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Monocytes play a key role in the inflammatory stage of the healing process. To allow monocyte migration to injured tissues, the balances between secreted matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) must be finely modulated. However, a reduction of blood supply and local oxygen tension can modify the phenotype of immune cells. Intriguingly, hypoxia might be targeted by new effective oxygenating devices such as 2H,3H-decafluoropentane- (DFP-) based oxygen-loaded nanodroplets (OLNs). Here, hypoxia effects on gelatinase/TIMP release from human peripheral monocytes were investigated, and the therapeutic potential of dextran-shelled OLNs was evaluated. Normoxic monocytes constitutively released ~500 ng/mL MMP-9, ~1.3 ng/mL TIMP-1, and ~0.6 ng/mL TIMP-2 proteins. MMP-2 was not detected. After 24 hours, hypoxia significantly altered MMP-9/TIMP-1 balance by reducing MMP-9 and increasing TIMP-1, without affecting TIMP-2 secretion. Interestingly OLNs, not displaying toxicity to human monocytes after cell internalization, effectively counteracted hypoxia, restoring a normoxia-like MMP-9/TIMP-1 ratio. The action of OLNs was specifically dependent on time-sustained oxygen diffusion up to 24 h from their DFP-based core. Therefore, OLNs appear as innovative, nonconventional, cost-effective, and nontoxic therapeutic tools, to be potentially employed to restore the physiological invasive phenotype of immune cells in hypoxia-associated inflammation.

In six male anesthetized, tracheotomized, and mechanically ventilated rabbits we derived indications on alveolar mechanics from in vivo imaging, using a "pleural window" technique (pleural space intact) that allows unrestrained movement of the same subpleural alveoli (N=60) on increasing alveolar pressure from 4 to 8 cmH2O. Absolute compliance (C(abs), ratio of change in alveolar surface area to the change in alveolar pressure) was significantly lower in smaller compared to larger alveoli. Specific compliance, C(sp), obtained by normalizing C(abs) to alveolar surface area, was essentially independent of alveolar size. Both C(abs) and C(sp) were affected by large variability likely reflecting the complex matching between elastic and surface forces. We hypothesize that the relative constancy of C(sp) might contribute to reduce interregional differences in parenchymal and surface forces in the lung tissue by contributing to assure a uniform stretching in a model of mechanically inter-dependent alveoli., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objectives: The objective of this study was to evaluate to what extent lung compliance is affected by the individual and combined action of lung resection and hydrothorax in an animal model., Methods: Anaesthetized and mechanically ventilated rabbits (weight range 2 ÷ 2.2 kg) were randomized in two groups: (i) experimental hydrothorax (from 2 to 8 ml) (n = 5) and (ii) right lower lobe lobectomy (n = 4) and right middle plus lower lobe resection (n = 2). To obtain lung compliance, we measured alveolar, oesophageal pressures and lung volume during slow inflation manoeuvres in control conditions and after hydrothorax or lung resection. Lung compliance was estimated as the change in lung volume divided by the change in transpulmonary pressure. Based on the changes in compliance of the whole lung, we calculated the corresponding changes in compliance of the right lung, which was directly exposed to unilateral hydrothorax and lobectomy., Results: Average total lung compliance in the control was 3.3 ± 0.8 (SD) ml/cmH2O. Eight millilitres of hydrothorax significantly decreased (P < 0.001) lung compliance to 2.7 ± 0.7 ml/cmH2O and increased pleural liquid pressure at the bottom of the cavity from -1 cmH2O up to ∼ 2.5-3 cmH2O. Resection of the right lower lobe significantly decreased (P < 0.001) lung compliance to 1.75 ± 0.3 ml/cmH2O. Resection of the right middle plus lower lobes significantly decreased (P < 0.001) lung compliance to 1.52 ± 0.4 ml/cmH2O., Conclusions: Following hydrothorax, the decrease in right lung compliance (∼ 45%) was much greater than that expected based on the estimated decrease in right lung volume (20%). We attribute this difference to the fact that hydrothorax causes the lung to be exposed to positive, rather than sub-atmospheric, pressure, causing atelectasis. Following lobectomy, right lung compliance decreased by 62 and 80% for estimated decreases in lung volume of 30 and 60%. This difference could reflect inaccuracy in the estimate of lung volume reduction based on resected weight and/or surgical damage. We conclude that potential detrimental effects of hydrothorax and lobar resection decrease lung compliance and expose the lung to the risk of over-distension when a chest drain is applied.

In six male anesthetized, tracheotomized, and mechanically ventilated rabbits, we imaged subpleural alveoli under microscopic view (60×) through a "pleural window" obtained by stripping the endothoracic fascia and leaving the parietal pleura intact. Three different imaging scale levels were identified for the analysis on increasing stepwise local distending pressure (P ld) up to 16.5 cmH2O: alveoli, alveolar cluster, and whole image field. Alveolar profiles were manually traced, clusters of alveoli of similar size were identified through a contiguity-constrained hierarchical agglomerative clustering analysis and alveolar surface density (ASD) was estimated as the percentage of air on the whole image field. Alveolar area distributions were remarkably right-skewed and showed an increase in median value with a large topology-independent heterogeneity on increasing P ld. Modeling of alveolar area distributions on increasing P ld led to hypothesize that absolute alveolar compliance (change in surface area over change in P ld) increases fairly linearly with increasing initial alveolar size, the corollary of this assumption being a constant specific compliance. Clusters were reciprocally interweaved due to their highly variable complex shapes. ASD was found to increase with a small coefficient of variation (CV <25%) with increasing P ld. The CV of lung volume at each transpulmonary pressure was further decreased (about 6%). The results of the study suggest that the considerable heterogeneity of alveolar size and of the corresponding alveolar mechanical behavior are homogenously distributed, resulting in a substantially homogenous mechanical behavior of lung units and whole organ.

Magnetic nanoparticles have emerged as important players in current research in modern medicine since they can be used in medicine for diagnosis and/or therapeutic treatment of diseases. Among many therapeutic applications of iron-based nanoparticles, drug delivery and photothermal therapy are of particular interest. At cellular level their uptake has been studied and the mechanism by which nanoparticles enter into the cell has important implication not only for their fate but also for their impact on the biological systems. We present here a dynamic investigation of interaction of biocompatible iron oxide nanoparticles coated with L-3,4-dihydroxyphenylalanine and labeled with tetra-methylrhodamine-5/6-isothiocyanate with lung epithelial cells. Our data show that after macropinocytosis-mediated internalization, nanoparticles in form of vesicles approach the nucleus and converge in the more acidic compartments of the cells in a microtubule-dependent manner. During progression the nanoparticles aggregate. Finally, we have demonstrated that a converging laser radiation on the cells, causes the increase in the local temperature and thus damages the cells, suggesting that these nanoparticles may be applied for photothermal therapy studies.

This study analyzed the time dependence decay of the mRNA of selected genes important for the hypoxia response. The genes chosen were the two isoforms of hypoxia-inducible factors, the three isoforms of the prolyl hydroxylase domain protein, the vascular endothelial growth factor and endothelial nitric oxide synthase. mRNA and proteins were extracted from lungs obtained from control, hypoxic and 15 minutes normoxic recovered rats and analyzed by Real-time RT-PCR or by the Western Blot technique. Results indicated that in normoxia isoform 2á was the more represented hypoxia-inducible factor mRNA, and among the prolyl hydroxylase domain transcripts, isoform 3 was the least abundant. Moreover, in chronic hypoxia only hypoxia-inducible factor 1α and prolyl hydroxylase domain protein 3 increased significantly, while after 15 minutes of recovery all the mRNAs tested were decreased except endothelial nitric oxide synthase mRNA. In terms of proteins, hypoxia-inducible 1α was the isoform more significant in the nucleus, while 2á predominated in the cytosol. While the former was steady even after a brief recovery from hypoxia, the latter underwent a strong degradation. In conclusion we showed the relevance of the decay in the mRNA and protein levels upon re-oxygenation in normoxia. We believe that this has to be considered in research studies dealing with recovery from hypoxia.

Background: As part of a project designing nanoparticles for the treatment of Alzheimer's disease, we have synthesized and characterized a small library of nanoparticles binding with high affinity to the β-amyloid peptide and showing features of biocompatibility in vitro, which are important properties for administration in vivo. In this study, we focused on biocompatibility issues, evaluating production of nitric oxide by cultured human umbilical vein endothelial cells and macrophages, used as models of cells which would be exposed to nanoparticles after systemic administration., Methods: The nanoparticles tested were liposomes and solid lipid nanoparticles carrying phosphatidic acid or cardiolipin, and PEGylated poly(alkyl cyanoacrylate) nanoparticles (PEG-PACA). We measured nitric oxide production using the Griess method as well as phosphorylation of endothelial nitric oxide synthase and intracellular free calcium, which are biochemically related to nitric oxide production. MTT viability tests and caspase-3 detection were also undertaken., Results: Exposure to liposomes did not affect the viability of endothelial cells at any concentration tested. Increased production of nitric oxide was detected only with liposomes carrying phosphatidic acid or cardiolipin at the highest concentration (120 μg/mL), together with increased synthase phosphorylation and intracellular calcium levels. Macrophages exposed to liposomes showed a slightly dose-dependent decrease in viability, with no increase in production of nitric oxide. Exposure to solid lipid nanoparticles carrying phosphatidic acid decreased viability in both cell lines, starting at the lowest dose (10 μg/mL), with increased production of nitric oxide detected only at the highest dose (1500 μg/mL). Exposure to PEG-PACA affected cell viability and production of nitric oxide in both cell lines, but only at the highest concentration (640 μg/mL)., Conclusion: Liposomal and PEG-PACA nanoparticles have a limited effect on vascular homeostasis and inflammatory response, rendering them potentially suitable for treatment of Alzheimer's disease. Moreover, they highlight the importance of testing such nanoparticles for production of nitric oxide in vitro in order to identify a therapeutic dose range suitable for use in vivo.

Nanoparticles (NPs) are materials with overall dimensions in the nanoscale range. They have unique physicochemical properties, and have emerged as important players in current research in modern medicine. In the last few decades, several types of NPs and microparticles have been synthesized and proposed for use as contrast agents for diagnostics and imaging and for drug delivery; for example, in cancer therapy. Yet specific targeting that will improve their delivery still represents an unsolved challenge. The mechanism by which NPs enter the cell has important implications not only for their fate but also for their impact on biological systems. Several papers in the literature discuss the potential risks related to NP exposure, and more recently the concept that even sublethal doses of NPs may elicit a cell response has been proposed. In this review, we intend to present an overall view of cell mechanisms that may be perturbed by cell-NP interaction. Published data, in fact, emphasize that NPs should no longer be viewed only as simple carriers for biomedical applications, but that they can also play an active role in mediating biological effects.

We have measured in human alveolar cells the cytoplasmic distribution of the fluorophore coumarin-6 carried by Solid Lipid Nanoparticles (SLNs) and observed a perinuclear accumulation of the fluorescence that can be described by a single exponential growth along an ideal line joining the plasma membrane to the nuclear border and by a sigmoidal relationship as a function of time. Intracellular distribution was affected by lowering the temperature from 37 to 4° C and by the disruption of cytoskeleton by cytochalasin D, but it was minimally perturbed by the inhibition of ATP dependent molecular motors. A biophysical model was developed for an accumulation of loaded particles against a diffusion gradient based on a mean field interaction energy, whose origin we ascribe to the actin structure of the cytoskeleton. The estimated value for the load diffusion coefficient was four and two orders of magnitude less than that of free coumarin-6 and of SLNs in aqueous solutions, respectively, suggesting that the load moves within the cell cytoplasm in a form still reminiscent of the nanocarrier structure., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We evaluated how the increase in lung interstitial pressure correlates with the pulmonary vascular response to chronic hypoxia. In control and hypoxic (30 days; 10% O₂) Wistar male rats, we measured: pulmonary interstitial pressure (P(ip)), cardiac and haemodynamic parameters by echocardiography, and performed lung morphometry on tissue specimens fixed in situ. In control animals, mean ± sd P(ip), air/tissue volume ratio and capillary vascularity index in the air-blood barrier were -12 ± 2.03 cmH₂O, 3.9 and 0.43, respectively. After hypoxia exposure, the corresponding values of these indices in apparently normal lung regions were 2.6 ± 1.7 cmH₂O, 3.6, and 0.5, respectively. In oedematous regions, the corresponding values were 12 ± 4 cmH₂O, 0.4 and 0.3, respectively. Furthermore, in normal regions, the density of pre-capillary vessels (diameter ~50-200 μm) increased and their thickness/internal diameter ratio decreased, while opposite results were found in oedematous regions. Pulmonary artery pressure increased in chronic hypoxia relative to the control (39.8 ± 5.9 versus 26.2 ± 2.2 mmHg). Heterogeneity in local lung vascular response contributes to developing pulmonary hypertension in chronic hypoxia. In oedematous regions, the decrease in capillary vascularity correlated with the remarkable increase in interstitial pressure and morphometry of the pre-capillary vessels suggested an increase in vascular resistance; the opposite was true in apparently normal regions.

The aim of present work was to elucidate the interaction of solid lipid nanoparticles (SLNs) with cellular plasma-membrane to gain insight of intracellular drug delivery. To this aim we followed the uptake of coumarin-6 (a drug model) either free in the extracellular medium or loaded on SLN (c-SLN). Alveolar epithelial cells were exposed to a biocompatible concentration of c-SLN (0.01 mg/ml of tripalmitin) prepared by warm microemulsion whose lipid matrix was constituted by low melting point molecules (fatty acids, triglycerides). Intracellular fluorescence and preferential accumulation in the perinuclear region were increased by 54.8% on comparing c-SLN to the same amount of free coumarin-6 in the medium. Lowering temperature from 37 ° to 4 °C decreased the intracellular signal intensity by about 48% equally for the free as well as for loaded drug, thus suggesting the inhibition of a similar non-endocytotic entrance pathway. No specific co-localization of the fluorescence with intracellular organelles was found. The c-SLN calorimetric profile obtained with differential scanning calorimetry (DSC), revealing transition within the range 58-62 °C, altered remarkably upon incubation with cells, suggesting a change in SLN structure after association with cells membranes. We propose that the uptake of the model drug loaded on SLN is only partly related to the endocytotic pathway; it occurs despite the loss of integrity of the original SLN structure and it appears to be more efficient when the drug is vehicled rather than being free in the culture medium.

Membrane rafts (MRs) are clusters of lipids, organized in a "quasicrystalline" liquid-order phase, organized on the cell surface and whose pattern of molecules and physicochemical properties are distinct from those of the surrounding plasma membrane. MRs may be considered an efficient and fairly rapid cell-activated mechanism to express or mask surface receptors aimed at triggering specific response pathways. This paper reports observations concerning the role of MRs in the control of lung extravascular water that ought to be kept at minimum to assure gas diffusion, supporting the hypothesis that MRs expression is a potential mechanism of sensing minor changes in the volume of extravascular water. We present the evidence that MRs expression specifically relates to signal-transduction processes evoked by mechanical stimuli arising in the interstitial lung compartment when a small increase in extravascular volume occurs. We further hypothesize that a differential expression of MRs might also reflect the damage to precise components of the extracellular matrix caused by the perturbation in water balance and thus can trigger a molecule-oriented specific matrix remodelling.

The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done.

Thoracic surgery that requires resection of a portion of lung or of a whole lung profoundly alters the mechanical and fluid dynamic setting of the lung-chest wall coupling, as well as the water balance in the pleural space and in the remaining lung. The most frequent postoperative complications are of a respiratory nature, and their incidence increases the more the preoperative respiratory condition seems compromised. There is an obvious need to identify risk factors concerning mainly the respiratory function, without neglecting the importance of other comorbidities, such as coronary disease. At present, however, a satisfactory predictor of postoperative cardiopulmonary complications is lacking; postoperative morbidity and mortality have remained unchanged in the last 10 years. The aim of this review is to provide a pathophysiologic interpretation of the main respiratory complications of a respiratory nature by relying on new concepts relating to lung fluid dynamics and mechanics. New parameters are proposed to improve evaluation of respiratory function from pre- to the early postoperative period when most of the complications occur., (Published by Elsevier Inc.)

We evaluated the response to mild hypoxia exposure of A549 alveolar human cells and of a continuous alveolar cell line from human excised lungs (A30) exposed to 5% O(2) for 5 and 24 h. No signs of increased peroxidation and of early apoptosis were detected. After 24 h of hypoxia total cell proteins/DNA ratio decreased significantly by about 20%. Similarly, we found a decrease in membrane phospholipid and cholesterol content. The membrane fluidity assessed by fluorescence anisotropy measurements was unchanged. We also prepared the detergent resistant membrane fraction (DRM) to analyze the distribution of the two types of lipid microdomains, caveolae and lipid rafts. The DRM content of Cav-1, marker of caveolae, was decreased, while CD55, marker of lipid rafts, increased in both cell lines. Total content of these markers in the membranes was unchanged indicating remodelling of their distribution between detergent-resistant and detergent-soluble fraction of the cellular membrane. The changes in protein markers distribution did not imply changes in the corresponding mRNA, except in the case of Cav-1 for A30 line. In the latter case we found a parallel decrease in Cav-1 and in the corresponding mRNA. We conclude that an exposure to a mild degree of hypoxia triggers a significant remodelling of the lipid microdomains expression, confirming that they are highly dynamic structures providing a prompt signalling platform to changes of the pericellular microenvironment., ((c) 2008 Wiley-Liss, Inc.)

The effects of two SCN5A mutations (Y1795C, Y1795H), previously identified in one Long QT syndrome type 3 (LQT3) and one Brugada syndrome (BrS) families, were investigated by means of numerical modeling of ventricular action potential (AP). A Markov model capable of reproducing a wild-type as well as a mutant sodium current (I(Na)) was identified and was included into the Luo-Rudy ventricular cell model for action potential (AP) simulation. The characteristics of endocardial, midmyocardial, and epicardial cells were reproduced by differentiating the transient outward current (I(TO)) and the ratio of slow delayed rectifier potassium (I(Ks)) to rapid delayed rectifier current (I(Kr)). Administration of flecainide and mexiletine was simulated by appropriately modifying I(Na), calcium current (I(Ca)), I(TO), and I(Kr). Y1795C prolonged AP in a rate-dependent manner, and early afterdepolarizations (EADs) appeared during bradycardia in epicardial and midmyocardial cells; flecainide and mexiletine shortened AP and abolished EADs. Y1795H resulted in minimal changes in the APs; flecainide but not mexiletine induced APs heterogeneity across the ventricular wall that accounts for the ST segment elevation induced by flecainide in Y1795H carriers. The AP abnormalities induced by Y1795H and Y1795C can explain the clinically observed surface ECG phenotype. For the first time by modeling the effects of flecainide and mexiletine, we are able to gather mechanistic insights on the response to drugs administration observed in affected patients.

Long QT syndrome (LQTS) and Brugada syndrome (BrS) are inherited diseases predisposing to ventricular arrhythmias and sudden death. Genetic studies linked LQTS and BrS to mutations in genes encoding for cardiac ion channels. Recently, two novel missense mutations at the same codon in the gene encoding the cardiac Na+ channel (SCN5A) have been identified: Y1795C (causing the LQTS phenotype) and Y1795H (causing the BrS phenotype). Functional studies in HEK293 cells showed that both mutations alter the inactivation of Na+ current and cause a sustained Na+ current upon depolarisation. In this paper, a nine state Markov model was used to simulate the Na+ current in wild-type Na+ cardiac channel and the current alterations observed in Y1795C and Y1795H mutant channels. The model includes three distinct closed states, a conducting open state and five inactivation states (one fast-, two intermediate- and two closed-inactivation). Transition rates between these states were identified on the basis of previously published voltage-clamp experiments. The model was able to reproduce the experimental Na+ current in mutant channels just by altering the assignment of model parameters with respect to wild-type case. Parameter assignment was validated by performing action potential clamp experiments and comparing experimental and simulated I(Na) current. The Markov model was subsequently introduced in the Luo-Rudy model of ventricular myocyte to investigate "in silico" the consequences on the ventricular cell action potential of the two mutations. Coherently with their phenotypes, the Y1795C mutation prolongs the action potential, while the Y1795H mutation causes only negligible changes in action potential morphology.

Short QT syndrome (SQTS) leads to an abbreviated QTc interval and predisposes patients to life-threatening arrhythmias. To date, two forms of the disease have been identified: SQT1, caused by a gain of function substitution in the HERG (I(Kr)) channel, and SQT2, caused by a gain of function substitution in the KvLQT1 (I(Ks)) channel. Here we identify a new variant, "SQT3", which has a unique ECG phenotype characterized by asymmetrical T waves, and a defect in the gene coding for the inwardly rectifying Kir2.1 (I(K1)) channel. The affected members of a single family had a G514A substitution in the KCNJ2 gene that resulted in a change from aspartic acid to asparagine at position 172 (D172N). Whole-cell patch-clamp studies of the heterologously expressed human D172N channel demonstrated a larger outward I(K1) than the wild-type (P<0.05) at potentials between -75 mV and -45 mV, with the peak current being shifted in the former with respect to the latter (WT, -75 mV; D172N, -65 mV). Coexpression of WT and mutant channels to mimic the heterozygous condition of the proband yielded an outward current that was intermediate between WT and D172N. In computer simulations using a human ventricular myocyte model the increased outward I(K1) greatly accelerated the final phase of repolarization, and shortened the action potential duration. Hence, unlike the known mutations in the two other SQTS forms (N588K in HERG and V307L in KvLQT1), simulations using the D172N and WT/D172N mutations fully accounted for the ECG phenotype of tall and asymmetrically shaped T waves. Although we were unable to test for inducibility of arrhythmia susceptibility due to lack of patients' consent, our computer simulations predict a steeper steady-state restitution curve for the D172N and WT/D172N mutation, compared with WT or to HERG or KvLQT1 mutations, which may predispose SQT3 patients to a greater risk of reentrant arrhythmias.

We identify a human mutation (E1053K) in the ankyrin-binding motif of Na(v)1.5 that is associated with Brugada syndrome, a fatal cardiac arrhythmia caused by altered function of Na(v)1.5. The E1053K mutation abolishes binding of Na(v)1.5 to ankyrin-G, and also prevents accumulation of Na(v)1.5 at cell surface sites in ventricular cardiomyocytes. Ankyrin-G and Na(v)1.5 are both localized at intercalated disc and T-tubule membranes in cardiomyocytes, and Na(v)1.5 coimmunoprecipitates with 190-kDa ankyrin-G from detergent-soluble lysates from rat heart. These data suggest that Na(v)1.5 associates with ankyrin-G and that ankyrin-G is required for Na(v)1.5 localization at excitable membranes in cardiomyocytes. Together with previous work in neurons, these results in cardiomyocytes suggest that ankyrin-G participates in a common pathway for localization of voltage-gated Na(v) channels at sites of function in multiple excitable cell types.

Na(v)1.5, the cardiac isoform of the voltage-gated Na+ channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Na(v)1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Na(v)1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na(v)1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na(v)1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2-dependent ubiquitination of Na(v)1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Na(v)1.5 and Nedd4-2. Na(v)1.5 current density was decreased by 65% upon Nedd4-2 cotransfection, whereas the PY-motif mutant channels were not affected. In contrast, a catalytically inactive Nedd4-2 had no effect, indicating that ubiquitination mediates this downregulation. However, Nedd4-2 did not alter the whole-cell or the single channel biophysical properties of Na(v)1.5. Consistent with the functional findings, localization at the cell periphery of Na(v)1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Na(v)1.5, suggesting that Nedd4-2 is a specific regulator of Na(v)1.5. These results demonstrate that Na(v)1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Na(v)1.5 by decreasing the channel density at the cell surface.

Platelets release glutamate upon activation and are an important clearance system of the amino acid from blood, through high-affinity glutamate uptake, similar to that described in brain synaptosomes. Since platelet glutamate uptake is decreased in neurodegenerative disorders, we performed a morphological and molecular characterization of platelet glutamate transporters. The three major brain glutamate transporters EAAT1, EAAT2 and EAAT3 are expressed in platelets, with similar molecular weight, although at lower density than brain. A Na(+)-dependent-high-affinity glutamate uptake was competitively inhibited by known inhibitors but not by dihydrokainic acid, suggesting platelet EAAT2 does not play a major role in glutamate uptake at physiological conditions. We observed decreased glutamate uptake V(max), without modification of transporter affinity, in aging, which could be linked to the selective decrease of EAAT1 expression and mRNA. Moreover, in AD patients we found a further EAAT1 reduction compared to age-matched controls, which could explain the decrease of platelet uptake previously described. Platelet glutamate transporters may be used as peripheral markers to investigate the role of glutamate in patients with neuropsychiatric disorders.

Mutations associated with sodium channel-linked inherited Long-QT syndrome often result in a gain of channel function by disrupting channel inactivation. A small fraction of channels fail to inactivate (burst) at depolarized potentials where normal (wild type) channels fully inactivate. These noninactivating channels give rise to a sustained macroscopic current. We studied the effects of protein kinase A stimulation on sustained current in wild type and three disease-linked C-terminal mutant channels (D1790G, Y1795C, and Y1795H). We show that protein kinase A stimulation differentially affects gating in the mutant channels. Wild type, Y1795C, and Y1795H channels are insensitive to protein kinase A stimulation, whereas "bursting" in the D1790G mutant is markedly enhanced by protein kinase A-dependent phosphorylation. Our results suggest that the charge at position 1790 of the C terminus of the channel modulates the response of the cardiac sodium channel to protein kinase A stimulation and that phosphorylation of residue 36 in the N terminus and residue 525 in the cytoplasmic linker joining domains I and II of the channel alpha subunit facilitate destabilization of inactivation and thereby increase sustained current.

In the last few years, a very active line of research took place after the first identification of SCN5A mutations associated with an inherited form of cardiac arrhythmias and sudden death, the LQT3 variant of the long QT syndrome. Subsequently, two allelic diseases additional to LQT3 were shown to be due to mutations in the same gene, the Brugada syndrome (BrS) and the Lev-Lenegre syndrome (progressive cardiac conduction defect). Genotype-phenotype correlation and in vitro expression studies provide evidence that structure-function relationships of the SCN5A protein are much more complex than initially anticipated. The biophysical characterization of the sodium channel defects associated with different phenotypes and the genotype-phenotype correlation studies brought to the attention of the scientific community a plethora of mechanisms by which even a single amino acid substitution may remarkably affect cardiac excitability. Finally, the evidence of patients harboring an SCN5A mutation and overlapping clinical presentations creates a need for a revision of the traditional classification of the above mentioned diseases. It is now appropriate to consider the "sodium channel syndrome" as a unique clinical entity that may manifest itself with a spectrum of possible phenotypes.

Mutations in the gene (SCN5A) encoding the alpha-subunit of the cardiac Na+ channel cause congenital long QT syndrome (LQT-3). Here we describe a novel LQT-3 mutation I1768V (I1768V) located in the sixth transmembrane spanning segment of domain IV. This mutation is unusual in that it is located within a transmembrane spanning domain and does not promote the typically observed sustained inward current corresponding to a gain of channel function (bursting). Rather, I1768V increases the rate of recovery from inactivation and increases the channel availability, observed as a positive shift of the steady-state inactivation curve (+7.6 mV). Using a Markovian model of the cardiac Na+ channel, we simulated these changes in gating behavior and demonstrated that a small increase in the rate of recovery from inactivation is sufficient to explain all of the experimentally observed current changes. The effect of these alterations in channel gating results in an increase in window current that may act to disrupt cardiac repolarization.

Little is known about the structure of the C terminus of the human cardiac voltage-gated sodium channel alpha subunit (SCN5A), but disease-linked mutations within this 244-amino acid intracellular region of the channel have marked effects on channel inactivation. Here we report a structural analysis of the C-terminal tail of the cardiac Na(+) channel that sheds new light on mechanisms that control its inactivation gating. Homology modeling of the SCN5A C terminus predicts predominant alpha-helical structure (six helices) in the proximal half of this intracellular tail but little structure in the distal half. Circular dichroism of isolated and purified C terminus supports this prediction. Whole cell and single channel patch clamp recordings of wild type and mutant alpha subunits co-expressed with the hbeta(1) subunit in HEK 293 cells indicate that truncation of the distal, nonstructured, C terminus (L1921stop mutant) reduces current density but does not affect channel gating (n = 6). In contrast, truncation of the sixth helix containing a concentration of positively charged residues along with the distal C terminus (S1885stop mutant) also reduces current density but, in addition, has profound and selective effects on inactivation (no effect on activation). Channel availability is shifted (-11 +/- 0.6 mV), and there is a 10-fold increase in the percentage of channels that burst (fail to inactivate) during prolonged depolarization (0.025% S1885stop (n = 7) versus 0.0028% wild type (n = 9), p < 0.005). These results suggest that the charged structured region of the SCN5A C terminus plays a major role in channel inactivation, stabilizing the inactivated state.

Variant 3 of the congenital long-QT syndrome (LQTS-3) is caused by mutations in the gene encoding the alpha subunit of the cardiac Na(+) channel. In the present study, we report a novel LQTS-3 mutation, E1295K (EK), and describe its functional consequences when expressed in HEK293 cells. The clinical phenotype of the proband indicated QT interval prolongation in the absence of T-wave morphological abnormalities and a steep QT/R-R relationship, consistent with an LQTS-3 lesion. However, biophysical analysis of mutant channels indicates that the EK mutation changes channel activity in a manner that is distinct from previously investigated LQTS-3 mutations. The EK mutation causes significant positive shifts in the half-maximal voltage (V(1/2)) of steady-state inactivation and activation (+5.2 and +3.4 mV, respectively). These gating changes shift the window of voltages over which Na(+) channels do not completely inactivate without altering the magnitude of these currents. The change in voltage dependence of window currents suggests that this alteration in the voltage dependence of Na(+) channel gating may cause marked changes in action potential duration because of the unique voltage-dependent rectifying properties of cardiac K(+) channels that underlie the plateau and terminal repolarization phases of the action potential. Na(+) channel window current is likely to have a greater effect on net membrane current at more positive potentials (EK channels) where total K(+) channel conductance is low than at more negative potentials (wild-type channels), where total K(+) channel conductance is high. These findings suggest a fundamentally distinct mechanism of arrhythmogenesis for congenital LQTS-3.

Background: Sodium channels isolated from mammalian brain are composed of alpha-, beta(1)-, and beta(2)-subunits. The composition of sodium channels in cardiac muscle, however, has not been defined, and disagreement exists over which beta-subunits are expressed in the myocytes. Some investigators have demonstrated beta(1) expression in heart. Others have not detected any auxiliary subunits. On the basis of Northern blot analysis of total RNA, beta(2) expression has been thought to be exclusive to neurons and absent from cardiac muscle., Methods and Results: The goal of this study was to define the subunit composition of cardiac sodium channels in myocytes. We show that cardiac sodium channels are composed of alpha-, beta(1)-, and beta(2)-subunits. Nav1.5 and Nav1.1 are expressed in myocytes and are associated with beta(1)- and beta(2)-subunits. Immunocytochemical localization of Nav1.1, beta(1), and beta(2) in adult heart sections showed that these subunits are expressed at the Z lines, as shown previously for Nav1.5. Coexpression of Nav1.5 with beta(2) in transfected cells resulted in no detectable changes in sodium current., Conclusions: Cardiac sodium channels are composed of alpha- (Nav1.1 or Nav1.5), beta(1)-, and beta(2)-subunits. Although beta(1)-subunits modulate cardiac sodium channel current, beta(2)-subunit function in heart may be limited to cell adhesion.