Ruben Coronel

Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome : a combined electrophysiological, genetic, histopathologic, and computational study

Background— The mechanism of ECG changes and arrhythmogenesis in Brugada syndrome (BS) patients is unknown. Methods and Results— A BS patient without clinically detected cardiac structural abnormalities underwent cardiac transplantation for intolerable numbers of implantable cardioverter/defibrillator discharges. The patient’s explanted heart was studied electrophysiologically and histopathologically. Whole-cell currents were measured in HEK293 cells expressing wild-type or mutated sodium channels from the patient. The right ventricular outflow tract (RVOT) endocardium showed activation slowing and was the origin of ventricular fibrillation without a transmural repolarization gradient. Conduction restitution was abnormal in the RVOT but normal in the left ventricle. Right ventricular hypertrophy and fibrosis with epicardial fatty infiltration were present. HEK293 cells expressing a G1935S mutation in the gene encoding the cardiac sodium channel exhibited enhanced slow inactivation compared with wild-type channels. Computer simulations demonstrated that conduction slowing in the RVOT might have been the cause of the ECG changes. Conclusions— In this patient with BS, conduction slowing based on interstitial fibrosis, but not transmural repolarization differences, caused the ECG signs and was the origin of ventricular fibrillation.

Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death

Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10−68; rs9388451, P = 5.1 × 10−17) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10−14). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10−81). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.

Ventricular tachyrdia in the infarcted, Langendorff-perfused human heart: Role of the arrangement of surviving cardiac fibers

Electrophysiologic and histologic studies were performed on Langendorff-perfused human hearts from patients who underwent heart transplantation because of extensive infarction. In nine hearts, 15 sustained ventricular tachycardias could be induced by programmed stimulation. In all hearts, mapping of epicardial and endocardial electrical activity during tachycardia was carried out. Histologic examination of the infarcted area between the site of latest activation of one cycle and the site of earliest activation of the next cycle revealed zones of viable myocardial tissue. In two hearts in which the time gap between latest and earliest activation was small, surviving myocardial tissue constituted a continuous tract that traversed the infarct. In three other hearts in which the time gap was large, surviving tissue consisted of parallel bundles that coursed separately over a few hundred micrometers, then merged into a single bundle and finally branched again. The direction of the fibers within the bundles was perpendicular to the direction of the activation front in that area. A similar type of inhomogeneous anisotrophy and activation delay was found in an infarcted papillary muscle removed from one of the explanted hearts and studied in a tissue bath during basic stimulation. Histologic examination of this preparation revealed that the delay was caused by a zigzag route of activation over branching and merging bundles of surviving myocytes separated by connective tissue.

Dispersion of refractoriness in canine ventricular myocardium. Effects of sympathetic stimulation.

In 18 dogs on total cardiopulmonary bypass, the average interval between local activations during artificially induced ventricular fibrillation (VF interval) was measured from extracellular electrograms, simultaneously recorded from up to 32 ventricular sites. VF intervals were used as an index of local refractoriness, based on the assumption that during ventricular fibrillation, cells are reexcited as soon as they have recovered their excitability. In support of this, microelectrode recordings in two hearts during ventricular fibrillation did not show a diastolic interval between successive action potentials. Refractory periods determined at a basic cycle length of 300 msec with the extrastimulus method correlated well with VF intervals measured at the same sites. Thus, this technique allows assessment of spatial dispersion of refractoriness during brief interventions such as sympathetic stimulation. The responses to left, right, and combined stellate ganglion stimulation varied substantially among individual hearts. This was observed both in dogs with an intact (n = 12) and decentralized (n = 6) autonomic nervous system. Individual ventricular sites could show effects of both left and right stellate ganglion stimulation (42% of tested sites) or show effects of left-sided stimulation only (31%) or right-sided stimulation only (14%). In 13% of sites, no effects of stellate stimulation were observed. Apart from these regional effects, the responses could be qualitatively different; that is, within the same heart, the VF interval prolonged at one site but shortened at another in response to the same intervention, although shortening was the general effect and prolongation the exception. Whenever sites responded to stellate ganglion stimulation with a shortening of VF interval, this shortening was approximately 10% for left, right, or combined stimulation, whether the autonomic nervous system was intact or decentralized. In six of 12 hearts in the intact group, there was a distinct regional effect of left stellate ganglion stimulation; in the other six hearts, the effects were distributed homogeneously over the ventricles. In three hearts, the effect of left stellate ganglion stimulation was strongest in the posterior wall, and in the other three hearts, in the anterior wall. The effects of right stellate ganglion stimulation were restricted to the anterior or lateral part of the left ventricle. Dispersion of VF intervals increased after left and combined stellate ganglion stimulation in the intact group and after right stellate ganglion stimulation in the decentralized group, but not significantly in every heart. This points to a marked individual variation with regard to the effects of sympathetic stimulation on electrophysiological properties of the heart.

Transmural repolarisation in the left ventricle in humans during normoxia and ischaemia

BACKGROUND Studies in isolated tissues and myocytes show different repolarisation properties in subepicardium, midmyocardium and subendocardium. Whether these differences are present in vivo and are relevant to humans has been the subject of controversy. Our objectives were (1) to ascertain whether transmural repolarisation gradients are present in humans, (2) to determine whether the greater sensitivity of subepicardial cells to ischaemia in vitro is manifest during early ischaemia in humans in vivo. METHODS AND RESULTS We studied 21 patients during routine coronary artery surgery. Unipolar activation recovery intervals (ARI) were recorded from five transmural locations between subepicardium and subendocardium in the left ventricular wall. A pacing protocol spanned a range of cycle lengths from a cycle length of 300 ms to the maximum permitted by the intrinsic atrial activity. Following the onset of cardiopulmonary bypass recordings were obtained before (control) and during a 3-min period of global ischaemia. During control transmural ARIs were homogeneous between 300 and 1500 ms (ventricular pacing) and 750 and 1500 ms (atrial spontaneous beats). During ischaemia, ARIs shortened similarly at all transmural electrode sites and transmural homogeneity was maintained. CONCLUSIONS Transmural repolarisation differences within the ventricular wall of the human heart were absent at cycle lengths within the physiological range but also during prolonged cycles. During early (global) ischaemia repolarisation changed equally in subepicardial and subendocardial regions and transmural homogeneity of repolarisation was preserved.

Intracellular Ca2+, Intercellular Electrical Coupling, and Mechanical Activity in Ischemic Rabbit Papillary Muscle: Effects of Preconditioning and Metabolic Blockade

During myocardial ischemia, electrical uncoupling and contracture herald irreversible damage. In the present study, we tested the hypothesis that an increase of intracellular Ca2+ is an important factor initiating these events. Therefore, we simultaneously determined tissue resistance, mechanical activity, pH(0), and intracellular Ca2+ (with the fluorescent indicator indo 1, Molecular Probes, Inc) in arterially perfused rabbit papillary muscles. Sustained ischemia was induced in three experimental groups: (1) control, (2) preparations preconditioned with two 5-minute periods of ischemia followed by reperfusion, and (3) preparations pretreated with 1 mmol/L iodoacetate to block anaerobic metabolism and minimize acidification during ischemia. In a fourth experimental group, intracellular Ca2+ was increased under nonischemic conditions by perfusing with 0.1 mmol/L ionomycin and 0.1 mumol/L gramicidin. Ca2+ transients and contractions rapidly disappeared after the induction of ischemia. In the control group, diastolic Ca2+ began to rise after 12.6 +/- 1.3 minutes of ischemia; uncoupling, after 14.5 +/- 1.2 minutes of ischemia; and contracture, after 12.6 +/- 1.5 minutes of ischemia (mean +/- SEM). Preconditioning significantly postponed Ca2+ rise, uncoupling, and contracture (21.5 +/- 4.0, 24.0 +/- 4.1, and 23.0 +/- 5.3 minutes of ischemia, respectively). Pretreatment with iodoacetate significantly advanced these events (1.9 +/- 0.7, 3.6 +/- 0.9, and 1.9 +/- 0.2 minutes of ischemia, respectively). In all groups, the onset of uncoupling always followed the start of Ca2+ rise, whereas the start of contracture was not different from the rise in Ca2+. Perfusion with ionomycin and gramicidin permitted estimation of a threshold [Ca2+] for electrical uncoupling of 685 +/- 85 nmol/L. In conclusion, the rise in intracellular Ca2+ is the main trigger for cellular uncoupling during ischemia. Contracture is closely associated with the increase of intracellular Ca2+ during ischemia.

Increased Na+/H+-exchange activity is the cause of increased [Na+]i and underlies disturbed calcium handling in the rabbit pressure and volume overload heart failure model

OBJECTIVE Cytosolic sodium ([Na+]i) is increased in heart failure (HF). We hypothesize that up-regulation of Na+/H+-exchanger (NHE) in heart failure is causal to the increase of [Na+]i and underlies disturbance of cytosolic calcium ([Ca2+]i) handling. METHODS Heart failure was induced in rabbits by combined volume and pressure overload. Age-matched animals served as control. [Na+]i, cytosolic calcium [Ca2+]i and cytosolic pH (pH(i)) were measured in isolated left ventricular midmural myocytes with SBFI, indo-1 and SNARF. SR calcium content was measured as the response of [Ca2+]i to rapid cooling (RC). Calcium after-transients were elicited by cessation of rapid stimulation (3 Hz) in the presence of 100 nmol/l noradrenalin. NHE and Na+/K+-ATPase activity were inhibited with 10 micromol/l cariporide and 100 micromol/l ouabain, respectively. RESULTS At all stimulation rates (0-3 Hz) [Na+]i and diastolic [Ca2+]i were significantly higher in HF than in control. With increasing frequency [Na+]i and diastolic [Ca2+]i progressively increased in HF and control, and the calcium transient amplitude (measured as total calcium released from SR) decreased in HF and increased in control. In HF (at 2 Hz), SR calcium content was reduced by 40% and the calcium gradient across the SR membrane by 60%. Fractional systolic SR calcium release was 90% in HF and 60% in control. In HF the rate of pH(i) recovery following acid loading was much faster at all pH(i) and NHE dependent sodium influx was almost twice as high as in control. In HF cariporide (10 micromol/l, 5 min) reduced [Na+]i and end diastolic [Ca2+]i to almost control values, and reversed the relation between calcium transient amplitude and stimulation rate from negative to positive. It increased SR calcium content and SR membrane gradient and decreased fractional systolic SR depletion to 60%. Cariporide greatly reduced the susceptibility to develop calcium after-transients. In control animals, cariporide had only minor effects on all these parameters. Increase of [Na+]i with ouabain in control myocytes induced abnormal calcium handling as found in HF. CONCLUSIONS In HF up-regulation of NHE activity is causal to increased [Na+]i and secondarily to disturbed diastolic, systolic and SR calcium handling. Specific inhibition of NHE partly normalized [Na+]i, end diastolic [Ca2+]i, and SR calcium handling and reduced the incidence of calcium after-transients. Chronic treatment with specific NHE inhibitors may provide a useful future therapeutic option in treatment of developing hypertrophy and heart failure.

Acute ischemia-induced gap junctional uncoupling and arrhythmogenesis.

Sudden cardiac death forms a major cause of mortality. Myocardial ischemia-induced ventricular fibrillation (VF) is frequently the underlying mechanism. Ventricular arrhythmias arise in two distinct phases during the first hour of ischemia. The first, the 1A phase, has been extensively studied, and few studies relate to the 1B phase. The latter is associated with intercellular electrical uncoupling, mediated by decreased conductance of gap junction channels. Although the relation between gap junctional uncoupling and decreased conduction velocity appears clear under normoxic conditions, additional factors contribute to conduction slowing during ischemia, and VF occurs preferentially at moderate levels of uncoupling. A potential mechanism of arrhythmias depends on temporary electrotonic depression of intrinsically viable tissue by the large bulk of the ischemic zone. This causes conduction slowing and conduction block in the surviving layers, leading to arrhythmias. These arrhythmias then resolve with progression of uncoupling. It is unknown whether either accelerated uncoupling or maintenance of gap junctional communication is antiarrhythmic. Ischemic preconditioning postpones both gap junctional uncoupling and occurrence of VF. Given the burden of sudden death and the large number of casualties in the low-risk population, there is, even in the era of implantable cardiac defibrillators, need for further understanding the mechanism of ischemia-induced VF.

Repolarization Gradients in the Canine Left Ventricle Before and After Induction of Short-Term Cardiac Memory

Background—Questions remain about the contributions of transmural versus apicobasal repolarization gradients to the configuration of the T wave in control settings and after the induction of short-term cardiac memory. Methods and Results—Short-term cardiac memory is seen as T-wave changes induced by altered ventricular activation that persists after restoration of sinus rhythm. We studied cardiac memory in anesthetized, open-chest dogs paced from the ventricle for 2 hours. Unipolar electrograms were recorded from as many as 98 epicardial and 144 intramural sites, and activation times and activation-recovery intervals (ARIs) were measured. In separate experiments, epicardial monophasic action potentials were recorded. We found no appreciable left ventricular intramural gradients in repolarization times (activation time+ARI) in either control conditions or after the induction of memory. In controls, there was a left ventricular apicobasal gradient, with the shortest repolarization times in anterobasal regions and longest repolarization times posteroapically. After induction of memory, repolarization times shortened uniformly throughout the ventricular wall. Monophasic action potential duration at 90% repolarization decreased by ≈10 ms after induction of memory. Conclusions—In the intact canine left ventricle at physiological rates, there is no transmural gradient in repolarization. Apicobasal gradients in repolarization time, with shortest repolarization times in anterobasal areas and longest repolarization times in posteroapical regions, are important in the genesis of the T wave. Repolarization times and monophasic action potentials at the 90% repolarization level shorten after the induction of memory. The deeper T wave in the ECG after induction of memory may be explained by the more rapid phase 3 of the action potential.

Slow and Discontinuous Conduction Conspire in Brugada Syndrome A Right Ventricular Mapping and Stimulation Study

Background—Brugada syndrome (BrS) is associated with lethal arrhythmias, which are linked to specific ST-segment changes (type-1 BrS-ECG) and the right ventricle (RV). The pathophysiological basis of the arrhythmias and type-1 BrS-ECG is unresolved. We studied the electrophysiological characteristics of the RV endocardium in BrS. Methods and Results—RV endocardial electroanatomical mapping and stimulation studies were performed in controls (n=12) and BrS patients with a type-1 (BrS-1, n=10) or type-2 BrS-ECG (BrS-2, n=12) during the studies. BrS-1 patients had prominent impairment of RV endocardial impulse propagation when compared with controls, as represented by: (1) prolonged activation-duration during sinus rhythm (86±4 versus 65±3 ms), (2) increased electrogram fractionation (1.36±0.04 versus 1.15±0.01 deflections per electrogram), (3) longer electrogram duration (83±3 versus 63±2 ms), (4) activation delays on premature stimulation (longitudinal: 160±26 versus 86±9 ms; transversal: 112±5 versus 58±6 ms), and (5) abnormal transversal conduction velocity restitution (42±8 versus 18±2 ms increase in delay at shortest coupling intervals). Wider and more fractionated electrograms were also found in BrS-2 patients. Repolarization was not different between groups. Conclusions—BrS-1 and BrS-2 patients are characterized by wide and fractionated electrograms at the RV endocardium. BrS-1 patients display additional conduction slowing during sinus rhythm and premature stimulation along with abnormal transversal conduction velocity restitution. These patients may thus exhibit a substrate for slow and discontinuous conduction caused by abnormal active membrane processes and electric coupling. Our findings support the emerging notion that BrS is not solely attributable to abnormal electrophysiological properties but requires the conspiring effects of conduction slowing and tissue discontinuities.