Expression and Function of a Biological Pacemaker in Canine Heart

Abstract

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...

Inhibitory G Protein Overexpression Provides Physiologically Relevant Heart Rate Control in Persistent Atrial Fibrillation

“…To date, there are only 3 in vivo examples of the concept: our original report of AV nodal gene transfer in sedated pigs with acute AF and 2 illustrations of genetic pacemaker activity. 7,16,17 In the current report, we continue the process of developing gene therapy as a potential option for cardiac arrhythmias. In a stepwise fashion, we first showed rate control in an acute model of AF, 7 and now we demonstrate rate control in a physiologically relevant model of persistent AF and severe chronic heart failure.…”

Gene Therapy for Cardiac Arrhythmias

“…If current, also called a "pacemaker current", is the inward current during the diastolic phase, resulting in spontaneous rhythmic activity. 19,20 The adenoviral gene transduction of HCN2 channel induced pacemaker ac- tivity in the atrium 21 and the left bundle branch. 22 These initial trials revealed that a gene transfer into cardiomyocytes evoked spontaneous activity, but the heart rate as a result of these biological pacemakers was relatively low.…”

Fibroblasts Can Be Genetically Modified to Produce Excitable Cells Capable of Electrical Coupling

“…1 For example, in the context of cardiac impulse generation and propagation, focal gene transfer has recently been reported to result in both the creation of biological pacemakers 2,3 and conduction slowing through the atrioventricular (AV) node. 4 The essential physiological properties of cardiomyocytes supporting efficient myocardial impulse propagation are an excitable cell membrane and capacity for gap junctional intercellular communication (GJIC).…”