Microglia-Mediated Inflammation and Neural Stem Cell Differentiation in Alzheimer’s Disease: Possible Therapeutic Role of KV1.3 Channel Blockade

Increase of deposits of amyloid beta peptides in the extracellular matrix is landmark during Alzheimer's Disease (AD) due to the imbalance in the production vs. clearance. This accumulation of amyloid beta deposits triggers microglial activation. Microglia plays a dual role in AD, a protective role by clearing the deposits of amyloid beta peptides increasing the phagocytic response (CD163, IGF-1 or BDNF) and a cytotoxic role, releasing free radicals (ROS or NO) and proinflammatory cytokines (TNF-alpha, IL-1beta) in response to reactive gliosis activated by the amyloid beta aggregates. Microglia activation correlated with an increase KV1.3 channels expression, protein levels and current density. Several studies highlight the importance of KV1.3 in the activation of inflammatory response and inhibition of neural progenitor cell proliferation and neuronal differentiation. However, little is known about the pathways of this activation in neural stem cells differentiation and proliferation and the role in amyloid beta accumulation. In recent studies using in vitro cells derived from mice models, it has been demonstrated that KV1.3 blockers inhibit microglia-mediated neurotoxicity in culture reducing the expression and production of the pro-inflammatory cytokines IL-1beta and TNF-alpha through the NF-kB and p38MAPK pathway. Overall, we conclude that KV1.3 blockers change the course of AD development, reducing microglial cytotoxic activation and increasing neural stem cell differentiation. However, further investigations are needed to establish the specific pathway and to validate the use of this blocker as therapeutic treatment in Alzheimer patients.This work was supported by a grant from the MICINN (PID2020-118814RB-I00), the Government of the Autonomous Community of the Basque Country (IT1165-19 and KK-2020/00110), and the Spanish Ministry of Science and Innovation (RTI2018-097839-B-100 to AV)

Adult and Developing Zebrafish as Suitable Models for Cardiac Electrophysiology and Pathology in Research and Industry

The electrophysiological behavior of the zebrafish heart is very similar to that of the human heart. In fact, most of the genes that codify the channels and regulatory proteins required for human cardiac function have their orthologs in the zebrafish. The high fecundity, small size, and easy handling make the zebrafish embryos/larvae an interesting candidate to perform whole animal experiments within a plate, offering a reliable and low-cost alternative to replace rodents and larger mammals for the study of cardiac physiology and pathology. The employment of zebrafish embryos/larvae has widened from basic science to industry, being of particular interest for pharmacology studies, since the zebrafish embryo/larva is able to recapitulate a complete and integrated view of cardiac physiology, missed in cell culture. As in the human heart, I-Kr is the dominant repolarizing current and it is functional as early as 48 h post fertilization. Finally, genome editing techniques such as CRISPR/Cas9 facilitate the humanization of zebrafish embryos/larvae. These techniques allow one to replace zebrafish genes by their human orthologs, making humanized zebrafish embryos/larvae the most promising in vitro model, since it allows the recreation of human-organ-like environment, which is especially necessary in cardiac studies due to the implication of dynamic factors, electrical communication, and the paracrine signals in cardiac functionThis work was supported by grants from the Gobierno Vasco PIBA2018-58 and GIC18/150. MH-V was supported by the Government of Extremadura (Grant No. TA18052

Electrical features of the diabetic myocardium. Arrhythmic and cardiovascular Safety considerations in diabetes

[EN] Diabetes is a chronic metabolic disease characterized by hyperglycemia in the absence of treatment. Among the diabetes-associated complications, cardiovascular disease is the major cause of mortality and morbidity in diabetic patients. Diabetes causes a complex myocardial dysfunction, referred as diabetic cardiomyopathy, which even in the absence of other cardiac risk factors results in abnormal diastolic and systolic function. Besides mechanical abnormalities, altered electrical function is another major feature of the diabetic myocardium. Both type 1 and type 2 diabetic patients often show cardiac electrical remodeling, mainly a prolonged ventricular repolarization visible in the electrocardiogram as a lengthening of the QT interval duration. The underlying mechanisms at the cellular level involve alterations on the expression and activity of several cardiac ion channels and their associated regulatory proteins. Consequent changes in sodium, calcium and potassium currents collectively lead to a delay in repolarization that can increase the risk of developing life-threatening ventricular arrhythmias and sudden death. QT duration correlates strongly with the risk of developing torsade de pointes, a form of ventricular tachycardia that can degenerate into ventricular fibrillation. Therefore, QT prolongation is a qualitative marker of proarrhythmic risk, and analysis of ventricular repolarization is therefore required for the approval of new drugs. To that end, the Thorough QT/QTc analysis evaluates QT interval prolongation to assess potential proarrhythmic effects. In addition, since diabetic patients have a higher risk to die from cardiovascular causes than individuals without diabetes, cardiovascular safety of the new antidiabetic drugs must be carefully evaluated in type 2 diabetic patients. These cardiovascular outcome trials reveal that some glucose-lowering drugs actually reduce cardiovascular risk. The mechanism of cardioprotection might involve a reduction of the risk of developing arrhythmia.This work was supported by grants from the Basque Government: Govierno Vasco PIBA2018-58, GIC18/150 and IT1196-19. AA received a predoctoral fellowship from the Basque Government

Molecular and Electrophysiological Role of Diabetes-Associated Circulating Inflammatory Factors in Cardiac Arrhythmia Remodeling in a Metabolic-Induced Model of Type 2 Diabetic Rat

Background: Diabetic patients have prolonged cardiac repolarization and higher risk of arrhythmia. Besides, diabetes activates the innate immune system, resulting in higher levels of plasmatic cytokines, which are described to prolong ventricular repolarization. Methods: We characterize a metabolic model of type 2 diabetes (T2D) with prolonged cardiac repolarization. Sprague-Dawley rats were fed on a high-fat diet (45% Kcal from fat) for 6 weeks, and a low dose of streptozotozin intraperitoneally injected at week 2. Body weight and fasting blood glucose were measured and electrocardiograms of conscious animals were recorded weekly. Plasmatic lipid profile, insulin, cytokines, and arrhythmia susceptibility were determined at the end of the experimental period. Outward K+ currents and action potentials were recorded in isolated ventricular myocytes by patch-clamp. Results: T2D animals showed insulin resistance, hyperglycemia, and elevated levels of plasma cholesterol, triglycerides, TNFα, and IL-1b. They also developed bradycardia and prolonged QTc-interval duration that resulted in increased susceptibility to severe ventricular tachycardia under cardiac challenge. Action potential duration (APD) was prolonged in control cardiomyocytes incubated 24 h with plasma isolated from diabetic rats. However, adding TNFα and IL-1b receptor blockers to the serum of diabetic animals prevented the increased APD. Conclusions: The elevation of the circulating levels of TNFα and IL-1b are responsible for impaired ventricular repolarization and higher susceptibility to cardiac arrhythmia in our metabolic model of T2D.This work was supported by grants from the Gobierno Vasco PIBA2018-58 and GIC18/150 and MICINN PID2020-118814RB-I00. JZ-A is a predoctoral Fellow of the UPV/EHU and had a STSM from the EU COST Action CA16225

Diabetesa gaixotasun inflamatorio gisa

Diabetesa gaixotasun kronikoa da, prebalentzia handikoa, zeinetan pankreak ez duen behar adina intsulina sortzen edo organismoak sortzen duen intsulina ez duen eraginkortasunez erabiltzen. Kontrolatu gabeko diabetesaren ondorioa hipergluzemia da, denborarekin organo eta sistema asko larriki kaltetzen dituena, nerbioak eta odol-hodiak batez ere. 2 motako diabetesa (DT2) diabetes motarik ohikoena da, non ehunek ez dioten intsulinari eraginkortasunez erantzuten. Egoera horri intsulinarekiko erresistentzia deritzo. Obesitatea, munduko asaldura metabolikorik ohikoena da, eta DT2 garatzeko arrisku-faktore nagusienetako bat. Obesitatea diabetesarekin lotzen duten mekanismoak oraindik argi ez badaude ere, azken urteotan hanturak indarra hartu du funtsezko faktore gisa. Paziente diabetikoek zitokina proinflamatorioen maila altuak dituzte, eta horrek gradu baxuko hantura kroniko orokortua eragiten du, gantz-ehunean sortzen duena. Hantura kroniko horrek alterazioak eragiten ditu intsulinaren seinaleztapen-jauzian eta, ondorioz, intsulinarekiko erresistentzia eta hipergluzemia dakar. Gaur egungo tratamenduaren helburu nagusia gluzemia normalizatzea da, farmako hipogluzemiatzaileen bidez. Hala ere, sistema immunearen modulazioak, TNF-α eta IL-b bezalako zitokinen blokeoaren bidez, medikamentuak garatzeko aukera berri interesgarriak irekitzen ditu.; Diabetes is a chronic, highly prevalent disease that occurs when the pancreas does not produce enough insulin or does not effectively use the insulin it produces. The effect of uncontrolled diabetes is hyperglycemia, which over time severely damages many organs and systems, especially nerves and blood vessels. Type 2 diabetes is the most common type of diabetes, in which the tissues do not respond effectively to insulin, a condition known as insulin resistance. Obesity is the most common metabolic disorder in the world and is one of the main risk factors for the development of T2DM. Although the mechanisms linking obesity and diabetes are still unclear, inflammation has emerged in the last years as a key factor. Diabetic patients have high levels of pro-inflammatory cytokines, originating in adipose tissue, leading to chronic generalized low-grade inflammation. This chronic inflammation causes alterations in insulin signaling, leading to insulin resistance and hyperglycemia. Although the main goal of current treatment is to normalize glycaemia withhypoglycemic drugs, modulation of the immune system, by blocking cytokines such as TNF-α or IL-1β, is an interesting therapeutic target that may open up new possibilities for drug development

Kv1.3 Channel Blockade Improves Inflammatory Profile, Reduces Cardiac Electrical Remodeling, and Prevents Arrhythmia in Type 2 Diabetic Rats

Purpose Kv1.3 channel regulates the activity of lymphocytes, macrophages, or adipose tissue and its blockade reduces inflammatory cytokine secretion and improves insulin sensitivity in animals with metabolic syndrome and in genetically obese mice. Thus, Kv1.3 blockade could be a strategy for the treatment of type 2 diabetes. Elevated circulating levels of TNF alpha and IL-1b mediate the higher susceptibility to cardiac arrhythmia in type 2 diabetic rats. We hypothesized that Kv1.3 channel blockade with the psoralen PAP1 could have immunomodulatory properties that prevent QTc prolongation and reduce the risk of arrhythmia in type 2 diabetic rats. Methods Type 2 diabetes was induced to Sprague-Dawley rats by high-fat diet and streptozotocin injection. Diabetic animals were untreated, treated with metformin, or treated with PAP1 for 4 weeks. Plasma glucose, insulin, cholesterol, triglycerides, and cytokine levels were measured using commercial kits. ECG were recorded weekly, and an arrhythmia-inducing protocol was performed at the end of the experimental period. Action potentials were recorded in isolated ventricular cardiomyocytes. Results In diabetic animals, PAP1 normalized glycaemia, insulin resistance, adiposity, and lipid profile. In addition, PAP1 prevented the diabetes-induced repolarization defects through reducing the secretion of the inflammatory cytokines IL-10, IL-12p70, GM-CSF, IFN gamma, and TNF alpha. Moreover, compared to diabetic untreated and metformin-treated animals, those treated with PAP1 had the lowest risk of developing the life-threatening arrhythmia Torsade de Pointes under cardiac challenge. Conclusion Kv1.3 inhibition improves diabetes and diabetes-associated low-grade inflammation and cardiac electrical remodeling, resulting in more protection against cardiac arrhythmia compared to metformin.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by grants from the Gobierno Vasco (PIBA2018-58 and GIC18/150), and MICINN (PID2020-118814RB-I00). V. F.-L. is a predoctoral Fellow of the UPV/EHU and A. A. is a predoctoral Fellow of the Gobierno Vasco. J.Z.-A. is a predoctoral Fellow of the UPV/EHU and had a STSM from the EU-CARDIOPROTECTION COST Action CA16225

Methylmercury Poisoning Induces Cardiac Electrical Remodeling and Increases Arrhythmia Susceptibility and Mortality

This study aims to investigate the cardiac electrical remodeling associated with intoxication by methylmercury (MeHg). We evaluated the chronic effects of MeHg on in vivo electrocardiograms and on ex vivo action potentials and depolarizing (ICa-L) and repolarizing (Ito) currents. The acute effect of MeHg was evaluated on HEK293 cells expressing human ERG, Kv4.3 and KCNQ1/KCNE1 channels. Chronic MeHg treatment increased QTc and Tpeak–Tend interval duration, prolonged action potential duration and decreased amplitude of Ito and ICa-L. In addition, heterologously expressed IhKv4.3, IhERG or IhKCNQ1/KCNE1 decreased after acute exposure to MeHg at subnanomolar range. The introduction of the in vitro effects of MeHg in a computer model of human ventricular action potentials triggered early afterdepolarizations and arrhythmia. In conclusion, cardiac electrical remodeling induced by MeHg poisoning is related to the reduction of Ito and ICa-L. The acute effect of MeHg on hKv4.3; hERG and hKCNQ1/KCNE1 currents and their transposition to in silico models show an association between MeHg intoxication and acquired Long QT Syndrome in humans. MeHg can exert its high toxicity either after chronic or acute exposure to concentrations as low as picomolar.This work was supported by grants from the Gobierno Vasco PIBA2018-58 and GIC18/150

The Crossroad of Ion Channels and Calmodulin in Disease

Calmodulin (CaM) is the principal Ca2+ sensor in eukaryotic cells, orchestrating the activity of hundreds of proteins. Disease causing mutations at any of the three genes that encode identical CaM proteins lead to major cardiac dysfunction, revealing the importance in the regulation of excitability. In turn, some mutations at the CaM binding site of ion channels cause similar diseases. Here we provide a summary of the two sides of the partnership between CaM and ion channels, describing the diversity of consequences of mutations at the complementary CaM binding domains.The Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Elkartek 2017 bG17 kk-2017/000843M50.17.EK.C6) and the Spanish Ministry of Economy, Industry and Competitiveness (BFU2015-66910 and RTI2018-097839) provided financial support for this work. E.N. is supported by a predoctoral grant of the Basque Government

Metformin Reduces Potassium Currents and Prolongs Repolarization in Non-Diabetic Heart

Metformin is the first choice drug for the treatment of type 2 diabetes due to positive results in reducing hyperglycaemia and insulin resistance. However, diabetic patients have higher risk of ventricular arrhythmia and sudden cardiac death, and metformin failed to reduce ventricular arrhythmia in clinical trials. In order to explore the mechanisms responsible for the lack of protective effect, we investigated in vivo the effect of metformin on cardiac electrical activity in non-diabetic rats; and in vitro in isolated ventricular myocytes, HEK293 cells expressing the hERG channel and human induced pluripotent stem cells derived cardiomyocytes (hIPS-CMs). Surface electrocardiograms showed that long-term metformin treatment (7 weeks) at therapeutic doses prolonged cardiac repolarization, reflected as QT and QTc interval duration, and increased ventricular arrhythmia during the caffeine/dobutamine challenge. Patch-clamp recordings in ventricular myocytes isolated from treated animals showed that the cellular mechanism is a reduction in the cardiac transient outward potassium current (Ito). In vitro, incubation with metformin for 24 h also reduced Ito, prolonged action potential duration, and increased spontaneous contractions in ventricular myocytes isolated from control rats. Metformin incubation also reduced IhERG in HEK293 cells. Finally, metformin incubation prolonged action potential duration at 30% and 90% of repolarization in hIPS-CMs, which is compatible with the reduction of Ito and IhERG. Our results show that metformin directly modifies the electrical behavior of the normal heart. The mechanism consists in the inhibition of repolarizing currents and the subsequent decrease in repolarization capacity, which prolongs AP and QTc duration.This work was supported by The University of the Basque Country (Grant number PPG17/13), Gobierno Vasco (PIBA2018-58) and MICIIN (PID2020-118814RB-I00). V.Z.R. is recipient of a Fundación Alfonso Martín Escudero (SPAIN) postdoctoral fellowship

High thyrotropin is critical for cardiac electrical remodeling and arrhythmia vulnerability in hypothyroidism

Background: Hypothyroidism, the most common endocrine disease, induces cardiac electrical remodeling that creates a substrate for ventricular arrhythmias. Recent studies report that high thyrotropin (TSH) levels are related to cardiac electrical abnormalities and increased mortality rates. The aim of the present work was to investigate the direct effects of TSH on the heart and its possible causative role in the increased incidence of arrhythmia in hypothyroidism. Methods: A new rat model of central hypothyroidism (low TSH levels) was created and characterized together with the classical propylthiouracil-induced primary hypothyroidism model (high TSH levels). Electrocardiograms were recorded in vivo, and ionic currents were recorded from isolated ventricular myocytes in vitro by the patch-clamp technique. Protein and mRNA were measured by Western blot and quantitative reverse transcription polymerase chain reaction in rat and human cardiac myocytes. Adult human action potentials were simulated in silico to incorporate the experimentally observed changes. Results: Both primary and central hypothyroidism models increased the L-type Ca2+ current (ICa-L) and decreased the ultra-rapid delayed rectifier K+ current (IKur) densities. However, only primary but not central hypothyroidism showed electrocardiographic repolarization abnormalities and increased ventricular arrhythmia incidence during caffeine/dobutamine challenge. These changes were paralleled by a decrease in the density of the transient outward K+ current (Ito) in cardiomyocytes from animals with primary but not central hypothyroidism. In vitro treatment with TSH for 24 hours enhanced isoproterenol-induced spontaneous activity in control ventricular cells and diminished Ito density in cardiomyocytes from control and central but not primary hypothyroidism animals. In human myocytes, TSH decreased the expression of KCND3 and KCNQ1, Ito, and the delayed rectifier K+ current (IKs) encoding proteins in a protein kinase A–dependent way. Transposing the changes produced by hypothyroidism and TSH to a computer model of human ventricular action potential resulted in enhanced occurrence of early afterdepolarizations and arrhythmia mostly in primary hypothyroidism, especially under b-adrenergic stimulation. Conclusions: The results suggest that suppression of repolarizing K+ currents by TSH underlies most of the electrical remodeling observed in hypothyroidism. This work demonstrates that the activation of the TSHreceptor/protein kinase A pathway in the heart is responsible for the cardiac electrical remodeling and arrhythmia generation seen in hypothyroidism.Fil: Fernandez Ruocco, Maria Julieta. Universidade Federal do Rio de Janeiro; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Gallego, Monica. Universidad del País Vasco; EspañaFil: Rodriguez de Yurre, Ainhoa. Universidade Federal do Rio de Janeiro; Brasil. Universidad del País Vasco; EspañaFil: Zayas Arrabal, Julian. Universidad del País Vasco; EspañaFil: Echeazarra, Leyre. Universidade Federal do Rio de Janeiro; BrasilFil: Alquiza, Amaia. Universidad del País Vasco; EspañaFil: Fernández López, Victor. Universidad del País Vasco; EspañaFil: Rodriguez Robledo, Juan M.. Universidad del País Vasco; EspañaFil: Brito, Oscar. Instituto Nacional de Cardiologia; BrasilFil: Schleier, Ygor. Universidade Federal do Rio de Janeiro; BrasilFil: Sepúlveda, Marisa Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Oshiyama, Natalia F.. University of Campinas. Center for Biomedical Engineering; BrasilFil: Vila Petroff, Martin Gerarde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Bassani, Rosana A.. University of Campinas. Center for Biomedical Engineering; BrasilFil: Medei, Emiliano H.. Universidade Federal do Rio de Janeiro; BrasilFil: Casis, Oscar. Universidad del País Vasco; Españ