Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction

Cardiac arrhythmias are associated with raised intracellular [Ca2+] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca2+-dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca2+-dependent phosphatase, calcineurin. Intracellular [Ca2+] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2. Raised [Ca2 +]i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca2+]i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca2+-independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca2+]i. PP2A had no role. Conduction velocity was reduced by raised [Ca2+]i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca2+] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.Centro de Investigaciones Cardiovasculare

Characterisation of nerve‐mediated ATP release from bladder detrusor muscle and its pathological implications

Background and Purpose. To characterise the molecular mechanisms that determine variability of atropine‐resistance of nerve‐mediated contractions in human and guinea‐pig detrusor smooth muscle Experimental Approach. Atropine‐resistance of nerve‐mediated contractions, and the role of P2X1 receptors, was measured in isolated preparations from guinea‐pigs and also humans with or without overactive bladder syndrome, from which the mucosa was removed. Nerve‐mediated ATP release was measured directly with amperometric ATP‐sensitive electrodes. Ecto‐ATPase activity of guinea‐pig and human detrusor samples was measured in vitro by measuring the concentration‐dependent rate of ATP breakdown. The transcription of ecto‐ATPase subtypes in human samples was measured by qPCR. Key Results Atropine resistance was greatest in guinea‐pig detrusor, absent in human tissue from normally‐functioning bladders and intermediate in human overactive bladder. Greater atropine resistance correlated with reduction of contractions by the ATP‐diphospho‐hydrolase apyrase, directly implicating ATP in their generation. E‐NTPDase‐1 was the most abundantly transcribed ecto‐ATPase of those tested and transcription was reduced in tissue from human overactive, compared to normal, bladders. E‐NTPDase‐1 enzymatic activity was inversely related to the magnitude of atropine resistance. Nerve‐mediated ATP release was continually measured and varied with stimulation frequency over the range 1‐16 Hz. Conclusion and Implications Atropine‐resistance in nerve‐mediated detrusor contractions is due to ATP release and its magnitude is inversely related to E‐NTPDase‐1 activity. ATP is released under different stimulation conditions compared to acetylcholine that implies different routes for their release</p

New targets for overactive bladder-ICI-RS 2109

Aim: To review evidence for novel drug targets that can manage overactive bladder (OAB) symptoms. Methods: A think tank considered evidence from the literature and their own research experience to propose new drug targets in the urinary bladder to characterize their use to treat OAB. Results: Five classes of agents or cellular pathways were considered. (a) Cyclic nucleotide–dependent (cyclic adenosine monophosphate and cyclic guanosine monophosphate) pathways that modulate adenosine triphosphate release from motor nerves and urothelium. (b) Novel targets for β3 agonists, including the bladder wall vasculature and muscularis mucosa. (c) Several TRP channels (TRPV1, TRPV4, TRPA1, and TRPM4) and their modulators in affecting detrusor overactivity. (d) Small conductance Ca2+-activated K+ channels and their influence on spontaneous contractions. (e) Antifibrosis agents that act to modulate directly or indirectly the TGF-β pathway—the canonical fibrosis pathway. Conclusions: The specificity of action remains a consideration if particular classes of agents can be considered for future development as receptors or pathways that mediate actions of the above mentioned potential agents are distributed among most organ systems. The tasks are to determine more detail of the pathological changes that occur in the OAB and how the specificity of potential drugs may be directed to bladder pathological changes. An important conclusion was that the storage, not the voiding, phase in the micturition cycle should be investigated and potential targets lie in the whole range of tissue in the bladder wall and not just detrusor

Contractile function of detrusor smooth muscle from children with posterior urethral valves – the role of fibrosis

IntroductionPosterior urethral valves (PUV) is the most common cause of congenital bladder outflow obstruction with persistent lower urinary tract and renal morbidities. There is a spectrum of functional bladder disorders ranging from hypertonia to bladder underactivity, but the aetiology of these clinical conditions remains unclear.Aims and objectivesWe tested the hypothesis that replacement of detrusor muscle with non-muscle cells and excessive deposition of connective tissue is an important factor in bladder dysfunction with PUV. We used isolated detrusor samples from children with PUV and undergoing primary or secondary procedures in comparison to age-matched data from children with functionally normal bladders. In vitro contractile properties, as well as passive stiffness, were measured and matched to histological assessment of muscle and connective tissue. We examined if a major pathway for fibrosis was altered in PUV tissue samples.MethodsIsometric contractions were measured in vitro in response to either stimulation of motor nerves to detrusor or exposure to cholinergic and purinergic receptor agonists. Passive mechanical stiffness was measured by rapid stretching of the tissue and recording changes to muscle tension. Histology measured the relative amounts of detrusor muscle and connective tissue. Multiplex quantitative immunofluorescence labelling using five epitope markers was designed to determine cellular pathways, in particular the Wnt-signalling pathway, responsible for any changes to excessive deposition of connective tissue.Results and DiscussionPUV tissue showed equally reduced contractile function to efferent nerve stimulation or exposure to contractile agonists. Passive muscle stiffness was increased in PUV tissue samples. The smooth muscle:connective tissue ratio was also diminished and mirrored the reduction of contractile function and the increase of passive stiffness. Immunofluorescence labelling showed in PUV samples increased expression of the matrix metalloproteinase, MMP-7; as well as cyclin-D1 expression suggesting cellular remodelling. However, elements of a fibrosis pathway associated with Wnt-signalling were either reduced (β-catenin) or unchanged (c-Myc). The accumulation of extracellular matrix, containing collagen, will contribute to the reduced contractile performance of the bladder wall. It will also increase tissue stiffness that in vivo would lead to reduced filling compliance.ConclusionsReplacement of smooth muscle with fibrosis is a major contributory factor in contractile dysfunction in the hypertonic PUV bladder. This suggests that a potential strategy to restore normal contractile and filling properties is development of the effective use of antifibrotic agents

Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction.

Cardiac arrhythmias are associated with raised intracellular [Ca2+] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca2+-dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca2+-dependent phosphatase, calcineurin. Intracellular [Ca2+] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Raised [Ca2+]i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca2+]i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca2+-independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca2+]i. PP2A had no role. Conduction velocity was reduced by raised [Ca2+]i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca2+] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1

Prediction of Paroxysmal Atrial Fibrillation From Complexity Analysis of the Sinus Rhythm ECG: A Retrospective Case/Control Pilot Study.

Paroxysmal atrial fibrillation (PAF) is the most common cardiac arrhythmia, conveying a stroke risk comparable to persistent AF. It poses a significant diagnostic challenge given its intermittency and potential brevity, and absence of symptoms in most patients. This pilot study introduces a novel biomarker for early PAF detection, based upon analysis of sinus rhythm ECG waveform complexity. Sinus rhythm ECG recordings were made from 52 patients with (n = 28) or without (n = 24) a subsequent diagnosis of PAF. Subjects used a handheld ECG monitor to record 28-second periods, twice-daily for at least 3 weeks. Two independent ECG complexity indices were calculated using a Lempel-Ziv algorithm: R-wave interval variability (beat detection, BD) and complexity of the entire ECG waveform (threshold crossing, TC). TC, but not BD, complexity scores were significantly greater in PAF patients, but TC complexity alone did not identify satisfactorily individual PAF cases. However, a composite complexity score (h-score) based on within-patient BD and TC variability scores was devised. The h-score allowed correct identification of PAF patients with 85% sensitivity and 83% specificity. This powerful but simple approach to identify PAF sufferers from analysis of brief periods of sinus-rhythm ECGs using hand-held monitors should enable easy and low-cost screening for PAF with the potential to reduce stroke occurrence

Contractile effects and receptor analysis of adenosine-receptors in human detrusor muscle from stable and neuropathic bladders

To measure the relative transcription of adenosine receptor subtypes and the contractile effects of adenosine and selective receptor-subtype ligands on detrusor smooth muscle from patients with neuropathic overactive (NDO) and stable bladders and also from guinea-pigs. Contractile function was measured at 37°C in vitro from detrusor smooth muscle strips. Contractions were elicited by superfusate agonists or by electrical field stimulation. Adenosine-receptor (A1, A2A, A2B, A3) transcription was measured by RT-PCR. Adenosine attenuated nerve-mediated responses with equivalent efficacy in human and guinea-pig tissue (pIC50 3.65–3.86); the action was more effective at low (1–8 Hz) compared to high (20– 40 Hz) stimulation frequencies in human NDO and guinea-pig tissue. With guinea-pig detrusor the action of adenosine was mirrored by the A1/A2-agonist N-ethylcarboxamido adenosine (NECA), partly abolished in turn by the A2B-selectve antagonist alloxazine, as well as the A1-selective agonist N6- cyclopentyladenosine (CPA). With detrusor from stable human bladders the effects of NECA and CPA were much smaller than that of adenosine. Adenosine also attenuated carbachol contractures, but mirrored by NECA (in turn blocked by alloxazine) only in guinea-pig tissue. Adenosine receptor subtype transcription was measured in human detrusor and was similar in both groups, except reduced A2A levels in overactive bladder. Suppression of the carbachol contracture in human detrusor is independent of A-receptor activation, in contrast to an A2B-dependent action with guinea-pig tissue. Adenosine also reduced nerve-mediated contractions, by an A1- dependent action suppressing ATP neurotransmitter action

Prediction of Paroxysmal Atrial Fibrillation From Complexity Analysis of the Sinus Rhythm ECG: A Retrospective Case/Control Pilot Study

Paroxysmal atrial fibrillation (PAF) is the most common cardiac arrhythmia, conveying a stroke risk comparable to persistent AF. It poses a significant diagnostic challenge given its intermittency and potential brevity, and absence of symptoms in most patients. This pilot study introduces a novel biomarker for early PAF detection, based upon analysis of sinus rhythm ECG waveform complexity. Sinus rhythm ECG recordings were made from 52 patients with (n= 28) or without (n= 24) a subsequent diagnosis of PAF. Subjects used a handheld ECG monitor to record 28-second periods, twice-daily for at least 3 weeks. Two independent ECG complexity indices were calculated using a Lempel-Ziv algorithm: R-wave interval variability (beat detection, BD) and complexity of the entire ECG waveform (threshold crossing, TC). TC, but not BD, complexity scores were significantly greater in PAF patients, but TC complexity alone did not identify satisfactorily individual PAF cases. However, a composite complexity score (h-score) based on within-patient BD and TC variability scores was devised. Theh-score allowed correct identification of PAF patients with 85% sensitivity and 83% specificity. This powerful but simple approach to identify PAF sufferers from analysis of brief periods of sinus-rhythm ECGs using hand-held monitors should enable easy and low-cost screening for PAF with the potential to reduce stroke occurrence

Accurate quantification of apoptosis progression and toxicity using a dielectrophoretic approach

A loss of ability of cells to undergo apoptosis (programmed cell death, whereby the cell ceases to function and destroys itself) is commonly associated with cancer, and many anti-cancer interventions aim to restart the process. Consequently, the accurate quantification of apoptosis is essential in understanding the function and performance of new anti-cancer drugs. Dielectrophoresis has previously been demonstrated to detect apoptosis more rapidly than other methods, and is low-cost, label-free and rapid, but has previously been unable to accurately quantify cells through the apoptotic process because cells in late apoptosis disintegrate, making cell tracking impossible. In this paper we use a novel method based on light absorbance and multi-population tracking to quantify the progress of apoptosis, benchmarking against conventional assays including MTT, trypan blue and Annexin-V. Analyses are performed on suspension and adherent cells, and using two apoptosis-inducing agents. IC50 measurements compared favourably to MTT and were superior to trypan blue, whilst also detecting apoptotic progression faster than Annexin-V