Iryna N. Shlapakova

Human Mesenchymal Stem Cells as a Gene Delivery System to Create Cardiac Pacemakers

Abstract— We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker to the heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by electroporation expressed high levels of Cs+-sensitive current (31.1±3.8 pA/pF at −150 mV) activating in the diastolic potential range with reversal potential of −37.5±1.0 mV, confirming the expressed current as If-like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation. Acetylcholine had no direct effect, but in the presence of isoproterenol, shifted activation 15 mV negative. Transfected hMSCs influenced beating rate in vitro when plated onto a localized region of a coverslip and overlaid with neonatal rat ventricular myocytes. The coculture beating rate was 93±16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161±4 bpm when hMSCs were expressing both EGFP+mHCN2 (P <0.05). We next injected 10 6 hMSCs transfected with either control plasmid or mHCN2 gene construct subepicardially in the canine left ventricular wall in situ. During sinus arrest, all control (EGFP) hearts had spontaneous rhythms (45±1 bpm, 2 of right-sided origin and 2 of left). In the EGFP+mHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rate=61±5 bpm; P <0.05). Moreover, immunostaining of the injected regions demonstrated the presence of hMSCs forming gap junctions with adjacent myocytes. These findings demonstrate that genetically modified hMSCs can express functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart or other electrical syncytia.

Expression and Function of a Biological Pacemaker in Canine Heart

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 (If). Spontaneous LA rhythms occurred after vagal stimulation-induced sinus arrest in 4 of 4 HCN2+GFP dogs and 0 of 3 GFP dogs (P <0.05). Native If in nonexpressed atrial myocytes was 7±4 pA at −130 mV (n=5), whereas HCN2+GFP LA had expressed pacemaker current (IHCN2) of 3823±713 pA at −125 mV (n=10) and 768±365 pA at −85 mV. Conclusions—HCN2 overexpression provides an If-based pacemaker sufficient to drive the heart when injected into a localized region of atrium, offering a promising gene therapy for pacemaker disease.

Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates.

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.

Xenografted Adult Human Mesenchymal Stem Cells Provide a Platform for Sustained Biological Pacemaker Function in Canine Heart

Background— Biological pacemaking has been performed with viral vectors, human embryonic stem cells, and adult human mesenchymal stem cells (hMSCs) as delivery systems. Only with human embryonic stem cells are data available regarding stability for >2 to 3 weeks, and here, immunosuppression has been used to facilitate survival of xenografts. The purpose of the present study was to determine whether hMSCs provide stable impulse initiation over 6 weeks without the use of immunosuppression, the “dose” of hMSCs that ensures function over this period, and the catecholamine responsiveness of hMSC-packaged pacemakers. Methods and Results— A full-length mHCN2 cDNA subcloned in a pIRES2-EGFP vector was electroporated into hMSCs. Transfection efficiency was estimated by GFP expression. IHCN2 was measured with patch clamp, and cells were administered into the left ventricular anterior wall of adult dogs in complete heart block and with backup electronic pacemakers. Studies encompassed 6 weeks. IHCN2 for all cells was 32.1±1.3 pA/pF (mean±SE) at −150 mV. Pacemaker function in intact dogs required 10 to 12 days to fully stabilize and persisted consistently through day 42 in dogs receiving ≥700 000 hMSCs (≈40% of which carried current). Rhythms were catecholamine responsive. Tissues from animals killed at 42 days manifested neither apoptosis nor humoral or cellular rejection. Conclusions— hMSCs provide a means for administering catecholamine-responsive biological pacemakers that function stably for 6 weeks and manifest no cellular or humoral rejection at that time. Cell doses >700 000 are sufficient for pacemaking when administered to left ventricular myocardium.

Wild-Type and Mutant HCN Channels in a Tandem Biological-Electronic Cardiac Pacemaker

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<0.05). mE324A manifested a positive shift of voltage dependence of gating kinetics of activation and deactivation compared with mHCN2 (P<0.05) in myocytes as well as 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<0.05). When spontaneous rate fell below 45 bpm, EPM intervened at that rate, triggering 83% of beats in control, contrasting (P<0.05) with 26% (mHCN2) and 36% (mE324A). On day 14, epinephrine (1 &mgr;g/kg per minute IV) induced a 50% heart rate increase in all mE324A, one third of mHCN2, and one fifth of control (P<0.05 mE324A versus control or mHCN2). 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.

Repolarization Gradients in the Canine Left Ventricle Before and After Induction of Short-Term Cardiac Memory

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.

Implantation of sinoatrial node cells into canine right ventricle: biological pacing appears limited by the substrate.

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.

Epicardial border zone overexpression of skeletal muscle sodium channel, SkM1, normalizes activation, preserves conduction and suppresses ventricular arrhythmia: an in silico, in vivo, in vitro study

Background— In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel (SCN5A) is largely inactivated, contributing to low action potential upstroke velocity (&OV0312; max), slow conduction, and reentry. We hypothesized that a fast inward current such as the skeletal muscle sodium channel (SkM1) operating more effectively at depolarized membrane potentials might restore fast conduction in epicardial border zones and be antiarrhythmic. Methods and Results— Computer simulations were done with a modified Hund-Rudy model. Canine myocardial infarcts were created by coronary ligation. Adenovirus expressing SkM1 and green fluorescent protein or green fluorescent protein alone (sham) was injected into epicardial border zones. After 5 to 7 days, dogs were studied with epicardial mapping, programmed premature stimulation in vivo, and cellular electrophysiology in vitro. Infarct size was determined, and tissues were immunostained for SkM1 and green fluorescent protein. In the computational model, modest SkM1 expression preserved fast conduction at potentials as positive as −60 mV; overexpression of SCN5A did not. In vivo epicardial border zone electrograms were broad and fragmented in shams (31.5±2.3 ms) and narrower in SkM1 (22.6±2.8 ms; P=0.03). Premature stimulation induced ventricular tachyarrhythmia/fibrillation >60 seconds in 6 of 8 shams versus 2 of 12 SkM1 (P=0.02). Microelectrode studies of epicardial border zones from SkM1 showed membrane potentials equal to that of shams and &OV0312; max greater than that of shams as membrane potential depolarized (P<0.01). Infarct sizes were similar (sham, 30±2.8%; SkM1, 30±2.6%; P=0.86). SkM1 expression in injected epicardium was confirmed immunohistochemically. Conclusions— SkM1 increases &OV0312; max of depolarized myocardium and reduces the incidence of inducible sustained ventricular tachyarrhythmia/fibrillation in canine infarcts. Gene therapy to normalize activation by increasing &OV0312; max at depolarized potentials may be a promising antiarrhythmic strategy.

HCN212-channel biological pacemakers manifesting ventricular tachyarrhythmias are responsive to treatment with If blockade

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 microM 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.

Cardiac Memory Evolves With Age in Association With Development of the Transient Outward Current

Background—Calcium-insensitive transient outward current (Ito) 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 Ito. 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<0.05). TVD began to increase at ≈40 days of age, reaching adult levels by ≈200 days. Microelectrode studies performed in 18 dogs (ages 3 to 94 days) after completing the CM protocol and 20 additional dogs (1 day old to adult) revealed that the epicardial action potential notch was absent in neonates, became apparent in the young, and was deepest in adults. The relationship between TVD and epicardial notch was such that as notch magnitude increased, TVD increased (r=−0.65, P<0.05). KChIP2 and Kv4.3 mRNA (measured via reverse transcription–polymerase chain reaction) also increased with age. 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 Ito.