J. Kevin Donahue

Inherited Arrhythmias A National Heart, Lung, and Blood Institute and Office of Rare Diseases Workshop Consensus Report About the Diagnosis, Phenotyping, Molecular Mechanisms, and Therapeutic Approaches for Primary Cardiomyopathies of Gene Mutations Affecting Ion Channel Function

The National Heart, Lung, and Blood Institute and Office of Rare Diseases at the National Institutes of Health organized a workshop (September 14 to 15, 2006, in Bethesda, Md) to advise on new research directions needed for improved identification and treatment of rare inherited arrhythmias. These included the following: (1) Na+ channelopathies; (2) arrhythmias due to K+ channel mutations; and (3) arrhythmias due to other inherited arrhythmogenic mechanisms. Another major goal was to provide recommendations to support, enable, or facilitate research to improve future diagnosis and management of inherited arrhythmias. Classifications of electric heart diseases have proved to be exceedingly complex and in many respects contradictory. A new contemporary and rigorous classification of arrhythmogenic cardiomyopathies is proposed. This consensus report provides an important framework and overview to this increasingly heterogeneous group of primary cardiac membrane channel diseases. Of particular note, the present classification scheme recognizes the rapid evolution of molecular biology and novel therapeutic approaches in cardiology, as well as the introduction of many recently described diseases, and is unique in that it incorporates ion channelopathies as a primary cardiomyopathy in consensus with a recent American Heart Association Scientific Statement.

Focal modification of electrical conduction in the heart by viral gene transfer

Modern treatment of cardiac arrhythmias is limited to pharmacotherapy, radiofrequency ablation, or implantable devices. Antiarrhythmic medications suppress arrhythmias, but their systemic effects are often poorly tolerated and their proarrhythmic tendencies increase mortality. Radiofrequency ablation can cure only a limited number of arrhythmias. Implantable devices can be curative for bradyarrhythmias and lifesaving for tachyarrhythmias, but require a lifetime commitment to repeated procedures, are a significant expense, and may lead to severe complications. One possibility is the use of gene therapy as an antiarrhythmic strategy. As an initial attempt to explore this option, we focused on genetic modification of the atrioventricular node. First, we developed an intracoronary perfusion model for gene delivery, building on our previous work in isolated cardiac myocytes and hearts perfused ex vivo. Using this method, we infected porcine hearts with Adβgal (recombinant adenovirus expressing Escherichia coli β-galactosidase) or with AdGi (adenovirus encoding the Gαi2 subunit). We hypothesized that excess Gαi2 would mimic the effects of β-adreneric antagonists, in effect creating a localized β-blockade. Gαi2 overexpression suppressed baseline atrioventricular conduction and slowed the heart rate during atrial fibrillation without producing complete heart block. In contrast, expression of the reporter gene β-galactosidase had no electrophysiological effects. Our results demonstrate the feasibility of using myocardial gene transfer strategies to treat common arrhythmias.

Magnetic Resonance–Based Anatomical Analysis of Scar-Related Ventricular Tachycardia. Implications for Catheter Ablation

In catheter ablation of scar-related monomorphic ventricular tachycardia (VT), substrate voltage mapping is used to electrically define the scar during sinus rhythm. However, the electrically defined scar may not accurately reflect the anatomical scar. Magnetic resonance–based visualization of the scar may elucidate the 3D anatomical correlation between the fine structural details of the scar and scar-related VT circuits. We registered VT activation sequence with the 3D scar anatomy derived from high-resolution contrast-enhanced MRI in a swine model of chronic myocardial infarction using epicardial sock electrodes (n=6, epicardial group), which have direct contact with the myocardium where the electrical signal is recorded. In a separate group of animals (n=5, endocardial group), we also assessed the incidence of endocardial reentry in this model using endocardial basket catheters. Ten to 12 weeks after myocardial infarction, sustained monomorphic VT was reproducibly induced in all animals (n=11). In the epicardial group, 21 VT morphologies were induced, of which 4 (19.0%) showed epicardial reentry. The reentry isthmus was characterized by a relatively small volume of viable myocardium bound by the scar tissue at the infarct border zone or over the infarct. In the endocardial group (n=5), 6 VT morphologies were induced, of which 4 (66.7%) showed endocardial reentry. In conclusion, MRI revealed a scar with spatially complex structures, particularly at the isthmus, with substrate for multiple VT morphologies after a single ischemic episode. Magnetic resonance–based visualization of scar morphology would potentially contribute to preprocedural planning for catheter ablation of scar-related, unmappable VT.

Oxidized CaMKII causes cardiac sinus node dysfunction in mice

Sinus node dysfunction (SND) is a major public health problem that is associated with sudden cardiac death and requires surgical implantation of artificial pacemakers. However, little is known about the molecular and cellular mechanisms that cause SND. Most SND occurs in the setting of heart failure and hypertension, conditions that are marked by elevated circulating angiotensin II (Ang II) and increased oxidant stress. Here, we show that oxidized calmodulin kinase II (ox-CaMKII) is a biomarker for SND in patients and dogs and a disease determinant in mice. In wild-type mice, Ang II infusion caused sinoatrial nodal (SAN) cell oxidation by activating NADPH oxidase, leading to increased ox-CaMKII, SAN cell apoptosis, and SND. p47-/- mice lacking functional NADPH oxidase and mice with myocardial or SAN-targeted CaMKII inhibition were highly resistant to SAN apoptosis and SND, suggesting that ox-CaMKII-triggered SAN cell death contributed to SND. We developed a computational model of the sinoatrial node that showed that a loss of SAN cells below a critical threshold caused SND by preventing normal impulse formation and propagation. These data provide novel molecular and mechanistic information to understand SND and suggest that targeted CaMKII inhibition may be useful for preventing SND in high-risk patients.

Targeted Modification of Atrial Electrophysiology by Homogeneous Transmural Atrial Gene Transfer

Background—Safe and effective myocardial gene transfer remains elusive. Heterogeneous ventricular gene delivery has been achieved in small mammals but generally with methods not readily transferable to the clinic. Atrium-specific gene transfer has not yet been reported. We hypothesized that homogeneous atrial gene transfer could be achieved by direct application of adenoviral vectors to the epicardial surface, use of poloxamer gel to increase virus contact time, and mild trypsinization to increase virus penetration. Methods and Results—We “painted” recombinant adenovirus encoding the reporter gene Escherichia coli &bgr;-galactosidase directly onto porcine atria. Investigational variables included poloxamer use, trypsin concentration, and safety. Using the painting method, we modified the atrial phenotype with an adenovirus expressing HERG-G628S, a long-QT-syndrome mutant. Our results showed that application of virus with poloxamer alone resulted in diffuse epicardial gene transfer with negligible penetration into the myocardium. Dilute trypsin concentrations allowed complete transmural gene transfer. After trypsin exposure, echocardiographic left atrial diameter did not change. Left atrial function decreased on postoperative day 3 but returned to baseline by day 7. Tissue tensile strength was affected only in the 1% trypsin group. HERG-G628S gene transfer prolonged atrial action potential duration and refractory period without affecting ventricular electrophysiology. Conclusions—We show complete transmural atrial gene transfer by this novel painting method. Adaptation of the method could allow application to other tissue targets. Use with functional proteins in the atria could cure or even prevent diseases such as atrial fibrillation or sinus node dysfunction.

Molecular ablation of ventricular tachycardia after myocardial infarction.

Ventricular tachycardia is a common and lethal complication after myocardial infarction. Here we show that focal transfer of a gene encoding a dominant-negative version of the KCNH2 potassium channel (KCNH2-G628S) to the infarct scar border eliminated all ventricular arrhythmias in a porcine model. No proarrhythmia or other negative effects were discernable. Our results demonstrate the potential viability of gene therapy for ablation of ventricular arrhythmias.

Connexin Gene Transfer Preserves Conduction Velocity and Prevents Atrial Fibrillation

Background— Several lines of evidence have suggested that maintenance of atrial fibrillation (AF) depends on reentrant mechanisms. Maintenance of reentry necessitates a sufficiently short refractory period and/or delayed conduction, and AF has been associated with both alterations. Fibrosis, cellular dysfunction, and gap junction protein alterations occur in AF and cause conduction delay. We performed this study to test the hypothesis that gap junction protein overexpression would improve conduction and prevent AF. Methods and Results— Thirty Yorkshire swine were randomized into 2 groups (sinus rhythm and AF), and each group into 3 subgroups: sham-operated control, gene therapy with adenovirus expressing connexin (Cx) 40, and gene therapy with adenovirus expressing Cx43 (n=5 per subgroup). All animals had epicardial gene painting; the AF group had burst atrial pacing. All animals underwent terminal study 7 days after gene transfer. Sinus rhythm animals had strong transgene expression but no atrial conduction changes. In AF animals, controls had reduced and lateralized Cx43 expression, and Cx43 gene transfer restored expression and cellular location to sinus rhythm control levels. In the AF group, both Cx40 and Cx43 gene transfer improved conduction and reduced AF relative to controls. Conclusions— Connexin gene therapy preserved atrial conduction and prevented AF.

Creation of a Genetic Calcium Channel Blocker by Targeted Gem Gene Transfer in the Heart

Calcium channel blockers are among the most commonly used therapeutic drugs. Nevertheless, the utility of calcium channel blockers for heart disease is limited because of the potent vasodilatory effect that causes hypotension, and other side effects attributable to blockade of noncardiac channels. Therefore, focal calcium channel blockade by gene transfer is highly desirable. With a view to creating a focally applicable genetic calcium channel blocker, we overexpressed the ras-related small G-protein Gem in the heart by somatic gene transfer. Adenovirus-mediated delivery of Gem markedly decreased L-type calcium current density in ventricular myocytes, resulting in the abbreviation of action potential duration. Furthermore, transduction of Gem resulted in a significant shortening of the electrocardiographic QTc interval and reduction of left ventricular systolic function. Focal delivery of Gem to the atrioventricular (AV) node significantly slowed AV nodal conduction (prolongation of PR and AH intervals), which was effective in the reduction of heart rate during atrial fibrillation. Thus, these results indicate that gene transfer of Gem functions as a genetic calcium channel blocker, the local application of which can effectively modulate cardiac electrical and contractile function.

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

Background—The need for new treatment strategies for cardiac arrhythmias has motivated our continuing development of gene therapeutic options. Previously, we reported a decreased heart rate in an acute model of atrial fibrillation after atrioventricular nodal gene transfer. Here, we expand those observations to persistent atrial fibrillation and severe heart failure. Methods and Results—After 3 weeks of atrial fibrillation, domestic swine received atrioventricular nodal gene transfer with adenoviruses encoding &bgr;-galactosidase (&bgr;-gal), wild-type G&agr;i2 (wtGi), or constitutively active mutant (cGi). Heart rates in awake, alert animals were not altered by &bgr;-gal or wtGi. cGi caused a sustained 15% to 25% decrease in heart rate. The wtGi effect became evident with sedation. A tachycardia-induced cardiomyopathy was present before gene transfer. In the &bgr;-gal group, cardiomyopathy worsened over time. In the wtGi group, the condition improved slightly, and in the cGi group, ejection fraction was near normal at the end of the study. TUNEL staining results corroborated this finding. Conclusions—cGi overexpression in the porcine atrioventricular node causes physiologically relevant heart rate control in persistent atrial fibrillation. These data advance the development of gene therapy as a potential treatment for common cardiac arrhythmias.

Selective Molecular Potassium Channel Blockade Prevents Atrial Fibrillation

Background— Safety and efficacy limit currently available atrial fibrillation (AF) therapies. We hypothesized that atrial gene transfer would allow focal manipulation of atrial electrophysiology and, by eliminating reentry, would prevent AF. Methods and Results— In a porcine AF model, we compared control animals to animals receiving adenovirus that encoded KCNH2-G628S, a dominant negative mutant of the IKr potassium channel &agr;-subunit (G628S animals). After epicardial atrial gene transfer and pacemaker implantation for burst atrial pacing, animals were evaluated daily for cardiac rhythm. Electrophysiological and molecular studies were performed at baseline and when animals were euthanized on either postoperative day 7 or 21. By day 10, none of the control animals and all of the G628S animals were in sinus rhythm. After day 10, the percentage of G628S animals in sinus rhythm gradually declined until all animals were in AF by day 21. The relative risk of AF throughout the study was 0.44 (95% confidence interval 0.33 to 0.59, P<0.01) among the G628S group versus controls. Atrial monophasic action potential was considerably longer in G628S animals than in controls at day 7, and KCNH2 protein levels were 61% higher in the G628S group than in control animals (P<0.01). Loss of gene expression at day 21 correlated with loss of action potential prolongation and therapeutic efficacy. Conclusions— Gene therapy with KCNH2-G628S eliminated AF by prolonging atrial action potential duration. The effect duration correlated with transgene expression.