Normal Ventricular Tissue Research Articles

Mechanistic investigation of Ca2+ alternans in human heart failure and its modulation by fibroblasts.

BackgroundHeart failure (HF) is characterized, among other factors, by a progressive loss of contractile function and by the formation of an arrhythmogenic substrate, both aspects partially related to intracellular Ca2+ cycling disorders. In failing hearts both electrophysiological and structural remodeling, including fibroblast proliferation, contribute to changes in Ca2+ handling which promote the appearance of Ca2+ alternans (Ca-alt). Ca-alt in turn give rise to repolarization alternans, which promote dispersion of repolarization and contribute to reentrant activity. The computational analysis of the incidence of Ca2+ and/or repolarization alternans under HF conditions in the presence of fibroblasts could provide a better understanding of the mechanisms leading to HF arrhythmias and contractile function disorders.Methods and findingsThe goal of the present study was to investigate in silico the mechanisms leading to the formation of Ca-alt in failing human ventricular myocytes and tissues with disperse fibroblast distributions. The contribution of ionic currents variability to alternans formation at the cellular level was analyzed and the results show that in normal ventricular tissue, altered Ca2+ dynamics lead to Ca-alt, which precede APD alternans and can be aggravated by the presence of fibroblasts. Electrophysiological remodeling of failing tissue alone is sufficient to develop alternans. The incidence of alternans is reduced when fibroblasts are present in failing tissue due to significantly depressed Ca2+ transients. The analysis of the underlying ionic mechanisms suggests that Ca-alt are driven by Ca2+-handling protein and Ca2+ cycling dysfunctions in the junctional sarcoplasmic reticulum and that their contribution to alternans occurrence depends on the cardiac remodeling conditions and on myocyte-fibroblast interactions.ConclusionIt can thus be concluded that fibroblasts modulate the formation of Ca-alt in human ventricular tissue subjected to heart failure-related electrophysiological remodeling. Pharmacological therapies should thus consider the extent of both the electrophysiological and structural remodeling present in the failing heart.

Mitochondrial depolarization and electrophysiological changes during ischemia in the rabbit and human heart.

Instability of the inner mitochondrial membrane potential (ΔΨm) has been implicated in electrical dysfunction, including arrhythmogenesis during ischemia-reperfusion. Monitoring ΔΨm has led to conflicting results, where depolarization has been reported as sporadic and as a propagating wave. The present study was designed to resolve the aforementioned difference and determine the unknown relationship between ΔΨm and electrophysiology. We developed a novel imaging modality for simultaneous optical mapping of ΔΨm and transmembrane potential (Vm). Optical mapping was performed using potentiometric dyes on preparations from 4 mouse hearts, 14 rabbit hearts, and 7 human hearts. Our data showed that during ischemia, ΔΨm depolarization is sporadic and changes asynchronously with electrophysiological changes. Spatially, ΔΨm depolarization was associated with action potential duration shortening but not conduction slowing. Analysis of focal activity indicated that ΔΨm is not different within the myocardium where the focus originates compared with normal ventricular tissue. Overall, our data suggest that during ischemia, mitochondria maintain their function at the expense of sarcolemmal electrophysiology, but ΔΨm depolarization does not have a direct association to ischemia-induced arrhythmias.

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Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart

The functional consequences of Connexin43 (Cx43) phosphorylation remain largely unexplored. Using an antibody that specifically recognizes Cx43 phosphorylated at serine residues 325, 328 and/or 330 (pS325/328/330-Cx43), we show that labeling of this form of Cx43 as well as of total Cx43 is restricted to the intercalated disk region of normal ventricular tissue. In ischemic heart, significant relocalization of total Cx43 to the lateral edges of myocytes was evident; however pS325/328/330-Cx43 remained predominately at the intercalated disk. Western blots indicated a eightfold decrease in pS325/328/330-Cx43 in ischemic tissue. Peptide-binding- and competition-experiments indicated that our antibody mainly detected Cx43 phosphorylated at S328 and/or S330 in heart tissue. To evaluate how this change in Cx43 phosphorylation contributes to ischemia-induced downregulation of intercellular communication, we stably transfected Cx43(-/-) cells with a Cx43 construct in which serine residues 325, 328 and 330 had been mutated to alanine (Cx43-TM). Cx43-TM was not efficiently processed to isoforms that have been correlated with gap junction assembly. Nevertheless, Cx43-TM cells were electrically coupled, although development of coupling was delayed. Fully opened channels were only rarely observed in Cx43-TM cells, and Lucifer-Yellow-dye-coupling was significantly reduced compared with wild-type cells. These data suggest that phosphorylation of Cx43 at serine residues 325, 328 and/or 330 influences channel permselectivity and regulates the efficiency of gap junction assembly.

Controlling the Gap

See related article, pages 558–566 Effective myocardial contraction depends in part on the efficient propagation of electrical signals through the tissue. The principal structure allowing for such rapid myocardial depolarization is the gap junction plaque, a collection of intercellular pores located primarily at intercalated discs. There, at myocyte poles, gap junctions function in close proximity to the complexes responsible for cell–cell tethering—desmosomes and fascia adherens junctions. Thus, the intercalated disc is the site of both intercellular coupling and adhesion. Gap junctions are formed by clusters of paired hexamers, one from each cell, which bridge to form a low-resistance intercellular channel facilitating the transmission of ions and small molecules (<1000 kD). Each hexamer (or connexon) is comprised of six individual connexins, a diverse protein family found in vascular, neural, and cardiac tissue. In humans, ventricular gap junctions are formed primarily from connexin43 (Cx43), a 43-kD protein encoded by chromosome 6.1 Cx43 is a dynamic molecule, with rapid turnover (t1/2 roughly 1 hour)2 and responsiveness to a variety of mechanical and chemical stimuli. Myocyte connectivity, as mediated by gap junctions, depends not only on net expression of Cx43, but on effective translocation of Cx43 to the cell surface, the phosphorylation state of both Cx43 and associated proteins,3 and intracellular pH.4 Ventricular Cx43 net protein expression is downregulated in various cardiac disease conditions such as end-stage ischemic,5 idiopathic,5 hypertrophic,1,6 and tachycardia-induced induced cardiomyopathies,7 myocardial ischemia and hibernation,8 and allograft rejection.9 …

Tedisamil and lidocaine enhance each other's antiarrhythmic activity against ischaemia-induced arrhythmias in rats.

1. Combinations of the action potential-widening drug tedisamil (Class III antiarrhythmic activity), and the inactivated state sodium channel blocker lidocaine (Class Ib antiarrhythmic activity) were assessed for antiarrhythmic actions in a rat model of ischaemia-induced arrhythmias and for electrophysiological actions in normal rat myocardial tissue. 2. Both tedisamil and lidocaine dose-dependently suppressed ischaemia-induced arrhythmias. The ED(50) values were 3.0+/-1.3 and 4.9+/-0.6 micro mol kg(-1) min(-1), respectively. 3. Combinations of the two drugs acted synergistically such that the ED(50) for tedisamil was reduced to 0.8+/-0.2 micro mol kg(-1) min(-1) in the presence of 2 micro mol kg(-1) min(-1) lidocaine. Similarly, the ED(50) for lidocaine was reduced to 0.7+/-0.2 micro mol kg(-1) min(-1) in the presence of 2 micro mol kg(-1) min(-1) tedisamil (both P<0.05). 4. In a separate series of experiments in which normal ventricular tissue was electrically stimulated, 2 micro mol kg(-1) min(-1) lidocaine produced a leftward shift in the dose-response curve for tedisamil's effect on effective refractory period (P<0.05). This dose of lidocaine had no effect on its own. These data indicate that the synergistic actions of combinations of tedisamil and lidocaine were mediated, at least in part, by extension of effective refractory period in normal myocardial tissue. 5. In contrast to the strategy of developing drugs that are selective for a single electrophysiological mechanism, the results of the present study suggest that effective antiarrhythmic drugs might be developed by optimising the combination of two complimentary electrophysiological mechanisms (i.e., action potential-prolonging activity and inactivated state sodium channel blockade).

Effect of phorbol ester on the release of atrial natriuretic peptide from the hypertrophied rat myocardium

1. To determine the cellular mechanisms of atrial natriuretic peptide (ANP) release from ventricular cardiomyocytes, the secretory and the cardiac effects of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase C activity in heart cells, were studied in isolated, perfused heart preparations from 2- and 21-month-old Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. TPA was added to the perfusion fluid for 30 min at a concentration of 46 nM after removal of atrial tissue. Additionally, atrial and ventricular levels of immunoreactive ANP (IR-ANP) and ANP mRNA, the distribution of ANP within ventricles as well as the relative contribution of atria and ventricles in the release of ANP were studied. 2. Ventricular hypertrophy that gradually developed in hypertensive rats resulted in remarkable augmentation of ANP gene expression, as reflected by elevated levels of immunoreactive ANP and ANP mRNA. The total amount of IR-ANP in the ventricles of the SHR rats increased 41 fold and ANP mRNA levels 12.9 fold from the age of 2 to 21 months. At the age of 21 months, levels of IR-ANP and ANP mRNA in the ventricles of SHR rats were 5.4 fold and 3.7 fold higher, respectively, than in the normotensive WKY rats. Immunohistochemical studies demonstrated ANP granules within the hypertrophic ventricles of the old SHR rats, but not within normal ventricular tissue. 3. In isolated perfused heart preparations, the severely hypertrophied ventricular tissue of SHR rats after atrialectomy secreted more ANP into the perfusate than did the control hearts. Interestingly, the ANP release from the hypertrophied ventricles of the old SHR rats increased considerably (from 413 + 30 to the maximum of 623 + 75 pgml-1, F = 10.8, P < 0.001, two-way analysis of variance), whereas only a small increase was seen in old WKY rats and no effect was observed in young animals of either strain. When intact rat hearts (without atrialectomy) were used, infusion of phorbol ester also increased the ANP secretion into the perfusate in young animals. 4. Our present results indicate that the phorbol ester TPA increases the release of ANP from the hypertrophied, but not from normal rat myocardium. Thus, hypertrophied rat ventricular myocytes appear to possess the cellular mechanisms necessary to secrete ANP by a regulated pathway. The results further suggest that protein kinase C activity may be involved in the the regulation of ANP secretion from ventricular cells, as has been shown earlier for atrial myocytes.

Electrophysiologic Effects of Bepridil in Normal and Infarcted Canine Myocardium

The electrophysiologic effects of bepridil, 10 mg/kg i.v., were determined in normal noninfarcted and in infarcted ventricular myocardium in 8 urethane-anesthetized dogs 4-6 days after anterior myocardial infarction. At drive cycle lengths of 400 and 333 ms, bepridil significantly increased relative (RRP) and effective (ERP) refractory periods in both normal ventricular tissue (mean increases, RRP 7-14%, ERP 5-6%, p less than 0.05-0.01) and in infarcted ventricular tissue (mean increases, RRP 12-15%, ERP 13-14%, p less than 0.01). Bepridil also selectively prolonged the local activation delay in infarcted ventricular myocardium (mean increases 37.5-45.1%, p less than 0.01), while ventricular excitation thresholds were not altered by bepridil in either normal or infarcted myocardium. Before bepridil administration, programmed ventricular stimulation initiated sustained ventricular tachycardias in 6 of the 8 postinfarction dogs tested. After bepridil, 2 of the 6 previously responsive animals were rendered noninducible, 3 animals responded to programmed stimulation with nonsustained tachyarrhythmias of relatively slower rates, and the one remaining dog responded with sustained ventricular tachycardia (VT). These data suggest that increases in refractoriness in both normal noninjured and in ischemically injured ventricular tissue, with a selective delay in conduction in ischemically injured tissue, contribute to the antiarrhythmic actions of bepridil in the setting of myocardial infarction.

Electrophysiologic actions of lidocaine in a canine model of chronic myocardial ischemic damage--arrhythmogenic actions of lidocaine.

Lidocaine facilitated the induction of ventricular arrhythmias by programmed electrical stimulation in 16 dogs, 5 to 14 days after a temporary (90-min) occlusion of the left anterior descending coronary artery. In these 16 animals, programmed stimulation failed to produce ventricular tachyarrhythmias in any animal before lidocaine administration (3 mg/kg), while after lidocaine administration, programmed stimulation produced nonsustained ventricular tachycardia in four animals (25%), sustained ventricular tachycardia in nine animals (56%), and ventricular fibrillation in one animal (6%). Delayed electrical activity in ischemically injured ventricular myocardium produced by premature ventricular stimuli (mean +/- SD = 179 +/- 34 ms) was delayed further by the administration of lidocaine (237 +/- 42 ms, p less than 0.01), resulting in continuous local electrical activity between the final premature ventricular stimulus and the initial beat of the resultant ventricular tachycardia. Lidocaine administration did not alter myocardial refractoriness in normal ventricular tissue, but it prolonged refractoriness in ischemically injured ventricular myocardium. These results show that lidocaine can have arrhythmogenic actions when administered in the presence of existing ischemic injury, possibly the result of increased delay in activation of ischemically injured ventricular myocardium with localized reentry of myocardial electrical activity.

Comparison of the adenine nucleotide metabolism of dog atrial and ventricular myocardium

Measurements of atrial and ventricular adenine nucleotides, nucleosides and bases in dog heart indicated that normal atrial ATP, ADP and AMP levels are about one-half those of normal ventricular tissue, but atrial and ventricular tissue levels of adenosine, inosine, and hypoxanthine were about equal. With ischemia, production of adenosine and inosine was less in atrial than in ventricular muscle. In the ventricle, 5′-nucleotidase is associated with external membranes, including those of the T-tubular system and the lack of a T-tubular network in atrium suggested that there might be fewer AMP hydrolyzing sites and hence a slower rate of adenosine production in response to ischemia. This possibility was ruled out since measurements of 5′-nucleotidase on crude tissue extracts indicated that atrial tissue has 2 to 3 times the activity of ventricular tissue. Furthermore, measurements of adenosine kinase activity indicate that atrial and ventricular activities of this enzyme are about equal; hence the lower adenosine levels in atria are also not due to a higher rate of adenosine rephosphorylation. Inhibition of 5′-nucleotidase by ATP and ADP can also be eliminated as a cause of the differences in atrial and ventricular adenosine formation since the concentrations of these compounds are lower in atrial than in ventricular muscle. Finally, the lower levels of IMP and inosine in atrial tissue indicate that deamination by adenylate deaminase is not the major pathway for AMP degradation in atrial tissue. It is possible that the lower substrate (AMP) concentration for 5′-nucleotidase is responsible for the lower adenosine production by atrial tissue in response to ischemia. However, to what extent this contributed to the lower atrial adenosine levels cannot be determined from these data.