Michael A. Beardslee

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.

Rapid Turnover of Connexin43 in the Adult Rat Heart

Remodeling of the distribution of gap junctions is an important feature of anatomic substrates of arrhythmias in patients with healed myocardial infarcts. Mechanisms underlying this process are poorly understood but probably involve changes in gap junction protein (connexin) synthesis, assembly into channels, and degradation. The half-life of the principal cardiac gap junction protein, connexin43 (Cx43), is only 1.5 to 2 hours in primary cultures of neonatal myocytes, but it is unknown whether rapid turnover of Cx43 occurs in the adult heart or is unique to disaggregated neonatal myocytes that are actively reestablishing connections in vitro. To characterize connexin turnover dynamics in the adult heart and to elucidate its potential role in remodeling of gap junctions, we measured Cx43 turnover kinetics and characterized the proteolytic pathways involved in Cx43 degradation in isolated perfused adult rat hearts. Hearts were labeled for 40 minutes with Krebs-Henseleit buffer containing [35S]methionine, and then chase perfusions were performed with nonradioactive buffer for 0, 60, 120, and 240 minutes. Quantitative immunoprecipitation assays of Cx43 radioactivity in 4 hearts at each time point yielded a monoexponential decay curve indicating a Cx43 half-life of 1.3 hours. Proteolytic pathways responsible for Cx43 degradation were elucidated by perfusing isolated rat hearts for 4 hours with specific inhibitors of either lysosomal or proteasomal proteolysis. Immunoblot analysis demonstrated significant increases ( approximately 30%) in Cx43 content in hearts perfused with either lysosomal or proteasomal pathway inhibitors. Most of the Cx43 in hearts perfused with lysosomal inhibitors consisted of phosphorylated isoforms, whereas nonphosphorylated Cx43 accumulated selectively in hearts perfused with a specific proteasomal inhibitor. These results indicate that Cx43 turns over rapidly in the adult heart and is degraded by multiple proteolytic pathways. Regulation of Cx43 degradation could play an important role in gap junction remodeling in response to cardiac injury.

Disparate Effects of Deficient Expression of Connexin43 on Atrial and Ventricular Conduction Evidence for Chamber-Specific Molecular Determinants of Conduction

BACKGROUND Myocardial conduction depends on intercellular transfer of current at gap junctions. Atrial myocytes express three different gap junction channel proteins-connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40)-- whereas ventricular myocytes express only Cx43 and Cx45. However, the physiological roles of individual connexins are unknown. We have previously shown that mice heterozygous for a null mutation in the gene encoding Cx43 (Cx43(+/-) mice) express 50% of the normal amount of Cx43 in ventricular myocardium and exhibit marked slowing of ventricular conduction. METHODS AND RESULTS To determine whether atrial conduction is affected in Cx43(+/-) mice, we measured atrial conduction velocity in isolated hearts, performed detailed ECG and electrophysiological studies in intact animals, and determined the amount of cardiac connexins in atrial and ventricular tissue. Ventricular conduction velocity was reduced by 38% in Cx43(+/-) mice compared with wild-types, but atrial conduction velocity in the same hearts was normal. QRS duration was significantly greater in Cx43(+/-) mice than in wild-types, but P-wave duration and amplitude did not differ. Atrial expression of Cx43 was reduced by 50%. CONCLUSIONS These results indicate that Cx43 is a principal conductor of intercellular current in the ventricle because ventricular conduction is significantly slowed when Cx43 content is reduced by only 50%. In contrast, a similar reduction in Cx43 content in atrial muscle has no effect on atrial conduction, suggesting that Cx40 (which is expressed in atrial but not ventricular myocytes) is a major electrical coupling protein in atrial muscle. Thus, Cx43 and Cx40 may be chamber-specific determinants of myocardial conduction.

Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes.

OBJECTIVES To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes. BACKGROUND Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response. METHODS Cultures were exposed to 0.1 micromol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy. RESULTS Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n=4, p < 0.05). This response was inhibited by the presence of 1.0 micromol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [35S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point). CONCLUSIONS Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.

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 …

Infective endocarditis resulting from CardioSEAL closure of a patent foramen ovale.

Patent foramen ovale and atrial septal aneurysm are associated with an increased risk of cryptogenic stroke and recurrent thromboembolic events. Percutaneous closure is a therapeutic option to medical therapy and surgical closure. We present the first case of endocarditis associated with a CardioSEAL device closing a patent foramen ovale. Cathet Cardiovasc Intervent 2002;55:217–220.

Noninvasive evaluation of a cutting balloon entrapped in a left main coronary artery using transesophageal echocardiography.

There are reports in the literature describing the utility of transesophageal echocardiography (TEE) in the evaluation of the coronary arteries. Studies have also shown the value of TEE in patients undergoing coronary intervention such as assessing coronary anatomy and flow following angioplasty. We report an interesting case where TEE helped to establish the location of a fractured cutting balloon device lodged in the left main coronary artery and obviated the need for cardiac surgery to remove the fractured balloon catheter. To our knowledge, this is the first case report of this type of complication utilizing a cutting balloon device.