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-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.

Abstract-Ventricular pacemaker current (I f ) shows distinct voltage dependence as a function of age, activating outside the physiological range in normal adult ventricle, but less negatively in neonatal ventricle. However, heterologously expressed HCN2 and HCN4, the putative molecular correlates of ventricular I f , exhibit only a modest difference in activation voltage. We therefore prepared an adenoviral construct (AdHCN2) of HCN2, the dominant ventricular isoform at either age, and used it to infect neonatal and adult rat ventricular myocytes to investigate the role of maturation on current gating. The expressed current exhibited an 18-mV difference in activation (V 1/2 Ϫ95.9Ϯ1.9 in adult; Ϫ77.6Ϯ1.6 mV in neonate), comparable to the 22-mV difference between native I f in adult and neonatal cultures (V 1/2 Ϫ98.7 versus Ϫ77.0 mV). This did not result from developmental differences in basal cAMP, because saturating cAMP in the pipette caused an equivalent positive shift in both preparations. In the neonate, AdHCN2 caused a significant increase in spontaneous rate compared with control (88Ϯ5 versus 48Ϯ4 bpm). In adult, where HCN2 activates more negatively, the effect was evident only during anodal excitation, requiring significantly less stimulus energy than control (2149Ϯ266 versus 3140Ϯ279 mV ⅐ ms).

Spontaneous electrical activity and indo 1 fluorescence ratios were recorded simultaneously in cultured pacemaker cells isolated from the rabbit sinoatrial node. Ryanodine (10 μM) reduced the amplitude of action potential-induced intracellular Ca2+([Formula: see text]) transients by 19 ± 3%, increased the time constant for their decay by 51 ± 5%, and slowed spontaneous firing by 32 ± 3%. 1,2-Bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM; 25 μM) inhibited the [Formula: see text] transients and slowed spontaneous firing by 28 ± 4%. Ryanodine did not alter hyperpolarization-activated or time-independent inward current, but it reduced the sum of L- and T-type Ca2+ currents ( I Ca,L and I Ca,T) in both the presence and absence of BAPTA-AM. In contrast, I Ca,L was unchanged by ryanodine. Slow inward current tails, presumed to be Na/Ca exchange current ( I Na/Ca), were abolished by BAPTA or ryanodine. The results suggest that a decrement of I Ca,T, due to reduction of the intracellular Ca2+ concentration or a direct effect of ryanodine on T-type Ca2+channels, contributes to the negative chronotropic effect. Another possibility, based primarily on theory and results in other preparations, is that a reduction of I Na/Ca, as a consequence of the smaller action potential-induced[Formula: see text] transients, contributes to the effect of ryanodine.

MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotidegated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between ؊60 and ؊95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a ␤ subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.MinK-related protein (MiRP1 or KCNE2) is purported to be a ␤ subunit for several voltage-gated potassium channels, including the rapid delayed rectifier (1), slow delayed rectifier (2), and transient outward current (3). In addition, we have reported that it can act as a ␤ subunit for the hyperpolarizationactivated, cyclic nucleotide-gated (HCN) 1 family of pacemaker channels (4). We found that co-expression of MiRP1 with HCN1 or HCN2 in Xenopus oocytes resulted in larger and more rapidly activating currents than when either HCN isoform was expressed alone but that MiRP1 did not shift the midpoint of activation of either isoform. Further, co-immunoprecipitation experiments involving HA-tagged MiRP1 and HCN1 demonstrated that the two proteins interact when co-expressed in oocytes.A more recent study demonstrated MiRP1 interaction with the HCN4 isoform. Here, co-expression with MiRP1, either in oocytes or Chinese hamster ovary cells, also resulted in increased current amplitude, but this was associated with slowing of kinetics and a negative shift of the midpoint of activation (5). However, another laboratory did not detect any effect of MiRP1 when co-expressed in HEK293 cells with either HCN4 or an HCN1-HCN4 tandem construct (6). Finally, a study of MiRP1 and HCN2 co-expression in Chinese hamster ovary cells reported a reduced time-dependent current component and increased instantaneous ...

Neuropeptide Y (NPY) is released from sympathetic neurons and exerts short-term (acute) effects on prejunctional nerve terminals and postjunctional cardiac ion channels. However, NPY also exerts long-term (trophic) effects on angiogenesis, cardiac hypertrophy, autonomic signaling, and cardiac ion channels, including effects on L-type Ca2+ and pacemaker channels.