Contractile and Structural Properties of Detrusor from Children with Neurogenic Lower Urinary Tract Dysfunction.

Neurogenic lower urinary tract (NLUT) dysfunction in paediatric patients can arise after congenital or acquired conditions that affect bladder innervation. With some patients, urinary tract dysfunction remains and is more difficult to treat without understanding the pathophysiology. We measured in vitro detrusor smooth muscle function of samples from such bladders and any association with altered Wnt-signalling pathways that contribute to both foetal development and connective tissue deposition. A comparator group was tissue from children with normally functioning bladders. Nerve-mediated and agonist-induced contractile responses and passive stiffness were measured. Histology measured smooth muscle and connective tissue proportions, and multiplex immunohistochemistry recorded expression of protein targets associated with Wnt-signalling pathways. Detrusor from the NLUT group had reduced contractility and greater stiffness, associated with increased connective tissue content. Immunohistochemistry showed no major changes to Wnt-signalling components except down-regulation of c-Myc, a multifunctional regulator of gene transcription. NLUT is a diverse term for several diagnoses that disrupt bladder innervation. While we cannot speculate about the reasons for these pathophysiological changes, their recognition should guide research to understand their ultimate causes and develop strategies to attenuate and even reverse them. The role of changes to the Wnt-signalling pathways was minor.

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 function of detrusor smooth muscle from children with posterior urethral valves - The role of fibrosis.

INTRODUCTION: Posterior 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 OBJECTIVES: We 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. METHODS: Isometric 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 DISCUSSION: PUV 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 (ß-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. CONCLUSIONS: Replacement 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.

Stretch- and carbachol-induced ATP release from bladder wall preparations of young and aged mice.

AIMS: Bladder wall stretch increases tissue tension and releases adenosine 5'-triphosphate (ATP) as part of a transduction process to sense bladder filling. Aging is associated with bladder fibrosis to produce a stiffer bladder wall: this may augment ATP release and contribute to age-dependent urgency. Muscarinic agonists also release ATP and present a potential target for antimuscarinic agents, but its age-dependency is unknown. This study aimed, in young and old mice, to: (a) quantify the relationship between bladder wall stiffness and stretch-dependent ATP release and; (b) characterize muscarinic agonist-dependent release. METHODS: ATP release from young (9-12 weeks) and aged (24 months) mouse bladder wall was measured in vitro, with a luciferin-luciferase assay, after stretch or carbachol exposure. Bladder wall stiffness, measured simultaneously during stretch, was compared to histological proportions of connective tissue and detrusor muscle. RESULTS: With young mice, stretch-activated ATP release required an intact mucosa and was positively associated with wall stiffness. ATP release by carbachol was about four-fold greater compared to stretch. With aged mice: ATP release varied a hundred-fold and no association with stiffness; carbachol release diminished; connective tissue and mucosa thickness increased. CONCLUSIONS: With young mice, stretch, or muscarinic agonists potently induce bladder wall ATP release. Stretch-dependent release is proportional to bladder wall stiffness, independent of the extent of stretch. With aged mice dependence of stretch-activated ATP release with stiffness was lost. The huge variability of release suggests that aged mice do not form a homogenous cohort and may underlie the heterogeneity in bladder filling sensations.

ICI-RS 2019 nocturia think tank: How can experimental science guide us in understanding the pathophysiology of nocturia?

INTRODUCTION: The following is a report on the proceedings of the 2019 International Consultation on Incontinence-Research Society nocturia think tank (NTT). OBJECTIVES: The objectives of the 2019 NTT were as follows: (a) to evaluate the role of urothelium in the pathophysiology of nocturia; (b) to determine whether nocturia is a circadian disorder; (c) to discuss the role of melatonin in nocturia; (d) to consider ambulatory urodynamic monitoring in evaluating patients with nocturia; (e) to explore studies of water handling in human compartments utilizing heavy water; and (f) to explore whether basic science is the key to understanding the treatment options for diminished bladder capacity in patients with nocturia. METHODS: A compendium of discussions of the role of experimental science in understanding the pathophysiology of nocturia is described herein. RESULTS AND CONCLUSIONS: Translational science will play an increasing role in understanding the pathophysiology of nocturia, which may result in improved treatment strategies.

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 ß3 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-ß 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.

Ten-Second Electrophysiology: Evaluation of the 3DEP Platform for high-speed, high-accuracy cell analysis.

Electrical correlates of the physiological state of a cell, such as membrane conductance and capacitance, as well as cytoplasm conductivity, contain vital information about cellular function, ion transport across the membrane, and propagation of electrical signals. They are, however, difficult to measure; gold-standard techniques are typically unable to measure more than a few cells per day, making widespread adoption difficult and limiting statistical reproducibility. We have developed a dielectrophoretic platform using a disposable 3D electrode geometry that accurately (r2 > 0.99) measures mean electrical properties of populations of ~20,000 cells, by taking parallel ensemble measurements of cells at 20 frequencies up to 45 MHz, in (typically) ten seconds. This allows acquisition of ultra-high-resolution (100-point) DEP spectra in under two minutes. Data acquired from a wide range of cells - from platelets to large cardiac cells - benchmark well with patch-clamp-data. These advantages are collectively demonstrated in a longitudinal (same-animal) study of rapidly-changing phenomena such as ultradian (2-3 hour) rhythmicity in whole blood samples of the common vole (Microtus arvalis), taken from 10 µl tail-nick blood samples and avoiding sacrifice of the animal that is typically required in these studies.

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

BACKGROUND AND PURPOSE: This study aims 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, were assessed 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-diphosphohydrolase 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 enzymic 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 of 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 with ACh, implying different routes for their release.

Casein Kinase 1 Underlies Temperature Compensation of Circadian Rhythms in Human Red Blood Cells.

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.

Rhythmic potassium transport regulates the circadian clock in human red blood cells.

Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. Here we show using electrophysiological and pharmacological approaches that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.

Mild external heating and reduction in spontaneous contractions of the bladder.

OBJECTIVES: To measure the effect of external heating on bladder wall contractile function, histological structure and expression of proteins related to tissue protection and apoptosis. MATERIAL AND METHODS: In vitro preparations of bladder wall and ex vivo perfused pig bladders were heated from 37 to 42°C, 46 and 50°C for 15 min. Isolated preparations were heated by radiant energy and perfused bladders were heated by altering perfusate temperature. Spontaneous contractions or pressure variations were recorded, as well as responses to the muscarinic agonist carbachol or motor nerve excitation in vitro during heating. Tissue histology in control and after heating was analysed using haematoxylin and eosin staining and 4'-6-diamidino-2-phenylindole (DAPI) nuclear labelling. The effects of heating on protein expression levels of (i) heat shock proteins HSP27-pSer82 and inducible-HSP70 and (ii) caspase-3 and its downstream DNA-repair substrate poly-[ADP-ribose] polymerase (PARP) were measured. RESULTS: Heating to 42°C reduced spontaneous contractions or pressure variations by ~70%; effects were fully reversible. There were no effects on carbachol or nerve-mediated responses. Tissue histology was unaffected by heating, and expression of heat shock proteins as well as caspase-3 and PARP were also unaltered. A TRPV1 antagonist had no effect on the reduction of spontaneous activity. Heating to 46°C had a similar effect on spontaneous activity and also reduced the carbachol contracture. Urothelial structure was damaged, caspase-3 levels were increased and inducible-HSP70 levels declined. At 50°C evoked contractions were abolished, the urothelium was absent and heat shock proteins and PARP expression was reduced with raised caspase-3 expression. CONCLUSIONS: Heating to 42°C caused a profound, reversible and reproducible attenuation of spontaneous activity, with no tissue damage and no initiation of apoptosis pathways. Higher temperatures caused tissue damage and activation of apoptotic mechanisms. Mild heating offers a novel approach to reducing bladder spontaneous activity.

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.

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.

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-ethylcarboxamidoadenosine (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.

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.

Protein kinase Cε-calcineurin cosignaling downstream of toll-like receptor 4 downregulates fibrosis and induces wound healing gene expression in cardiac myofibroblasts.

The pathways which regulate resolution of inflammation and contribute to positive remodeling of the myocardium following injury are poorly understood. Here we show that protein kinase C epsilon (PKCε) cooperates with the phosphatase calcineurin (CN) to potentiate induction of cardioprotective gene expression while suppressing expression of fibrosis markers. This was achieved by detailed analysis of the regulation of cyclooxygenase 2 (COX-2) expression as a marker gene and by using gene expression profiling to identify genes regulated by coexpression of CN-Aα/PKCε in adult rat cardiac myofibroblasts (ARVFs) on a larger scale. GeneChip analysis of CN-Aα/PKCε-coexpressing ARVFs showed that COX-2 provides a signature for wound healing and is associated with downregulation of fibrosis markers, including connective tissue growth factor (CTGF), fibronectin, and collagens Col1a1, Col3a1, Col6a3, Col11a1, Col12a1, and Col14a1, with concomitant upregulation of cardioprotection markers, including COX-2 itself, lipocalin 2 (LCN2), tissue inhibitor of metalloproteinase 1 (TIMP-1), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS). In primary rat cardiomyocyte cultures Toll-like receptor 4 (TLR4) agonist- or PKCε/CN-dependent COX-2 induction occurred in coresident fibroblasts and was blocked by selective inhibition of CN or PKC α/ε or elimination of fibroblasts. Furthermore, ectopic expression of PKCε and CN in ARVFs showed that the effects on COX-2 expression are mediated by specific NFAT sites within the COX-2 promoter as confirmed by site-directed mutagenesis and chromatin immunoprecipitation (ChIP). Therefore, PKCε may negatively regulate adverse myocardial remodeling by cooperating with CN to downregulate fibrosis and induce transcription of cardioprotective wound healing genes, including COX-2.

Relationship between gap-junctional conductance and conduction velocity in mammalian myocardium.

BACKGROUND: Gap junction resistivity, R(j), 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 R(j) and CV, each independently measured in human and guinea-pig myocardium. METHODS AND RESULTS: R(j) 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 R(j) and CV in human and guinea-pig myocardium, pre- and post-carbenoxolone (r(2)=0.946; P<0.01). In guinea-pig left ventricle, left atrium, and right atrium, carbenoxolone increased R(j) 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 R(j) 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.

p42/p44-MAPK and PI3K are sufficient for IL-6 family cytokines/gp130 to signal to hypertrophy and survival in cardiomyocytes in the absence of JAK/STAT activation.

The effect of differential signalling by IL-6 and leukaemia inhibitory factor (LIF) which signal by gp130 homodimerisation or LIFRß/gp130 heterodimerisation on survival and hypertrophy was studied in neonatal rat cardiomyocytes. Both LIF and IL-6 [in the absence of soluble IL-6 receptor (sIL-6Rα)] activated Erk1/2, JNK1/2, p38-MAPK and PI3K signalling peaking at 20min and induced cytoprotection against simulated ischemia-reperfusion injury which was blocked by the MEK1/2 inhibitor PD98059 but not the p38-MAPK inhibitor SB203580. In the absence of sIL-6R, IL-6 did not induce STAT1/3 phosphorylation, whereas IL-6/sIL-6R and LIF induced STAT1 and STAT3 phosphorylation. Furthermore, IL-6/sIL-6R induced phosphorylation of STAT1 Tyr(701) and STAT3 Tyr(705) were enhanced by SB203580. IL-6 and pheneylephrine (PE), but not LIF, induced cardiomyocyte iNOS expression and nitric oxide (NO) production. IL-6, LIF and PE induced cardiomyocyte hypertrophy, but with phenotypic differences in ANF and SERCA2 expression and myofilament organisation with IL-6 more resembling PE than LIF. Transfection of cardiomyocytes with full length or truncated chimaeric gp130 cytoplasmic domain/Erythropoietin receptor (EpoR) extracellular domain fusion constructs showed that the membrane proximal Box 1 and Box 2 containing region of gp130 was necessary and sufficient for MAPK and PI3K activation; hypertrophy; SERCA2 expression and iNOS/NO induction in the absence of JAK/STAT activation. In conclusion, IL-6 can signal in cardiomyocytes independent of sIL-6R and STAT1/3 and furthermore, that Erk1/2 and PI3K activation by IL-6 are both necessary and sufficient for induced cardioprotection. In addition, p38-MAPK may act as a negative feedback regulator of JAK/STAT activation in cardiomyocytes.

Cytoplasm resistivity of mammalian atrial myocardium determined by dielectrophoresis and impedance methods.

Many cardiac arrhythmias are caused by slowed conduction of action potentials, which in turn can be due to an abnormal increase of intracellular myocardial resistance. Intracellular resistivity is a linear sum of that offered by gap junctions between contiguous cells and the cytoplasm of the myocytes themselves. However, the relative contribution of the two components is unclear, especially in atrial myocardium, as there are no precise measurements of cytoplasmic resistivity, R(c). In this study, R(c) was measured in atrial tissue using several methods: a dielectrophoresis technique with isolated cells and impedance measurements with both isolated cells and multicellular preparations. All methods yielded similar values for R(c), with a mean of 138 ± 5 Ω·cm at 23°C, and a Q(10) value of 1.20. This value is about half that of total intracellular resistivity and thus will be a significant determinant of the actual value of action potential conduction velocity. The dielectrophoresis experiments demonstrated the importance of including divalent cations (Ca(2+) and Mg(2+)) in the suspension medium, as their omission reduced cell integrity by lowering membrane resistivity and increasing cytoplasm resistivity. Accurate measurement of R(c) is essential to develop quantitative computational models that determine the key factors contributing to the development of cardiac arrhythmias.