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 ϩ -sensitive current (31.1Ϯ3.8 pA/pF at Ϫ150 mV) activating in the diastolic potential range with reversal potential of Ϫ37.5Ϯ1.0 mV, confirming the expressed current as I f -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 93Ϯ16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161Ϯ4 bpm when hMSCs were expressing both EGFPϩmHCN2 (PϽ0.05). 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 (45Ϯ1 bpm, 2 of right-sided origin and 2 of left). In the EGFPϩmHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rateϭ61Ϯ5 bpm; PϽ0.05). 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.

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 HCN2ϩGFP dogs and 0 of 3 GFP dogs (PϽ0.05). Native I f in nonexpressed atrial myocytes was 7Ϯ4 pA at Ϫ130 mV (nϭ5), whereas HCN2ϩGFP LA had expressed pacemaker current (I HCN2 ) of 3823Ϯ713 pA at Ϫ125 mV (nϭ10) and 768Ϯ365 pA at Ϫ85 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. Key Words: arrhythmia Ⅲ pacemakers Ⅲ electrophysiology I mplantable electronic devices have represented state-ofthe-art therapy for high degrees of heart block since the 1960s. Such devices save lives, and refinements in design have made them far more palatable to patients than they had been originally. Nonetheless, the ideal pacemaker, in terms of both physiological function of the heart and adaptability to the human body, would be biological. [1][2][3][4][5] The search for such a pacemaker has centered on 3 gene therapy strategies: (1) upregulation of ␤ 2 -adrenergic receptors by transfecting cloned receptors that increase heart rate responses to adrenergic input 1,2 ; (2) viral infection producing dominant negative inhibition of inwardly rectifying potassium current (I K1 ), such that the balance of inward currents suffices to depolarize ventricular myocardial cells 5 ; and (3) adenoviral transfer of ␣ (hyperpolarization-activated cyclic nucleotide-gated [HCN2]) 3 and/or ␤ (minK-related peptide 1 [MiRP1]) 6 subunits of the endogenous human pacemaker current to induce autonomically responsive pacemaker function in ventricular myocytes. The first two approaches have seen proof of concept demonstrated in animal models. 1,5 The third approach might be less problematic and proarrhythmic in that it incorporates the endogenous pacemaker channel gene, which selectively activates only during diastole. The present study provides proof of concept that HCN2 overexpression locally in left atrium (LA) induces both current and in situ pacemaker function. MethodsProtocols were approved by the Columbia University Animal Care and Use Committee. Viral/Genetic PreparationWe prepared an adenoviral construct of mouse HCN2 (mHCN2, GenBank AJ225122) driven by the cytomegalovirus promoter, as previously described. 3 The construct AdHCN2 was purified through plaque assay, amplified to a large stock, and harvested and titrated after CsCl banding. The same procedure was used to construct an adenoviral vector of enhanced green fluorescent protei...

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 Ͼ2 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. I HCN2 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. I HCN2 for all cells was 32.1Ϯ1.3 pA/pF (meanϮSE) at Ϫ150 mV. Pacemaker function in intact dogs required 10 to 12 days to fully stabilize and persisted consistently through day 42 in dogs receiving Ն700 000 hMSCs (Ϸ40% 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 Ͼ700 000 are sufficient for pacemaking when administered to left ventricular myocardium.

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-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 Ϫ46.9 mV in mE324A versus Ϫ66.1 mV in mHCN2 (PϽ0.05). mE324A manifested a positive shift of voltage dependence of gating kinetics of activation and deactivation compared with mHCN2 (PϽ0.05) 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; PϽ0.05). When spontaneous rate fell below 45 bpm, EPM intervened at that rate, triggering 83% of beats in control, contrasting (PϽ0.05) with 26% (mHCN2) and 36% (mE324A). On day 14, epinephrine (1 g/kg per minute IV) induced a 50% heart rate increase in all mE324A, one third of mHCN2, and one fifth of control (PϽ0.05 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.

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Background-In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel (SCN5A) is largely inactivated, contributing to low action potential upstroke velocity (V 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 Ϫ60 mV; overexpression of SCN5A did not. In vivo epicardial border zone electrograms were broad and fragmented in shams (31.5Ϯ2.3 ms) and narrower in SkM1 (22.6Ϯ2.8 ms; Pϭ0.03). Premature stimulation induced ventricular tachyarrhythmia/fibrillation Ͼ60 seconds in 6 of 8 shams versus 2 of 12 SkM1 (Pϭ0.02). Microelectrode studies of epicardial border zones from SkM1 showed membrane potentials equal to that of shams and V max greater than that of shams as membrane potential depolarized (PϽ0.01). Infarct sizes were similar (sham, 30Ϯ2.8%; SkM1, 30Ϯ2.6%; Pϭ0.86). SkM1 expression in injected epicardium was confirmed immunohistochemically. Conclusions-SkM1 increases V max of depolarized myocardium and reduces the incidence of inducible sustained ventricular tachyarrhythmia/fibrillation in canine infarcts. Gene therapy to normalize activation by increasing V max at depolarized potentials may be a promising antiarrhythmic strategy. Key Words: arrhythmia Ⅲ gene therapy Ⅲ ion channels Ⅲ myocardial infarction Ⅲ tachyarrhythmias R eentry accounts for Ϸ85% of serious arrhythmias complicating ischemic heart disease. 1 Prevention and treatment are rooted in early 20th century research on reentry. [2][3][4] The goals are to create bidirectional conduction block (with drugs that block Na ϩ channels, surgery, or ablation), to prolong refractoriness so that reentry fails (with drugs that usually prolong repolarization), or both in combination. 5 These therapies have drawbacks ranging from incomplete success to toxicity, including proarrhythmia. Editorial p 6 Clinical Perspective p 27Less attention has been paid to another therapeutic approach suggested many years ago 2-5 : reentry should terminate if an activating waveform persists in conducting at normal velocity, even through depolarized tissues. Therefore, we hypothesized that "improving" the efficiency of propagation through depolarized regions by increasing the maximum Received July 22, 2008; accep...

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.