Enhancement of Gap Junction Function During Acute Myocardial Infarction Modifies Healing and Reduces Late Ventricular Arrhythmia Susceptibility.

OBJECTIVES: The purpose of this study was to investigate the effects of enhancing gap junction (GJ) coupling during acute myocardial infarction (MI) on the healed infarct scar morphology and late post-MI arrhythmia susceptibility. BACKGROUND: Increased heterogeneity of myocardial scarring after MI is associated with greater arrhythmia susceptibility. We hypothesized that short-term enhancement of GJ coupling during acute MI can produce more homogeneous infarct scars, reducing late susceptibility to post-MI arrhythmias. METHODS: Following arrhythmic characterization of a rat 4-week post-MI model (n = 24), another 27 Sprague-Dawley rats were randomized to receive rotigaptide to enhance GJ coupling (n = 13) or to saline control (n = 14) by osmotic minipump immediately prior to and for the first 7 days following surgically induced MI. At 4 weeks post-MI, hearts were explanted for ex vivo programmed electrical stimulation (PES) and optical mapping. Heterogeneity of infarct border zone (IBZ) scarring was quantified by histomorphometry. RESULTS: Despite no detectable differences in infarct size at 4 weeks post-MI, rotigaptide-treated hearts had reduced arrhythmia susceptibility during PES (inducibility score for rotigaptide: 2.4 ± 0.8; for control: 5.0 ± 0.6; p = 0.02) and less heterogeneous IBZ scarring (dispersion of IBZ complexity score: rotigaptide: 1.1 ± 0.1; control: 1.4 ± 0.1; p = 0.04), associated with an improvement in IBZ conduction velocity (rotigaptide: 43.1 ± 3.4 cm/s; control: 34.8 ± 2.0 cm/s; p = 0.04). CONCLUSIONS: Enhancement of GJ coupling for only 7 days at the time of acute MI produced more homogeneous IBZ scarring and reduced arrhythmia susceptibility at 4 weeks post-MI. Short-term GJ modulation at the time of MI may represent a novel treatment strategy to modify the healed infarct scar morphology and reduce late post-MI arrhythmic risk.

Fractionation of electrograms is caused by colocalized conduction block and connexin disorganization in the absence of fibrosis as AF becomes persistent in the goat model.

BACKGROUND: Electrogram fractionation and atrial fibrosis are both thought to be pathophysiological hallmarks of evolving persistence of atrial fibrillation (AF), but recent studies in humans have shown that they do not colocalize. The interrelationship and relative roles of fractionation and fibrotic change in AF persistence therefore remain unclear. OBJECTIVE: The aim of the study was to examine the hypothesis that electrogram fractionation with increasing persistence of AF results from localized conduction slowing or block due to changes in atrial connexin distribution in the absence of fibrotic change. METHODS: Of 12 goats, atrial burst pacemakers maintained AF in 9 goats for up to 3 consecutive 4-week periods. After each 4-week period, 3 goats underwent epicardial mapping studies of the right atrium and examination of the atrial myocardium for immunodetection of connexins 43 and 40 (Cx43 and Cx40) and quantification of connective tissue. RESULTS: Despite refractoriness returning to normal in between each 4-week period of AF, there was a cumulative increase in the prevalence of fractionated atrial electrograms during both atrial pacing (control and 1, 2, and 3 months period of AF 0.3%, 1.3% ± 1.5%, 10.6% ± 2%, and 17% ± 5%, respectively; analysis of variance, P < .05) and AF (0.3% ± 0.1%, 2.3% ± 1.2%, 14% ± 2%, and 23% ± 3%; P < .05) caused by colocalized areas of conduction block during both pacing (local conduction velocity <10 cm/s: 0.1% ± 0.1%, 0.3% ± 0.6%, 6.5% ± 3%, and 6.9% ± 4%; P < .05) and AF (1.5% ± 0.5%, 2.7% ± 1.1%, 10.1% ± 1.2%, and 13.6% ± 0.4%; P < .05), associated with an increase in the heterogeneity of Cx40 and lateralization of Cx43 (lateralization scores: 1.75 ± 0.89, 1.44 ± 0.31, 2.85 ± 0.96, and 2.94 ± 0.31; P < .02), but not associated with change in connective tissue content or net conduction velocity. CONCLUSION: Electrogram fractionation with increasing persistence of AF results from slow localized conduction or block associated with changes in atrial connexin distribution in the absence of fibrotic change.

Architectural correlates of myocardial conduction: changes to the topography of cellular coupling, intracellular conductance, and action potential propagation with hypertrophy in Guinea-pig ventricular myocardium.

BACKGROUND: We tested the hypothesis that alterations to action potential conduction velocity (CV) and conduction anisotropy in left ventricular hypertrophy are associated with topographical changes to gap-junction coupling and intracellular conductance by measuring these variables in the same preparations. METHODS AND RESULTS: Left ventricular papillary muscles were excised from aortic-banded or sham-operated guinea-pig hearts. With intracellular stimulating and recording microelectrodes, CV was measured in 3 dimensions with simultaneous conductance mapping with subthreshold stimuli and correlated with quantitative histomorphometry of myocardial architecture and connexin 43 distribution. In hypertrophied myocardium, CV in the longitudinal axis was smaller and transverse velocity was greater compared with control; associated with similar differences of intracellular conductance, consistent with more cell contacts per cell (5.7 ± 0.2 versus 8.1 ± 0.5; control versus hypertrophy), and more intercalated disks mediating side-to-side coupling (8.2 ± 0.2 versus 10.2 ± 0.4 per cell). Intercalated disk morphology and connexin 43 immunolabelling were not different in hypertrophy. Hypertrophied preparations showed local submillimeter (≈250 µm) regions with slow conduction and low intracellular conductance, which, although not affecting CV on the millimeter scale, were consistent with discontinuities from increased microscopical connective tissue content. CONCLUSIONS: With myocardial hypertrophy, altered longitudinal and transverse CV, and greater nonuniformity of CV anisotropy correspond to changes of intracellular conductance. These are associated with alteration of myocardial architecture, specifically the topography of cell-cell coupling and gap-junction connectivity.

Relationship between connexin expression and gap-junction resistivity in human atrial myocardium.

BACKGROUND: The relative roles of the gap-junctional proteins connexin40 (Cx40) and connexin43 (Cx43) in determining human atrial myocardial resistivity is unknown. In addressing the hypothesis that changing relative expression of Cx40 and Cx43 underlies an increase in human atrial myocardial resistivity with age, this relationship was investigated by direct ex vivo measurement of gap-junctional resistivity and quantitative connexin immunoblotting and immunohistochemistry. METHODS AND RESULTS: Oil-gap impedance measurements were performed to determine resistivity of the intracellular pathway (Ri), which correlated with total Cx40 quantification by Western blotting (rs=0.64, P<0.01, n=20). Specific gap-junctional resistivity (Rj) correlated not only with Western immunoquantification of Cx40 (rs=0.63, P=0.01, n=20), but also more specifically, with the Cx40 fraction localized to the intercalated disks on immunohistochemical quantification (rs=0.66, P=0.02, n=12). Although Cx43 expression showed no correlation with resistivity values, the proportional expression of the 2 connexins, (Cx40/[Cx40+Cx43]) correlated with Ri and Rj (rs=0.58, P<0.01 for Ri and rs=0.51, P=0.02 for Rj). Advancing age was associated with a rise in Ri (rs=0.77, P<0.0001), Rj (rs=0.65, P<0.001, n=23), Cx40 quantity (rs=0.54, P=0.01, n=20), and Cx40 gap-junction protein per unit area of en face disk (rs=0.61, P=0.02, n=12). CONCLUSIONS: Cx40 is associated with human right atrial gap-junctional resistivity such that increased total, gap-junctional, and proportional Cx40 expression increases gap-junctional resistivity. Accordingly, advancing age is associated with an increase in Cx40 expression and a corresponding increase in gap-junctional resistivity. These findings are the first to demonstrate this relationship and a mechanistic explanation for changing atrial conduction and age-related arrhythmic tendency.

The Renin-Angiotensin system mediates the effects of stretch on conduction velocity, connexin43 expression, and redistribution in intact ventricle.

UNLABELLED: Effect of Stretch on Conduction and Cx43. INTRODUCTION: In disease states such as heart failure, myocardial infarction, and hypertrophy, changes in the expression and location of Connexin43 (Cx43) occur (Cx43 remodeling), and may predispose to arrhythmias. Stretch may be an important stimulus to Cx43 remodeling; however, it has only been investigated in neonatal cell cultures, which have different physiological properties than adult myocytes. We hypothesized that localized stretch in vivo causes Cx43 remodeling, with associated changes in conduction, mediated by the renin-angiotensin system (RAS). METHODS AND RESULTS: In an open-chest canine model, a device was used to stretch part of the right ventricle (RV) by 22% for 6 hours. Activation mapping using a 312-electrode array was performed before and after stretch. Regional stretch did not change longitudinal conduction velocity (post-stretch vs baseline: 51.5 ± 5.2 vs 55.3 ± 8.1 cm/s, P = 0.24, n = 11), but significantly reduced transverse conduction velocity (28.7 ± 2.5 vs 35.4 ± 5.4 cm/s, P < 0.01). It also reduced total Cx43 expression, by Western blotting, compared with nonstretched RV of the same animal (86.1 ± 32.2 vs 100 ± 19.4%, P < 0.02, n = 11). Cx43 labeling redistributed to the lateral cell borders. Stretch caused a small but significant increase in the proportion of the dephosphorylated form of Cx43 (stretch 9.95 ± 1.4% vs control 8.74 ± 1.2%, P < 0.05). Olmesartan, an angiotensin II blocker, prevented the stretch-induced changes in Cx43 levels, localization, and conduction. CONCLUSION: Myocardial stretch in vivo has opposite effects to that in neonatal myocytes in vitro. Stretch in vivo causes conduction changes associated with Cx43 remodeling that are likely caused by local stretch-induced activation of the RAS.

Characteristics of spontaneous activity in the bladder trigone.

BACKGROUND: During bladder filling, the trigone contracts help keep the ureteral orifices open and the bladder neck shut. The trigone generates spontaneous activity as well as responding to neuromuscular transmitters, but the relationship between these phenomena are unclear. OBJECTIVES: To characterise the cellular mechanisms that regulate and modify spontaneous activity in trigone smooth muscle. DESIGN, SETTING, AND PARTICIPANTS: Muscle strips from the superficial trigone of male guinea-pigs were used for tension experiments and immunofluorescent studies. MEASUREMENTS: In isolated trigonal cells, intracellular Ca(2+) was measured by epifluorescence microscopy using the fluorescent Ca(2+) indicator Fura-2. RESULTS AND LIMITATIONS: Spontaneous intracellular Ca(2+) transients and contractions were observed in trigonal single cells and strips and were significantly higher compared to the bladder dome. Ca-free superfusate and verapamil terminated spontaneity. T-type Ca(2+) channel block with NiCl(2) depressed slightly Ca(2+) transients but not spontaneous contractions. Neither the BK(Ca) channel blocker iberiotoxin nor the SK(Ca) channel blocker apamin had any effect on single cell activity. By contrast, the Cl(-) channel blocker niflumic acid attenuated significantly both Ca(2+) transients and muscle contractions. Agonist stimulation (carbachol, phenylephrine) up-regulated activity. Gap junction labelling (Cx43) was approximately 5 times denser in the trigone than in detrusor smooth muscle. The gap junction blocker 18-beta-glycyrrhetinic acid modulated spontaneous contractions in the trigone but not in the bladder dome. CONCLUSIONS: Trigone myocytes employ membrane L-type-Ca(2+) channels and Cl(-) channels to generate spontaneous activity. Intercellular electrical coupling ensures its propagation and, thus, sustains contraction of the whole trigone.

Suburothelial myofibroblasts in the human overactive bladder and the effect of botulinum neurotoxin type A treatment.

BACKGROUND: An increasing body of evidence suggests a possible role of suburothelial myofibroblasts (MFs) in bladder mechanosensation and in the pathophysiology of detrusor overactivity (DO). OBJECTIVE: To determine whether markers of MFs, including gap junction protein connexin43 (Cx43) and c-kit have altered immunohistochemical expression in the suburothelium of patients with neurogenic DO (NDO) or idiopathic DO (IDO) and whether this is affected by successful treatment of DO with botulinum neurotoxin type A (BoNTA). DESIGN, SETTING, AND PARTICIPANTS: Patients with NDO (n=10) or IDO (n=11) were treated in a single-centre, open-label study of intradetrusor BoNTA injections. Control tissue was obtained from 10 patients undergoing pelvic-floor repair procedures who had no overactive bladder (OAB) symptoms. This study is registered with ClinicalTrials.gov, number NCT00662064. INTERVENTIONS: Bladder biopsies performed with flexible cystoscopes were obtained from control subjects and from NDO and IDO patients before BoNTA treatment and at 4 wk and 16 wk after treatment. They were studied with quantitative immunofluorescence using antibodies to connexin 43 (Cx43), vimentin, and c-kit. MEASUREMENTS: Differences in Cx43, vimentin, and c-kit immunoreactivity between control subjects and NDO or IDO patients (primary outcomes). Changes in NDO or IDO, Cx43 immunoreactivity, and c-kit immunoreactivity after BoNTA treatment (secondary outcomes). RESULTS AND LIMITATIONS: Cx43 immunoreactivity was increased in both IDO and NDO patients compared to controls, but remained unchanged after BoNTA treatment. C-kit immunoreactivity was similar in NDO/IDO patients and controls and remained unchanged after BoNTA treatment. CONCLUSIONS: Increased gap junction formation in the suburothelium has been demonstrated in biopsies from humans with DO. It is hypothesised that this change could have a significant role in the pathogenesis of the detrusor abnormality. Successful treatment of NDO or IDO does not appear to be associated with changes in the expression of Cx43 or c-kit on suburothelial MFs.