William Harvey Heart Center

London, United Kingdom

William Harvey Heart Center

London, United Kingdom

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Gomes J.,University College London | Finlay M.,University College London | Ahmed A.K.,University College London | Ciaccio E.J.,Columbia University | And 11 more authors.
European Heart Journal | Year: 2012

Aims Anecdotal observations suggest that sub-clinical electrophysiological manifestations of arrhythmogenic right ventricular cardiomyopathy (ARVC) develop before detectable structural changes ensue on cardiac imaging. To test this hypothesis, we investigated a murine model with conditional cardiac genetic deletion of one desmoplakin allele (DSP ±) and compared the findings to patients with non-diagnostic features of ARVC who carried mutations in desmoplakin. Methods and resultsMurine: the DSP (±) mice underwent electrophysiological, echocardiographic, and immunohistochemical studies. They had normal echocardiograms but delayed conduction and inducible ventricular tachycardia associated with mislocalization and reduced intercalated disc expression of Cx43. Sodium current density and myocardial histology were normal at 2 months of age. Human: ten patients with heterozygous mutations in DSP without overt structural heart disease (DSP) and 12 controls with supraventricular tachycardia were studied by high-density electrophysiological mapping of the right ventricle. Using a standard S1S2 protocol, restitution curves of local conduction and repolarization parameters were constructed. Significantly greater mean increases in delay were identified particularly in the outflow tract vs. controls (P< 0.01) coupled with more uniform wavefront progression. The odds of a segment with a maximal activationrepolarization interval restitution slope >1 was 99 higher (95 CI: 13; 351, P 0.017) in DSP vs. controls. Immunostaining revealed Cx43 mislocalization and variable Na channel distribution. ConclusionDesmoplakin disease causes connexin mislocalization in the mouse and man preceding any overt histological abnormalities resulting in significant alterations in conductionrepolarization kinetics prior to morphological changes detectable on conventional cardiac imaging. Haploinsufficiency of desmoplakin is sufficient to cause significant Cx43 mislocalization. Changes in sodium current density and histological abnormalities may contribute to a worsening phenotype or disease but are not necessary to generate an arrhythmogenic substrate. This has important implications for the earlier diagnosis of ARVC and risk stratification. © The Author 2011.


Sciarretta S.,IRCCS Neuromed | Marchitti S.,IRCCS Neuromed | Bianchi F.,IRCCS Neuromed | Moyes A.,William Harvey Heart Center | And 13 more authors.
Circulation Research | Year: 2013

Rationale: C2238 atrial natriuretic peptide (ANP) minor allele (substitution of thymidine with cytosine in position 2238) associates with increased risk of cardiovascular events. Objective: We investigated the mechanisms underlying the vascular effects of C2238-αANP. Methods and Results: In vitro, human umbilical vein endothelial cell were exposed to either wild-type (T2238)- or mutant (C2238)-αANP. Cell survival and apoptosis were tested by Trypan blue, annexin V, and cleaved caspase-3 assays. C2238-αANP significantly reduced human umbilical vein endothelial cell survival and increased apoptosis. In addition, C2238-αANP reduced endothelial tube formation, as assessed by matrigel. C2238-αANP did not differentially modulate natriuretic peptide receptor (NPR)-A/B activity with respect to T2238-αANP, as evaluated by intracellular cGMP levels. In contrast, C2238-αANP, but not T2238-αANP, markedly reduced intracellular cAMP levels in an NPR-C-dependent manner. Accordingly, C2238-αANP showed higher affinity binding to NPR-C, than T2238-αANP. Either NPR-C inhibition by antisense oligonucleotide or NPR-C gene silencing by small interfering RNA rescued survival and tube formation of human umbilical vein endothelial cell exposed to C2238-αANP. Similar data were obtained in human aortic endothelial cell with NPR-C knockdown. NPR-C activation by C2238-αANP inhibited the protein kinase A/Akt1 pathway and increased reactive oxygen species. Adenovirusmediated Akt1 reactivation rescued the detrimental effects of C2238-αANP. Overall, these data indicate that C2238-αANP affects endothelial cell integrity through NPR-C-dependent inhibition of the cAMP/protein kinase A/Akt1 pathway and increased reactive oxygen species production. Accordingly, C2238-αANP caused impairment of acetylcholine-dependent vasorelaxation ex vivo, which was rescued by NPR-C pharmacological inhibition. Finally, subjects carrying C2238 minor allele showed early endothelial dysfunction, which highlights the clinical relevance of our results. Conclusions: C2238-αANP reduces endothelial cell survival and impairs endothelial function through NPR-C signaling. NPR-C targeting represents a potential strategy to reduce cardiovascular risk in C2238 minor-allele carriers. © 2013 American Heart Association, Inc.


Machhada A.,University College London | Machhada A.,Biomedical Imaging Center | Ang R.,University College London | Ang R.,William Harvey Heart Center | And 8 more authors.
Heart Rhythm | Year: 2015

Background The central nervous origins of functional parasympathetic innervation of cardiac ventricles remain controversial. Objective This study aimed to identify a population of vagal preganglionic neurons that contribute to the control of ventricular excitability. An animal model of synuclein pathology relevant to Parkinson's disease was used to determine whether age-related loss of the activity of the identified group of neurons is associated with changes in ventricular electrophysiology. Methods In vivo cardiac electrophysiology was performed in anesthetized rats in conditions of selective inhibition of the dorsal vagal motor nucleus (DVMN) neurons by pharmacogenetic approach and in mice with global genetic deletion of all family members of the synuclein protein. Results In rats anesthetized with urethane (in conditions of systemic beta-adrenoceptor blockade), muscarinic and neuronal nitric oxide synthase blockade confirmed the existence of a tonic parasympathetic control of cardiac excitability mediated by the actions of acetylcholine and nitric oxide. Acute DVMN silencing led to shortening of the ventricular effective refractory period (vERP), a lowering of the threshold for triggered ventricular tachycardia, and prolongation of the corrected QT (QTc) interval. Lower resting activity of the DVMN neurons in aging synuclein-deficient mice was found to be associated with vERP shortening and QTc interval prolongation. Conclusion Activity of the DVMN vagal preganglionic neurons is responsible for tonic parasympathetic control of ventricular excitability, likely to be mediated by nitric oxide. These findings provide the first insight into the central nervous substrate that underlies functional parasympathetic innervation of the ventricles and highlight its vulnerability in neurodegenerative diseases. © 2015 Heart Rhythm Society.


Telezhkin V.,University College London | Telezhkin V.,University of Cardiff | Thomas A.M.,William Harvey Heart Center | Harmer S.C.,William Harvey Heart Center | And 2 more authors.
Pflugers Archiv European Journal of Physiology | Year: 2013

All Kv7 potassium channels require membrane phosphatidylinositol-4,5- bisphosphate (PI(4,5)P2) for their normal function and hence can be physiologically regulated by neurotransmitters and hormones that stimulate phosphoinositide hydrolysis. Recent mutational analysis indicates that a cluster of basic residues in the proximal C-terminus (K354/K358/R360/K362) is crucial for PI(4,5)P2 activation of cardiac Kv7.1 channels. Since this cluster is largely conserved in all Kv7 subunits, we tested whether homologous residues are also required for activation of Kv7.2 (a subunit of neuronal M-channels). We found that the mutation Kv7.2 (R325A) (corresponding to R360 in Kv7.1) reduced Kv7.2 current amplitude by ∼60 % (P < 0.02) without change in voltage sensitivity and reduced the sensitivity of Kv7.2 channels to dioctanoyl-phosphatidylinositol-4,5-bisphosphate by ∼eightfold (P < 0.001). Taking into account previous experiments (Zhang et al., Neuron 37:963-75, 2003) implicating Kv7.2 (H328), and since R325 and H328 are conserved in homologous positions in all other Kv7 channels, we suggest that this proximal C-terminal domain adjacent to the last transmembrane domain that contains R325 and H328 (in Kv7.2) might play a major role in the activation of all members of the Kv7 channel family by PI(4,5)P2. © 2013 The Author(s).


Maden C.H.,University College London | Gomes J.,University College London | Schwarz Q.,University College London | Davidson K.,University College London | And 3 more authors.
Developmental Biology | Year: 2012

The sympathetic nervous system (SNS) arises from neural crest (NC) cells during embryonic development and innervates the internal organs of vertebrates to modulate their stress response. NRP1 and NRP2 are receptors for guidance cues of the class 3 semaphorin (SEMA) family and are expressed in partially overlapping patterns in sympathetic NC cells and their progeny. By comparing the phenotypes of mice lacking NRP1 or its ligand SEMA3A with mice lacking NRP1 in the sympathetic versus vascular endothelial cell lineages, we demonstrate that SEMA3A signalling through NRP1 has multiple cell-autonomous roles in SNS development. These roles include neuronal cell body positioning, neuronal aggregation and axon guidance, first during sympathetic chain assembly and then to regulate the innervation of the heart and aorta. Loss of NRP2 or its ligand SEMA3F impaired sympathetic gangliogenesis more mildly than loss of SEMA3A/NRP1 signalling, but caused ectopic neurite extension along the embryonic aorta. The analysis of compound mutants lacking SEMA3A and SEMA3F or NRP1 and NRP2 in the SNS demonstrated that both signalling pathways cooperate to organise the SNS. We further show that abnormal sympathetic development in mice lacking NRP1 in the sympathetic lineage has functional consequences, as it causes sinus bradycardia, similar to mice lacking SEMA3A. © 2012 Elsevier Inc.


Telezhkin V.,University College London | Reilly J.M.,University College London | Thomass A.M.,William Harvey Heart Center | Tinkers A.,William Harvey Heart Center | Brown D.A.,University College London
Journal of Biological Chemistry | Year: 2012

M-channels are voltage-gated potassium channels that regulate cell excitability. They are heterotetrameric assemblies of Kv7.2 and Kv7.3 subunits. Their opening requires the presence of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2). However, the specificity of PI(4,5)P 2 as a binding and activating ligand is unknown. Here, we tested the ability of different phosphoinositides and lipid phosphates to activate or bind to M-channel proteins. Activation of functional channels was measured in membrane patches isolated from cells coexpressing Kv7.2 and Kv7.3 subunits. Channels were activated to similar extents (maximum open probability of ∼0.8 at 0 mV) by 0.1-300 μM dioctanoyl homologs of the three endogenous phosphoinositides, PI(4)P, PI(4,5)P 2, and PI(3,4,5)P 3, with sensitivity increasing with increasing numbers of phosphates. Non-acylated inositol phosphates had no effect up to 100 μM. Channels were also activated with increasing efficacy by 1-300 μM concentrations of the monoacyl monophosphates fingolimod phosphate, sphingosine 1-phosphate, and lysophosphatidic acid but not by phosphate-free fingolimod or sphingosine or by phosphate-masked phosphatidylcholine or phosphatidyl-glycerol. An overlay assay confirmed that a fusion protein containing the full-lengthCterminus of Kv7.2 could bind to a broad range of phosphoinositides and phospholipids.Amutated Kv7.2 C-terminal construct with reduced sensitivity to PI(4,5)P showed significantly less binding to most polyphosphoinositides. We concluded that M-channels bind to, and are activated by, a wide range of lipid phosphates, with a minimum requirement for an acyl chain and a phosphate headgroup. In this, they more closely resemble inwardly rectifying Kir6.2 potassium channels than the more PI(4,5)P 2-specific Kir2 channels. Not-withstanding, the data also support the view that the main endogenous activator of M-channels is PI(4,5)P 2. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.


Harmer S.C.,William Harvey Heart Center | Mohal J.S.,William Harvey Heart Center | Royal A.A.,William Harvey Heart Center | McKenna W.J.,University College London | And 2 more authors.
Biochemical Journal | Year: 2014

The KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1) gene encodes the Kv7.1 potassium channel which forms a complex with KCNE1 (potassium voltage-gated channel Isk-related family member 1) in the human heart to produce the repolarizing IKs (slow delayed rectifier potassium current). Mutations in KCNQ1 can perturb IKs function and cause LQT1 (long QT syndrome type 1). In LQT1, compound mutations are relatively common and are associated with increased disease severity. LQT1 compound mutations have been shown to increase channel dysfunction, but whether other disease mechanisms, such as defective channel trafficking, contribute to the increase in arrhythmic risk has not been determined. Using an imaging-based assay we investigated the effects of four compound heterozygous mutations (V310I/R594Q, A341V/P127T, T391I/Q530X and A525T/R518X), one homozygous mutation (W248F) and one novel compound heterozygous mutation (A178T/K422fs39X) (where fs denotes frameshift) on channel trafficking. By analysing the effects in the equivalent of a homozygous, heterozygous and compound heterozygous condition, we identify three different types of behaviour. A341V/P127T and W248F/W248F had no effect, whereas V310I/R594Q had a moderate, but not compound, effect on channel trafficking. In contrast, T391I/Q530X, A525T/R518X and A178T/K422fs39X severely disrupted channel trafficking when expressed in compound form. In conclusion, we have characterized the disease mechanisms for six LQT1 compound mutations and report that, for four of these, defective channel trafficking underlies the severe clinical phenotype. © The Authors Journal compilation © 2014 Biochemical Society.


Lambiase P.D.,University College London | Tinker A.,William Harvey Heart Center
Cell and Tissue Research | Year: 2015

Connexins are essential in the propagation of electrical activity throughout the heart and are an important determinant of conduction velocity. Their dysfunction is an important factor in the genesis of abnormal cardiac rhythm and is relevant to the pathogenesis of a wide variety of cardiac pathologies. Here, we review the basic biology of connexins in the heart but particularly focus on their abnormal function in cardiac disease. © 2014, Springer-Verlag Berlin Heidelberg.


Harmer S.C.,William Harvey Heart Center | Mohal J.S.,William Harvey Heart Center | Kemp D.,William Harvey Heart Center | Tinker A.,William Harvey Heart Center
Biochemical Journal | Year: 2012

The nonsense mutations R518X-KCNQ1 and Q530X-KCNQ1 cause LQT1 (long-QT syndrome type 1) and result in a complete loss of I Ks channel function. In the present study we attempted to rescue the function of these mutants, in HEK (human embryonic kidney)-293 cells, by promoting readthrough of their PTCs (premature termination codons) using the pharmacological agents G-418, gentamicin and PTC124. Gentamicin and G-418 acted to promote full-length channel protein expression from R518X at 100 μM and from Q530X at 1 mM. In contrast, PTC124 did not, at any dose tested, induce readthrough of either mutant. G-418 (1 mM) treatment also acted to significantly (P<0.05) increase current density and peak-tail current density, at +80 mV for R518X, but not Q530X, to 58±11% and 82±17% of the wild-type level respectively. However, the biophysical properties of the currents produced from R518X, while similar, were not identical with wild-type as the voltage-dependence of activation was significantly (P < 0.05) shifted by +25 mV. Overall, these findings indicate that although functional rescue of LQT1 nonsense mutations is possible, it is dependent on the degree of readthrough achieved and the effect on channel function of the amino acid substituted for the PTC. Such considerations will determine the success of future therapies. ©The Authors Journal compilation © 2012 Biochemical Society.


Aziz Q.,William Harvey Heart Center | Thomas A.M.,William Harvey Heart Center | Khambra T.,William Harvey Heart Center | Tinker A.,William Harvey Heart Center
Journal of Biological Chemistry | Year: 2012

The activity of ATP-sensitive potassium (K ATP) channels is governed by the concentration of intracellular ATP and ADP and is thus responsive to the metabolic status of the cell. Phosphorylation of K ATP channels by protein kinase A (PKA) or protein kinase C (PKC) results in the modulation of channel activity and is particularly important in regulating smooth muscle tone. At the molecular level the smooth muscle channel is composed of a sulfonylurea subunit (SUR2B) and a pore-forming subunit Kir6.1 and/or Kir6.2. Previously, Kir6.1/SUR2B channels have been shown to be inhibited by PKC, and Kir6.2/SUR2B channels have been shown to be activated or have no response to PKC. In this study we have examined the modulation of channel complexes formed of the inward rectifier subunit, Kir6.2, and the sulfonylurea subunit, SUR2B. Using a combination of biochemical and electrophysiological techniques we show that this complex can be inhibited by protein kinase C in a Ca 2+-dependent manner and that this inhibition is likely to be as a result of internalization. We identify a residue in the distal C terminus of Kir6.2 (Ser-372) whose phosphorylation leads to down-regulation of the channel complex. This inhibitory effect is distinct from activation which is seen with low levels of channel activity. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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