Regulation of Pacemaker Current in the Sinoatrial Node by Zonula Occludens-1

Figures

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  Figure 1.. ZO-1cKO mice exhibit prominent atrial bradyarrhythmia as well as AV dissociation and junctional rhythm. Upper Traces: Representative surface ECGs recorded in lightly anesthetized 8- to 12-week-old control (black) and ZO-1cKO (red) mice. Prior to recordings, each mouse was maintained on a heating pad (37°C) and acclimated for 10 min, then data were collected for 1–2 minutes. Control mice showed normal sinus rhythm with a P wave (indicated by *) before each QRS complex, while ZO-1cKO mice exhibited various arrhythmias, most notably frequent sinus pauses (e.g., ZO-1cKO_1 displaying a 451 ms pause and junctional escape beats indicated by circled E) and intermittent AV dissociation indicative of complete heart block and junctional rhythm (ZO-1cKO_2). A, Summary data derived from ECG recordings show that PP intervals were highly variable in KO mice compared with control (4 mice surveyed in each group). B, P wave duration was greater in KO (19 KO mice vs 18 control). C, Increased variability in PR and D, RR intervals in KO mice result from intermittent AV block and junctional rhythm with AV dissociation. E, Overall, ventricular rate was slower in KO mice (N=21) than control (N= 19).…

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  Figure 2.. ZO-1cKO mice feature enlarged SAN/atrial tissue area and caudal shift of the leading pacemaker region. Representative images of intact SAN/atrial preparation with demarcations of main features in Control and ZO-1cKO. The sinoatrial node (SAN) is identified between the right atria (RA) and left atria (LA) along the crista terminalis (CT; dashed black line between the RA and SVC/RVC). The SAN/Atria tissue area size was significantly increased in ZO-1cKO vs. control (ImageJ software, NIH, Bethesda, MD). The leading region of pacemaker activity (colored dots) was recorded on SAN tissues loaded with a voltage-sensitive dye (RH237) from 7 KO and 8 control mice and plotted on the representative images above for illustration. In ZO-1cKO, the leading region of pacemaker activity is shifted caudally and dispersed along the CT. Data normality was evaluated with Shapiro-Wilk test. Data plots are represented as mean ± SEM. Unpaired 2-tailed Student’s test with Welch correction was used to assess the p values.****p<0.0001, **p<0.01, *p<0.05, ns indicates not significant.

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  Figure 3.. Optical voltage mapping demonstrates impaired conduction velocity and interatrial conduction block in ZO-1cKO mice Upper panels: Representative voltage maps from control and ZO-1cKO SAN/atrial preparations. The earliest activation sites are shown in red and the latest in blue as denoted on the color bar. Sample voltage recordings over time, taken from selected locations in the SAN and LA, are shown to the right of each map, with the SAN traces in black and the LA in red. Note that in control, there is a regular one-to-one correspondence of the action potentials (APs) in the SAN and LA consistent with normal sinus rhythm. In the first KO example (ZO-1cKO_1), the APs are irregular. In the second KO example (ZO-1cKO_2), there is no conduction from SAN to LA, and evidence of spontaneous LA activity after a long pause. The voltage traces on the far right are shown on a different time scale to illustrate the delay and block in conduction observed in the KO tissue compared to control. A 50 ms interval is plotted vertically to allow comparison to the typical conduction time for SAN to LA in control mice. A, Summary plots show reduced SAN AP rate (bpm) in ZO-1cKO preparations and B,…

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  Figure 4.. Spontaneous action potentials in patch-clamped ZO-1cKO SAN cells have a reduced diastolic depolarization rate and frequency. Upper panels: Representative spontaneous AP traces from control (black) and ZO-1cKO (red) SAN cells obtained by perforated patch-clamp and displayed on two different time scales. APs occurred at regular intervals in control SAN cells whereas irregular and reduced AP frequency is present in ZO-1cKO. A, Summary data show that ZO-1cKO cells exhibited a slower spontaneous firing rate (frequency, AP/min) than control cells, indicating impaired SAN cell automaticity. No significant changes were observed in B, maximum diastolic potential, C, peak potential or D, maximum upstroke velocity. E, consistent with our observations, slowed firing rate found in ZO-1cKO SAN cells results from reduced diastolic depolarization rate (DDR) compared to control cells. F, AP duration was reduced at 50% repolarization (APD50), but not at 90% (APD90). Data plots were summarized from recordings in 18 cells from 9 ZO-1 cKO mice and 20 cells from 12 control mice. Nested t test was used to calculate p values. **p<0.01, *p<0.05, ns indicates not significant.

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  Figure 5.. Stimulated AP recordings from patch-clamped isolated SAN cells show increased threshold and reduced APD50 in ZO-1 cKO SAN cells. Upper panels show representative traces of stimulated APs recorded in SAN cells isolated from control (black) and ZO-1cKO (red) mice. A, ZO-1cKO paced SAN cells exhibited an increased pacing threshold compared to control cells, but no significant differences were observed in B, resting membrane potential (RMP), C, overshoot and D, AP amplitude. Similar to spontaneous AP recordings, ZO-1cKO SAN cells had reduced APD50 compared to control (E) but no changes in APD90 (F). Data plots were summarized from recordings in 42 cells from 11 ZO-1cKO mice and 39 cells from 14 control mice. Plots are represented as mean ± SEM. Nested t test was used to calculate p values. **p<0.01, *p<0.05.

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  Figure 6.. Pacemaker current (If) and HCN4 expression are reduced in ZO-1cKO SAN. A, Representative If current families (normalized to cell capacitance) from control (black) and ZO-1cKO (red) SAN cells. If was recorded by applying 3 s hyperpolarizing voltage steps from −50 to −160 mV in 10 mV increments from a holding of potential of −50 mV. B, summary plot showing reduction in mean If current-voltage relationship in ZO-1cKO SAN cells. C, there was no change in normalized conductance. D, If density at −140 mV was found significantly reduced in ZO-1 cKO SAN cells compared to control. No significant differences were observed in E, the rapid phase of current decay (ζfast) and F, cell capacitance. Data plots were summarized from recordings in 38 cells from 10 ZO-1 cKO mice and 41 cells from 10 control mice. G, HCN4 gene expression normalized to GAPDH was found significantly reduced in ZO-1cKO SAN/atrial tissue (N=5 for each group). Data plots were analyzed by two-way ANOVA or two-tailed Mann-Whitney test and presented as mean ± SEM. ****p<0.0001, **p<0.01, *p<0.05, ns indicates not significant.