It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper we show that non-cytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A and KB cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm 2 in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1 -100 ms, while membrane resealing may occur over tens of seconds. Patchclamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for AMO-3, a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making ~3 nm holes in living cell membranes.

Background Early repolarization pattern in the ECG has been associated with increased risk for ventricular tachycardia/fibrillation (VT/VF), particularly when manifest in inferior leads. This study examines the mechanisms underlying VT/VF in the early repolarization syndrome (ERS). Method Transmembrane action potentials (AP) were simultaneously recorded from 2 epicardial and 1 endocardial site of coronary-perfused canine left-ventricular (LV) wedge preparations, together with a pseudo-ECG. Transient outward current (Ito) was recorded from epicardial myocytes isolated from inferior and lateral LV of the same heart. Results J wave area (pseudo-ECG), epicardial AP notch magnitude and index were larger in inferior vs. lateral wall preparations at baseline and after exposure to provocative agents (NS5806+verapamil+acetylcholine (ACh)). Ito density was greater in myocytes from inferior vs. lateral wall (18.4±2.3pA/pF vs. 11.6±2.0pA/pF;p<0.05). A combination of NS5806 (7μM) and verapamil (3μM) or pinacidil (4μM), used to pharmacologically model the genetic defects responsible for ERS, resulted in prominent J-point and ST-segment elevation. ACh (3μM), simulating increased vagal tone, precipitated phase-2-reentry-induced polymorphic VT/VF. Using identical protocols, inducibility of arrhythmias was 3-fold higher in inferior vs. lateral wedges. Quinidine (10μM) or isoproterenol (1μM) restored homogeneity and suppressed VT/VF. Conclusion Our data support the hypothesis that 1) ERS is caused by a preferential accentuation of the AP notch in LV epicardium; 2) this repolarization defect is accentuated by elevated vagal tone; 3) higher intrinsic levels of Ito account for the greater sensitivity of the inferior LV wall to development of VT/VF; 4) quinidine and isoproterenol exert ameliorative effects by reversing the repolarization abnormality.

Background Chronic iron-overload (CIO) is associated with blood disorders such as thalassemias and hemochromatosis. A major prognostic indicator of survival in patients with CIO is iron-mediated cardiomyopathy, characterized by contractile dysfunction and electrical disturbances including slow heart rate (HR; bradycardia) and heart block. Methods and Results We have used a mouse model of CIO to investigate the effects of iron on sinoatrial node (SAN) function. As in humans, CIO reduced HR (~20%) in conscious mice as well as in anesthetized mice with autonomic nervous system blockade and in isolated Langendorff-perfused mouse hearts, suggesting bradycardia originates from altered intrinsic SAN pacemaker function. Indeed, spontaneous action potential frequencies in SAN myocytes with CIO were reduced in association with decreased L-type Ca2+ current (ICa,L) densities and positive (rightward) voltage shifts in ICa,L activation. Pacemaker current (If) current was not affected by CIO. Since ICa,L in SAN myocytes (as well as in atrial and conducting system myocytes) activates at relatively negative potentials due to the presence of CaV1.3 channels (in addition to CaV1.2 channels), our data suggest that elevated iron preferentially suppresses CaV1.3 channel function. Consistent with this suggestion, CIO reduced CaV1.3 mRNA levels by ~40% in atrial tissue (containing SAN) and did not lower HR in CaV1.3 knockout mice. CIO also induced PR interval prolongation, heart block, and atrial fibrillation, conditions also seen in CaV1.3 knockout mice. Conclusion Our results demonstrate that CIO selectively reduces CaV1.3-mediated ICa,L leading to bradycardia, slowing of electrical conduction and atrial fibrillation as seen in iron-overload patients.

Recent studies have shown that Kir2 channels display differential sensitivity to intracellular polyamines, and have raised a number of questions about several properties of inward rectification important to the understanding of their physiological roles. In this study, we have carried out a detailed characterization of steady-state and kinetic properties of block of Kir2.1-3 channels by spermine. High-resolution recordings from outside-out patches showed that in all Kir2 channels current-voltage relationships display a 'crossover' effect upon change in extracellular K + . Experiments at different concentrations of spermine allowed for the characterization of two distinct shallow components of rectification, with the voltages for half-block negative (V .3 channels were not significantly different. The voltage dependence of spermine unblock was similar in all Kir2 channels, but the rates of unblock were ∼7-fold and ∼16-fold slower in Kir2.3 channels than those in Kir2.1 and Kir2.2 when measured at high and physiological extracellular K + , respectively. In all Kir2 channels, the instantaneous phase of activation was present. The instantaneous phase was difficult to resolve at high extracellular K + but it became evident and accounted for nearly 30-50% of the total current when recorded at physiological extracellular K + . In conclusion, the data are consistent with the universal mechanism of rectification in Kir2 channels, but also point to significant, and physiologically important, quantitative differences between Kir2 isoforms.

Background Wenxin Keli (WK), a Chinese herb extract, is reported to be effective in the treatment of atrial and ventricular cardiac arrhythmias. Recent studies suggest that WK inhibits the transient outward current (Ito). Objective The present study examines the effectiveness of WK, alone and in combination with quinidine, to suppress arrhythmogenesis in an experimental model of Brugada syndrome (BrS). Methods and Results Action potential and ECG recordings were obtained from epicardial and endocardial sites of coronary-perfused canine right ventricular wedge preparations. The Ito agonist NS5806 (10–15 μM) was used to pharmacologically mimic a genetic predisposition to BrS. The Ito agonist induced Phase 2 reentry (P2R) in 13/19 preparations and polymorphic ventricular tachycardia (pVT) in 11/19 wedge preparations. WK (10g/L) suppressed P2R and pVT in 100% (3/3) of preparations. A lower concentration of WK (5g/L) suppressed P2R in 60% (3/5) and pVT in 50% (2/4), but in combination with a low concentration of quinidine (5 μM) was 100% effective in suppressing P2R and pVT. Quinidine alone suppressed P2R and pVT in 60% (3/5) and 50% (2/4), respectively and in combination with WK (5g/L) suppressed P2R and pVT by 80% (4/5) and 75% (3/4), respectively. WK reduced Ito, ICa and contractility in single cardiomyocytes, but dose-dependently increased contractility in intact wedge preparations, an effect mimicked by tyramine. Conclusions Our data provide support for the hypothesis that Wenxin Keli, particularly in combination with quinidine, effectively suppresses arrhythmogenesis in an experimental model of BrS via inhibition of Ito and indirect adrenergic sympathomimetic effects.

Rationale: The fast transient outward K؉ current (I to,f ) plays a critical role in early repolarization of the heart. I to,f is consistently downregulated in cardiac disease. Despite its importance, the regulation of I to,f in disease remains poorly understood.Objective: Because the transcription factor nuclear factor (NF)-B is activated in cardiac hypertrophy and disease, we studied the role of NF-B in mediating I to,f reductions induced by hypertrophy. Methods and Results:

Previous studies have shown that cardiac inward rectifier potassium current ( IK1) channels are heteromers of distinct Kir2 subunits and suggested that species- and tissue-dependent expression of these subunits may underlie variability of IK1. In this study, we investigated the contribution of the slowly activating Kir2.3 subunit and free intracellular polyamines (PAs) to variability of IK1 in the mouse heart. The kinetics of activation was measured in Kir2 concatemeric tetramers with known subunit stoichiometry. Inclusion of only one Kir2.3 subunit to a Kir2.1 channel led to an approximate threefold slowing of activation kinetics, with greater slowing on subsequent additions of Kir2.3 subunits. Activation kinetics of IK1 in both ventricles and both atria was found to correspond to fast-activating Kir2.1/Kir2.2 channels, suggesting no major contribution of Kir2.3 subunits. In contrast, IK1 displayed significant variation in both the current density and inward rectification, suggesting involvement of intracellular PAs. The total levels of PAs were similar across the mouse heart. Measurements of the free intracellular PAs in isolated myocytes, using transgenically expressed Kir2.1 channels as PA sensors, revealed “microheterogeneity” of IK1 rectification as well as lower levels of free PAs in atrial myocytes compared with ventricular cells. These findings provide a quantitative explanation for the regional heterogeneity of IK1.

Obstructive sleep apnoea (OSA) affects 9–24% of the adult population. OSA is associated with atrial disease, including atrial enlargement, fibrosis and arrhythmias. Despite the link between OSA and cardiac disease, the molecular changes in the heart which occur with OSA remain elusive. To study OSA‐induced cardiac changes, we utilized a recently developed rat model which closely recapitulates the characteristics of OSA. Male Sprague Dawley rats, aged 50–70 days, received surgically implanted tracheal balloons which were inflated to cause transient airway obstructions. Rats were given 60 apnoeas per hour of either 13 sec. (moderate apnoea) or 23 sec. (severe apnoea), 8 hrs per day for 2 weeks. Controls received implants, but no inflations were made. Pulse oximetry measurements were taken at regular intervals, and post‐apnoea ECGs were recorded. Rats had longer P wave durations and increased T wave amplitudes following chronic OSA. Proteomic analysis of the atrial tissue homogenates revealed that three of the nine enzymes in glycolysis, and two proteins related to oxidative phosphorylation, were down regulated in the severe apnoea group. Several sarcomeric and pro‐hypertrophic proteins were also up regulated with OSA. Chronic OSA causes proteins changes in the atria which suggest impairment of energy metabolism and enhancement of hypertrophy.