Background-Ca2ϩ leak from the sarcoplasmic reticulum (SR) may play an important role in triggering and/or maintaining atrial arrhythmias, including atrial fibrillation (AF). Protein kinase A (PKA) hyperphosphorylation of the cardiac ryanodine receptor (RyR2) resulting in dissociation of the channel-stabilizing subunit calstabin2 (FK506-binding protein or FKBP12.6) causes SR Ca 2ϩ leak in failing hearts and can trigger fatal ventricular arrhythmias. Little is known about the role of RyR2 dysfunction in AF, however. Methods and Results-Left and right atrial tissue was obtained from dogs with AF induced by rapid right atrial pacing (nϭ6 for left atrial, nϭ4 for right atrial) and sham instrumented controls (nϭ6 for left atrial, nϭ4 for right atrial). Right atrial tissue was also collected from humans with AF (nϭ10) and sinus rhythm (nϭ10) and normal cardiac function. PKA phosphorylation of immunoprecipitated RyR2 was determined by back-phosphorylation and by immunoblotting with a phosphospecific antibody. The amount of calstabin2 bound to RyR2 was determined by coimmunoprecipitation. RyR2 channel currents were measured in planar lipid bilayers. Atrial tissue from both the AF dogs and humans with chronic AF showed a significant increase in PKA phosphorylation of RyR2, with a corresponding decrease in calstabin2 binding to the channel. Channels isolated from dogs with AF exhibited increased open probability under conditions simulating diastole compared with channels from control hearts, suggesting that these AF channels could predispose to a diastolic SR Ca 2ϩ leak. Conclusions-SR Ca 2ϩ leak due to RyR2 PKA hyperphosphorylation may play a role in initiation and/or maintenance of

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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Sex-related differences in basal right ventricular endocardial AP configuration persist in castrated rabbits, suggesting that extragonadal factors contribute to the differences in ventricular repolarization. In this model, drugs that block I(Kr) but not I(Ks) prolong repolarization in a way that suggests that protection from excess prolongation in males is attributable to testosterone, whereas the risk of excess prolongation of repolarization in females is related to sex-determined factors in addition to estrogen.