The electrophysiological effects of Endothelin-1 in human atrial myocytes

Introduction: Chronic heart failure (CHF) is associated with an increased incidence of atrial fibrillation (AF) and elevated levels of catecholamines and endothelin-1 (ET-1), each of which affects the atrial L-type calcium current (ICaL) and consequently action potentials. Hypotheses: ET-1 modulates the effects of isoproterenol (ISO) on ICaL and action potentials in human atrial myocytes. Methods: Atrial myocytes were isolated enzymatically from samples of right atrial appendage obtained from consenting patients in sinus rhythm undergoing cardiac surgery. The nystatin-perforated whole cell patch clamp technique was used at 37ºC to record ICaL and action potentials in voltage-clamp and current-clamp mode respectively. Results: The current-voltage relationship of ICaL was bell-shaped, peaking at +10 mV with a current density of -4.8±0.4 pA/pF (mean± s.e.m., n=89 cells, 34 patients). ISO, 0.1 nM to 1 µM, increased peak ICaL in a concentration-dependent manner (n=4-46 cells) with a maximum response of 250± 53% above control and an approximate EC50 of 0.06 µM. Isoproterenol at 0.05 µM significantly increased peak ICaL from -4.7± 0.4 to -12.2± 0.9 pA/pF (P<0.05, Students t-test; n=64 cells). This adrenergic effect was reversed by ET-1 at all concentrations tested from 0.01 to 10 nM and was partially reversible upon ET-1 washout and in the presence of the specific ET-A receptor antagonist, FR139317 (n=5-12 cells). Neither ET-1 alone nor the ET-B receptor agonist Sarafotoxin S6c, at 10 nM, had an effect on ICaL. Isoproterenol (0.05 µM) prolonged the action potential duration at 50% repolarisation (APD50) from 30± 7 to 46± 7 ms (P< 0.05, n=15 cells), but had no effect on APD90 nor the cellular ERP. These adrenergic effects on APD50 and SDs were also abolished by ET-1 at 10 nM (P< 0.05, n=15 cells). Superfusion with ET-1 (10 nM) alone had no significant effect on APD50, APD90, nor ERP (n=21 cells). There were no significant interactions between these electrophysiological effects and diseases states or chronic pre-operative drug therapy. Spontaneous activity, defined as a depolarisation occurring during phase 3 of action potential repolarisation or a depolarisation of greater than 3 mV amplitude during phase 4, frequently interrupted action potential recordings during, but not prior to, superfusion with ISO. Using a repetitive stimulation protocol, ISO at 0.05 µM produced spontaneous depolarisations in 5 of 7 cells studied (P< 0.05, chi-2 test). Endothelin-1 at 10 nM abolished these depolarisations in all 5 cells (P< 0.05). Superfusion with ET-1 (10 nM) alone was associated with spontaneous depolarisations in significantly fewer cells (P< 0.05, n=2 of 13 cells). In a retrospective univariate analysis, patient comorbidity and pre-operative drug therapy were not found to influence the electrophysiological effects observed. Conclusions: ET-1 reversed adrenergically induced increases in peak ICaL, APD50 and SDs in human atrial myocytes. This anti-adrenergic effect may be expected to influence the occurrence of AF in patients irrespective of comorbidity or pre-operative drug therapy

The Short QT Syndrome Proposed Diagnostic Criteria

ObjectivesWe aimed to develop diagnostic criteria for the short QT syndrome (SQTS) to facilitate clinical evaluation of suspected cases.BackgroundThe SQTS is a cardiac channelopathy associated with atrial fibrillation and sudden cardiac death. Ten years after its original description, a consensus regarding an appropriate QT interval cutoff and specific diagnostic criteria have yet to be established.MethodsThe MEDLINE database was searched for all reported cases of SQTS in the English language, and all relevant data were extracted. The distribution of QT intervals and electrocardiographic (ECG) features in affected cases were analyzed and compared to data derived from ECG analysis from general population studies.ResultsA total of 61 reported cases of SQTS were identified. Index events, including sudden cardiac death, aborted cardiac arrest, syncope, and/or atrial fibrillation occurred in 35 of 61 (57.4%) cases. The cohort was predominantly male (75.4%) and had a mean QTc value of 306.7 ms with values ranging from 248 to 381 ms in symptomatic cases. In reference to the ECG characteristics of the general population, and in consideration of clinical presentation, family history, and genetic findings, a highly sensitive diagnostic scoring system was developed.ConclusionsBased on a comprehensive review of 61 reported cases of the SQTS, formal diagnostic criteria have been proposed that will facilitate diagnostic evaluation in suspected cases of SQTS. Diagnostic criteria may lead to a greater recognition of this condition and provoke screening of at-risk family members

Post-operative atrial fibrillation is influenced by beta-blocker therapy but not by pre-operative atrial cellular electrophysiology

We investigated whether post-cardiac surgery (CS) new-onset atrial fibrillation (AF) is predicted by pre-CS atrial cellular electrophysiology, and whether the antiarrhythmic effect of beta-blocker therapy may involve pre-CS pharmacological remodeling. Atrial myocytes were obtained from consenting patients in sinus rhythm, just prior to CS. Action potentials and ion currents were recorded using whole-cell patch-clamp technique. Post-CS AF occurred in 53 of 212 patients (25%). Those with post-CS AF were older than those without (67 ± 2 vs 62 ± 1 years, P = 0.005). In cells from patients with post-CS AF, the action potential duration at 50% and 90% repolarization, maximum upstroke velocity, and effective refractory period (ERP) were 13 ± 4 ms, 217 ± 16 ms, 185 ± 10 V/s, and 216 ± 14 ms, respectively (n = 30 cells, 11 patients). Peak L-type Ca2+ current, transient outward and inward rectifier K+ currents, and the sustained outward current were −5.0 ± 0.5, 12.9 ± 2.4, −4.1 ± 0.4, and 9.7 ± 1.0 pA/pF, respectively (13-62 cells, 7-19 patients). None of these values were significantly different in cells from patients without post-CS AF (P > 0.05 for each, 60-279 cells, 29-86 patients), confirmed by multiple and logistic regression. In patients treated >7 days with a beta-blocker pre-CS, the incidence of post-CS AF was lower than in non-beta-blocked patients (13% vs 27%, P = 0.038). Pre-CS beta-blockade was associated with a prolonged pre-CS atrial cellular ERP (P = 0.001), by a similar degree (∼20%) in those with and without post-CS AF. Conclusion: Pre-CS human atrial cellular electrophysiology does not predict post-CS AF. Chronic beta-blocker therapy is associated with a reduced incidence of post-CS AF, unrelated to a pre-CS ERP-prolonging effect of this treatment

Cardiac Resynchronization Therapy in a Patient with Persistent Left Superior Vena Cava Draining into the Coronary Sinus and Absent Innominate Vein: A Case Report and Review of Literature

Introduction: Persistent left superior vena cava (PLSVC) is a rare congenital anomaly of the superior venous system that may be discovered at the time of cardiac implantable electronic device (CIED) implantation. Methods and Results: We present a subject who needed cardiac resynchronization therapy (CRT)-CIED implantation and was discovered to have PLSVC with absent innominate vein during the implant procedure. We were able to successfully implant a CRT-CIED using a right-sided approach via the right superior vena cava (SVC). We present a description of our implant technique and a brief review of the different aspects of CIED implantation in subjects with variants of PLSVC. Conclusions: Superior venous anomalies such as PLSVC can make CIED implantation technically challenging. However, with increasing operator experience, cardiac imaging and appropriate tools successful CIED implantation is possible in almost all cases

C2C12 myoblasts differentiate into myotubes expressing contractile apparatus and mitochondrial fission machinery.

<p>C2C12 myoblasts were cultured and induced to differentiate for 24–96 h at 37°C in an atmosphere of 10% CO₂. <b>Panel A:</b> C2C12 myoblasts differentiate into myotubes. Cells were plated onto coverslips and allowed to reach 70–80% confluence before inducing differentiation. Live images were captured at 405 nm (to visualize DNA, blue), 515 nm (to visualize oxidized hET, green) and 633 nm (to visualize mitochondira, red) on a laser-scanning confocal microscope. <b>Panel B:</b> Immunoblots confirming upregulated expression of mitochondrial fission protein DRP1 and contractile protein myosin heavy chain during differentiation in C2C12 myotubes. Cells were harvested, and 50 µg of protein (pooled from 5 separate experiments) were processed for gel electrophoresis. Primary antibodies used were against DRP1, MF-20, and HSP70. <b>Panel C:</b> Translocation of DRP1 to the mitochondrial membrane in C2C12 myotubes. Differentiated C2C12 myotubes were fixed, and incubated with primary antibodies against MF20, DRP1 and/or Tom20 and then corresponding Alexa Fluor secondary antibodies. Confocal microscopy images were captured as described before.</p

DRP1K38E expression and oxidative stress promote mitochondrial fusion, SR colocalisation and increased SR:mitochondrial contacts.

<p>C2C12 myoblasts were induced to differentiate for 48 h and then were infected with DRP1K38E or treated with BSO or NAC. No treatment or empty adenoviral vectors served as control. <b>Panel A:</b> Representative electron microscopy images (x15000) of C2C12 myotubes expressing DRP1K38E or having been incubated with BSO, NAC or both. Arrowheads mark mitochondria-ER association sites. <b>Panel B:</b> Objective mean quantitative data for mitochondrial 2D area on 2-D electron microscopy. <b>Panel C:</b> Objective mean quantitative data for the occurrence of visible MAM on 2-D electron microscopy. * Denotes <i>p<0.001 ANOVA and Fisher</i>'<i>s exact test</i>, n = 50 all groups.</p

Rapid stimulation promotes mitochondrial fusion, SR colocalisation and increased frequency of SR: mitochondrial contact sites.

<p>C2C12 myocytes were induced to differentiate for 72 h, and then were left unstimulated (control) or were subjected to EFS at a frequency of 0 Hz, 1 Hz or 5 Hz for 24 h. <b>Panel A:</b> Representative electron micrographs (x25000) of differentiated C2C12 myotubes following 0 Hz (control), 1 Hz or 5 Hz field stimulation. Arrowheads mark mitochondria-SR contact sites. <b>Panel B:</b> Objective mean quantitative data for (<b>i</b>) mitochondrial 2D area and (<b>ii</b>) the occurrence of visible MAM on 2-D electron microscopy. (<b>i</b>) Mitochondria 2D size is length multiplied by width. (<b>ii</b>) Frequency of mitochondria-SR contact sites is the ratio of the number of mitochondria-SR contacts to the number of mitochondria. <b>Panel C:</b> Confocal images of Tom20 and PDI distribution in electric field stimulated C2C12 myotubes. To illustrate colocalisation of mitochondria and ER markers, the corresponding line scans of Tom20 and PDI are drawn on the right. <i>#</i> Denotes <i>p<0.001 Fisher</i>'<i>s exact test and one-way ANOVA,</i> n = 50 all groups.</p

Oxidative stress promotes mitochondrial fusion and results in altered SR calcium handling.

<p><b>Panel A:</b> Confocal microscopy images of differentiated C2C12 myotubes having been previously treated with BSO or diamide, and loaded with TMRE. <b>Panel B:</b> Representative tracings of the fluorescence profile of myotubes cultured under oxidative stress and loaded with fluo-4 prior to the application of caffeine to trigger SR calcium release (BSO, Diamide v control). <b>Panel C:</b> Objective mean quantitative data for fluorescence resulting from caffeine induced calcium release (BSO, diamide v. control). <b>Panel D:</b> Objective mean quantitative data for fluorescence kinetics profile resulting from caffeine induced calcium release (BSO, diamide v. control). <b>Panel E:</b> Histogram displaying the mean quantitative data relating to the normalization of fluorescence following caffeine application (BSO, diamide v. control). <b>*</b> Denotes <i>p <0.05</i>–<i>0.001 ANOVA,</i> n = 40 per group.</p

The reducing agent NAC reverses the alterations in SR calcium release induced by ‘forced fusion’.

<p><b>Panel A:</b> Confocal microscopy images of differentiated C2C12 myotubes expressing DRP1K38E or treated with NAC, and loaded with TMRE. <b>Panel B:</b> Representative tracings of the fluorescence profile of myotubes loaded with fluo-4 prior to the application of caffeine to trigger SR calcium release (DRP1K38E, DRP1K38E+NAC v control). <b>Panel C:</b> Objective mean quantitative data for fluorescence resulting from caffeine induced calcium release (DRP1K38E, DRP1K38E+NAC v. control). <b>Panel D:</b> Objective mean quantitative data for fluorescence kinetics profile resulting from caffeine induced calcium release (DRP1K38E, DRP1K38E+NAC v. control). <b>Panel E:</b> Histogram displaying the mean quantitative data relating to the normalization of fluorescence following caffeine application (DRP1K38E, DRP1K38E+NAC v. control). <b>*</b> Denotes <i>p<0.001 ANOVA,</i> n = 40 per group.</p