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Background: Sinus node (SN) dysfunction is observed in some long-QT syndrome (LQTS) patients, but has not been studied as a function of LQTS genotype. LQTS6 involves mutations in the hERG β-subunit MiRP1, which also interacts with hyperpolarization-activated, cyclic nucleotide gated (HCN) channels-the molecular correlate of SN pacemaker current (If ). An LQTS registry search identified a 55-year male with M54T MiRP1 mutation, history of sinus bradycardia (39-56 bpm), and prolonged QTc., Objective: We tested if LQTS6 incorporates sinus bradycardia due to abnormal If ., Methods: We transiently co-transfected neonatal rat ventricular myocytes (to study currents in a myocyte background) with human HCN4 (hHCN4, primary SN isoform) or human HCN2 (hHCN2) and one of the following: empty vector, wild-type hMiRP1 (WT), M54T hMiRP1 (M54T). Current amplitude, voltage dependence, and kinetics were measured by whole cell patch clamp., Results: M54T co-expression decreased HCN4 current density by 80% compared to hHCN4 alone or with WT, and also slowed HCN4 activation at physiologically relevant voltages. Neither WT nor M54T altered HCN4 voltage dependence. A computer simulation predicts that these changes in HCN4 current would decrease rate and be additive with published effects of M54T mutation on hERG kinetics on rate., Conclusions: We conclude that M54T LQTS6 mutation can cause sinus bradycardia through effects on both hERG and HCN currents. Patients with other LQTS6 mutations should be examined for SN dysfunction, and the effect on HCN current determined., (© 2013 Wiley Periodicals, Inc.)

Biological pacing has been proposed as a physiologic counterpart to electronic pacing, and the sinoatrial node (SAN) is the general standard for biological pacemakers. We tested the expression of SAN pacemaker cell activity when implanted autologously in the right ventricle (RV). We induced complete heart block and implanted electronic pacemakers in the RV of adult mongrel dogs. Autologous SAN cells isolated enzymatically were studied by patch clamp to confirm SAN identity. SAN cells (400,000) were injected into the RV subepicardial free wall and dogs were monitored for 2 weeks. Pacemaker function was assessed by overdrive pacing and IV epinephrine challenge. SAN cells expressed a time-dependent inward current (I(f)) activating on hyperpolarization: density = 4.3 ± 0.6 pA/pF at -105 mV. Four of the six dogs demonstrated >50% of beats originating from the implant site at 24 h. Biological pacemaker rates on days 7-14 = 45-55 bpm and post-overdrive escape times = 1.5-2.5 s. Brisk catecholamine responsiveness occurred. Dogs implanted with autologous SAN cells manifest biological pacing properties dissimilar from those of the anatomic SAN. This highlights the importance of cell and substrate interaction in generating biological pacemaker function.

Background: Biological pacemakers based on the HCN2 channel isoform respond to beta-adrenergic and muscarinic stimulation, suggesting a capacity to respond to autonomic input., Objective: The purpose of this study was to investigate autonomic response to emotional arousal in canines implanted with murine HCN2-based biological pacemakers using gene therapy., Methods: An electronic pacemaker was implanted with its lead in the right ventricular apical endocardium (VVI 35 bpm). An adenoviral HCN2/GFP construct (Ad-HCN2, n = 7) or saline (control, n = 5) was injected into the left bundle branch on day 2 after radiofrequency ablation of the atrioventricular node to induce complete atrioventricular block. Emotional arousal was achieved by presenting food following an overnight fast. Autonomic control was evaluated with Poincaré plots of R-R(N) against R-R(N+1) intervals to characterize heart rate variability (HRV) and with continuous RR interval assessment via 24-hour ambulatory ECG. The 24-hour ECG and Poincaré plot shape were analyzed., Results: During day 1 after biological pacemaker implantation, Poincaré HRV parameters and RR intervals were unchanged with food presentation. However, on day 7, food presentation was accompanied by an increase in HRV (SD1, p < 0.07, and SD2, p < 0.05) and shortening of RR interval (P < .05) in dogs with Ad-HCN2 but not in controls., Conclusion: This is the first demonstration that biological pacemakers are capable of responding to natural arousal stimuli to elicit appropriate chronotropic responses, a potential advantage over electronic pacemakers., (Copyright © 2010 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

We examined whether coupling of a ventricular myocyte to a non-myocyte cell expressing HCN2 could create a two-cell syncytium capable of generating sustained pacing. Three non-myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2-induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell-cell coupling between heterologous pairs increased with time in co-culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2-expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell-cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of approximately 0.6 to 1.7 Hz (1.09 +/- 0.05 Hz). Addition of carbenoxolone (200 microM), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 microM 9-amino-1,2,3,4-tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2-expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell-based biological pacemakers in vivo once an understanding of delivery and target cell geometry is defined.

The pacemaker current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, contributes to the initiation and regulation of cardiac rhythm. Previous experiments creating HCN-based biological pacemakers in vivo found that an engineered HCN2/HCN1 chimeric channel (HCN212) resulted in significantly faster rates than HCN2, interrupted by 1-5 s pauses. To elucidate the mechanisms underlying the differences in HCN212 and HCN2 in vivo functionality as biological pacemakers, we studied newborn rat ventricular myocytes over-expressing either HCN2 or HCN212 channels. The HCN2- and HCN212-over-expressing myocytes manifest similar voltage dependence, current density and sensitivity to saturating cAMP concentrations, but HCN212 has faster activation/deactivation kinetics. Compared with HCN2, myocytes expressing HCN212 exhibit a faster spontaneous rate and greater incidence of irregular rhythms (i.e. periods of rapid spontaneous rate followed by pauses). To explore these rhythm differences further, we imposed consecutive pacing and found that activation kinetics of the two channels are slower at faster pacing frequencies. As a result, time-dependent HCN current flowing during diastole decreases for both constructs during a train of stimuli at a rapid frequency, with the effect more pronounced for HCN2. In addition, the slower deactivation kinetics of HCN2 contributes to more pronounced instantaneous current at a slower frequency. As a result of the frequency dependence of both instantaneous and time-dependent current, HCN2 exhibits more robust negative feedback than HCN212, contributing to the maintenance of a stable pacing rhythm. These results illustrate the benefit of screening HCN constructs in spontaneously active myocyte cultures and may provide the basis for future optimization of HCN-based biological pacemakers.

The prevention and treatment of life-threatening bradyarrhythmias have been revolutionized in the last half century by electronic pacemakers. Because this represents a palliative therapy, attempts have begun to effect a cure with the novel tools of gene and cell therapy. Over time, the strategies used have coalesced to focus on achieving a stable and autonomically responsive cardiac rhythm in a setting that ultimately would require no implanted hardware. In this report, we review the history of the disease process being treated, approaches now in progress, and the demands that must be met if biological therapies are to be successful.

The sinoatrial node performs its task as a cardiac impulse generator throughout the life of the organism, but this important function is not a constant. Rather, there are significant developmental changes in the expression and function of ion channels and other cellular elements, which lead to a postnatal slowing of heart rate and may be crucial to the reliable functioning of the node during maturation. In this review, we provide an overview of current knowledge regarding these changes, with the main focus placed on maturation of the ion channel expression profile. Studies on Na(+) and pacemaker currents have shown that their contribution to automaticity is greater in the newborn than in the adult, but this age-dependent decrease is at least partially opposed by an increased contribution of L-type Ca(2+) current. Whereas information regarding age-dependent changes in other transmembrane currents within the sinoatrial node are lacking, there are data on other relevant parameters. These include an increase in the nodal content of fibroblasts and in the area of nonexpression of connexin43, considered a molecular marker of nodal tissue. Although much remains to be done before a comprehensive view of the developmental biology of the node is available, important evidence in support of a molecular interpretation of developmental slowing of the intrinsic sinoatrial rate is beginning to emerge.

Two distinct intracellular mechanisms have been proposed to affect the firing rate of cardiac pacemaker cells: one involves modulation of the I(f) current by the second messenger cAMP, and one relies upon disruption or alteration of SR Ca2+ transients during activity. Although both mechanisms are necessary for proper automaticity and autonomic rate control, the specific contribution of each to pacemaking is still debated. We investigated if the two processes can be separated based on potentially different effects on action potential characteristics during rate modulation. To identify specific I(f)-mediated effects, we used the selective I(f) blocker ivabradine and found that ivabradine (3 microM) slows rate (-16.2%) by selectively reducing (-31.9%) the steepness of early diastolic depolarization (EDD). On the other hand ryanodine (3 microM), used to evaluate the effects of abolishment of SR Ca2+ transients, slowed rate (-31.3%) by depolarizing the take-off potential (TOP, 18.1%) without affecting EDD. We therefore used these two parameters to identify I(f)-based or SR Ca2+ transients-based processes and analyzed the effects on action potential's characteristics of Rp-cAMPs (50 microM), a membrane permeable cAMP analogue directly activating f-channels; we found that Rp-cAMPs accelerates rate by increasing EDD (+42.3%) without modifying TOP. Finally, rate modulation was achieved by muscarinic (ACh 0.01 microM) or beta-adrenergic (Iso 1 microM) stimulation; in both cases, rate changes were associated with modifications of EDD (ACh, -29.3% and Iso, +47.6%) and not of TOP. We conclude that rate-related changes in the EDD induced by autonomic agonists are mediated by I(f) and not by processes involving SR Ca2+ transients.

Pacemaker current (If) exists in both neonatal and adult ventricles, but activates at more negative voltages in the adult. This study uses whole-cell patch clamp to investigate the factors that may contribute to the maturational shift of If, comparing neonatal rat ventricular myocytes that were cultured for 4-6 days either alone, in co-culture with sympathetic nerves, or with neurotransmitters chronically present in culture. If recorded from nerve-muscle co-cultures had a significantly more negative and shallower activation-voltage relation than that from control muscle cultures, which was reflected in the midpoint potential (V50) and slope factor (K) of activation. This effect of innervation was prevented by the sustained presence in the culture of the alpha1-adrenergic antagonist prazosin (Pz) at 10(-7) M. In parallel experiments, myocytes treated with noradrenaline (NA) at 10(-7) M or neuropeptide Y (NPY) at 10(-7) M during culture had the same If activation as control cells, but cells treated with NA and NPY together had a significantly more negative and shallower activation curve. Maximum conductance and reversal potential were unchanged. The effect of chronic exposure to NA + NPY was prevented by the sustained presence of either Pz or the NPY Y2 selective antagonist T4-[NPY(33-36)]4 (3.5 x 10(-7) M) in the culture, indicating a requirement for both alpha1-adrenergic and NPY Y2 activation. Substituting NA with the alpha1A-adrenergic selective agonist A61603 (5(-10) x 10(-9) M), in the presence of NPY, did not alter If, suggesting the involvement of alpha1B- rather than alpha1A-adrenoceptors. Further, sequential exposure to NPY followed by NA was effective in reproducing the action of chronic simultaneous exposure to these agonists, but sequential exposure to NA followed by NPY was ineffective. The results are consistent with past studies indicating that NPY affects the functional expression of the alpha1B-adrenergic cascade and suggest that sympathetic innervation induces a negative shift of If in ventricle via a combined action at alpha1B-adrenergic and NPY Y2 receptors. This effect of innervation probably contributes to the developmental maturation of If activation.

Myocytes were isolated from newborn and adult rat ventricle. Using the whole-cell patch clamp, the two cell populations were compared for the presence of the hyperpolarization-activated pacemaker current if. As in other mammalian species, the threshold voltage in acutely dissociated adult rat myocytes was extremely negative (-113 +/- 5 mV; n=12). In contrast, threshold in newborn cells was relatively positive, regardless of whether measured in acutely dissociated (-72 +/- 2 mV; n=6) or cultured cells (-70 +/- 2 mV; n=9). Current density was not reduced in the adult. These results suggest that with development the ventricle assumes its non-pacemaker function, at least in part, by a shift of the voltage dependence of if outside the physiological range.