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.

Heterogeneous repolarization creates ventricular tachycardia circuits in healed myocardial infarction scar.

Arrhythmias originating in scarred ventricular myocardium are a major cause of death, but the underlying mechanism allowing these rhythms to exist remains unknown. This gap in knowledge critically limits identification of at-risk patients and treatment once arrhythmias become manifest. Here we show that potassium voltage-gated channel subfamily E regulatory subunits 3 and 4 (KCNE3, KCNE4) are uniquely upregulated at arrhythmia sites within scarred myocardium. Ventricular arrhythmias occur in areas with a distinctive cardiomyocyte repolarization pattern, where myocyte tracts with short repolarization times connect to myocytes tracts with long repolarization times. We found this unique pattern of repolarization heterogeneity only in ventricular arrhythmia circuits. In contrast, conduction abnormalities were ubiquitous within scar. These repolarization heterogeneities are consistent with known functional effects of KCNE3 and KCNE4 on the slow delayed-rectifier potassium current. We observed repolarization heterogeneity using conventional cardiac electrophysiologic techniques that could potentially translate to identification of at-risk patients. The neutralization of the repolarization heterogeneities could represent a potential strategy for the elimination of ventricular arrhythmia circuits.

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 I(Kr) potassium channel alpha-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.

Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker.

BACKGROUND: Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity. METHODS AND RESULTS: Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Na(v)1.5, K(v)4.3, K(v)1.5, ERG, K(ir)2.1, K(ir)6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Ca(v)1.3, Ca(v)3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of K(v)4.2, K(ir)6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking. CONCLUSIONS: Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.

Computer three-dimensional reconstruction of the atrioventricular node.

Because of its complexity, the atrioventricular node (AVN), remains 1 of the least understood regions of the heart. The aim of the study was to construct a detailed anatomic model of the AVN and relate it to AVN function. The electric activity of a rabbit AVN preparation was imaged using voltage-dependent dye. The preparation was then fixed and sectioned. Sixty-five sections at 60- to 340-microm intervals were stained for histology and immunolabeled for neurofilament (marker of nodal tissue) and connexin43 (gap junction protein). This revealed multiple structures within and around the AVN, including transitional tissue, inferior nodal extension, penetrating bundle, His bundle, atrial and ventricular muscle, central fibrous body, tendon of Todaro, and valves. A 3D anatomically detailed mathematical model (approximately 13 million element array) of the AVN and surrounding atrium and ventricle, incorporating all cell types, was constructed. Comparison of the model with electric activity recorded in experiments suggests that the inferior nodal extension forms the slow pathway, whereas the transitional tissue forms the fast pathway into the AVN. In addition, it suggests the pacemaker activity of the atrioventricular junction originates in the inferior nodal extension. Computer simulation of the propagation of the action potential through the anatomic model shows how, because of the complex structure of the AVN, reentry (slow-fast and fast-slow) can occur. In summary, a mathematical model of the anatomy of the AVN has been generated that allows AVN conduction to be explored.

Differential expression of ion channel transcripts in atrial muscle and sinoatrial node in rabbit.

The aim of the study was to identify ion channel transcripts expressed in the sinoatrial node (SAN), the pacemaker of the heart. Functionally, the SAN can be divided into central and peripheral regions (center is adapted for pacemaking only, whereas periphery is adapted to protect center and drive atrial muscle as well as pacemaking) and the aim was to study expression in both regions. In rabbit tissue, the abundance of 30 transcripts (including transcripts for connexin, Na(+), Ca(2+), hyperpolarization-activated cation and K(+) channels, and related Ca(2+) handling proteins) was measured using quantitative PCR and the distribution of selected transcripts was visualized using in situ hybridization. Quantification of individual transcripts (quantitative PCR) showed that there are significant differences in the abundance of 63% of the transcripts studied between the SAN and atrial muscle, and cluster analysis showed that the transcript profile of the SAN is significantly different from that of atrial muscle. There are apparent isoform switches on moving from atrial muscle to the SAN center: RYR2 to RYR3, Na(v)1.5 to Na(v)1.1, Ca(v)1.2 to Ca(v)1.3 and K(v)1.4 to K(v)4.2. The transcript profile of the SAN periphery is intermediate between that of the SAN center and atrial muscle. For example, Na(v)1.5 messenger RNA is expressed in the SAN periphery (as it is in atrial muscle), but not in the SAN center, and this is probably related to the need of the SAN periphery to drive the surrounding atrial muscle.

Site of origin and molecular substrate of atrioventricular junctional rhythm in the rabbit heart.

During failure of the sinoatrial node, the heart can be driven by an atrioventricular (AV) junctional pacemaker. The position of the leading pacemaker site during AV junctional rhythm is debated. In this study, we present evidence from high-resolution fluorescent imaging of electrical activity in rabbit isolated atrioventricular node (AVN) preparations that, in the majority of cases (11 out of 14), the AV junctional rhythm originates in the region extending from the AVN toward the coronary sinus along the tricuspid valve (posterior nodal extension, PNE). Histological and immunohistochemical investigation showed that the PNE has the same morphology and unique pattern of expression of neurofilament160 (NF160) and connexins (Cx40, Cx43, and Cx45) as the AVN itself. Block of the pacemaker current, If, by 2 mmol/L Cs+ increased the AV junctional rhythm cycle length from 611+/-84 to 949+/-120 ms (mean+/-SD, n=6, P<0.001). Immunohistochemical investigation showed that the principal If channel protein, HCN4, is abundant in the PNE. As well as the AV junctional rhythm, the PNE described in this study may also be involved in the slow pathway of conduction into the AVN as well as AVN reentry, and the predominant lack of expression of Cx43 as well as the presence of Cx45 in the PNE shown could help explain its slow conduction.

Structure-function relationship in the AV junction.

In the normal heart, the atrioventricular node (AVN) is part of the sole pathway between the atria and ventricles. Under normal physiological conditions, the AVN controls appropriate frequency-dependent delay of contractions. The AVN also plays an important role in pathology: it protects ventricles during atrial tachyarrhythmia, and during sinoatrial node failure an AV junctional pacemaker can drive the heart. Finally, the AV junction provides an anatomical substrate for reentry. Using fluorescent imaging with voltage-sensitive dyes and immunohistochemistry, we have investigated the structure-function relationship of the AV junction during normal conduction, reentry, and junctional rhythm. We identified molecular and structural heterogeneity that provides a substrate for the dual-pathway AVN conduction. We observed heterogeneity of expression of three isoforms of connexins: Cx43, Cx45, and Cx40. We identified the site of origin of junctional rhythm at the posterior extension of the AV node in 79% (n = 14) of the studied hearts. This structure was similar to the compact AV node as determined by morphologic and molecular investigations. In particular, both the posterior extension and the compact node express the pacemaking channel HCN4 (responsible for the I(F) current) and neurofilament 160. In the rabbit heart, AV junction conduction, reentrant arrhythmia, and spontaneous rhythm are governed by heterogeneity of expression of several isoforms of gap junctions and ion channels. Uniform neurofilament expression suggests that AV nodal posterior extensions are an integral part of the cardiac pacemaking and conduction system. On the other hand, differential expression of Cx isoforms in this region provides an explanation of longitudinal dissociation, dual-pathway electrophysiology, and AV nodal reentrant arrhythmogenesis.

c-Src kinase inhibition reduces arrhythmia inducibility and connexin43 dysregulation after myocardial infarction.

OBJECTIVES: The aim of this study was to evaluate the role of tyrosine kinase cellular-Src (c-Src) inhibition on connexin43 (Cx43) regulation in a mouse model of myocardial infarction (MI). BACKGROUND: MI is associated with decreased expression of Cx43, the principal gap junction protein responsible for propagating current in ventricles. Activated c-Src has been linked to Cx43 dysregulation. METHODS: MI was induced in 12-week-old mice by coronary artery occlusion. MI mice were treated with c-Src inhibitors (PP1 or AZD0530), PP3 (an inactive analogue of PP1), or saline. Treated hearts were compared to sham mice by echocardiography, optical mapping, telemetry electrocardiographic monitoring, and inducibility studies. Tissues were collected for immunoblotting, quantitative polymerase chain reaction, and immunohistochemistry. RESULTS: Active c-Src was elevated in PP3-treated MI mice compared to sham at the scar border (280%, p = 0.003) and distal ventricle (346%, p = 0.013). PP1 treatment restored active c-Src to sham levels at the scar border (86%, p = 0.95) and distal ventricle (94%, p = 1.0). PP1 raised Cx43 expression by 69% in the scar border (p = 0.048) and by 73% in the distal ventricle (p = 0.043) compared with PP3 mice. PP1-treated mice had restored conduction velocity at the scar border (PP3: 32 cm/s, PP1: 41 cm/s, p < 0.05) and lower arrhythmic inducibility (PP3: 71%, PP1: 35%, p < 0.05) than PP3 mice. PP1 did not change infarct size, electrocardiographic pattern, or cardiac function. AZD0530 treatment demonstrated restoration of Cx43 comparable to PP1. CONCLUSIONS: c-Src inhibition improved Cx43 levels and conduction velocity and lowered arrhythmia inducibility after MI, suggesting a new approach for arrhythmia reduction following MI.

Connexin43 gene transfer reduces ventricular tachycardia susceptibility after myocardial infarction.

OBJECTIVES: The aim of this study was to evaluate the links between connexin43 (Cx43) expression, myocardial conduction velocity, and ventricular tachycardia in a model of healed myocardial infarction. BACKGROUND: Post-infarction ventricular arrhythmias frequently cause sudden death. Impaired myocardial conduction has previously been linked to ventricular arrhythmias. Altered connexin expression is a potential source of conduction slowing identified in healed scar border tissues. The functional effect of increasing border-zone Cx43 has not been previously evaluated. METHODS: Twenty-five Yorkshire pigs underwent anterior infarction by transient left anterior descending coronary artery occlusion, followed by weekly testing for arrhythmia inducibility. Twenty animals with reproducibly inducible sustained monomorphic ventricular tachycardia were randomized 2:1:1 to receive AdCx43, Adßgal, or no gene transfer. One week later, animals underwent follow-up electrophysiologic study and tissue assessment for several functional and molecular measures. RESULTS: Animals receiving AdCx43 had less electrogram fractionation and faster conduction velocity in the anterior-septal border zone. Only 40% of AdCx43 animals remained inducible for ventricular tachycardia, while 100% of controls were inducible after gene transfer. AdCx43 animals had 2-fold higher Cx43 protein levels in the anterior-septal infarct border, with similar percents of phosphorylated and intercalated disk-localized Cx43 compared with controls. CONCLUSIONS: These data mechanistically link Cx43 expression to slow conduction and arrhythmia susceptibility in the healed scar border zone. Targeted manipulation of Cx43 levels improved conduction velocity and reduced ventricular tachycardia susceptibility. Cx43 gene transfer represents a novel treatment strategy for post-infarction arrhythmias.

Ventricular tachycardia from the healed myocardial infarction scar: validation of an animal model and utility of gene therapy.

Life-threatening ventricular arrhythmias generally occur in the setting of structural heart disease. Current clinical options for patients at risk for these rhythm disturbances are limited. We developed a porcine model of inducible ventricular tachycardia originating in the border region of a healed myocardial infarction scar. After validating the model, we assessed gene transfer techniques, focusing on local modification of border zone tissues. We found that gene transfer of the dominant negative KCNH2-G628S mutation to the anteroseptal infarct border caused localized prolongation of effective refractory period in the target region and eliminated all ventricular arrhythmia inducibility. In this work, we characterize the animal model and review the gene transfer results.

Ion channel transcript expression at the rabbit atrioventricular conduction axis.

BACKGROUND: Little is known about the distribution of gap junctions and ion channels in the atrioventricular node, even though the physiology and pathology of the atrioventricular node is ultimately dependent on them. METHODS AND RESULTS: The abundance of 30 transcripts for markers, gap junctions, ion channels, and Ca(2+)-handling proteins in different regions of the rabbit atrioventricular node (nodal extension and proximal and distal penetrating bundle of His as well as atrial and ventricular muscle) was measured using a novel quantitative polymerase chain reaction technique and in situ hybridization. The expression profile of the nodal extension (slow pathway into penetrating bundle) was similar to that of the sinoatrial node. For example, in the nodal extension, in contrast to the atrial muscle and as expected for a slowly conducting tissue with pacemaker activity, there was no or reduced expression of Cx43, Na(v)1.5, Ca(v)1.2, K(v)1.4, KChIP2, and RYR3 and high expression of Ca(v)1.3 and HCN4. The expression profile of the penetrating bundle was less specialized. In situ hybridization revealed a transitional zone with reduced expression of Cx43, Na(v)1.5, and KChIP2 that may form the fast pathway into the penetrating bundle. CONCLUSIONS: At the atrioventricular node, the expression of gap junctions and ion channels in the nodal extension (slow pathway) and a transitional zone (putative fast pathway) as well as the penetrating bundle (output pathway) is specialized and heterogeneous and roughly matches the electrophysiology of the different regions.

P2 purinergic receptor mRNA in rat and human sinoatrial node and other heart regions.

It is known that adenosine 5'-triphosphate (ATP) is a cotransmitter in the heart. Additionally, ATP is released from ischemic and hypoxic myocytes. Therefore, cardiac-derived sources of ATP have the potential to modify cardiac function. ATP activates P2X(1-7) and P2Y(1-14) receptors; however, the presence of P2X and P2Y receptor subtypes in strategic cardiac locations such as the sinoatrial node has not been determined. An understanding of P2X and P2Y receptor localization would facilitate investigation of purine receptor function in the heart. Therefore, we used quantitative PCR and in situ hybridization to measure the expression of mRNA of all known purine receptors in rat left ventricle, right atrium and sinoatrial node (SAN), and human right atrium and SAN. Expression of mRNA for all the cloned P2 receptors was observed in the ventricles, atria, and SAN of the rat. However, their abundance varied in different regions of the heart. P2X(5) was the most abundant of the P2X receptors in all three regions of the rat heart. In rat left ventricle, P2Y(1), P2Y(2), and P2Y(14) mRNA levels were highest for P2Y receptors, while in right atrium and SAN, P2Y(2) and P2Y(14) levels were highest, respectively. We extended these studies to investigate P2X(4) receptor mRNA in heart from rats with coronary artery ligation-induced heart failure. P2X(4) receptor mRNA was upregulated by 93% in SAN (P < 0.05), while a trend towards an increase was also observed in the right atrium and left ventricle (not significant). Thus, P2X(4)-mediated effects might be modulated in heart failure. mRNA for P2X(4-7) and P2Y(1,2,4,6,12-14), but not P2X(2,3) and P2Y(11), was detected in human right atrium and SAN. In addition, mRNA for P2X(1) was detected in human SAN but not human right atrium. In human right atrium and SAN, P2X(4) and P2X(7) mRNA was the highest for P2X receptors. P2Y(1) and P2Y(2) mRNA were the most abundant for P2Y receptors in the right atrium, while P2Y(1), P2Y(2), and P2Y(14) were the most abundant P2Y receptor subtypes in human SAN. This study shows a widespread distribution of P2 receptor mRNA in rat heart tissues but a more restricted presence and distribution of P2 receptor mRNA in human atrium and SAN. This study provides further direction for the elucidation of P2 receptor modulation of heart rate and contractility.