization was observed in Rgs5-/-hearts including widened P wave in surface ECG recording, increased action potential duration (APD) and atrial effective refractory periods (AERP), all of them showed significant difference with WT mice (P<0.05). At the cellular level, whole-cell patch clamp recorded markedly decreased densities of repolarizing K+currents including IKur and Ito in Rgs5-/-atrial cardiomyocytes, compared to those of WT mice (P<0.05).2. We observed that Rgs5-/-mice would render the atria more susceptible to electrically induced ATA within activation of IKAch by1μM acetylcholine (Ach). Compared with those from WT mice, isolated perfused hearts from Rgs5-/-mice had significantly (P<0.01) abbreviated atrial effective refractory periods (AERPs) during 10Hz,8Hz,6.7Hz, and5Hz programmed electrical stimulation procedures, and significantly (P<0.05) prolonged sinus nodal recovery time in the presence of1μM acetylcholine. Fast fourier transform analysis was performed on recordings of ATA; the dominant frequency was increased in Rgs5-/-mice following administration of acetylcholine. In addition, whole patch clamp analyses of single atrial myocytes revealed that the acetylcholine-regulated potassium current was stronger in Rgs5-/-mice than in WT mice.3. Rgs5-/-mouse hearts showed significantly prolonged cardiac repolarization, including prolonged QT interval and action potential duration (APD). Consistent with these findings, TAP recordings at Rgs5-/-samples showed increased APD90compared to WT group over all paced CL, and EADs were occurred at CL of400ms and200ms in Rgs5-/-group; measurement of K+currents in ventricular myocytes of Rgs5-/-mice revealed significant reduction of the outward voltage-dependent K+currents, including Ipeak, Ito Ikur, and Iss, compared to that in wild-type mice. Transcript and protein expression levels of Kv4.2, Kv4.3, Kv1.5, and Kv2.1were downregulated in Rgs5-/mouse ventricles compared with those in wild-type mice (P<0.05).4. During a24-h ECG recording, the mean RR interval and SDNN showed similar between Rgs5-/-and WT mice (P>0.05), but QTV and QTVI were increased in Rgs5-/-group; the QTv had positive correlation with SDNN in WT mice (r=0.62, P<0.01), but not in Rgs5-/-mice (r=0.01, P>0.05);the increased APD and ERP were stable at different sites throughout the ventricular chamber, and these alterations showed increased spatial heterogeneity in Rgs5-/-mice, compared with WT mice; Rgs5-/-markedly steepened the slopes of the APD restitution curves at all10sites in the heart (P<0.01) but also increased the spatial dispersions of Smax (COV-Smax) compared to WT mice; the incidence and duration of VA were also increased in Rgs5-/-mouse hearts; the fibrosis and diameter of ventricular chamber showed similar between Rgs5-/-and WT heart (P>0.05).Conclusions1. These results suggest that Rgs5is an important regulator of arrhythmogenesis in the mouse atrium and that the enhanced susceptibility to atrial tachyarrhythmias in Rgs5-/-mice may contribute to abnormalities of atrial repolarization.2. these results suggest that Rgs5is a critical cholinergic regulator in atrial tissue; Rgs5-/-mice have enhanced susceptibility to atrial tachyarrhythmia due to increased acetylcholine-regulated potassium currents.3. The results strongly indicate that Rgs5-/-induced prolonged repolarization, which were closely related to the remodeling of voltage-dependent K+currents.4. Rgs5absence has major influences on APD restitution and repolarizing heterogeneity, these observation may provide a further insight into understanding the Rgs5related ventricular arrhythmic developing.