Rasheda A. Chowdhury

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.

Relationship Between Gap-Junctional Conductance and Conduction Velocity in Mammalian Myocardium

Background—Gap junction resistivity, Rj, has been proposed as a key determinant of conduction velocity (CV). However, studies in connexin-gene knockout mice demonstrated significant CV slowing only with near-complete connexin deletion, and these findings led to the concept of a significant redundancy of myocardial gap junctions. We challenged this prevailing concept and addressed the hypothesis that there is a continuous relationship between Rj and CV, each independently measured in human and guinea-pig myocardium. Methods and Results—Rj and CV were directly measured by oil-gap impedance and microelectrode techniques in human left ventricular myocardium from patients with hypertrophic cardiomyopathy and in guinea-pig atrial and ventricular myocardium before and during pharmacological uncoupling with 20-µmol/L carbenoxolone. There was a continuous relationship between Rj and CV in human and guinea-pig myocardium, pre- and post-carbenoxolone (r2=0.946; P<0.01). In guinea-pig left ventricle, left atrium, and right atrium, carbenoxolone increased Rj by 28±9%, 26±16%, and 25±14% and slowed CV by 17±3%, 23±8%, and 11±4% respectively (all P<0.05 versus control). As a clinically accessible measure of local microscopic myocardial conduction slowing in vivo in the intact human heart, carbenoxolone prolonged electrogram duration in the right atrium (39.7±4.2 to 42.3±4.3 ms; P=0.01) and right ventricle (48.1±2.5 to 53.3±5.3 ms; P<0.01). Conclusions—There is a continuous relationship between Rj and CV that is consistent between cardiac chambers and across species, indicating that naturally occurring variations in cellular coupling can account for variations in CV, and that the concept that there is massive redundancy of coupling is not tenable.

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.

The Renin–Angiotensin System Mediates the Effects of Stretch on Conduction Velocity, Connexin43 Expression, and Redistribution in Intact Ventricle

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).

Characterisation of Connexin Expression and Electrophysiological Properties in Stable Clones of the HL-1 Myocyte Cell Line

Abstract The HL-1 atrial line contains cells blocked at various developmental stages. To obtain homogeneous sub-clones and correlate changes in gene expression with functional alterations, individual clones were obtained and characterised for parameters involved in conduction and excitation-contraction coupling. Northern blots for mRNAs coding for connexins 40, 43 and 45 and calcium handling proteins (sodium/calcium exchanger, L- and T-type calcium channels, ryanodine receptor 2 and sarco-endoplasmic reticulum calcium ATPase 2) were performed. Connexin expression was further characterised by western blots and immunofluorescence. Inward currents were characterised by voltage clamp and conduction velocities measured using microelectrode arrays. The HL-1 clones had similar sodium and calcium inward currents with the exception of clone 2 which had a significantly smaller calcium current density. All the clones displayed homogenous propagation of electrical activity across the monolayer correlating with the levels of connexin expression. Conduction velocities were also more sensitive to inhibition of junctional coupling by carbenoxolone (∼80%) compared to inhibition of the sodium current by lidocaine (∼20%). Electrical coupling by gap junctions was the major determinant of conduction velocities in HL-1 cell lines. In summary we have isolated homogenous and stable HL-1 clones that display characteristics distinct from the heterogeneous properties of the original cell line.

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.

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.

Characterisation of re-entrant circuit (or rotational activity) in vitro using the HL1-6 myocyte cell line

Fibrillation is the most common arrhythmia observed in clinical practice. Understanding of the mechanisms underlying its initiation and maintenance remains incomplete. Functional re-entries are potential drivers of the arrhythmia. Two main concepts are still debated, the “leading circle” and the “spiral wave or rotor” theories. The homogeneous subclone of the HL1 atrial-derived cardiomyocyte cell line, HL1-6, spontaneously exhibits re-entry on a microscopic scale due to its slow conduction velocity and the presence of triggers, making it possible to examine re-entry at the cellular level. We therefore investigated the re-entry cores in cell monolayers through the use of fluorescence optical mapping at high spatiotemporal resolution in order to obtain insights into the mechanisms of re-entry. Re-entries in HL1-6 myocytes required at least two triggers and a minimum colony area to initiate (3.5 to 6.4 mm2). After electrical activity was completely stopped and re-started by varying the extracellular K+ concentration, re-entries never returned to the same location while 35% of triggers re-appeared at the same position. A conduction delay algorithm also allows visualisation of the core of the re-entries. This work has revealed that the core of re-entries is conduction blocks constituted by lines and/or groups of cells rather than the round area assumed by the other concepts of functional re-entry. This highlights the importance of experimentation at the microscopic level in the study of re-entry mechanisms.

Concurrent micro- to macro-cardiac electrophysiology in myocyte cultures and human heart slices

The contact cardiac electrogram is derived from the extracellular manifestation of cellular action potentials and cell-to-cell communication. It is used to guide catheter based clinical procedures. Theoretically, the contact electrogram and the cellular action potential are directly related, and should change in conjunction with each other during arrhythmogenesis, however there is currently no methodology by which to concurrently record both electrograms and action potentials in the same preparation for direct validation of their relationships and their direct mechanistic links. We report a novel dual modality apparatus for concurrent electrogram and cellular action potential recording at a single cell level within multicellular preparations. We further demonstrate the capabilities of this system to validate the direct link between these two modalities of voltage recordings.

Rethinking multiscale cardiac electrophysiology with machine learning and predictive modelling

We review some of the latest approaches to analysing cardiac electrophysiology data using machine learning and predictive modelling. Cardiac arrhythmias, particularly atrial fibrillation, are a major global healthcare challenge. Treatment is often through catheter ablation, which involves the targeted localised destruction of regions of the myocardium responsible for initiating or perpetuating the arrhythmia. Ablation targets are either anatomically defined, or identified based on their functional properties as determined through the analysis of contact intracardiac electrograms acquired with increasing spatial density by modern electroanatomic mapping systems. While numerous quantitative approaches have been investigated over the past decades for identifying these critical curative sites, few have provided a reliable and reproducible advance in success rates. Machine learning techniques, including recent deep-learning approaches, offer a potential route to gaining new insight from this wealth of highly complex spatio-temporal information that existing methods struggle to analyse. Coupled with predictive modelling, these techniques offer exciting opportunities to advance the field and produce more accurate diagnoses and robust personalised treatment. We outline some of these methods and illustrate their use in making predictions from the contact electrogram and augmenting predictive modelling tools, both by more rapidly predicting future states of the system and by inferring the parameters of these models from experimental observations.