In myotonic dystrophy type 2 (DM2), an association has been reported between early and severe myotonia and recessive chloride channel (CLCN1) mutations. No DM2 cases have been described with sodium channel gene (SCN4A) mutations. The aim is to describe a DM2 patient with severe and early onset myotonia and co-occurrence of a novel missense mutation in SNC4A. A 26-year-old patient complaining of hand cramps and difficulty relaxing her hands after activity was evaluated at our department. Neurophysiology and genetic analysis for DM1, DM2, CLCN1 and SCN4A mutations were performed. Genetic testing was positive for DM2 (2650 CCTG repeat) and for a variant c.215C>T (p.Pro72Leu) in the SCN4A gene. The variation affects the cytoplasmic N terminus domain of Nav1.4, where mutations have never been reported. The biophysical properties of the mutant Nav1.4 channels were evaluated by whole-cell voltage-clamp analysis of heterologously expressed mutant channel in tsA201 cells. Electrophysiological studies of the P72L variant showed a hyperpolarizing shift (-5 mV) of the voltage dependence of activation that may increase cell excitability. This case suggests that SCN4A mutations may enhance the myotonic phenotype of DM2 patients and should be screened for atypical cases with severe myotonia.

Introduction and Aims: Clinical studies have demonstrated the risk of chronic kidney disease after the occurrence of an acute kidney injury (AKI).Experimental works indicate that AKI result in incomplete repair, persistent tubulointerstitial inflammation and fibrosis.Cysteine-rich protein 61 (Cyr61), a secreted matrix-associated protein, has been found to be up-regulated in the kidney ischemia reperfusion injury (IRI) animal model.The present study aimed to investigate the role of Cyr61 in the kidney after IRI.Methods: Using mouse unilateral IRI model, we analyzed gene and protein expression of Cyr61.We further investigated the effect of blockade of Cyr61 in unilateral IRI mice by treating polyclonal anti-Cyr61 antibody or non-specific IgG.In addition, we used proximal tubular epithelial (NRK-52E) cells for cell culture studies.Results: After IRI, kidney Cyr61 expression increased significantly in both mRNA and protein level.Immunofluorescence staining indicated Cyr61 was predominantly expressed in renal proximal tubular epithelial cells.This was supported by in vitro studies showing hypoxia condition stimulate Cyr61 expression in NRK-52E cells.Daily treatment with anti-Cyr61 antibody produced a decrease in the renal type 1 collagen, PAI-1, MCP-1, and IL-1 gene expression, as well as α-SMA protein production at day 14 after IRI.The degree of collagen fibril accumulation, evaluated by picrosirius red staining, and macrophage infiltration were both attenuated by the Cyr61 blockade on day 7 and 14.Concurrently, renal VEGF-A gene expression was enhanced and vessel density was more preserved at day 14 in the treatment group.Conclusions: Renal Cyr61 expression by tubular epithelial cells is enhanced after IRI.Our findings suggest that Cyr61 contributes to the renal inflammation, vascular rarefaction, and fibrosis after ischemic AKI.

Fluorescence correlation spectroscopy (FCS) was first developed for biophysical studies in analogy with photon scattering correlation spectroscopy. Although it is mainly devoted to the study of freely diffusing particles, FCS is actually able to discern between different kinds of motions, such as diffusion, anomalous diffusion, or drift motions. The frontier application of FCS nowadays is in medical studies both within cells and on the cell membranes, and in the investigation of single molecules in solid matrices. In this field, FCS originated also image correlation spectroscopy methods. The whole field can be unified under the name of fluorescence fluctuation spectroscopy (FFS). We present here a short review of the theoretical bases of FFS under a unified vision and discuss some applications to the study of dynamics of nanoparticles in cells and to the investigation of the photodynamics of immobilized dyes.

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.

Exposure to silicon nanoparticles (Si‐NPs) may occur in professional working conditions or for people undergoing a diagnostic screening test. Despite the fact that silicon is known as a non‐toxic material, in the first case the risk is mostly related to the inhalation of nanoparticles, thus the most likely route of entry is across the lung alveolar epithelium. In the case of diagnostic imaging, nanoparticles are usually injected intravenously and Si‐NPs could impact on the endothelial wall. In our study we investigated the interaction between selected Si‐based NPs and an epithelial lung cell line. Our data showed that, despite the overall silicon biocompatibility, however accurate studies of the potential toxicity induced by the nanostructure and engineered surface characteristics need to be accurately investigated before Si nanoparticles can be safely used for in vivo applications as bio‐imaging, cell staining and drug delivery.

Iron oxide Nanoparticles represents promising nanocarrier for magnetic resonance imaging (MRI), targeted drug and gene delivery. In our study we investigated the interaction between lung alveolar epithelial cells and iron oxide NPs coated with L‐Dihydroxyphenylalanina (L‐Dopa)‐TRITC. Our data suggest that particles crossed the plasma membrane with an energy‐dependent process.

Solid Lipid Nanoparticles (SLN) are well defined nanocarriers that reach the close proximity of the cell membrane and bring their content into the cell. We studied the distribution of the SLN within the cell cytoplasm trying to understand the internalization mechanism of these nanoparticles. Alveolar epithelial lung and mouse fibroblast cells were incubated with SLN. Our data suggest that SLN may be a compatible tool but still deserve further investigation.

The EC BONSAI Project intends to develop multifunctional nanoparticles with tailored optical and/or magnetic properties for visualizing complex cellular structures (in tissues and organs), receptors, tumor cells and masses. In this framework, here we will report on recent advances on the preparation of luminescent silicon nanoparticles, magnetic iron oxide nanoparticles and Au nanorods for bio-imaging applications.

The overall objective of BONSAI project (FP6, EC) is the development of ultrasensitive bio-imaging techniques based on novel multifunctional nanoparticles (NPs) with tailored optical and magnetic properties for visualizing complex cellular structures (in tissues and organs), receptors, tumour cells and masses. An important aspect to take into consideration involves the cellular responses to the impact of exogenous structures represented by NPs themselves. As a preliminary sign of reaction, we start to investigate the basic cytotoxicity.

Vecchietti S, Grandi E, Severi S, Rivolta I, Napolitano C, Priori SG, Cavalcanti S. In silico assessment of Y1795C and Y1795H SCN5A mutations: implication for inherited arrhythmogenic syndromes. Am J Physiol Heart Circ Physiol 292: H56–H65, 2007. First published September 15, 2006; doi:10.1152/ajpheart.00270.2006.—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 (INa) 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 (ITO) and the ratio of slow delayed rectifier potassium (IKs) to rapid delayed rectifier current (IKr). Administration of flecainide and mexiletine was simulated by appropriately modifying INa, calcium current (ICa), ITO, and IKr. 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.

Background: The role of structural heart disease and sodium channel dysfunction in the induction of electrical instability in Brugada syndrome is still debated. Methods and Results: We studied 18 consecutive patients (15 males, 3 females; mean age 42.0±12.4 years) with clinical phenotype of Brugada syndrome and normal cardiac structure and function on noninvasive examinations. Clinical presentation was ventricular fibrillation in 7 patients, sustained polymorphic ventricular tachycardia in 7, and syncope in 4. All patients underwent cardiac catheterization, coronary and ventricular angiography, biventricular endomyocardial biopsy, and DNA screening of the SCN5A gene. Biopsy samples were processed for histology, electron microscopy, and molecular screening for viral genomes. Microaneurysms were detected in the right ventricle in 7 patients and also in the left ventricle in 4 of them. Histology showed a prevalent or localized right ventricular myocarditis in 14 patients, with detectable viral genomes in 4; right ventricular cardiomyopathy in 1 patient; and cardiomyopathic changes in 3. Genetic studies identified 4 carriers of SCN5A gene mutations that cause in vitro abnormal function of mutant proteins. In these patients, myocyte cytoplasm degeneration was present at histology, whereas terminal dUTP nick end-labeling assay showed a significant increase of apoptotic myocytes in right and left ventricle versus normal controls (P=0.014 and P=0.013, respectively). Conclusions: Despite an apparently normal heart at noninvasive evaluation, endomyocardial biopsy detected structural alterations in all 18 patients with Brugada syndrome. Mutations in the SCN5A gene, identified in 4 of the 18 patients, may have induced concealed structural abnormalities of myocardiocytes that accounted for paroxysmal arrhythmic manifestations.

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.