Volume 9, Issue 10, Pages 1681-1688.e1 (October 2012) Escape capture bigeminy: Phenotypic marker of cardiac sodium channel voltage sensor mutation R222Q  Krishnakumar Nair, MBBS, Roman Pekhletski, PhD, Louise Harris, MBChB, Melanie Care, MD, Chantal Morel, MD, Talha Farid, MBBS, Peter H. Backx, PhD, Elod Szabo, MD, Kumaraswamy Nanthakumar, MD  Heart Rhythm  Volume 9, Issue 10, Pages 1681-1688.e1 (October 2012) DOI: 10.1016/j.hrthm.2012.06.029 Copyright © 2012 Heart Rhythm Society Terms and Conditions

Figure 1 A: Pedigree of family. The pedigree shows 6 affected members. Squares indicate male family members, and circles indicate female family members. Filled squares and circles indicate that the patients are affected. A filled left lower quadrant indicates dilated cardiomyopathy. A filled right upper quadrant indicates conduction disease. The proband is the female member with both dilated cardiomyopathy and conduction disease. B: CARTO local activation time (LAT) map of the coupled beat. The left side depicts the LAT map of the second beat with earliest activation close to the right ventricular apex on the interventricular septum. Inset depicts surface lead V5 and electrograms from mapping catheter (M1M2) indicating the window of interest. On the right side, surface electrocardiogram leads are shown. The next 3 channels represent His bundle electrograms, and the last 10 channels are electrograms from mapping catheter placed along the right side of the interventricular septum with electrode T1 being closest to the apex and electrode T10 being farthest from the apex. The first beat is preceded by an anterograde His bundle/right bundle potential (downward looking black arrow), seen best in channels T3 and T4. The second beat does not have an anterograde His bundle potential but is in fact associated with a retrograde His/right bundle electrogram (upward looking gray arrow) seen best in channel T3. C: Effect of a class IB sodium channel blocker. i: Baseline junctional capture escape capture bigeminy; ii: Postintravenous lidocaine, suppression of bigeminy. Heart Rhythm 2012 9, 1681-1688.e1DOI: (10.1016/j.hrthm.2012.06.029) Copyright © 2012 Heart Rhythm Society Terms and Conditions

Figure 2 The electrocardiographic signature—escape capture bigeminy. A pattern of bigeminy is demonstrated on the electrocardiograms of 5 members of a family. Uniquely, the coupling interval of the second beat was constant between 320 and 420 ms in all cases. Heart Rhythm 2012 9, 1681-1688.e1DOI: (10.1016/j.hrthm.2012.06.029) Copyright © 2012 Heart Rhythm Society Terms and Conditions

Figure 3 A: Electrocardiogram shows left bundle branch block and RBBB ventricular ectopics with the same coupling interval to the preceding sinus beat indicating septal breakthroughs from either side of the interventricular septum. B: An RBBB ectopic with 2 different axes indicating different locations on the same side of the septum. The first 4 leads represent surface electrocardiogram leads—I, aVF, V1, and V6. The next channel is the right ventricular channel, and the last 2 channels represent electrograms from the mapping catheter placed on the left side of the interventricular septum. C: The first 12 leads represent surface electrocardiogram and the next 5 channels represent electrograms from a CRISTA catheter placed along the left side of the interventricular septum. The earliest site of activation was only 15 ms ahead of surface QRS at the mid interventricular septum with no difference between the right and left side of the septum. RBBB = right bundle branch block. Heart Rhythm 2012 9, 1681-1688.e1DOI: (10.1016/j.hrthm.2012.06.029) Copyright © 2012 Heart Rhythm Society Terms and Conditions

Figure 4 INa measurements of WT and R222Q SCN5A expressed in Chinese hamster ovary K1 cells. Typical WT (A) and R222Q (B) currents (INa) recorded in response to voltage steps to ∼80 to +100 by 10 mV from a holding potential of ∼80 mV. INa is shown for every second voltage step for clarity. C: Normalized peak INa (n = 8 for both groups) as a function of step voltage for WT and R222Q. D: The estimates of the maximal conductance (GMax) for WT and R222Q (N = 12) show no differences between the groups. Conductance was calculated as described in the online supplement. WT = wild type. Heart Rhythm 2012 9, 1681-1688.e1DOI: (10.1016/j.hrthm.2012.06.029) Copyright © 2012 Heart Rhythm Society Terms and Conditions

Figure 5 Steady-state activation and inactivation curves. Normalized conductance (see online supplement) derived from the peak INa measurements as summarized in Figure 3 (ie, INa activation) was used to generate steady-state activation curves (n = 8) for WT channels (closed triangle) and R222Q channels (open circles). Normalized conductance plotted as a function of the prepulse voltage (see online supplement) was used to generate steady-state inactivation curves (n = 8), which are shown for WT channels (open triangles) and R222Q channels (closed circles). Black lines are the best fits of the data to a Boltzmann function with 2 voltage components (see online supplement). Parameters and fit results are summarized in Table 3. The inset shows the overlap region between the fitted activation and inactivation curves (WT—orange and R222Q—green background). Data were corrected for junction potentials. Heart Rhythm 2012 9, 1681-1688.e1DOI: (10.1016/j.hrthm.2012.06.029) Copyright © 2012 Heart Rhythm Society Terms and Conditions

Figure 6 Fast inactivation and recovery from inactivation. A: Typical normalized INa traces recorded in response to ∼40 mV step for WT (pink) and R222Q (gray) SCNA5 channels. The decline of INa was fit to a biexponential (fast and slow time constants: τfast and τslow) decay function.4 Notice that the decline of the R222Q channels is slower than WT. B: The fast time constants (τfast) as a function of voltage (n = 8). C: A typical protocol and superimposed current traces used to measure the recovery from fast inactivation. In these studies, after applying steps to +20 mV (prepulse), the membrane potential was repolarized for variable periods of time to ∼100 mV followed by a second step to +20 mV (test pulse). D: The recovery is determined by taking the ratio of the INa peak in the test pulse to the peak INa in the prepulse as a function of time. Clearly, the recovery from inactivation does not differ between R222Q and WT channels. R222Q whole-cell current (bottom) shown in the panel; the 1st (green trace) and last (red trace) pulses are highlighted for clarity; interpulse interval was set to 2 seconds. Heart Rhythm 2012 9, 1681-1688.e1DOI: (10.1016/j.hrthm.2012.06.029) Copyright © 2012 Heart Rhythm Society Terms and Conditions