From channels to systems: Ca(2+) -sensitive K(+) currents, alternans and cardiac arrhythmia.

C.L.-H.’s work is funded by the Medical Research Council (MR/M001288/1), Wellcome Trust (105727/Z/14/Z), British Heart Foundation (PG/08/116), the McVeigh Benefaction and SADS UK

Murine Electrophysiological Models of Cardiac Arrhythmogenesis

Cardiac arrhythmias can follow disruption of the normal cellular electrophysiological processes underlying excitable activity and their tissue propagation as coherent wavefronts from the primary sinoatrial node pacemaker, through the atria, conducting structures and ventricular myocardium. These physiological events are driven by interacting, voltage-dependent, processes of activation, inactivation, and recovery in the ion channels present in cardiomyocyte membranes. Generation and conduction of these events are further modulated by intracellular Ca$^{2+}$ homeostasis, and metabolic and structural change. This review describes experimental studies on murine models for known clinical arrhythmic conditions in which these mechanisms were modified by genetic, physiological, or pharmacological manipulation. These exemplars yielded molecular, physiological, and structural phenotypes often directly translatable to their corresponding clinical conditions, which could be investigated at the molecular, cellular, tissue, organ, and whole animal levels. Arrhythmogenesis could be explored during normal pacing activity, regular stimulation, following imposed extra-stimuli, or during progressively incremented steady pacing frequencies. Arrhythmic substrate was identified with temporal and spatial functional heterogeneities predisposing to reentrant excitation phenomena. These could arise from abnormalities in cardiac pacing function, tissue electrical connectivity, and cellular excitation and recovery. Triggering events during or following recovery from action potential excitation could thereby lead to sustained arrhythmia. These surface membrane processes were modified by alterations in cellular Ca$^{2+}$ homeostasis and energetics, as well as cellular and tissue structural change. Study of murine systems thus offers major insights into both our understanding of normal cardiac activity and its propagation, and their relationship to mechanisms generating clinical arrhythmias.Medical Research Council, Wellcome Trust, McVeigh Benefaction, British Heart Foundation, Helen Kirkland Trust, Sudden Arrhythmic Death Syndrome - SADS U

Effects of ageing on pro-arrhythmic ventricular phenotypes in incrementally paced murine Pgc1β-/- hearts

A range of chronic clinical conditions accompany cardiomyocyte energetic dysfunction and constitute independent risk factors for cardiac arrhythmia. We investigated pro-arrhythmic and arrhythmic phenotypes in energetically deficient C57BL mice with genetic ablation of the mitochondrial promoter peroxisome proliferator activated receptor-γ coactivator-1β (Pgc-1β), a known model of ventricular arrhythmia. Pro-arrhythmic and cellular action potential (AP) characteristics were compared in intact Langendorff-perfused hearts from young (12-16 week) and aged (>52 week), WT and Pgc-1β-/- mice. Simultaneous electrocardiographic and intracellular microelectrode recordings were made through successive trains of 100 regular stimuli at progressively incremented heart rates. Aged Pgc-1β-/- hearts displayed an increased incidence of arrhythmia compared to other groups. Young and aged Pgc-1β-/- hearts showed higher incidences of alternans in both AP activation (maximum AP upshoot velocity (dV/dt)max and latency), recovery (action potential duration (APD90) and resting membrane potential (RMP)) characteristics compared to WT hearts. This was particularly apparent at lower pacing frequencies. These findings accompanied reduced (dV/dt)max and increased AP latency values in the Pgc-1β-/- hearts. APs observed prior to termination of the protocol showed lower (dV/dt)max and longer AP latencies, but indistinguishable APD90 and RMPs in arrhythmic compared to non-arrhythmic hearts. APD restitution analysis showed that Pgc-1β-/- and WT hearts showed similar limiting gradients. However, Pgc-1β-/- hearts had shortened plateau AP wavelengths, particularly in aged Pgc-1β-/- hearts. Pgc-1β-/- hearts therefore show pro-arrhythmic instabilities attributable to altered AP conduction and activation rather than recovery characteristics.We acknowledge the financial support from the Medical Research Council (MR/M001288/1); the Wellcome Trust (105727/Z/14/Z); the British Heart Foundation (PG/14/79/31102 and PG/15/12/31280), Sudden arrhythmic death syndrome (SADS) UK; The McVeigh Benefaction and by the Fundamental Research Grant Scheme (FRGS/2/2014/SKK01/PERDANA/02/1), Ministry of Education, Malaysia

Ion channels, long QT syndrome and arrhythmogenesis in ageing.

Ageing is associated with increased prevalences of both atrial and ventricular arrhythmias, reflecting disruption of the normal sequence of ion channel activation and inactivation generating the propagated cardiac action potential. Experimental models with specific ion channel genetic modifications have helped clarify the interacting functional roles of ion channels and how their dysregulation contributes to arrhythmogenic processes at the cellular and systems level. They have also investigated interactions between these ion channel abnormalities and age-related processes in producing arrhythmic tendency. Previous reviews have explored the relationships between age and loss-of-function Nav 1.5 mutations in producing arrhythmogenicity. The present review now explores complementary relationships arising from gain-of-function Nav 1.5 mutations associated with long QT3 (LQTS3). LQTS3 patients show increased risks of life-threatening ventricular arrhythmias particularly after 40 years of age consistent with such interactions between the ion channel abnormailities and ageing. In turn clinical evidence suggests that ageing is accompanied by structural, particularly fibrotic, as well as electrophysiological change. These abnormalities may result from biochemical changes producing low-grade inflammation resulting from increased production of reactive oxygen species and superoxide. Experimental studies offer further insights into the underlying mechanisms underlying these phenotypes. Thus, studies in genetically modified murine models for LQTS implicated action potential recovery processes in arrhythmogenesis resulting from functional ion channel abnormalities. In addition, ageing WT murine models demonstrated both ion channel alterations and fibrotic changes with ageing. Murine models then suggested evidence for interactions between ageing and ion channel mutations and provided insights into potential arrhythmic mechanisms inviting future exploration.KJ is funded by the Fundamental Research Grant Scheme (FRGS/2/2014/SKK01/PERDANA/02/1), Ministry of Education, Malaysia and the Research Support Fund, Faculty of Health and Medical Science, University of Surrey. KC was funded by the Physiological Society, United Kingdom. HV is funded by the Wellcome Trust Research Training Fellowship (105727/Z/14/Z) and Sudden Arrhythmic Death Syndrome (SADS), UK. SA is funded by a Medical Research Council Research Fellowship (MR/M001288/1). AG is funded by the McVeigh Benefaction and Sudden Arrhythmic Death Syndrome (SADS), UK. CLHH is funded by the Wellcome Trust, Medical Research Council, British Heart Foundation and McVeigh Benefaction

Flecainide exerts paradoxical effects on sodium currents and atrial arrhythmia in murine RyR2-P2328S hearts.

AIMS: Cardiac ryanodine receptor mutations are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), and some, including RyR2-P2328S, also predispose to atrial fibrillation. Recent work associates reduced atrial Nav 1.5 currents in homozygous RyR2-P2328S (RyR2(S/S) ) mice with slowed conduction and increased arrhythmogenicity. Yet clinically, and in murine models, the Nav 1.5 blocker flecainide reduces ventricular arrhythmogenicity in CPVT. We aimed to determine whether, and how, flecainide influences atrial arrhythmogenicity in RyR2(S/S) mice and their wild-type (WT) littermates. METHODS: We explored effects of 1 μm flecainide on WT and RyR2(S/S) atria. Arrhythmic incidence, action potential (AP) conduction velocity (CV), atrial effective refractory period (AERP) and AP wavelength (λ = CV × AERP) were measured using multi-electrode array recordings in Langendorff-perfused hearts; Na(+) currents (INa ) were recorded using loose patch clamping of superfused atria. RESULTS: RyR2(S/S) showed more frequent atrial arrhythmias, slower CV, reduced INa and unchanged AERP compared to WT. Flecainide was anti-arrhythmic in RyR2(S/S) but pro-arrhythmic in WT. It increased INa in RyR2(S/S) atria, whereas it reduced INa as expected in WT. It increased AERP while sparing CV in RyR2(S/S) , but reduced CV while sparing AERP in WT. Thus, RyR2(S/S) hearts have low λ relative to WT; flecainide then increases λ in RyR2(S/S) but decreases λ in WT. CONCLUSIONS: Flecainide (1 μm) rescues the RyR2-P2328S atrial arrhythmogenic phenotype by restoring compromised INa and λ, changes recently attributed to increased sarcoplasmic reticular Ca(2+) release. This contrasts with the increased arrhythmic incidence and reduced INa and λ with flecainide in WT.This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC, UK) under a David Phillips Fellowship held by JAF (BB/FO23863/1) and by the Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge Joint Research Grants Scheme.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/apha.1250

Sodium channel biophysics, late sodium current and genetic arrhythmic syndromes

Arrhythmias arise from breakdown of orderly action potential (AP) activation, propagation and recovery driven by interactive opening and closing of successive voltage-gated ion channels, in which one or more Na$^{+}$ current components play critical parts. Early peak, Na$^{+}$ currents (I$_{Na}$) reflecting channel activation drive the AP upstroke central to cellular activation and its propagation. Sustained late Na$^{+}$ currents (I$_{Na-L}$) include contributions from a component with a delayed inactivation timecourse influencing AP duration (APD) and refractoriness, potentially causing pro-arrhythmic phenotypes. The magnitude of I$_{Na-L}$ can be analysed through overlaps or otherwise in the overall voltage dependences of the steady-state properties and kinetics of activation and inactivation of the Na$^{+}$ conductance. This was useful in analysing repetitive firing associated with paramyotonia congenita in skeletal muscle. Similarly, genetic cardiac Na$^{+}$ channel abnormalities increasing I$_{Na-L}$ are implicated in triggering phenomena of automaticity, early and delayed afterdepolarisations and arrhythmic substrate. This review illustrates a wide range of situations that may accentuate I$_{Na-L}$. These include (1) overlaps between steady-state activation and inactivation increasing $\textit{window current}$, (2) kinetic deficiencies in Na$^{+}$ channel inactivation leading to $\textit{bursting phenomena}$ associated with repetitive channel openings and (3) $\textit{non-equilibrium gating}$ processes causing channel re-opening due to more rapid recoveries from inactivation. All these biophysical possibilities were identified in a selection of abnormal human SCN5A genotypes. The latter presented as a broad range of clinical arrhythmic phenotypes, for which effective therapeutic intervention would require specific identification and targeting of the diverse electrophysiological abnormalities underlying their increased I$_{Na-L}$.KC was funded by the Physiological Society, United Kingdom at the University of Surrey. KJ is funded by the Fundamental Research Grant Scheme (FRGS/2/2014/SKK01/PERDANA/02/1), Ministry of Education, Malaysia, and the Research Support Fund, Faculty of Health and Medical Science, University of Surrey. ML is funded by the British Heart Foundation (PG/14/80/31106, PG/16/67/32340) and Medical Research Council (G10002647). CLHH is funded by the Medical Research Council (MR/M001288/1), Wellcome Trust (105727/Z/14/Z), British Heart Foundation (PG/14/79/31102), the McVeigh Benefaction and SADS UK

Arrhythmic substrate, slowed propagation and increased dispersion in conduction direction in the right ventricular outflow tract of murine Scn5a+/- hearts.

AIM: To test a hypothesis attributing arrhythmia in Brugada Syndrome to right ventricular (RV) outflow tract (RVOT) conduction abnormalities arising from Nav 1.5 insufficiency and fibrotic change. METHODS: Arrhythmic properties of Langendorff-perfused Scn5a+/- and wild-type mouse hearts were correlated with ventricular effective refractory periods (VERPs), multi-electrode array (MEA) measurements of action potential (AP) conduction velocities and dispersions in conduction direction (CD), Nav 1.5 expression levels, and fibrotic change, as measured at the RVOT and RV. Two-way anova was used to test for both independent and interacting effects of anatomical region and genotype on these parameters. RESULTS: Scn5a+/- hearts showed greater arrhythmic frequencies during programmed electrical stimulation at the RVOT but not the RV. The Scn5a+/- genotype caused an independent increase of VERP regardless of whether the recording site was the RVOT or RV. Effective AP conduction velocities (CV†s), derived from fitting regression planes to arrays of observed local activation times were reduced in Scn5a+/- hearts and at the RVOT independently. AP conduction velocity magnitudes derived by averaging MEA results from local vector analyses, CV*, were reduced by the Scn5a+/- genotype alone. In contrast, dispersions in conduction direction, were greater in the RVOT than the RV, when the atrioventricular node was used as the pacing site. The observed reductions in Nav 1.5 expression were attributable to Scn5a+/-, whereas increased levels of fibrosis were associated with the RVOT. CONCLUSIONS: The Scn5a+/- RVOT recapitulates clinical findings of increased arrhythmogenicity through reduced CV† reflecting reduced CV* attributable to reduced Nav 1.5 expression and increased CD attributable to fibrosis

W::Neo: A Novel Dual-Selection Marker for High Efficiency Gene Targeting in Drosophila

We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR)-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a “founder” knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W) and neomycin resistance (Neo). In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila

Synthesis of titanate nanostructures using amorphous precursor material and their adsorption/photocatalytic properties

This paper reports on a new and swift hydrothermal chemical route to prepare titanate nanostructures (TNS) avoiding the use of crystalline TiO2 as starting material. The synthesis approach uses a commercial solution of TiCl3 as titanium source to prepare an amorphous precursor, circumventing the use of hazardous chemical compounds. The influence of the reaction temperature and dwell autoclave time on the structure and morphology of the synthesised materials was studied. Homogeneous titanate nanotubes with a high length/diameter aspect ratio were synthesised at 160^{\circ}C and 24 h. A band gap of 3.06\pm0.03 eV was determined for the TNS samples prepared in these experimental conditions. This value is red shifted by 0.14 eV compared to the band gap value usually reported for the TiO2 anatase. Moreover, such samples show better adsorption capacity and photocatalytic performance on the dye rhodamine 6G (R6G) photodegradation process than TiO2 nanoparticles. A 98% reduction of the R6G concentration was achieved after 45 minutes of irradiation of a 10 ppm dye aqueous solution and 1 g/L of TNS catalyst.Comment: 29 pages, 10 figures, accepted for publication in Journal of Materials Scienc