Mass Spectrometry Based Comparative Proteomics Using One Dimensional and Two Dimensional SDS-PAGE of Rat Atria Induced with Obstructive Sleep Apnea.

Proteomics involves large-scale comprehensive study of specific proteomes which have been widely used in the field of biomarker discovery, drug development, disease diagnosis and therapy. Comprehensive proteomics involve two or more proteomics approaches that are confirmatory, complementary, and/or synergistic. Obstructive sleep apnea (OSA) is a sleep disorder which causes respiratory cessation (due to upper airway collapse). Here we describe a comprehensive MS based label-free quantitative proteomic analysis of the OSA induced rat atria homogenates and matched controls by using 1 dimensional SDS PAGE (1-D PAGE) and 2 dimensional SDS PAGE (2-D PAGE) separation of the proteins, enzymatic digestion and analysis by nanoliquid chromatography tandem-mass spectrometry (LC-MS/MS). The outcomes from the 1D-PAGE and 2D-PAGE studies not only identified dysregulated proteins due to OSA, but also confirmed and complemented each other.

Identification of dysregulation of atrial proteins in rats with chronic obstructive apnea using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

Obstructive sleep apnea (OSA) affects an estimated 20% of adults worldwide and has been associated with electrical and structural abnormalities of the atria, although the molecular mechanisms are not well understood. Here, we used two-dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. We found enzymes involved in the glycolysis, beta-oxidation, electron transport chain and Krebs cycle to be down-regulated. The data suggested that the dysregulated proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia. Our results are consistent with our previous 1D-PAGE and nanoLC-MS/MS study (Channaveerappa et al, J Cell Mol Med. 2017), where we found that some aerobic and anaerobic glycolytic and Krebs cycle enzymes were down-regulated, suggesting that apnea may be a result of paucity of oxygen and production of ATP and reducing equivalents (NADH). The 2D-PAGE study not only complements our current study, but also advances our understanding of the OSA. The complete mass spectrometry data are available via ProteomeXchange with identifier PXD011181.

Identification of an INa-dependent and Ito-mediated proarrhythmic mechanism in cardiomyocytes derived from pluripotent stem cells of a Brugada syndrome patient.

Brugada syndrome (BrS) is an inherited cardiac arrhythmia commonly associated with SCN5A mutations, yet its ionic mechanisms remain unclear due to a lack of cellular models. Here, we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a BrS patient (BrS1) to evaluate the roles of Na+ currents (INa) and transient outward K+ currents (Ito) in BrS induced action potential (AP) changes. To understand the role of these current changes in repolarization we employed dynamic clamp to "electronically express" IK1 and restore normal resting membrane potentials and allow normal recovery of the inactivating currents, INa, ICa and Ito. HiPSC-CMs were generated from BrS1 with a compound SCN5A mutation (p. A226V & p. R1629X) and a healthy sibling control (CON1). Genome edited hiPSC-CMs (BrS2) with a milder p. T1620M mutation and a commercial control (CON2) were also studied. CON1, CON2 and BrS2, had unaltered peak INa amplitudes, and normal APs whereas BrS1, with over 75% loss of INa, displayed a loss-of-INa basal AP morphology (at 1.0 Hz) manifested by a reduced maximum upstroke velocity (by ~80%, p < 0.001) and AP amplitude (p < 0.001), and an increased phase-1 repolarization pro-arrhythmic AP morphology (at 0.1 Hz) in ~25% of cells characterized by marked APD shortening (~65% shortening, p < 0.001). Moreover, Ito densities of BrS1 and CON1 were comparable and increased from 1.0 Hz to 0.1 Hz by ~ 100%. These data indicate that a repolarization deficit could be a mechanism underlying BrS.

Biophysical comparison of sodium currents in native cardiac myocytes and human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for safety pharmacology and to investigate genetic diseases affecting cardiac ion channels. It is unclear whether adult myocytes or hiPSC-CMs are the better platform for cardiac safety pharmacology. We examined the biophysical and molecular properties of INa in adult myocytes and hiPSC-CMs. METHODS: hiPSC-CMs were plated at low density. Atrial and ventricular cells were obtained from dog hearts. Whole cell patch clamp was used to record INa. RESULTS: Voltage clamp recordings showed a large INa in all three cell types but different densities. Small differences in steady-state inactivation and recovery from inactivation were noted in the three cell types. Application of lidocaine to the three cell types showed a similar pattern of block of INa under voltage clamp; however, lidocaine produced different effects on AP waveform under current clamp. AP clamp experiments showed that application of ventricular or atrial cell waveforms to the same hiPSC-CM elicited a large INa while application of a sinoatrial node waveform elicited no INa. Molecular analysis of Na+ channel subunits showed SCN5A and SCN1B-4B were expressed in adult cells and iPSC-CMs. However, iPSC-CMs express both fetal (exon 6A) and adult (exon 6) isoforms of SCN5A. DISCUSSION: There are major differences in INa density and smaller differences in other biophysical properties of INa in adult atrial, ventricular, and hiPSC-CMs. The depolarized maximum diastolic potential coupled with the presence of phase 4 depolarization limits the contribution of INa in hiPSC-CM action potentials. Our results suggest that hiPSC-CMs may be useful for drug screening of Na+ channel inhibitors under voltage clamp but not current clamp.

Atrial electrophysiological and molecular remodelling induced by obstructive sleep apnoea.

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.

Biophysical and molecular comparison of sodium current in cells isolated from canine atria and pulmonary vein.

The collar of the pulmonary vein (PV) is the focal point for the initiation of atrial arrhythmias, but the mechanisms underlying how PV cells differ from neighboring left atrial tissue are unclear. We examined the biophysical and molecular properties of INa in cells isolated from the canine pulmonary sleeve and compared the properties to left atrial tissue. PV and left atrial myocytes were isolated and patch clamp techniques were used to record INa. Action potential recordings from either tissue type were made using high-resistance electrodes. mRNA was determined using quantitative RT-PCR and proteins were determined by Western blot. Analysis of the action potential characteristics showed that PV tissue had a lower Vmax compared with left atrial tissue. Fast INa showed that current density was slightly lower in PV cells compared with LA cells (-96 ± 18.7 pA/pF vs. -120 ± 6.7 pA/pF, respectively, p < 0.05). The recovery from inactivation of INa in PV cells was slightly slower but no marked difference in steady-state inactivation was noted. Analysis of late INa during a 225-ms pulse showed that late INa was significantly smaller in PV cells compared to LA cells at all measured time points into the pulse. These results suggest PV cells have lower density of both peak and late INa. Molecular analysis of Nav1.5 and the four beta subunits showed lower levels of Nav1.5 as well as Navß1 subunits, confirming the biophysical findings. These data show that a lower density of INa may lead to depression of excitability and predispose the PV collar to re-entrant circuits under pathophysiological conditions.

Mechanisms underlying atrial-selective block of sodium channels by Wenxin Keli: Experimental and theoretical analysis.

INTRODUCTION: Atrial-selective inhibition of cardiac sodium channel current (INa) and INa-dependent parameters has been shown to contribute to the safe and effective management of atrial fibrillation. The present study was designed to examine the basis for the atrial-selective actions of Wenxin Keli. METHODS: Whole cell INa was recorded at room temperature in canine atrial and ventricular myocytes. Trains of 40 pulses were elicited over a range of pulse durations and interpulse intervals to determine tonic and use-dependent block. A Markovian model for INa that incorporates interaction of Wenxin Keli with different states of the channel was developed to examine the basis for atrial selectivity of the drug. RESULTS: Our data indicate that Wenxin Keli does not bind significantly to either closed or open states of the sodium channel, but binds very rapidly to the inactivated state of the channel and dissociates rapidly from the closed state. Action potentials recorded from atrial and ventricular preparations in the presence of 5g/L Wenxin Keli were introduced into the computer model in current clamp mode to simulate the effects on maximum upstroke velocity (Vmax). The model predicted much greater inhibition of Vmax in atrial vs. ventricular cells at rapid stimulation rates. CONCLUSION: Our findings suggest that atrial selectivity of Wenxin Keli to block INa is due to more negative steady-state inactivation, less negative resting membrane potential, and shorter diastolic intervals in atrial vs. ventricular cells at rapid activation rates. These actions of Wenxin Keli account for its relatively safe and effective suppression of atrial fibrillation.

Reductions in the Cardiac Transient Outward K+ Current Ito Caused by Chronic ß-Adrenergic Receptor Stimulation Are Partly Rescued by Inhibition of Nuclear Factor κB.

The fast transient outward potassium current (Ito,f) plays a critical role in the electrical and contractile properties of the myocardium. Ito,f channels are formed by the co-assembly of the pore-forming α-subunits, Kv4.2 and Kv4.3, together with the accessory ß-subunit KChIP2. Reductions of Ito,f are common in the diseased heart, which is also associated with enhanced stimulation of ß-adrenergic receptors (ß-ARs). We used cultured neonatal rat ventricular myocytes to examine how chronic ß-AR stimulation decreases Ito,f. To determine which downstream pathways mediate these Ito,f changes, adenoviral infections were used to inhibit CaMKIIδc, CaMKIIδb, calcineurin, or nuclear factor κB (NF-κB). We observed that chronic ß-AR stimulation with isoproterenol (ISO) for 48 h reduced Ito,f along with mRNA expression of all three of its subunits (Kv4.2, Kv4.3, and KChIP2). Inhibiting either CaMKIIδc nor CaMKIIδb did not prevent the ISO-mediated Ito,f reductions, even though CaMKIIδc and CaMKIIδb clearly regulated Ito,f and the mRNA expression of its subunits. Likewise, calcineurin inhibition did not prevent the Ito,f reductions induced by ß-AR stimulation despite strongly modulating Ito,f and subunit mRNA expression. In contrast, NF-κB inhibition partly rescued the ISO-mediated Ito,f reductions in association with restoration of KChIP2 mRNA expression. Consistent with these observations, KChIP2 promoter activity was reduced by p65 as well as ß-AR stimulation. In conclusion, NF-κB, and not CaMKIIδ or calcineurin, partly mediates the Ito,f reductions induced by chronic ß-AR stimulation. Both mRNA and KChIP2 promoter data suggest that the ISO-induced Ito,f reductions are, in part, mediated through reduced KChIP2 transcription caused by NF-κB activation.

Physiological roles of the transient outward current Ito in normal and diseased hearts.

The Ca(2+)-independent transient outward K(+) current (I(to)) plays a critical role in underlying phase 1 of repolarization of the cardiac action potential and, as a result, is central to modulating excitation-contraction coupling and propensity for arrhythmia. Additionally, I(to) and its molecular constituents are consistently reduced in cardiac hypertrophy and heart failure. In this review, we discuss the physiological role of I(to) as well as the molecular basis of this current in human and canine hearts, in which I(to) has been thoroughly studied. In particular, we discuss the role of Ito; in the action potential and the mechanisms by which I(to) modulates excitation-contraction coupling. We also describe the effects of mutations in the subunits constituting the Ito channel as well as the role of I(to) in the failing myocardium. Finally, we review pharmacological modulation of I(to) and discuss the evidence supporting the hypothesis that restoration of I(to) in the setting of heart failure may be therapeutically beneficial by enhancing excitation-contraction coupling and cardiac function.

Regional variation of the inwardly rectifying potassium current in the canine heart and the contributions to differences in action potential repolarization.

The inward rectifier potassium current, IK1, contributes to the terminal phase of repolarization of the action potential (AP), as well as the value and stability of the resting membrane potential. Regional variation in IK1 has been noted in the canine heart, but the biophysical properties have not been directly compared. We examined the properties and functional contribution of IK1 in isolated myocytes from ventricular, atrial and Purkinje tissue. APs were recorded from canine left ventricular midmyocardium, left atrial and Purkinje tissue. The terminal rate of repolarization of the AP in ventricle, but not in Purkinje, depended on changes in external K(+) ([K(+)]o). Isolated ventricular myocytes had the greatest density of IK1 while atrial myocytes had the lowest. Furthermore, the outward component of IK1 in ventricular cells exhibited a prominent outward component and steep negative slope conductance, which was also enhanced in 10 mM [K(+)]o. In contrast, both Purkinje and atrial cells exhibited little outward IK1, even in the presence of 10 mM [K(+)]o, and both cell types showed more persistent current at positive potentials. Expression of Kir2.1 in the ventricle was 76.9-fold higher than that of atria and 5.8-fold higher than that of Purkinje, whereas the expression of Kir2.2 and Kir2.3 subunits was more evenly distributed in Purkinje and atria. Finally, AP clamp data showed distinct contributions of IK1 for each cell type. IK1 and Kir2 subunit expression varies dramatically in regions of the canine heart and these regional differences in Kir2 expression likely underlie regional distinctions in IK1 characteristics, contributing to variations in repolarization in response to in [K(+)]o changes.

Mechanisms underlying the development of the electrocardiographic and arrhythmic manifestations of early repolarization syndrome.

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 early repolarization syndrome (ERS). Transmembrane action potentials (APs) were simultaneously recorded from 2 epicardial sites 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 the inferior and lateral LV of the same heart. 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 (7 µM) and verapamil (3 µM) or pinacidil (4 µM), used to pharmacologically model the genetic defects responsible for ERS, resulted in prominent J-point and ST-segment elevation. ACh (3 µM), 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 (10 µM) or isoproterenol (1 µM) restored homogeneity and suppressed VT/VF. Our data support the hypothesis that 1) ERS is caused by a preferential accentuation of the AP notch in the 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; and 4) quinidine and isoproterenol exert ameliorative effects by reversing the repolarization abnormality.

Developmental changes in expression and biophysics of ion channels in the canine ventricle.

BACKGROUND: Developmental changes in the electrical characteristics of the ventricular myocardium are not well defined. This study examines the contribution of inwardly rectifying K(+) current (IK1), transient outward K(+) current (Ito), delayed rectifier K(+) currents (IKr and IKs) and sodium channel current (INa) to repolarization in the canine neonate myocardium. METHODS: Single myocytes isolated from the left ventricle of 2-3week old canine neonate hearts were studied using patch-clamp techniques. RESULTS: Neonate cells were ~6-fold smaller than those of adults (28.8±8.8 vs. 176±6.7pF). IK1 was larger in neonate myocytes and displayed a substantial inward component and an outward component with negative slope conductance, peaking at -60mV (4.13 pA/pF). IKr tail currents (at -40mV), were small (<20pA). IKs could not be detected, even after exposure to isoproterenol (100nM). Ito was also absent in the neonate, consistent with the absence of a phase 1 in the action potential. Peak INa, late INa and ICa were smaller in the neonate compared with adults. KCND3, KCNIP2 and KCNQ1 mRNA expression was half, while KCNH2 was equal and KCNJ2 was greater in the neonate when compared with adults. CONCLUSIONS: Two major repolarizing K(+) currents (IKs and Ito) present in adult ventricular cells are absent in the 2week old neonate. Peak and late INa are significantly smaller in the neonate. Our results suggest that the absence of these two currents in the neonate heart may increase the susceptibility to arrhythmias under certain long QT conditions.

Mesenchymal stem cell transplantation mitigates electrophysiological remodeling in a rat model of myocardial infarction.

INTRODUCTION: Transplantation of mesenchymal stem cells (MSCs) has shown therapeutic potential for cardiovascular diseases, but the electrophysiological implications are not understood. The purpose of this study was to evaluate the impact of MSC transplantation on adverse electrophysiological remodeling in the heart following myocardial infarction (MI). METHODS AND RESULTS: Three weeks after coronary ligation to induce MI in rats, MSCs or culture medium were directly injected into each infarct. One to two weeks later, hearts were excised, Langendorff-perfused, and optically mapped using the potentiometric fluorescent dye Di-4-ANEPPS. Quantitative real-time PCR was also performed to assess gene expression. Optical mapping showed that post-MI reduction in conduction velocity (from 0.70 ± 0.04 m/s in 12 normal controls to 0.47 ± 0.02 m/s in 11 infarcted hearts, P < 0.05) was attenuated with MSC transplantation (0.65 ± 0.04 m/s, n = 18, P < 0.05). Electrophysiological changes correlated with higher vascular density and better-preserved ventricular geometry in MSC-transplanted hearts. A number of ion channel genes showed changes in RNA expression following infarction. In particular, the expression of Kir2.1, which mediates the inward rectifier potassium current, I(K1), was reduced in infarcted tissues (n = 7) to 13.8 ± 3.7% of normal controls, and this post-MI reduction was attenuated with MSC transplantation (44.4 ± 11.2%, n = 7, P < 0.05). CONCLUSION: In addition to promoting angiogenesis and limiting adverse structural remodeling in infarcted hearts, MSC transplantation also alters ion channel expression and mitigates electrophysiological remodeling. Further understanding of the electrophysiological impact of MSC transplantation to the heart may lead to the development of cell-based therapies for post-MI arrhythmias.

Effect of Wenxin Keli and quinidine to suppress arrhythmogenesis in an experimental model of Brugada syndrome.

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 potassium outward current (I(to)). OBJECTIVE: To examine the effectiveness of WK, alone and in combination with quinidine, to suppress arrhythmogenesis in an experimental model of Brugada syndrome (BrS). METHODS: Action potential and electrocardiographic 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. RESULTS: The Ito agonist induced Phase 2 reentry (P2R) in 13/19 preparations and polymorphic ventricular tachycardia (pVT) in 11/19 wedge preparations. WK (10 g/L) suppressed P2R and pVT in 100% (3/3) of preparations. A lower concentration of WK (5 g/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 (5 g/L) suppressed P2R and pVT by 80% (4/5) and 75% (3/4), respectively. WK reduced Ito, the L-type calcium current, 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 WK, particularly in combination with quinidine, effectively suppresses arrhythmogenesis in an experimental model of BrS via inhibition of Ito and indirect adrenergic sympathomimetic effects.

Regulation of murine cardiac function by phosphodiesterases type 3 and 4.

Cyclic nucleotide phosphodiesterases (PDEs) encompass a large group of enzymes that regulate intracellular levels of two-second messengers, cAMP and cGMP, by controlling the rates of their degradation. More than 60 isoforms, subdivided into 11 gene families (PDE1-11), exist in mammals with at least six families (PDE1-5 and PDE8) identified in mammalian hearts. The two predominant families implicated in regulating contraction strength of the heart are PDE3 and PDE4. Studies using transgenic models in combination with family-specific PDE inhibitors have demonstrated that PDE3A, PDE4B, and PDE4D isoforms regulate cardiac contractility by modulating cAMP levels in various subcellular compartments. These studies have further uncovered contributions of PDE4B and PDE4D in preventing ventricular arrhythmias.

Iron overload decreases CaV1.3-dependent L-type Ca2+ currents leading to bradycardia, altered electrical conduction, and atrial fibrillation.

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 (bradycardia) and heart block. METHODS AND RESULTS: We used a mouse model of CIO to investigate the effects of iron on sinoatrial node (SAN) function. As in humans, CIO reduced heart rate (≈20%) in conscious mice as well as in anesthetized mice with autonomic nervous system blockade and in isolated Langendorff-perfused mouse hearts, suggesting that 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 Ca(2+) current (I(Ca,L)) densities and positive (rightward) voltage shifts in I(Ca,L) activation. Pacemaker current (I(f)) was not affected by CIO. Because I(Ca,L) in SAN myocytes (as well as in atrial and conducting system myocytes) activates at relatively negative potentials due to the presence of Ca(V)1.3 channels (in addition to Ca(V)1.2 channels), our data suggest that elevated iron preferentially suppresses Ca(V)1.3 channel function. Consistent with this suggestion, CIO reduced Ca(V)1.3 mRNA levels by ≈40% in atrial tissue (containing SAN) and did not lower heart rate in Ca(V)1.3 knockout mice. CIO also induced PR-interval prolongation, heart block, and atrial fibrillation, conditions also seen in Ca(V)1.3 knockout mice. CONCLUSIONS: Our results demonstrate that CIO selectively reduces Ca(V)1.3-mediated I(Ca,L), leading to bradycardia, slowing of electrical conduction, and atrial fibrillation as seen in patients with iron overload.

Nuclear factor kappaB downregulates the transient outward potassium current I(to,f) through control of KChIP2 expression.

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)-κB is activated in cardiac hypertrophy and disease, we studied the role of NF-κB in mediating I(to,f) reductions induced by hypertrophy. METHODS AND RESULTS: Culturing neonatal rat ventricular myocytes in the presence of phenylephrine (PE) plus propranolol (Pro), to selectively activate α(1)-adrenergic receptors, caused reductions in I(to,f), as well as KChIP2 and Kv4.3 expression, while increasing Kv4.2 expression. Inhibition of NF-κB, via overexpression of a phosphorylation-deficient mutant of IκBα (IκBαSA) prevented PE/Pro-induced reductions in I(to,f) and KChIP2 mRNA, without affecting Kv4.2 or Kv4.3 expression, suggesting NF-κB mediates the I(to,f) reductions by repressing KChIP2. Indeed, overexpression of the NF-κB activator IκB kinase-ß also decreased KChIP2 expression and I(to,f) (despite increasing Kv4.2), whereas IκBαSA overexpression elevated KChIP2 and decreased Kv4.2 levels. In addition, the classic NF-κB activator tumor necrosis factor α also induced NF-κB-dependent reductions of KChIP2 and I(to,f). Finally, inhibition of calcineurin did not prevent PE/Pro-induced reductions in KChIP2. CONCLUSIONS: NF-κB regulates KChIP2 and Kv4.2 expression. The reductions in I(to,f) observed following α-adrenergic receptor stimulation or tumor necrosis factor α application require NF-κB-dependent decreases in KChIP2 expression.

Pulmonary vascular and cardiac effects of peroxynitrite decomposition in newborn rats.

Evidence implicates oxidative stress as playing a prominent role in the pathogenesis of pulmonary hypertension, to which peroxynitrite anion (ONOO(-)) may make a major contribution. Hypothesizing that removal of ONOO(-) would attenuate chronic neonatal pulmonary hypertension, we examined the effects of a ONOO(-) decomposition catalyst (FeTPPS) on pulmonary arteries in vitro, on primary cultured pulmonary artery smooth muscle cell (PASMC) and cardiomyocyte survival and growth, and on central hemodynamics in rat pups exposed to hypoxia (13% O(2)) for 7 days from birth. Daily FeTPPS (30 mg/kg ip) reduced lung nitrotyrosine content, attenuated vascular remodeling, and normalized pulmonary vascular resistance in hypoxia-exposed animals. FeTPPS attenuated proliferation and increased apoptosis of neonatal PASMCs in vitro. Isolated neonatal pulmonary arteries treated with FeTPPS showed reduced agonist-induced force development and enhanced endothelium-dependent and -independent relaxation, possibly via increased nitrate. However, we observed endothelial dysfunction, enhanced lung tissue phosphodiesterase 5 activity, and biventricular cardiac hypertrophy in air-exposed animals receiving FeTPPS. Further, in contrast to PASMCs, FeTPPS enhanced survival of newborn cardiomyocytes. We conclude that decomposition of ONOO(-) with FeTPPS attenuates chronic hypoxia-induced pulmonary hypertension; however, it may negatively influence the modulation of normal pulmonary arterial relaxation function, cell survival, and growth.

Functional consequences of Kir2.1/Kir2.2 subunit heteromerization.

Kir2 subunits form channels that underlie classical strongly inwardly rectifying potassium currents. While homomeric Kir2 channels display a number of distinct and physiologically important properties, the functional properties of heteromeric Kir2 assemblies, as well as the stoichiometries and the arrangements of Kir2 subunits in native channels, remain largely unknown. Therefore, we have implemented a concatemeric approach, whereby all four cloned Kir2 subunits were linked in tandem, in order to study the effects of Kir2.1 and Kir2.2 heteromerization on properties of the resulting channels. Kir2.2 subunits contributed stronger to single-channel conductance than Kir2.1 subunits, and channels containing two or more Kir2.2 subunits displayed conductances indistinguishable from that of a Kir2.2 homomeric channel. In contrast, single-channel kinetics was a more discriminating property. The open times were significantly shorter in Kir2.2 channels compared with Kir2.1 channels and decreased nearly proportionally to the number of Kir2.2 subunits in the heteromeric channel. Similarly, the sensitivity to block by barium also depended on the proportions of Kir2.1 to Kir2.2 subunits. Overall, the results showed that Kir2.1 and Kir2.2 subunits exert neither a dominant nor an anomalous effect on any of the properties of heteromeric channels. The data highlight opportunities and challenges of using differential properties of Kir2 channels in deciphering the subunit composition of native inwardly rectifying potassium currents.

Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

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 noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) 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. Patch-clamp 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 an amphiphilic phenylene ethynylene antimicrobial oligomer (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 approximately 3 nm holes in living cell membranes.