Biomedical subjects

Ilaria Rivolta

Publications and source records attributed to Ilaria Rivolta.

11 recordsLinked to original sources

PubMed2006

In silico assessment of Y1795C and Y1795H SCN5A mutations: implication for inherited arrhythmogenic syndromes.

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.

Action Potentials↗

PubMed2006

Computer simulation of wild-type and mutant human cardiac Na+ current.

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.

Action Potentials↗

PubMed2005

Cardiac histological substrate in patients with clinical phenotype of Brugada syndrome.

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.

Adult↗

PubMed2005

A novel form of short QT syndrome (SQT3) is caused by a mutation in the KCNJ2 gene.

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.

Action Potentials↗

PubMed2004

Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes.

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.

Amino Acid Sequence↗

PubMed2004

Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination.

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.

Amino Acid Motifs↗

PubMed2004

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer's disease.

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.

Aged↗

PubMed2003

Modulation of cardiac sodium channel gating by protein kinase A can be altered by disease-linked mutation.

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.

Cell Line↗

PubMed2003

Cardiac sodium channel diseases.

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.

Heart Diseases↗

PubMed2002

A novel SCN5A mutation associated with long QT-3: altered inactivation kinetics and channel dysfunction.

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.

Adolescent↗

PubMed2001

Secondary structure of the human cardiac Na+ channel C terminus: evidence for a role of helical structures in modulation of channel inactivation.

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.

Amino Acid Sequence↗