Cardiomyocyte ionic currents in intact young and aged murine Pgc-1β-/- atrial preparations.

INTRODUCTION: Recent studies reported that energetically deficient murine Pgc-1β-/- hearts replicate age-dependent atrial arrhythmic phenotypes associated with their corresponding clinical conditions, implicating action potential (AP) conduction slowing consequent upon reduced AP upstroke rates. MATERIALS AND METHODS: We tested a hypothesis implicating Na+ current alterations as a mechanism underlying these electrophysiological phenotypes. We applied loose patch-clamp techniques to intact young and aged, WT and Pgc-1β-/-, atrial cardiomyocyte preparations preserving their in vivo extracellular and intracellular conditions. RESULTS AND DISCUSSION: Depolarising steps activated typical voltage-dependent activating and inactivating inward (Na+) currents whose amplitude increased or decreased with the amplitudes of the activating, or preceding inactivating, steps. Maximum values of peak Na+ current were independently influenced by genotype but not age or interacting effects of genotype and age on two-way ANOVA. Neither genotype, nor age, whether independently or interactively, influenced voltages at half-maximal current, or steepness factors, for current activation and inactivation, or time constants for recovery from inactivation following repolarisation. In contrast, delayed outward (K+) currents showed similar activation and rectification properties through all experimental groups. These findings directly demonstrate and implicate reduced Na+ in contrast to unchanged K+ current, as a mechanism for slowed conduction causing atrial arrhythmogenicity in Pgc-1β-/- hearts

Photopigment quenching is Ca2+ dependent and controls response duration in salamander L-cone photoreceptors

The time scale of the photoresponse in photoreceptor cells is set by the slowest of the steps that quench the light-induced activity of the phototransduction cascade. In vertebrate photoreceptor cells, this rate-limiting reaction is thought to be either shutoff of catalytic activity in the photopigment or shutoff of the pigment's effector, the transducin-GTP–phosphodiesterase complex. In suction pipette recordings from isolated salamander L-cones, we found that preventing changes in internal [Ca2+] delayed the recovery of the light response and prolonged the dominant time constant for recovery. Evidence that the Ca2+-sensitive step involved the pigment itself was provided by the observation that removal of Cl− from the pigment's anion-binding site accelerated the dominant time constant for response recovery. Collectively, these observations indicate that in L-cones, unlike amphibian rods where the dominant time constant is insensitive to [Ca2+], pigment quenching rate limits recovery and provides an additional mechanism for modulating the cone response during light adaptation

Sarcoplasmic reticular Ca 2+ -ATPase inhibition paradoxically upregulates murine skeletal muscle Na v 1.4 function

Abstract: Skeletal muscle Na+ channels possess Ca2+- and calmodulin-binding sites implicated in Nav1.4 current (INa) downregulation following ryanodine receptor (RyR1) activation produced by exchange protein directly activated by cyclic AMP or caffeine challenge, effects abrogated by the RyR1-antagonist dantrolene which itself increased INa. These findings were attributed to actions of consequently altered cytosolic Ca2+, [Ca2+]i, on Nav1.4. We extend the latter hypothesis employing cyclopiazonic acid (CPA) challenge, which similarly increases [Ca2+]i, but through contrastingly inhibiting sarcoplasmic reticular (SR) Ca2+-ATPase. Loose patch clamping determined Na+ current (INa) families in intact native murine gastrocnemius skeletal myocytes, minimising artefactual [Ca2+]i perturbations. A bespoke flow system permitted continuous INa comparisons through graded depolarizing steps in identical stable membrane patches before and following solution change. In contrast to the previous studies modifying RyR1 activity, and imposing control solution changes, CPA (0.1 and 1 µM) produced persistent increases in INa within 1–4 min of introduction. CPA pre-treatment additionally abrogated previously reported reductions in INa produced by 0.5 mM caffeine. Plots of peak current against voltage excursion demonstrated that 1 µM CPA increased maximum INa by ~ 30%. It only slightly decreased half-maximal activating voltages (V0.5) and steepness factors (k), by 2 mV and 0.7, in contrast to the V0.5 and k shifts reported with direct RyR1 modification. These paradoxical findings complement previously reported downregulatory effects on Nav1.4 of RyR1-agonist mediated increases in bulk cytosolic [Ca2+]. They implicate possible local tubule-sarcoplasmic triadic domains containing reduced [Ca2+]TSR in the observed upregulation of Nav1.4 function following CPA-induced SR Ca2+ depletion

Finite element analysis predicts Ca 2+ microdomains within tubular-sarcoplasmic reticular junctions of amphibian skeletal muscle

Abstract: A finite element analysis modelled diffusional generation of steady-state Ca2+ microdomains within skeletal muscle transverse (T)-tubular-sarcoplasmic reticular (SR) junctions, sites of ryanodine receptor (RyR)-mediated SR Ca2+ release. It used established quantifications of sarcomere and T-SR anatomy (radial diameter d=220nm; axial distance w=12nm). Its boundary SR Ca2+ influx densities,Jinflux, reflected step impositions of influxes, Φinflux=Jinfluxπd24, deduced from previously measured Ca2+ signals following muscle fibre depolarization. Predicted steady-state T-SR junctional edge [Ca2+], [Ca2+]edge, matched reported corresponding experimental cytosolic [Ca2+] elevations given diffusional boundary effluxΦefflux=D[Ca2+]edgeλ(πdw), established cytosolic Ca2+ diffusion coefficients (D=4×107nm2/s) and exit length λ=9.2nm. Dependences of predicted [Ca2+]edge upon Jinflux then matched those of experimental [Ca2+] upon Ca2+ release through their entire test voltage range. The resulting model consistently predicted elevated steady-state T-SR junctional ~ µM-[Ca2+] elevations radially declining from maxima at the T-SR junction centre along the entire axial T-SR distance. These [Ca2+] heterogeneities persisted through 104- and fivefold, variations in D and w around, and fivefold reductions in d below, control values, and through reported resting muscle cytosolic [Ca2+] values, whilst preserving the flux conservation (Φinflux=Φefflux) condition, Ca2+edge=λdJinflux4Dw. Skeletal muscle thus potentially forms physiologically significant ~ µM-[Ca2+] T-SR microdomains that could regulate cytosolic and membrane signalling molecules including calmodulin and RyR, These findings directly fulfil recent experimental predictions invoking such Ca2+ microdomains in observed regulatory effects upon Na+ channel function, in a mechanism potentially occurring in similar restricted intracellular spaces in other cell types

Sarcoplasmic reticular Ca 2+ -ATPase inhibition paradoxically upregulates murine skeletal muscle Na v 1.4 function

Abstract: Skeletal muscle Na+ channels possess Ca2+- and calmodulin-binding sites implicated in Nav1.4 current (INa) downregulation following ryanodine receptor (RyR1) activation produced by exchange protein directly activated by cyclic AMP or caffeine challenge, effects abrogated by the RyR1-antagonist dantrolene which itself increased INa. These findings were attributed to actions of consequently altered cytosolic Ca2+, [Ca2+]i, on Nav1.4. We extend the latter hypothesis employing cyclopiazonic acid (CPA) challenge, which similarly increases [Ca2+]i, but through contrastingly inhibiting sarcoplasmic reticular (SR) Ca2+-ATPase. Loose patch clamping determined Na+ current (INa) families in intact native murine gastrocnemius skeletal myocytes, minimising artefactual [Ca2+]i perturbations. A bespoke flow system permitted continuous INa comparisons through graded depolarizing steps in identical stable membrane patches before and following solution change. In contrast to the previous studies modifying RyR1 activity, and imposing control solution changes, CPA (0.1 and 1 µM) produced persistent increases in INa within 1–4 min of introduction. CPA pre-treatment additionally abrogated previously reported reductions in INa produced by 0.5 mM caffeine. Plots of peak current against voltage excursion demonstrated that 1 µM CPA increased maximum INa by ~ 30%. It only slightly decreased half-maximal activating voltages (V0.5) and steepness factors (k), by 2 mV and 0.7, in contrast to the V0.5 and k shifts reported with direct RyR1 modification. These paradoxical findings complement previously reported downregulatory effects on Nav1.4 of RyR1-agonist mediated increases in bulk cytosolic [Ca2+]. They implicate possible local tubule-sarcoplasmic triadic domains containing reduced [Ca2+]TSR in the observed upregulation of Nav1.4 function following CPA-induced SR Ca2+ depletion

Ryanodine receptor modulation by caffeine challenge modifies Na + current properties in intact murine skeletal muscle fibres

Abstract: We investigated effects of the ryanodine receptor (RyR) modulator caffeine on Na+ current (INa) activation and inactivation in intact loose-patch clamped murine skeletal muscle fibres subject to a double pulse procedure. INa activation was examined using 10-ms depolarising, V1, steps to varying voltages 0–80 mV positive to resting membrane potential. The dependence of the subsequent, INa inactivation on V1 was examined by superimposed, V2, steps to a fixed depolarising voltage. Current-voltage activation and inactivation curves indicated that adding 0.5 and 2 mM caffeine prior to establishing the patch seal respectively produced decreased (within 1 min) and increased (after ~2 min) peak INa followed by its recovery to pretreatment levels (after ~40 and ~30 min respectively). These changes accompanied negative shifts in the voltage dependence of INa inactivation (within 10 min) and subsequent superimposed positive activation and inactivation shifts, following 0.5 mM caffeine challenge. In contrast, 2 mM caffeine elicited delayed negative shifts in both activation and inactivation. These effects were abrogated if caffeine was added after establishing the patch seal or with RyR block by 10 μM dantrolene. These effects precisely paralleled previous reports of persistently (~10 min) increased cytosolic [Ca2+] with 0.5 mM, and an early peak rapidly succeeded by persistently reduced [Ca2+] likely reflecting gradual RyR inactivation with ≥1.0 mM caffeine. The latter findings suggested inhibitory effects of even resting cytosolic [Ca2+] on INa. They suggest potentially physiologically significant negative feedback regulation of RyR activity on Nav1.4 properties through increased or decreased local cytosolic [Ca2+], Ca2+-calmodulin and FKBP12

Ryanodine receptor modulation by caffeine challenge modifies Na + current properties in intact murine skeletal muscle fibres

Abstract: We investigated effects of the ryanodine receptor (RyR) modulator caffeine on Na+ current (INa) activation and inactivation in intact loose-patch clamped murine skeletal muscle fibres subject to a double pulse procedure. INa activation was examined using 10-ms depolarising, V1, steps to varying voltages 0–80 mV positive to resting membrane potential. The dependence of the subsequent, INa inactivation on V1 was examined by superimposed, V2, steps to a fixed depolarising voltage. Current-voltage activation and inactivation curves indicated that adding 0.5 and 2 mM caffeine prior to establishing the patch seal respectively produced decreased (within 1 min) and increased (after ~2 min) peak INa followed by its recovery to pretreatment levels (after ~40 and ~30 min respectively). These changes accompanied negative shifts in the voltage dependence of INa inactivation (within 10 min) and subsequent superimposed positive activation and inactivation shifts, following 0.5 mM caffeine challenge. In contrast, 2 mM caffeine elicited delayed negative shifts in both activation and inactivation. These effects were abrogated if caffeine was added after establishing the patch seal or with RyR block by 10 μM dantrolene. These effects precisely paralleled previous reports of persistently (~10 min) increased cytosolic [Ca2+] with 0.5 mM, and an early peak rapidly succeeded by persistently reduced [Ca2+] likely reflecting gradual RyR inactivation with ≥1.0 mM caffeine. The latter findings suggested inhibitory effects of even resting cytosolic [Ca2+] on INa. They suggest potentially physiologically significant negative feedback regulation of RyR activity on Nav1.4 properties through increased or decreased local cytosolic [Ca2+], Ca2+-calmodulin and FKBP12

Atypical scrapie in sheep from a UK research flock which is free from classical scrapie

Background: In the wake of the epidemic of bovine spongiform encephalopathy the British government established a flock of sheep from which scrapie-free animals are supplied to laboratories for research. Three breeds of sheep carrying a variety of different genotypes associated with scrapie susceptibility/resistance were imported in 1998 and 2001 from New Zealand, a country regarded as free from scrapie. They are kept in a purpose-built Sheep Unit under strict disease security and are monitored clinically and post mortem for evidence of scrapie. It is emphasised that atypical scrapie, as distinct from classical scrapie, has been recognised only relatively recently and differs from classical scrapie in its clinical, neuropathological and biochemical features. Most cases are detected in apparently healthy sheep by post mortem examination.Results: The occurrence of atypical scrapie in three sheep in (or derived from) the Sheep Unit is reported. Significant features of the affected sheep included their relatively high ages (6 y 1 mo, 7 y 9 mo, 9 y 7 mo respectively), their breed (all Cheviots) and their similar PRNP genotypes (AFRQ/AFRQ, AFRQ/ALRQ, and AFRQ/AFRQ, respectively). Two of the three sheep showed no clinical signs prior to death but all were confirmed as having atypical scrapie by immunohistochemistry and Western immunoblotting. Results of epidemiological investigations are presented and possible aetiologies of the cases are discussed.Conclusion: By process of exclusion, a likely explanation for the three cases of atypical scrapie is that they arose spontaneously and were not infected from an exterior source. If correct, this raises challenging issues for countries which are currently regarded as free from scrapie. It would mean that atypical scrapie is liable to occur in flocks worldwide, especially in older sheep of susceptible genotypes. To state confidently that both the classical and atypical forms of scrapie are absent from a population it is necessary for active surveillance to have taken place

The evaluation of exposure risks for natural transmission of scrapie within an infected flock

Background: Although the epidemiology of scrapie has been broadly understood for many years, attempts to introduce voluntary or compulsory controls to eradicate the disease have frequently failed. Lack of precision in defining the risk factors on farm has been one of the challenges to designing control strategies. This study attempted to define which parts of the annual flock management cycle represented the greatest risk of infection to naive lambs exposed to the farm environment at different times.Results: In VRQ/VRQ lambs exposed to infected sheep at pasture or during lambing, and exposed to the buildings in which lambing took place, the attack rate was high and survival times were short. Where exposure was to pasture alone the number of sheep affected in each experimental group was reduced, and survival times were longer and related to length of exposure.Conclusion: At the flock level, eradication and control strategies for scrapie must take into account the need to decontaminate buildings used for lambing, and to reduce (or prevent) the exposure of lambs to infected sheep, especially in the later stages of incubation, and at lambing. The potential for environmental contamination from pasture should also be considered. Genotype selection may still prove to be the only viable tool to prevent infection from contaminated pasture, reduce environmental contamination and limit direct transmission from sheep to sheep