Patrick Lugenbiel

Connexin 43 gene therapy prevents persistent atrial fibrillation in a porcine model

AIMS Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, and effective treatment of AF still remains an unmet medical need. AF is associated with atrial conduction disturbances caused by electrical and/or structural remodelling. We hypothesized that AF suppresses expression of the gap junction protein connexin (Cx) 43 and that Cx43 gene transfer to both atria would prevent persistent AF. The first aim of this study was to assess whether AF is associated with connexin remodelling in a porcine model. A strategy to suppress persistent AF by gene therapy was then developed and evaluated in vivo. METHODS AND RESULTS AF was induced in domestic pigs via atrial burst pacing, causing a 62.4% reduction in atrial Cx43 protein. Adenoviruses encoding for Cx43 (AdCx43) or green fluorescent protein (AdGFP) were injected into both atria, followed by epicardial electroporation to enhance transgene expression. Combining direct injection of adenoviruses with electroporation achieved GFP reporter gene expression in ∼50% of atrial cells in vivo. AdCx43-treated animals exhibited a 2.5-fold increase in atrial Cx43 protein content and did not develop persistent AF during the observation period of 14 days. In contrast, control animals developed persistent AF within 7.4 ± 0.5 days. Rapid ventricular heart rates during AF led to deterioration of cardiac function in control pigs but not in pigs treated with AdCx43. CONCLUSION Our results highlight the contribution of Cx43 to the pathophysiology of AF and demonstrate the viability of gene therapy for prevention of atrial arrhythmias.

Upregulation of K(2P)3.1 K+ Current Causes Action Potential Shortening in Patients With Chronic Atrial Fibrillation

Background— Antiarrhythmic management of atrial fibrillation (AF) remains a major clinical challenge. Mechanism-based approaches to AF therapy are sought to increase effectiveness and to provide individualized patient care. K2P3.1 (TASK-1 [tandem of P domains in a weak inward-rectifying K+ channel–related acid-sensitive K+ channel-1]) 2-pore-domain K+ (K2P) channels have been implicated in action potential regulation in animal models. However, their role in the pathophysiology and treatment of paroxysmal and chronic patients with AF is unknown. Methods and Results— Right and left atrial tissue was obtained from patients with paroxysmal or chronic AF and from control subjects in sinus rhythm. Ion channel expression was analyzed by quantitative real-time polymerase chain reaction and Western blot. Membrane currents and action potentials were recorded using voltage- and current-clamp techniques. K2P3.1 subunits exhibited predominantly atrial expression, and atrial K2P3.1 transcript levels were highest among functional K2P channels. K2P3.1 mRNA and protein levels were increased in chronic AF. Enhancement of corresponding currents in the right atrium resulted in shortened action potential duration at 90% of repolarization (APD90) compared with patients in sinus rhythm. In contrast, K2P3.1 expression was not significantly affected in subjects with paroxysmal AF. Pharmacological K2P3.1 inhibition prolonged APD90 in atrial myocytes from patients with chronic AF to values observed among control subjects in sinus rhythm. Conclusions— Enhancement of atrium-selective K2P3.1 currents contributes to APD shortening in patients with chronic AF, and K2P3.1 channel inhibition reverses AF-related APD shortening. These results highlight the potential of K2P3.1 as a novel drug target for mechanism-based AF therapy.

Suppression of persistent atrial fibrillation by genetic knockdown of caspase 3: a pre-clinical pilot study

AIMS Atrial fibrillation (AF) is linked to cardiomyocyte apoptosis, leading to atrial remodelling and reduction in electrical conduction velocity. We hypothesized that genetic suppression of an apoptotic key enzyme, caspase 3, would prevent the development of persistent AF by reducing apoptosis which may serve as an arrhythmogenic substrate. METHODS AND RESULTS Atrial fibrillation was induced in domestic pigs by atrial burst pacing via an implanted cardiac pacemaker. Study animals were then assigned to receive either Ad-siRNA-Cas3 gene therapy to inactivate caspase 3 or green fluorescent protein (Ad-GFP) as a control. Adenoviruses were applied using a hybrid technique employing right and left atrial virus injection followed by epicardial electroporation to increase expression of plasmid DNA. In pigs treated with Ad-siRNA-Cas3, the onset of AF was suppressed or significantly delayed compared with controls (10.3 ± 1.2 days vs. 6.0 ± 1.6 days; P= 0.04). Electrical mapping revealed prolonged atrial conduction in the control group that was prevented by Ad-siRNA-Cas3 gene therapy. On the molecular level, Ad-siRNA-Cas3 application resulted in down-regulation of caspase 3 expression and suppression of apoptotic activity. CONCLUSION Knockdown of caspase 3 by atrial Ad-siRNA-Cas3 gene transfer suppresses or delays the onset of persistent AF by reduction in apoptosis and prevention of intra-atrial conduction delay in a porcine model. These results highlight the significance of apoptosis in the pathophysiology of AF and demonstrate short-term efficacy of gene therapy for suppression of AF.

Cloning, functional characterization, and remodeling of K2P3.1 (TASK-1) potassium channels in a porcine model of atrial fibrillation and heart failure

BACKGROUND Effective treatment of atrial fibrillation (AF) remains an unmet need. Human K2P3.1 (TASK-1) K(+) channels display atrial-specific expression and may serve as novel antiarrhythmic targets. In rodents, inhibition of K2P3.1 causes prolongation of action potentials and QT intervals. We used a porcine model to further elucidate the significance of K2P3.1 in large mammals. OBJECTIVE The purpose of this study was to study porcine (p)K2P3.1 channel function and cardiac expression and to analyze pK2P3.1 remodeling in AF and heart failure (HF). METHODS The porcine K2P3.1 ortholog was amplified and characterized using voltage-clamp electrophysiology. K2P3.1 mRNA expression and remodeling were studied in domestic pigs during AF and HF induced by atrial burst pacing. RESULTS Porcine K2P3.1 cDNA encodes a channel protein with 97% identity to human K2P3.1. K(+) currents recorded from Xenopus oocytes expressing pK2P3.1 were functionally and pharmacologically similar to their human counterparts. In the pig, K2P3.1 mRNA was predominantly expressed in atrial tissue. AF and HF were associated with reduction of K2P3.1 mRNA levels by 85.1% (right atrium) and 77.0% (left atrium) at 21-day follow-up. In contrast, ventricular K2P3.1 expression was low and not significantly affected by AF/HF. CONCLUSION Porcine K2P3.1 channels exhibit atrial expression and functional properties similar to their human orthologs, supporting a general role as antiarrhythmic drug targets. K2P3.1 down-regulation in AF with HF may indicate functional relevance of the channel that remains to be validated in prospective interventional studies.

Cardiac expression and atrial fibrillation-associated remodeling of K2P2.1 (TREK-1) K+ channels in a porcine model

AIMS Effective management of atrial fibrillation (AF) often remains an unmet need. Cardiac two-pore-domain K(+) (K2P) channels are implicated in action potential regulation, and their inhibition has been proposed as a novel antiarrhythmic strategy. K2P2.1 (TREK-1) channels are expressed in the human heart. This study was designed to identify and functionally express porcine K2P2.1 channels. In addition, we sought to analyze cardiac expression and AF-associated K2P2.1 remodeling in a clinically relevant porcine AF model. MAIN METHODS Three pK2P2.1 isoforms were identified and amplified. Currents were recorded using voltage clamp electrophysiology in the Xenopus oocyte expression system. K2P2.1 remodeling was studied by quantitative real time PCR and Western blot in domestic pigs during AF induced by atrial burst pacing. KEY FINDINGS Human and porcine K2P2.1 proteins share 99% identity. Residues involved in phosphorylation or glycosylation are conserved. Porcine K2P2.1 channels carried outwardly rectifying K(+) currents similar to their human counterparts. In pigs, K2P2.1 was expressed ubiquitously in the heart with predominance in the atrial tissue. AF was associated with time-dependent reduction of K2P2.1 protein in the RA by 70% (7 days of AF) and 80% (21 days of AF) compared to control animals in sinus rhythm. K2P2.1 expression in the left atrium, AV node, and ventricles was not affected by AF. SIGNIFICANCE Similarities between porcine and human K2P2.1 channels indicate that the pig may represent a valid model for mechanistic and preclinical studies. AF-related atrial K2P2.1 remodeling has potential implications for arrhythmia maintenance and antiarrhythmic therapy.

Atrial Fibrillation Complicated by Heart Failure Induces Distinct Remodeling of Calcium Cycling Proteins

Atrial fibrillation (AF) and heart failure (HF) are two of the most common cardiovascular diseases. They often coexist and account for significant morbidity and mortality. Alterations in cellular Ca2+ homeostasis play a critical role in AF initiation and maintenance. This study was designed to specifically elucidate AF-associated remodeling of atrial Ca2+ cycling in the presence of mild HF. AF was induced in domestic pigs by atrial burst pacing. The animals underwent electrophysiologic and echocardiographic examinations. Ca2+ handling proteins were analyzed in right atrial tissue obtained from pigs with AF (day 7; n = 5) and compared to sinus rhythm (SR) controls (n = 5). During AF, animals exhibited reduction of left ventricular ejection fraction (from 73% to 58%) and prolonged atrial refractory periods. AF and HF were associated with suppression of protein kinase A (PKA)RII (-62%) and Ca2+-calmodulin-dependent kinase II (CaMKII) δ by 37%, without changes in CaMKIIδ autophosphorylation. We further detected downregulation of L-type calcium channel (LTCC) subunit α2 (-75%), sarcoplasmic reticulum Ca2+-ATPase (Serca) 2a (-29%), phosphorylated phospholamban (Ser16, -92%; Thr17, -70%), and phospho-ryanodine receptor 2 (RyR2) (Ser2808, -62%). Na+-Ca2+ exchanger (NCX) levels were upregulated (+473%), whereas expression of Ser2814-phosphorylated RyR2 and LTCCα1c subunits was not significantly altered. In conclusion, AF produced distinct arrhythmogenic remodeling of Ca2+ handling in the presence of tachycardia-induced mild HF that is different from AF without structural alterations. The changes may provide a starting point for personalized approaches to AF treatment.

Biological Heart Rate Reduction Through Genetic Suppression of Gαs Protein in the Sinoatrial Node

Background Elevated heart rate represents an independent risk factor for cardiovascular outcome in patients with heart disease. In the sinoatrial node, rate increase is mediated by β1 adrenoceptor mediated activation of the Gαs pathway. We hypothesized that genetic inactivation of the stimulatory Gαs protein in the sinoatrial node would provide sinus rate control and would prevent inappropriate heart rate acceleration during β-adrenergic activation. Methods and Results Domestic pigs (n=10) were evenly assigned to receive either Ad-small interfering RNA (siRNA)-Gαs gene therapy to inactivate Gαs or adenovirus encoding for green fluorescent protein (Ad-GFP) as control. Adenoviruses were applied through virus injection into the sinoatrial node followed by epicardial electroporation, and heart rates were evaluated for 7 days. Genetic inhibition of Gαs protein significantly reduced mean heart rates on day 7 by 16.5% compared with control animals (110±8.8 vs 131±9.4 beats per minute; P<0.01). On β-adrenergic stimulation with isoproterenol, we observed a tendency toward diminished rate response in the Ad-siRNA-Gαs group (Ad-siRNA-Gαs, +79.3%; Ad-GFP, +61.7%; n=3 animals per group; P= 0.294). Adverse effects of gene transfer on left ventricular ejection fraction (LVEF) were not detected following treatment (LVEFAd-siRNA-Gαs, 66%; LVEFAd-GFP, 60%). Conclusions In this preclinical proof-of-concept study targeted Ad-siRNA-Gαs gene therapy reduced heart rates during normal sinus rhythm compared with Ad-GFP treatment and prevented inappropriate rate increase after β-adrenergic stimulation. Gene therapy may provide an additional therapeutic option for heart rate reduction in cardiac disease. (J Am Heart Assoc. 2012;1:jah3-e000372 doi: 10.1161/JAHA.111.000372)

Therapeutic targeting of two-pore-domain potassium (K2P) channels in the cardiovascular system

The improvement of treatment strategies in cardiovascular medicine is an ongoing process that requires constant optimization. The ability of a therapeutic intervention to prevent cardiovascular pathology largely depends on its capacity to suppress the underlying mechanisms. Attenuation or reversal of disease-specific pathways has emerged as a promising paradigm, providing a mechanistic rationale for patient-tailored therapy. Two-pore-domain K(+) (K(2P)) channels conduct outward K(+) currents that stabilize the resting membrane potential and facilitate action potential repolarization. K(2P) expression in the cardiovascular system and polymodal K2P current regulation suggest functional significance and potential therapeutic roles of the channels. Recent work has focused primarily on K(2P)1.1 [tandem of pore domains in a weak inwardly rectifying K(+) channel (TWIK)-1], K(2P)2.1 [TWIK-related K(+) channel (TREK)-1], and K(2P)3.1 [TWIK-related acid-sensitive K(+) channel (TASK)-1] channels and their role in heart and vessels. K(2P) currents have been implicated in atrial and ventricular arrhythmogenesis and in setting the vascular tone. Furthermore, the association of genetic alterations in K(2P)3.1 channels with atrial fibrillation, cardiac conduction disorders and pulmonary arterial hypertension demonstrates the relevance of the channels in cardiovascular disease. The function, regulation and clinical significance of cardiovascular K(2P) channels are summarized in the present review, and therapeutic options are emphasized.

Feasibility Study on Cardiac Arrhythmia Ablation Using High-Energy Heavy Ion Beams

High-energy ion beams are successfully used in cancer therapy and precisely deliver high doses of ionizing radiation to small deep-seated target volumes. A similar noninvasive treatment modality for cardiac arrhythmias was tested here. This study used high-energy carbon ions for ablation of cardiac tissue in pigs. Doses of 25, 40, and 55 Gy were applied in forced-breath-hold to the atrioventricular junction, left atrial pulmonary vein junction, and freewall left ventricle of intact animals. Procedural success was tracked by (1.) in-beam positron-emission tomography (PET) imaging; (2.) intracardiac voltage mapping with visible lesion on ultrasound; (3.) lesion outcomes in pathohistolgy. High doses (40–55 Gy) caused slowing and interruption of cardiac impulse propagation. Target fibrosis was the main mediator of the ablation effect. In irradiated tissue, apoptosis was present after 3, but not 6 months. Our study shows feasibility to use high-energy ion beams for creation of cardiac lesions that chronically interrupt cardiac conduction.

TREK-1 (K2P2.1) K+ channels are suppressed in patients with atrial fibrillation and heart failure and provide therapeutic targets for rhythm control

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Concomitant heart failure (HF) poses a particular therapeutic challenge and is associated with prolonged atrial electrical refractoriness compared with non-failing hearts. We hypothesized that downregulation of atrial repolarizing TREK-1 (K2P2.1) K+ channels contributes to electrical remodeling during AF with HF, and that TREK-1 gene transfer would provide rhythm control via normalization of atrial effective refractory periods in this AF subset. In patients with chronic AF and HF, atrial TREK-1 mRNA levels were reduced by 82% (left atrium) and 81% (right atrium) compared with sinus rhythm (SR) subjects. Human findings were recapitulated in a porcine model of atrial tachypacing-induced AF and reduced left ventricular function. TREK-1 mRNA (−66%) and protein (−61%) was suppressed in AF animals at 14-day follow-up compared with SR controls. Downregulation of repolarizing TREK-1 channels was associated with prolongation of atrial effective refractory periods versus baseline conditions, consistent with prior observations in humans with HF. In a preclinical therapeutic approach, pigs were randomized to either atrial Ad-TREK-1 gene therapy or sham treatment. Gene transfer effectively increased TREK-1 protein levels and attenuated atrial effective refractory period prolongation in the porcine AF model. Ad-TREK-1 increased the SR prevalence to 62% during follow-up in AF animals, compared to 35% in the untreated AF group. In conclusion, TREK-1 downregulation and rhythm control by Ad-TREK-1 transfer suggest mechanistic and potential therapeutic significance of TREK-1 channels in a subgroup of AF patients with HF and prolonged atrial effective refractory periods. Functional correction of ionic remodeling through TREK-1 gene therapy represents a novel paradigm to optimize and specify AF management.