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Aim: We compared the clinical and physiological consequences of the novel mutation R878C in a highly conserved pore residue in domain II (S5-S6) of human, hNa v 1.5, cardiac Na + channels.Methods: Full clinical evaluation of pedigree members through three generations of a Chinese family combined with SCN5A sequencing from genomic DNA was compared with patch and voltage-clamp results from two independent expression systems.Results: The four mutation carriers showed bradycardia, and slowed sinoatrial, atrioventricular and intraventricular conduction. Two also showed sick sinus syndrome; two had ST elevation in leads V1 and V2. Unlike WT-hNa v 1.5, whole-cell patch-clamped HEK293 cells expressing R878C-hNa v 1.5 showed no detectable Na + currents (i Na ), even with substitution of a similarly charged lysine residue. Voltage-clamped Xenopus oocytes injected with either 0.04 or 1.5 lg lL )1 R878C-hNa v 1.5 cRNA similarly showed no i Na , yet WT-hNa v 1.5 cRNA diluted to 0.0004-0.0008 ng lL )1 resulted in expression of detectable i Na . i Na was simply determined by the amount of injected WT-hNa v 1.5: doubling the dose of WT-hNa v 1.5 cRNA doubled i Na . i Na amplitudes and activation and inactivation characteristics were similar irrespective of whether WT-hNa v 1.5 cRNA was given alone or combined with equal doses of R878C-hNa v 1.5 cRNA therefore excluding dominant negative phenotypic effects. Na + channel function in HEK293 cells transfected with R878C-hNa v 1.5 was not restored by exposure to mexiletine (200 lm) and lidocaine (100 lm). Fluorescence confocal microscopy using E3-Nav1.5 antibody demonstrated persistent membrane expression of both WT and R878C-hNa v 1.5. Modelling studies confirmed that such i Na reductions reproduced the SSS phenotype. Conclusion: Clinical consequences of the novel R878C mutation correlate with results of physiological studies.

Recently published optical mapping studies in larger mammals, including human, have identified functionally discrete sino-atrial exit pathways of activation. This is in line with earlier mapping studies in dog and human but in contrast with findings in mouse and rabbit, where a propagation wavefront pattern of activation has been described. It underpins the complex 3D organization of the cardiac pacemaking and conduction system in larger species, where sinoatrial and atrioventricular nodal physiology both demonstrate identifiable activation pathways, which coincide with anatomical landmarks and histological architecture. So that, in addition to muscle fiber orientation and cell coupling, these intrinsic factors act to determine excitation pathways. This complex 3D organization increases the effect of source-to-sink mismatch both by greater variability of the space constant of tissue and by the 3D projection of this effect in all directions. Mathematical modeling provides a means to study these interactions and newer models should incorporate these additional factors and their effect in the 3D structure of large mammal physiology.

Key points The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN dysfunction, or ‘sick sinus syndrome’, can cause excessively slow heart rates and pauses, leading to exercise limitation and syncope, currently treated by implantation of an electronic pacemaker.‘Biopacemaking’ utilises gene therapy to restore pacemaker activity by manipulating gene expression. Overexpressing the HCN pacemaker ion channel has been widely used with limited success.We utilised bradycardic rat subsidiary atrial pacemaker tissue to evaluate alternative gene targets: the Na+/Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18 known to be involved in SAN embryonic development.TBX18 overexpression restored normal SAN function, as assessed by increased rate, improved heart rate stability and restoration of isoprenaline response. TBX3 and NCX1 were not effective in accelerating the rate of subsidiary atrial pacemaker tissue.Gene therapy targeting TBX18 could therefore have the potential to restore pacemaker function in human sick sinus syndrome obviating electronic pacemakers. AbstractThe sinoatrial node (SAN) is the primary pacemaker of the heart. Disease of the SAN, sick sinus syndrome, causes heart rate instability in the form of bradycardia and pauses, leading to exercise limitation and syncope. Biopacemaking aims to restore pacemaker activity by manipulating gene expression, and approaches utilising HCN channel overexpression have been widely used. We evaluated alternative gene targets for biopacemaking to restore normal SAN pacemaker physiology within bradycardic subsidiary atrial pacemaker (SAP) tissue, using the Na+/Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18. TBX18 expression in SAP tissue restored normal SAN function, as assessed by increased rate (SAN 267.5 ± 13.6 bpm, SAP 144.1 ± 8.6 bpm, SAP‐TBX18 214.4 ± 14.4 bpm; P < 0.001), improved heart rate stability (standard deviation of RR intervals fell from 39.3 ± 7.2 ms to 6.9 ± 0.8 ms, P < 0.01; root mean square of successive differences of RR intervals fell from 41.7 ± 8.2 ms to 6.1 ± 1.2 ms, P < 0.01; standard deviation of points perpendicular to the line of identity of Poincaré plots (SD1) fell from 29.5 ± 5.8 ms to 7.9 ± 2.0 ms, P < 0.05) and restoration of isoprenaline response (increases in rates of SAN 65.5 ± 1.3%, SAP 28.4 ± 3.4% and SAP‐TBX18 103.3 ± 10.2%; P < 0.001). These changes were driven by a TBX18‐induced switch in the dominant HCN isoform in SAP tissue, with a significant upregulation of HCN2 (from 1.01 × 10−5 ± 2.2 × 10−6 to 2.8 × 10−5 ± 4.3 × 10−6 arbitrary units, P < 0.001). Biophysically detailed computer modelling incorporating isoform‐specific HCN channel electrophysiology confirmed that the measured changes in HCN abundance could account for the observed changes in beating rates. TBX3 and NCX1 were not effective in accelerating the rate of SAP tissue.

In this paper, we proposed a kernel differenceweighted k-nearest neighbor classifier (KDF-WKNN)