High resolution optical mapping reveals conduction slowing in connexin43 deficient mice.

UNLABELLED: Analysis of mice with genetically altered expression of cardiac connexins can provide insights into the role of individual gap junction channel proteins in cell-to-cell communication, impulse propagation, and arrhythmias. However, conflicting results have been reported regarding conduction velocity slowing in mice heterozygous for a null mutation in the gene encoding connexin43 (Cx43). METHODS: High-resolution optical mapping was used to record action potentials from 256 sites, simultaneously, on the ventricular surface of Langendorff perfused hearts from 15 heterozygous (Cx43+/-) and 8 wildtype (Cx43+/+) mice (controls). A sensitive method for measuring epicardial conduction velocity was developed to minimize confounding influences of subepicardial breakthrough and virtual electrode effects. RESULTS: Epicardial conduction velocity was significantly slower (23 to 35%, P<0.01) in Cx43+/- mice compared to wildtype. There was no change in conduction patterns or anisotropic ratio (Cx43+/- 1.54+/-0.33; Cx43+/+ 1.57+/-0.17) suggesting that Cx43 expression was reduced uniformly throughout myocardium. The magnitude of reductions in conduction velocity and Cx43 protein expression (45%) were similar in mice in which the null allele occurred in a pure C57BL/6J genetic background versus a mixed (C57BL/6J X 129) background. Action potential duration did not differ between mice of different genotypes. CONCLUSIONS: A approximately 50% reduction of Cx43 expression causes significant conduction velocity slowing in the Cx43+/- mouse heart. The apparent lack of conduction velocity changes reported in previous studies may be related to technical factors rather than variations in genetic background. High-resolution optical mapping is a powerful tool for investigating molecular determinants of propagation and arrhythmias in genetically engineered mice.

Cryoablation of ventricular tachycardia guided by return cycle mapping after entrainment.

BACKGROUND: Although the implantable cardioverter-defibrillator effectively prevents sudden cardiac death, patients are still prone to recurrence of ventricular tachyarrhythmias. Electrophysiologically guided surgery is the most effective modality in abolishing ventricular tachycardia, having a lower recurrence rate than pharmacologic therapy or catheter ablation. Return cycle mapping after entrainment has been shown to localize the central common pathway, which is the target region for ablation, without pacing at the pathway or recording the potentials from the pathway. METHODS: To determine the accuracy and usefulness of return cycle mapping in surgery for ventricular tachycardia, we cryoablated 8 morphologies of ventricular tachycardia induced in postinfarction dogs with the guidance of return cycle mapping. The ventricular tachycardia was entrained from 3 to 5 different epicardial sites at a paced cycle length 10 to 20 ms shorter than the ventricular tachycardia cycle length and the epicardium was mapped with 61 unipolar electrodes during cessation of entrainment to construct return cycle maps. The return cycle was determined by subtracting the first activation time from the second activation time after the last stimulus in each electrode location, and the maps were then displayed on a computer. RESULTS: The total analysis process was completed within 3 minutes by means of a computer with custom-made programs. The activation map during ventricular tachycardia did not localize the central common pathway in any morphology of ventricular tachycardia, because the pattern of activation was concentric and diastolic potentials were not recorded. Cryoablation of the region where the isotemporal lines of the return cycle equal to the ventricular tachycardia cycle length intersected resulted in termination of ventricular tachycardia in all morphologies. The intersection was 26 +/- 9 mm from the earliest activation site. Epicardial mapping with 253 electrodes during cryothermia showed that the region localized by return cycle mapping was the central common pathway sandwiched between the lines of conduction block and that the cryolesion connected the lines of block, blocked the rotating wave front, and resulted in termination of the ventricular tachycardia. CONCLUSION: Return cycle mapping provides an accurate and rapid means of localizing the central common pathway without the need for recording potentials from the pathway or pacing at the pathway in ablation for ventricular tachycardia.

Morphological and membrane characteristics of spider and spindle cells isolated from rabbit sinus node.

This study reports the comparative quantitative, morphological, and electrophysiological properties of two pacemaker cell types, spider and spindle-shaped cells, isolated from the rabbit sinoatrial node. Isolated nodal cells were studied with perforated and ruptured patch whole cell recording techniques. The basic spontaneous cycle length of the spider cells was 381 +/- 12 ms, and the basic spontaneous cycle length of the spindle cells was 456 +/- 17 ms (n = 12, P < 0.05). The spider cells had a more positive maximum diastolic potential (-54 +/- 1 mV) compared with the spindle cells (-68 +/- 1mV, P < 0.05). The overshoot and action potential amplitudes were also smaller in the spider cells. The hyperpolarization-activated inward (I(f)) current density, measured from their tail currents, was 15 +/- 1.3 pA/pF for the spider cells and 9 +/- 0.7 pA/pF for the spindle cells (P < 0.01). I(f) current activation voltage was more positive in the spider cells than the spindle cells. Isoproterenol (1 microM) decreased the spontaneous cycle length of the spider cells by 28 +/- 3% and the spindle cells by 20 +/- 1.5% (P < 0.05). Acetylcholine (0.5 microM) hyperpolarized the membrane potential of the spider cells to -86 +/- 0.7 mV and the spindle cells to -76 +/- 0.8 mV (P < 0.05). In summary, there are at least two distinct pacemaker cell types in the sinus node with different electrophysiological characteristics.

Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia.

Electrical uncoupling at gap junctions during acute myocardial ischemia contributes to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca(2+) and H(+) and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling, but other mechanisms may play a role. We tested the hypothesis that uncoupling induced by acute ischemia is associated with changes in phosphorylation of the major cardiac gap junction protein, connexin43 (Cx43). Adult rat hearts perfused on a Langendorff apparatus were subjected to ischemia or ischemia/reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in the amount and distribution of phosphorylated and nonphosphorylated isoforms of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. In control hearts, virtually all Cx43 identified immunohistochemically at apparent intercellular junctions was phosphorylated. During ischemia, however, Cx43 underwent progressive dephosphorylation with a time course similar to that of electrical uncoupling. The total amount of Cx43 did not change, but progressive reduction in total Cx43 immunofluorescent signal and concomitant accumulation of nonphosphorylated Cx43 signal occurred at sites of intercellular junctions. Functional recovery during reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia is associated with dephosphorylation of Cx43, accumulation of nonphosphorylated Cx43 within gap junctions, and translocation of Cx43 from gap junctions into intracellular pools.

Spatial distribution and frequency dependence of arrhythmogenic vagal effects in canine atria.

INTRODUCTION: Prior studies in isolated canine atria demonstrated that acetylcholine-induced reentrant atrial fibrillation (AF) was triggered by multifocal activity in the area of normal impulse origin (sinus node-crista terminalis). The aim of this study was to investigate the activation sequence in AF induced by vagal stimulation (VS) in intact dog hearts. METHODS AND RESULTS: VS (10 to 50 Hz, 1 msec, 15 V, 5-sec trains) induced single or multiple atrial premature depolarizations (APDs), and/or AF in 8 of 10 open chest dogs. Occurrence of APDs and AF increased with increasing VS intensity. Epicardial mapping (254 unipolar electrodes) of both atria showed that APDs as a rule emerged from ectopic sites, often from the right atrial appendage. Activation mapping of the first 10 cycles of AF showed that only a small number (<3 to 4) of unstable reentrant circuits were possible at the same moment. Moreover, most sustained VS-induced AFs were accounted for by a single leading stable reentrant circuit that activated the remainder of the atria. CONCLUSION: (1) Occurrence of vagally induced APDs and AF increases with increasing frequency of VS. (2) VS-induced focal ectopic APDs are widely distributed over the atria. (3) A single APD can be sufficient for initiation of reentrant AF. (4) Despite its high rate of sustained AF, it may be maintained by single stable reentrant circuit. (5) The atrial septum can play an important role in both the initiation and the maintenance of VS-induced AF.

Effects of measurement error and sampling resolution on estimates of atrial tissue recovery parameters.

We studied the effect of sampling resolution and measurement error on estimates of tissue recovery parameters using experimental and simulated data. Action potential duration (APD) was estimated from monophasic action potentials recorded at 250 sites (delta x = 3.5 mm) on the endocardium of the canine right atrium (n = 8) during control and acetylcholine perfusion. APD distributions were also simulated using a random number generator, then scaled and filtered to physiological values. The following parameters were estimated at increasing APD sampling interval and measurement error: mean APD, standard deviation of APD, mean APD gradient, standard deviation of APD gradient, APD wavelength, and APD correlation length. We found that large errors can result from APDs collected at inadequate sampling intervals and adequate sampling intervals may be 3-6 times less than the Nyquist interval. Large parameter errors also resulted from data with relatively low levels of measurement error. The effect of measurement error was dependent upon the standard deviation of APD, sampling resolution, and APD wavelength. Inadequate sampling resolution was the largest source of error in experimental parameter estimates. Estimates of mean and standard deviation of APD gradient decreased with spacing as estimates of correlation length and wavelength increased. Careful selection of spacing interval, taking into account the spatial complexity of recovery, as well as considerably low measurement errors will produce accurate estimates of gradients, correlation length, and wavelength.

Sinus nodal function and risk for atrial fibrillation after coronary artery bypass graft surgery.

BACKGROUND: Nonsurgical patients with sinus node dysfunction are at high risk for atrial tachyarrhythmias, but whether a similar relation exists for atrial fibrillation after coronary artery bypass graft surgery is not clear. The purpose of this study was to evaluate sinus nodal function before and after coronary artery bypass graft surgery and to evaluate its relation with the risk for postoperative atrial arrhythmias. METHODS: Sixty patients without complications having elective coronary artery bypass graft surgery underwent sinus nodal function testing by measurement of sinoatrial conduction time (SACT) and corrected sinus nodal recovery time (CSNRT). Patients were categorized based on whether postoperative atrial fibrillation developed. RESULTS: Twenty patients developed atrial fibrillation between postoperative days 1 through 3. For patients remaining in sinus rhythm (n = 40), sinoatrial conduction times were no different and corrected sinus nodal recovery times were shorter after surgery when compared with measurements obtained after anesthesia induction. Sinus node function test results before surgery were similar between the sinus rhythm and the atrial fibrillation groups. After surgery, patients who later developed atrial fibrillation had longer sinoatrial conduction times compared with the sinus rhythm group (P = 0.006), but corrected sinus nodal recover time was not different between these groups. A sinoatrial conduction time > 96 ms measured at this time point was associated with a 7.3-fold increased risk of postoperative atrial fibrillation (sensitivity, 62%; specificity, 81%; positive and negative predictive values, 56% and 85%, respectively; area under the receiver operator characteristic curve, 0.72). CONCLUSIONS: These data show that sinus nodal function is not adversely affected by uncomplicated coronary artery bypass surgery. Patients who later developed atrial fibrillation, however, had prolonged sinoatrial conduction immediately after surgery compared with patients remaining in sinus rhythm. These results suggest that injury to atrial conduction tissue at the time of surgery predisposes to postoperative atrial fibrillation and that assessment of sinoatrial conduction times could provide a means of identifying patients at high risk for postoperative atrial fibrillation.

The development of the Maze procedure for the treatment of atrial fibrillation.

The Maze procedure was developed for the treatment of atrial fibrillation over a period of several years. Extensive experimental and clinical studies of the underlying electrophysiology of the arrhythmia were performed, and numerous surgical techniques and principles were tried before the Maze procedure was conceived. Few cardiac surgical procedures have undergone more extensive research and experimental trials before being applied clinically. This article gives a brief summary of the work leading up to the eventual Maze-III procedure that is now in clinical use.

Accelerated onset and increased incidence of ventricular arrhythmias induced by ischemia in Cx43-deficient mice.

BACKGROUND: Myocardial ischemia causes profound changes in both active membrane currents and passive electrical properties. Because these complex changes develop and progress concomitantly, it has not been possible to elucidate the relative contributions of any one component to arrhythmogenesis induced by acute ischemia. Cx43+/- mice express 50% of the normal level of connexin43 (Cx43), the major ventricular electrical coupling protein, but are otherwise identical to wild-type (Cx43+/+) mice. Comparison of arrhythmogenesis in Cx43+/- and +/+ mice can provide insights into the role of changes in electrical coupling as an independent variable in the complex setting of acute ischemia. METHODS AND RESULTS: Acute ischemia was induced in isolated perfused mouse hearts by occlusion of the left anterior descending coronary artery. Spontaneous ventricular tachyarrhythmias (VT) occurred in more than twice as many Cx43+/- hearts than Cx43+/+ hearts. VT was induced in nearly 3 times as many Cx43+/- hearts. Multiple runs and prolonged runs of spontaneous VT were more frequent in Cx43+/- hearts. Onset of the first run of VT occurred significantly earlier in Cx43+/- hearts. Premature ventricular beats were also more frequent in Cx43+/- hearts. The size of the hypoperfused region was equivalent in both groups. CONCLUSIONS: Reduced expression of Cx43 accelerates the onset and increases the incidence, frequency, and duration of ventricular tachyarrhythmias after coronary artery occlusion. Thus diminished electrical coupling per se plays a critical role in arrhythmogenesis induced by acute ischemia.

The closed heart MAZE: a nonbypass surgical technique.

BACKGROUND: The MAZE-III is the surgical treatment of choice for medically refractory atrial fibrillation. Although a number of nonsurgical techniques are evolving to duplicate the transmural atrial lesions of the MAZE-III, the surgical atriotomy remains the gold standard for conduction block. It was the objective of this study to surgically create the atrial incisions of the MAZE-III without the use of cardiopulmonary bypass. METHODS: A technique was developed to create and intersect the linear incisions of the MAZE-III on 10 beating canine hearts without the use of cardiopulmonary bypass using a "tunnel" of atrial tissue. The effectiveness of the procedure was tested by atrial burst pacing. RESULTS: This technique was successfully performed on 10 mongrel dogs without operative mortality. Preoperatively, sustained atrial fibrillation (>30 seconds) was induced in all animals. Postoperatively, all the animals remained in sinus rhythm even after burst pacing. CONCLUSIONS: In an experimental canine model, the MAZE-III can be performed on beating hearts without the assistance of cardiopulmonary bypass using a "tunnel" technique. This technique allows for the immediate assessment of electrophysiologic and mechanical function after the MAZE-III, or any other type of procedure using the "maze principle" and may find future application in the clinical arena.

Radial approach: a new concept in surgical treatment for atrial fibrillation I. Concept, anatomic and physiologic bases and development of a procedure.

BACKGROUND: The maze procedure cures atrial fibrillation; however, it isolates the pulmonary vein area and results in discordant activation in certain adjacent left atrial segments, which affects left atrial function. To preserve a more physiologic atrial transport function, we developed a new concept of surgical treatment for atrial fibrillation-the radial approach. The atrial incisions radiate from the sinus node toward the atrioventricular annular margins to allow a more physiologic atrial activation sequence and parallel the atrial coronary arteries to preserve blood supply to most atrial segments. METHODS: We examined the atrial coronary arteries and the activation sequence during sinus rhythm in normal canine hearts to design the atrial incisions according to the concept of a radial approach. RESULTS: The pattern of coronary artery distribution was centripetal, branching from the right coronary or left circumflex coronary artery at the right or left atrioventricular groove and spreading toward the sinus node. The endocardial mapping of the atria disclosed some important findings in designing the atrial incisions of the radial approach: the activation sequence at the left atrial septum and at the posterior left atrium between the pulmonary vein orifices. The atrial incisions were designed according to these findings. CONCLUSIONS: The radial approach may represent a more physiologic atrial transport function.

Radial approach: a new concept in surgical treatment for atrial fibrillation. II. Electrophysiologic effects and atrial contribution to ventricular filling.

BACKGROUND: In a previous study the atrial incisions that follow the concept of the radial approach were designed according to the activation sequence during sinus rhythm and the atrial coronary artery anatomy in normal dogs. The purpose of the present study was to determine whether the radial approach provides a more physiologic activation sequence and atrial transport function than the maze procedure. METHODS: Ten dogs that had undergone the radial approach (n = 5) or the maze procedure (n = 5) were studied 6 weeks postoperatively. Sinus node function and inducibility of atrial fibrillation were examined before and after operation. The atria were mapped endocardially with 212 electrodes, and atrial activation sequences during sinus rhythm and right atrial pacing were examined. Atrial transport function was assessed by transepicardial Doppler echocardiography. RESULTS: No dogs developed sinus node dysfunction postoperatively. Both the radial approach and the maze procedure equally prevented sustained atrial fibrillation. The atrial activation sequence was more synchronous after the radial approach than after the maze procedure. There was no electrically isolated region after the radial approach. The total activation time of the left atrium was significantly shorter after the radial approach than after the maze procedure (53.6+/-9.8 versus 70.5+/-9.6 ms, p<0.05). The ratio of peak flow velocity of the E wave to the A wave (peak E/A) of the transmitral Doppler flow was significantly smaller after the radial approach than after the maze procedure (1.7+/-0.4 versus 3.5+/-1.7, p<0.05). The atrial filling fraction of the transmitral Doppler flow was significantly larger after the radial approach than after the maze procedure (29.9%+/-7.3% versus 14.8%+/-5.0%, p<0.01). There was no significant difference in peak E/A and atrial filling fraction of the transtricuspid Doppler flow between the two procedures. CONCLUSIONS: The radial approach provides a more synchronous activation sequence and atrial transport function, and thus may represent a more physiologic alternative to the maze procedure as a surgical treatment for atrial fibrillation.

Nontransmural laser treatment incompletely denervates canine myocardium.

BACKGROUND: Clinical experience with transmyocardial laser revascularization (TMLR) has reproducibly demonstrated an improvement in angina class. Denervation has been implicated as a mechanism whereby this clinical effect may be achieved. Because endovascular techniques for TMLR are currently under development, we investigated the impact of nontransmural endoventricular laser treatment on cardiac nerves in a canine model. METHODS AND RESULTS: Five mongrel dogs underwent creation of nontransmural endoventricular channels in the anterior left ventricle with a Holmium:YAG laser. Cardiac afferent nerve function was assessed in control and treatment regions by the epicardial application of bradykinin, a potent algesic, at initial thoracotomy before laser treatment, and at repeat thoracotomy 2 weeks later. The resulting central nervous system-mediated decrease in systemic mean arterial pressure seen in all animals at baseline was reduced by 90% at 2 weeks in the laser-treated territory but was preserved in controls. Immunoblot analysis of tissue samples taken from laser-treated regions demonstrated a 66% reduction in tyrosine hydroxylase, a sympathetic nerve-specific enzyme, as assessed by densitometry. Enzyme content was unchanged in control regions. CONCLUSIONS: These data suggest that nontransmural endoventricular laser treatment only partially denervates the heart. This may have implications for the clinical efficacy of the endovascular approach in the relief of angina pectoris.

An experimental model of small intestinal submucosa as a growing vascular graft.

OBJECTIVE: The ideal vascular graft for use in children with congenital heart disease should not only be biocompatible and nonthrombogenic and present no infectious risk, but ideally it should grow at the same rate as the recipient. METHODS: We have tested autologous small intestine submucosa as a superior vena cava interposition graft in 11 piglets. The grafts were prepared from segments of jejunum, rendered nonthrombogenic by heparin bonding. The superior vena cava from the level of the azygos vein to the superior vena cava-right atrial junction was replaced. RESULTS: One early and 1 late death were not related to the graft material. At 90 days, the weight of the 9 survivors increased by 630%, from a mean of 10.3 +/- 2.0 kg to a mean of 59.2 +/- 16.7 kg (P < .001). The grafts increased in circumference by 184%, from a mean of 36.8 +/- 4.4 mm to a mean of 61.4 +/- 12.1 mm (P < .001) at late follow-up. Their length increased by 147%, from a mean of 9.9 +/- 2.1 mm at implantation to a mean of 15.8 +/- 5.5 mm at explantation (P = .002 ). At the time of explantation, all 11 grafts were patent and free of thrombus. Cavograms showed no anastomotic stricture or aneurysm formation in 7 of 9 cases. The luminal surface of all grafts was smooth, shiny, and indistinguishable from that of the native cava. Light microscopy showed a loosely textured collagen framework, with a dense capillary network and complete luminal coverage by a single, continuous cell layer displaying the ultrastructural features characteristic of endothelial cells. CONCLUSION: Small intestine submucosa provides a collagen framework that becomes remodeled, grows, and acquires a nonthrombogenic endothelial lining. This makes it potentially well suited as a cardiovascular substitute in children.

Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit.

A novel in vivo experimental strategy, involving cell type-specific expression of a dominant-negative K+ channel pore-forming alpha subunit, was developed and exploited to probe the molecular identity of the cardiac transient outward K+ current (I(to)). A point mutation (W to F) was introduced at position 362 in the pore region of Kv4.2 to produce a nonconducting mutant (Kv4.2W362F) subunit. Coexpression of Kv4.2W362F with Kv4.2 (or Kv4.3) attenuates the wild-type currents, and the effect is subfamily specific; ie, Kv4.2W362F does not affect heterologously expressed Kv1.4 currents. With the use of the alpha-myosin heavy chain promoter to direct cardiac-specific expression, several lines of Kv4.2W362F transgenic mice were generated. Electrophysiological recordings reveal that I(to) is selectively eliminated in ventricular myocytes isolated from transgenic mice expressing Kv4.2W362F, thereby demonstrating directly that the Kv 4 subfamily underlies I(to) in the mammalian heart. Functional knockout of I(to) leads to marked increases in action potential durations in ventricular myocytes and to prolongation of the QT interval in surface ECG recordings. In addition, a novel rapidly activating and inactivating K+ current, which is not detectable in myocytes from nontransgenic littermates, is evident in Kv4.2W362F-expressing ventricular cells. Importantly, these results demonstrate that electrical remodeling occurs in the heart when the expression of endogenous K- channels is altered.

RR interval dynamics before atrial fibrillation in patients after coronary artery bypass graft surgery.

BACKGROUND: Atrial fibrillation/flutter (AF) is a frequent complication of coronary artery bypass graft surgery (CABG) that leads to increased costs and morbidity. We hypothesized that heart rate variability (HRV), an indicator of cardiac sympathovagal balance, is altered before the onset of postoperative AF. Because nonlinear methods of HRV analysis provide information about heart rate dynamics not evident from usual HRV measures, we also hypothesized that approximate entropy (ApEn), a nonlinear measure of HRV, might have predictive value. METHODS AND RESULTS: Analysis of HRV was performed in 3 sequential 20-minute intervals preceding the onset of postoperative AF (24 episodes in 18 patients). These data were compared with corresponding intervals in 18 sex- and age-matched postoperative control subjects who did not develop AF. Patients had left ventricular ejection fractions >45% before surgery and were not receiving beta-blockers during ambulatory ECG monitoring after surgery. Logistic regression demonstrated that on the basis of averaged values for the three 20-minute intervals, increased heart rate and decreased ApEn were independently associated with AF. Heart rate dynamics before AF was associated with either lower (n= 19) or higher (n=5) RR interval variation by traditional measures of HRV or quantitative Poincaré analysis, suggesting the possibility of divergent autonomic conditions before AF onset. CONCLUSIONS: In the hour before AF after CABG surgery, higher heart rate and lower heart rate complexity compared with values in control patients were independent predictors of AF. Decreased ApEn occurs in patients with either increased or decreased HRV by traditional measures and may provide a useful tool for risk stratification or investigation of mechanisms.