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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 (If) 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: The concept that the interval between the peak (T(peak)) and the end (T(end)) of the T wave (T(p-e)) is a measure of transmural dispersion of repolarization time is widely accepted but has not been tested rigorously by transmural mapping of the intact heart. OBJECTIVES: The purpose of this study was to test the relationship of T(p-e) to transmural dispersion of repolarization by correlating local repolarization times at endocardial, midmural, and epicardial sites in the left and right ventricles with the T wave of the ECG. METHODS: Local activation times, activation-recovery intervals, and repolarization times were measured at 98 epicardial sites and up to 120 midmural and endocardial sites in eight open-chest dogs. In four of the dogs, long-term cardiac memory was induced by 3 weeks of ventricular pacing at 130 bpm because previous data suggest that, in this setting, delayed epicardial repolarization increases transmural dispersion. The other four dogs were sham operated. RESULTS: In sham dogs, T(p-e) was 41 +/- 2.2 ms (X +/- SEM), whereas the transmural dispersion of repolarization time was 2.7 +/- 4.2 ms (not significant between endocardium and epicardium). Cardiac memory was associated with evolution of a transmural gradient of 14.5 +/- 1.9 ms (P

Background— Questions remain about the contributions of transmural versus apicobasal repolarization gradients to the configuration of the T wave in control settings and after the induction of short-term cardiac memory. Methods and Results— Short-term cardiac memory is seen as T-wave changes induced by altered ventricular activation that persists after restoration of sinus rhythm. We studied cardiac memory in anesthetized, open-chest dogs paced from the ventricle for 2 hours. Unipolar electrograms were recorded from as many as 98 epicardial and 144 intramural sites, and activation times and activation-recovery intervals (ARIs) were measured. In separate experiments, epicardial monophasic action potentials were recorded. We found no appreciable left ventricular intramural gradients in repolarization times (activation time+ARI) in either control conditions or after the induction of memory. In controls, there was a left ventricular apicobasal gradient, with the shortest repolarization times in anterobasal regions and longest repolarization times posteroapically. After induction of memory, repolarization times shortened uniformly throughout the ventricular wall. Monophasic action potential duration at 90% repolarization decreased by ≈10 ms after induction of memory. Conclusions— In the intact canine left ventricle at physiological rates, there is no transmural gradient in repolarization. Apicobasal gradients in repolarization time, with shortest repolarization times in anterobasal areas and longest repolarization times in posteroapical regions, are important in the genesis of the T wave. Repolarization times and monophasic action potentials at the 90% repolarization level shorten after the induction of memory. The deeper T wave in the ECG after induction of memory may be explained by the more rapid phase 3 of the action potential.

Background— Calcium-insensitive transient outward current ( I to ) is important to the development of cardiac memory (CM), which itself reflects the capacity of the heart to remodel electrophysiologically. We used cardiac pacing to test the hypothesis that CM evolution can be explained by developmental maturation of I to . Methods and Results— Acutely anesthetized dogs from 1 day old to adult were paced from the left ventricle (VP, n=29) or left atrial appendage (AP, n=12) to induce CM. T-wave vector displacement (TVD) obtained during VP was greater than with AP (adults, 0.39±0.06 mV; neonates, 0.04±0.01 mV; P r =−0.65, P Conclusions— The inducibility of CM gradually increases with age in association with evolution of the epicardial action potential notch and mRNA expression for KChIP2 and Kv4.3. This suggests that the capacity of the heart to remodel electrophysiologically and to manifest memory during development depends in part on evolution of the determinants of I to .

Background— We hypothesized that administration of the HCN2 gene to the left bundle-branch (LBB) system of intact dogs would provide pacemaker function in the physiological range of heart rates. Methods and Results— An adenoviral construct incorporating HCN2 and green fluorescent protein (GFP) as a marker was injected via catheter under fluoroscopic control into the posterior division of the LBB. Controls were injected with an adenoviral construct of GFP alone or saline. Animals were monitored electrocardiographically for up to 7 days after surgery, at which time they were anesthetized and subjected to vagal stimulation to permit emergence of escape pacemakers. Hearts were then removed and injection sites visually identified and removed for microelectrode study of action potentials, patch clamp studies of pacemaker current, and/or immunohistochemical studies of HCN2. For 48 hours postoperatively, 7 of 7 animals subjected to 24-hour ECG monitoring showed multiple ventricular premature depolarizations and/or ventricular tachycardia attributable to injection-induced injury. Thereafter, sinus rhythm prevailed. During vagal stimulation, HCN2-injected dogs showed rhythms originating from the left ventricle, the rate of which was significantly more rapid than in the controls. Excised posterior divisions of the LBB from HCN2-injected animals manifested automatic rates significantly greater than the controls. Isolated tissues showed immunohistochemical and biophysical evidence of overexpressed HCN2. Conclusions— A gene-therapy approach for induction of biological pacemaker activity within the LBB system provides ventricular escape rhythms that have physiologically acceptable rates. Long-term stability and feasibility of the approach remain to be tested.

Background— We hypothesized that localized overexpression of the hyperpolarization-activated, cyclic nucleotide-gated (HCN2) pacemaker current isoform in canine left atrium (LA) would constitute a novel biological pacemaker. Methods and Results— Adenoviral constructs of mouse HCN2 and green fluorescent protein (GFP) or GFP alone were injected into LA, terminal studies performed 3 to 4 days later, hearts removed, and myocytes examined for native and expressed pacemaker current ( I f ). Spontaneous LA rhythms occurred after vagal stimulation-induced sinus arrest in 4 of 4 HCN2+GFP dogs and 0 of 3 GFP dogs ( P I f in nonexpressed atrial myocytes was 7±4 pA at −130 mV (n=5), whereas HCN2+GFP LA had expressed pacemaker current ( I HCN2 ) of 3823±713 pA at −125 mV (n=10) and 768±365 pA at −85 mV. Conclusions— HCN2 overexpression provides an I f -based pacemaker sufficient to drive the heart when injected into a localized region of atrium, offering a promising gene therapy for pacemaker disease.

Objectives This study sought to test the hypothesis that hyperpolarization-activated cyclic nucleotide-gated (HCN)-based biological pacing might be improved significantly by hyperpolarizing the action potential (AP) threshold via coexpression of the skeletal muscle sodium channel 1 (SkM1). Background Gene-based biological pacemakers display effective in vivo pacemaker function. However, approaches used to date have failed to manifest optimal pacemaker properties, defined as basal beating rates of 60 to 90 beats/min, a brisk autonomic response achieving maximal rates of 130 to 160 beats/min, and low to absent electronic backup pacing. Methods We implanted adenoviral SkM1, HCN2, or HCN2/SkM1 constructs into left bundle branches (LBB) or left ventricular (LV) epicardium of atrioventricular-blocked dogs. Results During stable peak gene expression on days 5 to 7, HCN2/SkM1 LBB-injected dogs showed highly stable in vivo pacemaker activity superior to SkM1 or HCN2 alone and superior to LV-implanted dogs with regard to beating rates (resting approximately 80 beats/min; maximum approximately 130 beats/min), no dependence on electronic backup pacing, and enhanced modulation of pacemaker function during circadian rhythm or epinephrine infusion. In vitro isolated LV of dogs overexpressing SkM1 manifested a significantly more negative AP threshold. Conclusions LBB-injected HCN2/SkM1 potentially provides a more clinically suitable biological pacemaker strategy than other reported constructs. This superiority is attributable to the more negative AP threshold and injection into the LBB. (J Am Coll Cardiol 2013;61:1192-201) (C) 2013 by the American College of Cardiology Foundation

Background— Biological pacing performed solely via HCN2 gene transfer in vivo results in relatively slow idioventricular rates and only moderate autonomic responsiveness. We induced biological pacing using the Ca 2+ -stimulated adenylyl cyclase AC1 gene expressed alone or in combination with HCN2 and compared outcomes with those with single-gene HCN2 transfer. Methods and Results— We implanted adenoviral HCN2, AC1, or HCN2/AC1 constructs into the left bundle branches of atrioventricular-blocked dogs. During steady-state gene expression (days 5–7), differences between AC1, HCN2/AC1, and HCN2 alone were evident in basal beating rate, escape time, and dependence on electronic backup pacing. In HCN2, AC1, and HCN2/AC1, these parameters were as follows: basal beating rate: 50±1.5, 60±5.0, and 129±28.9 bpm ( P P P Conclusion— AC1 or HCN2/AC1 overexpression in left bundle branches provides highly efficient biological pacing and greater sensitivity to autonomic modulation than HCN2 alone.

Background— In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel is largely inactivated, contributing to slow conduction and reentry. We have demonstrated that adenoviral delivery of the skeletal muscle Na + channel (SkM1) to epicardial border zones normalizes conduction and reduces induction of ventricular tachycardia/ventricular fibrillation. We now studied the impact of canine mesenchymal stem cells (cMSCs) in delivering SkM1. Methods and Results— cMSCs were isolated and transfected with SkM1. Coculture experiments showed cMSC/SkM1 but not cMSC alone and maintained fast conduction at depolarized potentials. We studied 3 groups in the canine 7d infarct: sham, cMSC, and cMSC/SkM1. In vivo epicardial border zones electrograms were broad and fragmented in sham, narrower in cMSCs, and narrow and unfragmented in cMSC/SkM1 ( P P P >0.05). Conclusion— cMSCs provide efficient delivery of SkM1 current. The interventions performed (cMSCs or cMSC/SkM1) were neither antiarrhythmic nor proarrhythmic. Comparing outcomes with cMSC/SkM1 and viral gene delivery highlights the criticality of the delivery platform to SkM1 antiarrhythmic efficacy.

Aims Reentry accounts for most life-threatening arrhythmias, complicating myocardial infarction, and therapies that consistently prevent reentry from occurring are lacking. In this study, we compare antiarrhythmic effects of gene transfer of green fluorescent protein (GFP; sham), the skeletal muscle sodium channel (SkM1), the liver-specific connexin (Cx32), and SkM1/Cx32 in the subacute canine infarct. Methods and results Immediately after ligation of the left anterior descending artery, viral constructs were implanted in the epicardial border zone (EBZ). Five to 7 days later, efficient restoration of impulse propagation (narrow QRS and local electrogram duration) occurred in SkM1, Cx32, and SkM1/Cx32 groups ( P < 0.05 vs. GFP). Programmed electrical stimulation from the EBZ induced sustained ventricular tachycardia (VT)/ventricular fibrillation (VF) in 15/22 GFP dogs vs. 2/12 SkM1, 6/14 Cx32, and 8/10 SkM1/Cx32 ( P < 0.05 SkM1 vs. GFP). GFP, SkM1, and SkM1/Cx32 had predominantly polymorphic VT/VF, whereas in Cx32 dogs, monomorphic VT predominated ( P < 0.05 for Cx32 vs. GFP). Tetrazolium red staining showed significantly larger infarcts in Cx32- vs. GFP-treated animals ( P < 0.05). Conclusion Whereas SkM1 gene transfer reduces the incidence of inducible VT/VF, Cx32 therapy to improve gap junctional conductance results in larger infarct size, a different VT morphology, and no antiarrhythmic efficacy.

Background Left ventricular pacing (LVP) in canine heart alters ventricular activation, leading to reduced transient outward potassium current (I to ), loss of the epicardial action potential notch, and T-wave vector displacement. These repolarization changes, referred to as cardiac memory, are initiated by locally increased angiotensin II (AngII) levels. In HEK293 cells in which Kv4.3 and KChIP2, the channel subunits contributing to I to , are overexpressed with the AngII receptor 1 (AT1R), AngII induces a decrease in I to as the result of internalization of a Kv4.3/KChIP2/AT1R macromolecular complex. Objective To test the hypothesis that in canine heart in situ, 2h LVP-induced decreases in membrane KChIP2, AT1R, and I to are prevented by blocking subunit trafficking. Methods We used standard electrophysiological, biophysical, and biochemical methods to study 4 groups of dogs: (1) Sham, (2) 2h LVP, (3) LVP + colchicine (microtubule-disrupting agent), and (4) LVP + losartan (AT1R blocker). Results The T-wave vector displacement was significantly greater in LVP than in Sham and was inhibited by colchicine or losartan. Epicardial biopsies showed significant decreases in KChIP2 and AT1R proteins in the membrane fraction after LVP but not after sham treatment, and these decreases were prevented by colchicine or losartan. Colchicine but not losartan significantly reduced microtubular polymerization. In isolated ventricular myocytes, AngII-induced I to reduction and loss of action potential notch were blocked by colchicine. Conclusions LVP-induced reduction of KChIP2 in plasma light membranes depends on an AngII-mediated pathway and intact microtubular status. Loss of I to and the action potential notch appear to derive from AngII-initiated trafficking of channel subunits.

Background Left ventricular pacing (LVP) to induce cardiac memory (CM) in dogs results in a decreased transient outward K current (I to ) and reduced mRNA and protein of the I to channel accessory subunit, KChIP2. The KChIP2 decrease is attributed to a decrease in its transcription factor, cyclic adenosine monophosphate response element binding protein (CREB). Objective This study sought to determine the mechanisms responsible for the CREB decrease that is initiated by LVP. Methods CM was quantified as T-wave vector displacement in 18 LVP dogs. In 5 dogs, angiotensin II receptor blocker, saralasin, was infused before and during pacing. In 3 dogs, proteasomal inhibitor, lactacystin, was injected into the left anterior descending artery before LVP. Epicardial biopsy samples were taken before and after LVP. Neonatal rat cardiomyocytes (NRCM) were incubated with H 2 O 2 (50 μmol/l) for 1 hour with or without lactacystin. Results LVP significantly displaced the T-wave vector and was associated with increased lipid peroxidation and increased tissue angiotensin II levels. Saralasin prevented T-vector displacement and lipid peroxidation. CREB was significantly decreased after 2 hours of LVP and was comparably decreased in H 2 O 2 –treated NRCM. Lactacystin inhibited the CREB decrease in LVP dogs and H 2 O 2 -treated NRCM. LVP and H 2 O 2 both induced CREB ubiquitination, and the H 2 O 2 -induced CREB decrease was prevented by knocking down ubiquitin. Conclusion LVP initiates myocardial angiotensin II production and reactive oxygen species synthesis, leading to CREB ubiquitination and its proteasomal degradation. This sequence of events would explain the pacing-induced reduction in KChIP2, and contribute to altered repolarization and the T-wave changes of cardiac memory.

Biological pacemakers based on the HCN2 channel isoform respond to beta-adrenergic and muscarinic stimulation, suggesting a capacity to respond to autonomic input. 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. 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. 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

Skeletal muscle sodium channel (Nav1.4) expression in border zone myocardium increases action potential upstroke velocity in depolarized isolated tissue. Because resting membrane potential in the 1-week canine infarct is reduced, we hypothesized that conduction velocity (CV) is greater in Nav1.4 dogs compared with in control dogs.The purpose of this study was to measure CV in the infarct border zone border in dogs with and without Nav1.4 expression.Adenovirus was injected in the infarct border zone in 34 dogs. The adenovirus incorporated the Nav1.4- and a green fluorescent protein (GFP) gene (Nav1.4 group, n = 16) or only GFP (n = 18). After 1 week, upstroke velocity and CV were measured by sequential microelectrode recordings at 4 and 7 mM [K(+)] in superfused epicardial slabs. High-density in vivo epicardial activation mapping was performed in a subgroup (8 Nav1.4, 6 GFP) at three to four locations in the border zone. Microscopy and antibody staining confirmed GFP or Nav1.4 expression.Infarct sizes were similar between groups (30.6% +/- 3% of left ventricle mass, mean +/- standard error of the mean). Longitudinal CV was greater in Nav1.4 than in GFP sites (58.5 +/- 1.8 vs. 53.3 +/- 1.2 cm/s, 20 and 15 sites, respectively; P.05). Transverse CV was not different between the groups. In tissue slabs, dV/dt(max) was higher and CV was greater in Nav1.4 than in control at 7 mM [K(+)] (P.05). Immunohistochemical Nav1.4 staining was seen at the longitudinal ends of the myocytes.Nav1.4 channels in myocardium surviving 1 week infarction increases longitudinal but not transverse CV, consistent with the increased dV/dt(max) and with the cellular localization of Nav1.4.

Background A potential concern about biological pacemakers is their possible malfunction, which might create ventricular tachycardias (VTs). Objective The purpose of this study was to test our hypothesis that should VTs complicate implantation of HCN-channel-based biological pacemakers, they would be suppressed by inhibitors of the pacemaker current, I f . Methods We created a chimeric channel (HCN212) containing the N- and C-termini of mouse HCN2 and the transmembrane region of mouse HCN1 and implanted it in HEK293 cells. Forty-eight hours later, in whole-cell patch clamp recordings, mean steady state block induced by 3 μM ivabradine (IVB) showed HCN1=HCN212 > HCN2 currents. The HCN212 adenoviral construct was then implanted into the canine left bundle branch in 11 dogs. Complete AV block was created via radiofrequency ablation, and a ventricular demand electronic pacemaker was implanted (VVI 45 bpm). Electrocardiogram, 24-hour Holter monitoring, and pacemaker log record check were performed for 11 days. Results All dogs developed rapid VT (>120 bpm, maximum rate=285 ± 37 bpm) at 0.9 ± 0.3 days after implantation that persisted through 5 ± 1 days. IVB, 1 mg/kg over 5 minutes, was administered during rapid VT, and three dogs received a second dose 24 hours later. While VT terminated with IBV in all instances within 3.4 ± 0.6 minutes, no effect of IVB on sinus rate was noted. Conclusion We conclude that (1) I f -associated tachyarrhythmias—if they occur with HCN-based biological pacemakers—can be controlled with I f -inhibiting drugs such as IVB; (2) in vitro , IVB appears to have a greater steady state inhibiting effect on HCN1 and HCN212 isoforms than on HCN4; and (3) VT originating from the HCN212 injection site is suppressed more readily than sinus rhythm. This suggests a selectivity of IVB at the concentration attained for ectopic over HCN4-based pacemaker function. This might confer a therapeutic benefit.

OBJECTIVE: The contribution of regional electrophysiologic heterogeneity to the T-wave changes of long-term cardiac memory (CM) is not known. We mapped activation and repolarization in dogs after induction of CM and in sham animals. METHODS AND RESULTS: CM was induced by three weeks of AV-sequential pacing at the anterior free wall of the left ventricle (LV), midway between apex and base in 5 dogs. In 4 sham controls a pacemaker was implanted but ventricular pacing was not performed. At 3 weeks, unipolar electrograms were recorded (98 epicardial, 120 intramural and endocardial electrodes) during atrial stimulation (cycle length 450 ms). Activation times (AT) and repolarization times (RT) were measured and activation recovery intervals (ARIs) calculated. CM was associated with 1) deeper T waves on ECG, with no change in QT interval; 2) longer activation time at the site of stimulation in CM (29.7+/-1.0, X+/-SEM) than sham (23.9+/-1.3 ms p

Background— Biological pacemakers (BPM) implanted in canine left bundle branch function competitively with electronic pacemakers (EPM). We hypothesized that BPM engineered with the use of mE324A mutant murine HCN2 (mHCN2) genes would improve function over mHCN2 and that BPM/EPM tandems confer advantage over either approach alone. Methods and Results— In cultured neonatal rat myocytes, activation midpoint was −46.9 mV in mE324A versus −66.1 mV in mHCN2 ( P P Xenopus oocytes. In intact dogs in complete atrioventricular block, saline (control), mHCN2, or mE324A virus was injected into left bundle branch, and EPM were implanted (VVI 45 bpm). Twenty-four–hour ECGs were monitored for 14 days. With EPM discontinued, there was no difference in duration of overdrive suppression among groups. However, basal heart rates in controls were less than those in mHCN2, which did not differ from those in E324A (45 versus 57 versus 53 bpm; P P P Conclusions— mE324A induces faster, more positive pacemaker current activation than mHCN2 and stable, catecholamine-sensitive rhythms in situ that compete with EPM comparably but more catecholamine responsively than mHCN2. BPM/EPM tandems function reliably, reduce the number of EPM beats, and confer sympathetic responsiveness to the tandem.