The Role of MicroRNA Regulation of Cardiac Ion Channel in Arrhythmia

La fibrillation auriculaire (FA) est le trouble du rythme le plus fréquemment observé en pratique clinique. Elle constitue un risque important de morbi-mortalité. Le traitement de la FA reste un défi majeur en lien avec les nombreux effets secondaires associés aux approches thérapeutiques actuelles. Dans ce contexte, une meilleure compréhension des mécanismes sous-jacents à la FA est essentielle pour le développement de nouvelles thérapies offrant un meilleur rapport bénéfice/risque pour les patients. La FA est caractérisée par i) un remodelage électrique délétère associé le plus souvent ii) à un remodelage structurel du myocarde favorisant la récurrence et le maintien de l’arythmie. La diminution de la période réfractaire effective au sein du tissu auriculaire est un élément clef du remodelage électrique. Le remodelage structurel, quant à lui, se manifeste principalement par une fibrose tissulaire qui altère la propagation de l’influx électrique dans les oreillettes. Les mécanismes moléculaires impliqués dans la mise en place de ces deux substrats restent mal connus. Récemment, le rôle des microARNs (miARNs) a été pointé du doigt dans de nombreuses pathologies notamment cardiaques. Dans ce contexte les objectifs principaux de ce travail ont été i) d'acquérir une compréhension approfondie du rôle des miARNs dans la régulation de l’expression des canaux ioniques et ii) de mieux comprendre le rôle de ces molécules dans l’installation d’un substrat favorable a la FA. Nous avons, dans un premier temps, effectué une analyse bio-informatique combinée à des approches expérimentales spécifiques afin d’identifier clairement les miARNs démontrant un fort potentiel de régulation des gènes codant pour l’expression des canaux ioniques cardiaques humains. Nous avons identifié un nombre limité de miARNs cardiaques qui possédaient ces propriétés. Sur la base de ces résultats, nous avons démontré que l’altération de l'expression des canaux ioniques, observée dans diverse maladies cardiaques (par exemple, les cardiomyopathies, l’ischémie myocardique, et la fibrillation auriculaire), peut être soumise à ces miARNs suggérant leur implication dans l’arythmogénèse. La régulation du courant potassique IK1 est un facteur déterminant du remodelage électrique auriculaire associée à la FA. Les mécanismes moléculaires sous-jacents sont peu connus. Nous avons émis l’hypothèse que l'altération de l’expression des miARNs soit corrélée à l’augmentation de l’expression d’IK1 dans la FA. Nous avons constaté que l’expression de miR-26 est réduite dans la FA et qu’elle régule IK1 en modulant l’expression de sa sous-unité Kir2.1. Nous avons démontré que miR-26 est sous la répression transcriptionnelle du facteur nucléaire des lymphocytes T activés (NFAT) et que l’activité accrue de NFATc3/c4, aboutit à une expression réduite de miR-26. En conséquence IK1 augmente lors de la FA. Nous avons enfin démontré que l’interférence in vivo de miR-26 influence la susceptibilité à la FA en régulant IK1, confirmant le rôle prépondérant de miR-26 dans le remodelage auriculaire électrique. La fibrose auriculaire est un constituant majeur du remodelage structurel associé à la FA, impliquant l'activation des fibroblastes et l’influx cellulaire du Ca2 +. Nous avons cherché à déterminer i) si le canal perméable au Ca2+, TRPC3, jouait un rôle dans la fibrose auriculaire en favorisant l'activation des fibroblastes et ii) étudié le rôle potentiel des miARNs dans ce contexte. Nous avons démontré que les canaux TRPC3 favorisent l’influx du Ca2 +, activant la signalisation Ca2 +-dépendante ERK et en conséquence activent la prolifération des fibroblastes. Nous avons également démontré que l’expression du TRPC3 est augmentée dans la FA et que le blocage in vivo de TRPC3 empêche le développement de substrats reliés à la FA. Nous avons par ailleurs validé que miR-26 régule les canaux TRPC3 en diminuant leur expression dans les fibroblastes. Enfin, nous avons montré que l'expression réduite du miR-26 est également due à l’activité augmentée de NFATc3/c4 dans les fibroblastes, expliquant ainsi l’augmentation de TRPC3 lors de la FA, confirmant la contribution de miR-26 dans le processus de remodelage structurel lié à la FA. En conclusion, nos résultats mettent en évidence l'importance des miARNs dans la régulation des canaux ioniques cardiaques. Notamment, miR-26 joue un rôle important dans le remodelage électrique et structurel associé à la FA et ce, en régulant IK1 et l’expression du canal TRPC3. Notre étude démasque ainsi un mécanisme moléculaire de contrôle de la FA innovateur associant des miARNs. miR-26 en particulier représente apres ces travaux une nouvelle cible thérapeutique prometteuse pour traiter la FA.

Granulocyte Colony-stimulating Factor Reduces Mortality by Suppressing Ventricular Arrhythmias in Acute Phase of Myocardial Infarction in Rats

Our aim was to evaluate the effects of granulocyte colony-stimulating factor (G-CSF) on early cardiac arrhythmias after myocardial infarction (MI) and the impact on survival. Male Wistar rats received repeated doses of 50 mu g/kg G-CSF (MI-GCSF group) or vehicle (MI group) at 7, 3, and 1 days before surgery. MI was induced by permanent occlusion of left corollary artery. The electrocardiogram was obtained before occlusion and then for 30 minutes after surgery. Events and duration of ventricular arrhythmias were analyzed. The levels of connexin43 (Cx43) were measured by Western blot immediately before MI production. Survival was significantly increased in MI-GCSF pretreated group (74% versus 52.0% MI. P < 0.05). G-CSF pretreatment also significantly reduced the ventricular premature beats when compared with the untreated-MI group (201 +/- 47 versus 679 +/- 117, P < 0.05). The number and the duration of ventricular tachycardia were smaller in the MI-G-CSF group, as well as the number of ventricular fibrillation episodes (10% versus 69% in NIL P < 0.05). Cx43 levels were significantly increased by G-CSF treatment (1.27 +/- 0.13 versus 0.86 +/- 0.11; P < 0.05). The MI size 24 hours after occlusion was reduced by G-CSF pretreatment (36 +/- 3% versus 44 +/- 2% of left ventricle in MI group; P < 0.05). The increase of Cx43 expression in the heart may explain the reduced incidence in ventricular arrhythmias in the early phases after coronary artery occlusion in rats, thus increasing survival after MI.

Sudden Cardiac Death in Brazil: Study Based on Physicians' Perceptions of the Public Health Care System

Background: There are no available statistical data about sudden cardiac death in Brazil. Therefore, this study has been conducted to evaluate the incidence of sudden cardiac death in our population and its implications. Methods: The research methodology was based on Thurstone's Law of Comparative Judgment, whose premise is that the more an A stimulus differs from a B stimulus, the greater will be the number of people who will perceive this difference. This technique allows an estimation of actual occurrences from subjective perceptions, when compared to official statistics. Data were collected through telephone interviews conducted with Primary and Secondary Care physicians of the Public Health Service in the Metropolitan Area of Sao Paulo (MASP). Results: In the period from October 19, 2009, to October 28, 2009, 196 interviews were conducted. The incidence of 21,270 cases of sudden cardiac death per year was estimated by linear regression analysis of the physicians responses and data from the Mortality Information System of the Brazilian Ministry of Health, with the following correlation and determination coefficients: r = 0.98 and r2= 0.95 (95% confidence interval 0.81.0, P < 0.05). The lack of waiting list for specialized care and socioadministrative problems were considered the main barriers to tertiary care access. Conclusions: The incidence of sudden cardiac death in the MASP is high, and it was estimated as being higher than all other causes of deaths; the extrapolation technique based on the physicians perceptions was validated; and the most important bureaucratic barriers to patient referral to tertiary care have been identified. (PACE 2012; 35:13261331)

Computational Modeling of Cardiac Excitation-Contraction Coupling in Physiological and Pathological Conditions

The cardiomyocyte is a complex biological system where many mechanisms interact non-linearly to regulate the coupling between electrical excitation and mechanical contraction. For this reason, the development of mathematical models is fundamental in the field of cardiac electrophysiology, where the use of computational tools has become complementary to the classical experimentation. My doctoral research has been focusing on the development of such models for investigating the regulation of ventricular excitation-contraction coupling at the single cell level. In particular, the following researches are presented in this thesis: 1) Study of the unexpected deleterious effect of a Na channel blocker on a long QT syndrome type 3 patient. Experimental results were used to tune a Na current model that recapitulates the effect of the mutation and the treatment, in order to investigate how these influence the human action potential. Our research suggested that the analysis of the clinical phenotype is not sufficient for recommending drugs to patients carrying mutations with undefined electrophysiological properties. 2) Development of a model of L-type Ca channel inactivation in rabbit myocytes to faithfully reproduce the relative roles of voltage- and Ca-dependent inactivation. The model was applied to the analysis of Ca current inactivation kinetics during normal and abnormal repolarization, and predicts arrhythmogenic activity when inhibiting Ca-dependent inactivation, which is the predominant mechanism in physiological conditions. 3) Analysis of the arrhythmogenic consequences of the crosstalk between β-adrenergic and Ca-calmodulin dependent protein kinase signaling pathways. The descriptions of the two regulatory mechanisms, both enhanced in heart failure, were integrated into a novel murine action potential model to investigate how they concur to the development of cardiac arrhythmias. These studies show how mathematical modeling is suitable to provide new insights into the mechanisms underlying cardiac excitation-contraction coupling and arrhythmogenesis.

Computational Modelling of Cardiac Electrophysiology: from Cell to Bedside

Heart diseases are the leading cause of death worldwide, both for men and women. However, the ionic mechanisms underlying many cardiac arrhythmias and genetic disorders are not completely understood, thus leading to a limited efficacy of the current available therapies and leaving many open questions for cardiac electrophysiologists. On the other hand, experimental data availability is still a great issue in this field: most of the experiments are performed in vitro and/or using animal models (e.g. rabbit, dog and mouse), even when the final aim is to better understand the electrical behaviour of in vivo human heart either in physiological or pathological conditions. Computational modelling constitutes a primary tool in cardiac electrophysiology: in silico simulations, based on the available experimental data, may help to understand the electrical properties of the heart and the ionic mechanisms underlying a specific phenomenon. Once validated, mathematical models can be used for making predictions and testing hypotheses, thus suggesting potential therapeutic targets. This PhD thesis aims to apply computational cardiac modelling of human single cell action potential (AP) to three clinical scenarios, in order to gain new insights into the ionic mechanisms involved in the electrophysiological changes observed in vitro and/or in vivo. The first context is blood electrolyte variations, which may occur in patients due to different pathologies and/or therapies. In particular, we focused on extracellular Ca2+ and its effect on the AP duration (APD). The second context is haemodialysis (HD) therapy: in addition to blood electrolyte variations, patients undergo a lot of other different changes during HD, e.g. heart rate, cell volume, pH, and sympatho-vagal balance. The third context is human hypertrophic cardiomyopathy (HCM), a genetic disorder characterised by an increased arrhythmic risk, and still lacking a specific pharmacological treatment.

Cardiac sodium channel Na(v)1.5 and interacting proteins: Physiology and pathophysiology

The cardiac voltage-gated Na(+) channel Na(v)1.5 generates the cardiac Na(+) current (INa). Mutations in SCN5A, the gene encoding Na(v)1.5, have been linked to many cardiac phenotypes, including the congenital and acquired long QT syndrome, Brugada syndrome, conduction slowing, sick sinus syndrome, atrial fibrillation, and dilated cardiomyopathy. The mutations in SCN5A define a sub-group of Na(v)1.5/SCN5A-related phenotypes among cardiac genetic channelopathies. Several research groups have proposed that Na(v)1.5 may be part of multi-protein complexes composed of Na(v)1.5-interacting proteins which regulate channel expression and function. The genes encoding these regulatory proteins have also been found to be mutated in patients with inherited forms of cardiac arrhythmias. The proteins that associate with Na(v)1.5 may be classified as (1) anchoring/adaptor proteins, (2) enzymes interacting with and modifying the channel, and (3) proteins modulating the biophysical properties of Na(v)1.5 upon binding. The aim of this article is to review these Na(v)1.5 partner proteins and to discuss how they may regulate the channel's biology and function. These recent investigations have revealed that the expression level, cellular localization, and activity of Na(v)1.5 are finely regulated by complex molecular and cellular mechanisms that we are only beginning to understand.

Heart failure in two West Highland white terriers caused by congenital angiomatous cardiac anomaly

The paper describes the clinical and pathological characteristics of an unusual cystic congenital cardiac anomaly that caused clinical signs of congestive heart failure, respiratory distress and cardiac arrhythmias in two West Highland white terrier puppies. In both dogs a definitive diagnosis was made postmortem.

Sudden cardiac death in individuals with normal hearts: an update

Sudden death (SD) is a tragic event and a world-wide health problem. Every year, near 4-5 million people experience SD. SD is defined as the death occurred in 1h after the onset of symptoms in a person without previous signs of fatality. It can be named "recovered SD" when the case received medical attention, cardiac reanimation effective defibrillation or both, surviving the fatal arrhythmia. Cardiac channelopathies are a group of diseases characterized by abnormal ion channel function due to genetic mutations in ion channel genes, providing increased susceptibility to develop cardiac arrhythmias and SD. Usually the death occurs before 40 years of age and in the autopsy the heart is normal. In this review we discuss the main cardiac channelopathies involved in sudden cardiac death along with current management of cases and family members that have experienced such tragic event.

Late cardiac sodium current can be assessed using automated patch-clamp

The cardiac late Na (+) current is generated by a small fraction of voltage-dependent Na (+) channels that undergo a conformational change to a burst-gating mode, with repeated openings and closures during the action potential (AP) plateau. Its magnitude can be augmented by inactivation-defective mutations, myocardial ischemia, or prolonged exposure to chemical compounds leading to drug-induced (di)-long QT syndrome, and results in an increased susceptibility to cardiac arrhythmias. Using CytoPatch™ 2 automated patch-clamp equipment, we performed whole-cell recordings in HEK293 cells stably expressing human Nav1.5, and measured the late Na (+) component as average current over the last 100 ms of 300 ms depolarizing pulses to -10 mV from a holding potential of -100 mV, with a repetition frequency of 0.33 Hz. Averaged values in different steady-state experimental conditions were further corrected by the subtraction of current average during the application of tetrodotoxin (TTX) 30 μM. We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na (+) current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% ( n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% ( n = 28) of initial values. Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p. In human iPSC-derived cardiomyocytes, veratridine 1 μM reversibly increased APD90 2.12 ± 0.41-fold (mean ± SEM, n = 6). This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.

Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia.

Long QT syndrome (LQT) is an autosomal dominant disorder that can cause sudden death from cardiac arrhythmias. We recently discovered that mutations in HERG, a K+-channel gene, cause chromosome 7-linked LQT. Heterologous expression of HERG in Xenopus oocytes revealed that HERG current was similar to a well-characterized cardiac delayed rectifier K+ current, IKr, and led to the hypothesis that mutations in HERG reduced IKr, causing prolonged myocellular action potentials. To define the mechanism of LQT, we injected oocytes with mutant HERG complementary RNAs, either singly or in combination with wild-type complementary RNA. Some mutations caused loss of function, whereas others caused dominant negative suppression of HERG function. These mutations are predicted to cause a spectrum of diminished IKr and delayed ventricular repolarization, consistent with the prolonged QT interval observed in individuals with LQT.

Iontophoresis for modulation of cardiac drug delivery in dogs.

Cardiac arrhythmias are a frequent cause of death and morbidity. Conventional antiarrhythmia therapy involving oral or intravenous medication is often ineffective and complicated by drug-associated side effects. Previous studies from our laboratory have demonstrated the advantages of cardiac drug-polymer implants for enhanced efficacy for cardiac arrhythmia therapy compared with conventional administration. However, these studies were based on systems that deliver drugs at a fixed release rate. Modulation of the drug delivery rate has the advantage of regulating the amount of the drug delivered depending upon the disease state of the patient. We hypothesized that iontophoresis could be used to modulate cardiac drug delivery. In this study, we report our investigations of a cardiac drug implant in dogs that is capable of iontophoretic modulation of the administration of the antiarrhythmic agent sotalol. We used a heterogeneous cation-exchange membrane (HCM) as an electrically sensitive and highly efficient rate-limiting barrier on the cardiac-contacting surface of the implant. Thus, electric current is passed only through the HCM and not the myocardium. The iontophoretic cardiac implant demonstrated in vitro drug release rates that were responsive to current modulation. In vivo results in dogs have confirmed that iontophoresis resulted in regional coronary enhancement of sotalol levels with current-responsive increases in drug concentrations. We also observed acute current-dependent changes in ventricular effective refractory periods reflecting sotalol-induced refractoriness due to regional drug administration. In 30-day dog experiments, iontophoretic cardiac implants demonstrated robust sustained function and reproducible modulation of drug delivery kinetics.

Prediction of Sudden Cardiac Arrest for Patients with Congestive Heart Failure

Thesis (Ph.D.)--University of Washington, 2016-07

Rapidly Switching Multidirectional Defibrillation: Reversal Of Ventricular Fibrillation With Lower Energy Shocks.

Cardiac arrest after open surgery has an incidence of approximately 3%, of which more than 50% of the cases are due to ventricular fibrillation. Electrical defibrillation is the most effective therapy for terminating cardiac arrhythmias associated with unstable hemodynamics. The excitation threshold of myocardial microstructures is lower when external electrical fields are applied in the longitudinal direction with respect to the major axis of cells. However, in the heart, cell bundles are disposed in several directions. Improved myocardial excitation and defibrillation have been achieved by applying shocks in multiple directions via intracardiac leads, but the results are controversial when the electrodes are not located within the cardiac chambers. This study was designed to test whether rapidly switching shock delivery in 3 directions could increase the efficiency of direct defibrillation. A multidirectional defibrillator and paddles bearing 3 electrodes each were developed and used in vivo for the reversal of electrically induced ventricular fibrillation in an anesthetized open-chest swine model. Direct defibrillation was performed by unidirectional and multidirectional shocks applied in an alternating fashion. Survival analysis was used to estimate the relationship between the probability of defibrillation and the shock energy. Compared with shock delivery in a single direction in the same animal population, the shock energy required for multidirectional defibrillation was 20% to 30% lower (P < .05) within a wide range of success probabilities. Rapidly switching multidirectional shock delivery required lower shock energy for ventricular fibrillation termination and may be a safer alternative for restoring cardiac sinus rhythm.

Intracardiac Cavopulmonary Connection in Patients with Univentricular Heart Using Intra-atrial Lateral Tunnel and Intra-atrial Conduit Techniques

Background: In this study, we analyzed the time course of hemodynamic efficiency and follow-up in Fontan candidates who underwent the bidirectional Glenn procedure for staged intracardiac cavopulmonary connection (ICPC). Methods: Between 1991 and 2008, 52 patients with univentricular heart (mean age, 3.3 years; range, 2-8 years; 27 female patients [51.9%]) underwent ICPC. The cardiac malformations were as follows: tricuspid atresia, 25 cases (48.0%); common ventricle, 16 cases (30.7%); and pulmonary atresia with intact ventricular septum, 11 cases (21.1%). The intracardiac cavopulmonary procedure was indicated for all 52 cases. In 42 patients (80.7%), an intra-atrial lateral tunnel was constructed with a bovine pericardium patch. In the last 10 consecutive cases (19.3%), we performed a modified surgical technique in which we implanted an intra-atrial corrugated bovine pericardium tube sutured around the superior and inferior vena cava ostium. In all cases, a 4-mm fenestration was made to reduce the intratunnel pressure. All 52 patients had previously undergone a Glenn operation. Results: There were 2 hospital deaths (3.8%) and no recorded late deaths. During the follow-up, all patients were medicated with antiplatelet drugs. To evaluate the hemodynamic performance, we used Doppler echocardiography, computed tomography, and magnetic nuclear resonance studies. There were no prosthesis thromboses during this follow-up period. To evaluate cardiac arrhythmias, we conducted a Holter study. The last 10 patients with an intra-atrial conduit (IAC) presented with sinus rhythm and no arrhythmias during the last 4 years. The 50 surviving patients (96.1%) have been followed up for 6 to 204 months; all these patients are free of reoperation. Conclusion: The Glenn operation, which is performed at an early age, prepares the pulmonary bed to receive the ICPC. The midterm results of the intracardiac Fontan procedure seem to be good. The modified surgical procedure (IAC) can be a good alternative technique to the Fontan procedure in suitable patients.

Study of the Extent of the Information of Cardiologists from Sao Paulo City, Brazil, Regarding a Low-Prevalence Entity: Brugada Syndrome

Objective: To determine the degree of knowledge that cardiologists from Sao Paulo, Brazil, have regarding a low-prevalent entity associated with a high rate of sudden death-Brugada syndrome. Methods: Two hundred forty-four cardiologists were interviewed by an instrument divided in two parts: in the first, we recorded gender, age, and data related to academic profile. The second-answered only by the professionals that manifested having some degree of knowledge on the syndrome-had 28 questions that evaluated their knowledge. The answers were spontaneous and they did not have a chance to consult. We used uni- and multivariate analysis on the average percentage of right and wrong answers, and the influence of the academic profile. Results: The predominant gender was the male gender (61.1%), the average age was 44.32 +/- 10.83 years, 40% with more than 20 years after obtaining their degree, 44% were educated in public institutions, 69% had a residency in cardiology, 20% had overseas practice, 12% had postdegree, 41% were linked to an educational institution, 24% with publication(s) in an indexed journal, 17.2% were authors of chapters in books, 2.5% had edited books, and 10% were linked to the Brazilian Society of Cardiac Arrhythmias. The average percentage of right answers was 45.7%. Conclusion: The sample studied revealed a little knowledge on the entity. A residency in cardiology was the factor of greater significance in the percentage of right answers. Other significant factors were the link of the interviewed person to an educational institution, or the Brazilian Society of Cardiac Arrhythmias, and having a specialist degree.