Deborah L. Lerner

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 Ca2+ 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.

The in vivo role of p38 MAP kinases in cardiac remodeling and restrictive cardiomyopathy

Stress-induced mitogen-activated protein kinase (MAP) p38 is activated in various forms of heart failure, yet its effects on the intact heart remain to be established. Targeted activation of p38 MAP kinase in ventricular myocytes was achieved in vivo by using a gene-switch transgenic strategy with activated mutants of upstream kinases MKK3bE and MKK6bE. Transgene expression resulted in significant induction of p38 kinase activity and premature death at 7–9 weeks. Both groups of transgenic hearts exhibited marked interstitial fibrosis and expression of fetal marker genes characteristic of cardiac failure, but no significant hypertrophy at the organ level. Echocardiographic and pressure-volume analyses revealed a similar extent of systolic contractile depression and restrictive diastolic abnormalities related to markedly increased passive chamber stiffness. However, MKK3bE-expressing hearts had increased end-systolic chamber volumes and a thinned ventricular wall, associated with heterogeneous myocyte atrophy, whereas MKK6bE hearts had reduced end-diastolic ventricular cavity size, a modest increase in myocyte size, and no significant myocyte atrophy. These data provide in vivo evidence for a negative inotropic and restrictive diastolic effect from p38 MAP kinase activation in ventricular myocytes and reveal specific roles of p38 pathway in the development of ventricular end-systolic remodeling.

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.

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.

c-Jun N-Terminal Kinase Activation Mediates Downregulation of Connexin43 in Cardiomyocytes

Abstract— Loss of gap junctions and impaired intercellular communication are characteristic features of pathological remodeling in heart failure as a result of stress or injury, yet the underlying regulatory mechanism has not been identified. Here, we report that in cultured myocytes, rapid loss of the gap junction protein connexin43 (Cx43) occurs in conjunction with the activation of c-Jun N-terminal kinase (JNK), a stress-activated protein kinase, on stress stimulation. To investigate the specific role of JNK activation in the regulation of connexin in cardiomyocytes, an activated mutant of mitogen-activated protein kinase kinase 7 (mutant D), a JNK-specific upstream activator, was expressed in myocytes by adenovirus-mediated gene transfer. JNK activation in infected cardiomyocytes resulted in significant reduction of Cx43 expression at both mRNA and protein levels and impaired cell-cell communication. To evaluate the role of JNK in the regulation of Cx43 expression and gap junction structure in vivo, a Cre-LoxP–mediated gene-switch system was used to establish a transgenic animal model with targeted activation of JNK in ventricular myocardium. The transgenic hearts exhibited significant downregulation of Cx43 expression and loss of gap junctions in myocardium that may contribute to the cardiac dysfunction and premature death phenotype. Our report represents the first evidence, both in vitro and in vivo, implicating JNK as an important mediator of stress-induced Cx43 downregulation and impaired intercellular communication in the failing heart.

The role of altered intercellular coupling in arrhythmias induced by acute myocardial ischemia

Time for primary review 28 days. Sudden cardiac death occurs with unacceptably high incidence in patients with ischemic heart disease and cardiomyopathy. As Zipes and Wellens [1] have emphasized, sudden death arises from highly variable interactions between anatomic and/or functional myocardial substrates, transient initiating events and cellular/tissue arrhythmia mechanisms. In our view, a key strategy for developing mechanistic insights into sudden death is to first define the role of individual factors (including specific gene products) that contribute to arrhythmias, and to then understand how these factors interact to cause sudden death. One of the most common disease settings leading to sudden cardiac death is the acute coronary syndromes. Acute ischemia is marked by alterations in cell metabolism, cell signaling, intercellular communication and electrical impulse propagation [2]. These changes produce a cascade of events that are adaptive in the sense that mechanisms are activated to mitigate injury, forestall cell death and isolate irreversibly injured myocytes from their viable neighbors, but also maladaptive in that they can create a substrate that supports the initiation and maintenance of malignant ventricular arrhythmias. Among the electrophysiologically relevant changes that occur rapidly after the onset of ischemia are reductions in tissue pH, increases in interstitial K+ and intracellular Ca2+ concentrations and changes in active and passive membrane properties, all of which interact in a complex milieu to slow conduction, alter excitability and refractoriness, promote electrical uncoupling, and generate spontaneous electrical activity [1–4]. In this review, we focus on the specific role of diminished intercellular electrical coupling in the pathogenesis of lethal arrhythmias induced by acute ischemia. Until recently, it has been difficult to isolate the contribution of diminished coupling per se to arrhythmogenesis in the complex setting of acute ischemia. However, the advent of techniques to manipulate gene expression and characterize cardiac …

Spontaneous and inducible ventricular arrhythmias after myocardial infarction in mice.

INTRODUCTION Remodeling of gap junctions has been implicated in development of ventricular arrhythmias following myocardial infarction (MI) but the specific contribution of reduced electrical coupling is not known. We addressed this question using hearts from mice heterozygous for a connexin43 null allele (Cx43(+/-)). METHODS To determine whether Cx43-deficient mice exhibit increased spontaneous ventricular arrhythmias in the setting of chronic ischemic heart disease, radiofrequency transmitters were implanted in wild-type and Cx43(+/-) mice 2 days or 9 weeks after left anterior descending coronary artery ligation or sham operations. ECGs were recorded from unanesthetized, unrestrained mice 1 and 10 weeks after MI. Isolated, perfused hearts excised 1 and 10 weeks after MI were subjected to programmed electrical stimulation to induce arrhythmias. RESULTS AND CONCLUSIONS Hearts with infarcts exhibited more spontaneous and inducible arrhythmias, but there was no significant difference between wild-type and Cx43-deficient mice. Fewer hearts exhibited spontaneous ventricular tachycardia (VT) in vivo than were inducible in vitro, suggesting that structural and functional substrates for inducible VT in isolated hearts may not be sufficient for initiation and maintenance of sustained VT in vivo. Previous studies have shown that Cx43-deficient mice exhibit more VT than wild-type mice during acute regional ischemia. Mice with MI exhibit increased arrhythmias. However, reduced coupling in Cx43-deficient mice does not significantly enhance spontaneous or inducible VT after MI.

Chapter 21 – Gap Junction Distribution and Regulation in the Heart

Propagation of electrical impulses and exchange of metabolic and signaling information in the heart requires intercellular transfer of ions and small molecules. This process occurs at gap junctions, specialized sarcolemmal regions containing arrays of densely packed transmembrane channels that dock in the intercellular space to directly connect the cytoplasmic compartments of adjacent cells. Gap junctions are dynamic organelles that associate with extracellular matrix and intracellular scaffolding and signaling proteins. Such interactions likely regulate gap junction channel distribution, function, and turnover. Since the first gap junction channel genes were cloned and sequenced more than 15 years ago, much has been learned about molecular and structural features of gap junction channels as determinants of impulse propagation. This chapter focuses on current knowledge about how gap junction channels are regulated and how specific gap junction distributions contribute to the characteristic conduction properties of different cardiac tissues. For additional information on the role of gap junctions in cardiac electrophysiology, refer to other chapters in this textbook concerning the molecular structure and function of connexins (see Chapter 8 ), hybrid gap junction channels (see Chapter 14 ), pharmacology of cardiac gap junctions (see Chapter 19 ), and mouse models of cardiac arrhythmias (see Chapter 48 ).

Reversible down-regulation of connexin43 expression in acute cardiac allograft rejection

We tested the hypothesis that cardiac allograft dysfunction in acute cardiac rejection may be related, in part, to diminished expression of connexin43, a gap junction channel protein that facilitates intercellular communication and coordinates electrical and mechanical cardiac function. We measured connexin43 levels using quantitative confocal immunofluorescence microscopy of endocardial biopsies from heart transplant recipients with histologic evidence of either no rejection or acute cellular rejection. Expression of connexin43 diminished significantly during acute cellular rejection and returned to baseline levels following resolution of rejection. Reversible down-regulation of connexin43 may contribute to ventricular dysfunction in allograft rejection.

Prevention and treatment of upper airway obstruction in infants and children

This review examines some of the recent advances made in the prevention and treatment of upper airway obstruction in infancy and childhood. In some instances, the advances are the result of experimental studies that corroborate or refute therapeutic notions that had been adopted prematurely. Studies performed in the past few years, for instance, have demonstrated that both systemic and local corticosteroid treatments are indeed effective in the treatment of viral croup. In contrast, other studies carried out in the same period raise doubts about the usefulness of these medications in the prevention of postextubation laryngeal edema. In other instances, the advances are the result of pioneering efforts to correct anatomical defects, usually congenital, that cause severe airway obstruction. Tracheal and laryngeal stenoses and craniofacial deformities, which only 5 years ago would have been palliated by tracheotomy, undergo now routine primary correction. Despite all these advances, upper airway obstruction remains an important source of morbidity and mortality in early childhood.