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Voltage-gated ion channels are essential for membrane potential maintenance, homeostasis, electrical signal production and controlling the Ca2+ flow through the membrane. Among all ion channels, the key regulators of neuronal excitability are the voltage-gated potassium channels (KV), the largest family of K+ channels. Due to the ROS high levels in the aging brain, K+ channels might be affected by oxidative agents and be key in aging and neurodegeneration processes. This review provides new insight about channelopathies in the most studied neurodegenerative disorders, such as Alzheimer Disease, Parkinson's Disease, Huntington Disease or Spinocerebellar Ataxia. The main affected KV channels in these neurodegenerative diseases are the KV1, KV2.1, KV3, KV4 and KV7. Moreover, in order to prevent or repair the development of these neurodegenerative diseases, previous KV channel modulators have been proposed as therapeutic targets.
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Despite the increasing availability of genomic data and enhanced data analysis procedures, predicting the severity of associated diseases remains elusive in the absence of clinical descriptors. To address this challenge, we have focused on the KV7.2 voltage-gated potassium channel gene (KCNQ2), known for its link to developmental delays and various epilepsies, including self-limited benign familial neonatal epilepsy and epileptic encephalopathy. Genome-wide tools often exhibit a tendency to overestimate deleterious mutations, frequently overlooking tolerated variants, and lack the capacity to discriminate variant severity. This study introduces a novel approach by evaluating multiple machine learning (ML) protocols and descriptors. The combination of genomic information with a novel Variant Frequency Index (VFI) builds a robust foundation for constructing reliable gene-specific ML models. The ensemble model, MLe-KCNQ2, formed through logistic regression, support vector machine, random forest and gradient boosting algorithms, achieves specificity and sensitivity values surpassing 0.95 (AUC-ROC > 0.98). The ensemble MLe-KCNQ2 model also categorizes pathogenic mutations as benign or severe, with an area under the receiver operating characteristic curve (AUC-ROC) above 0.67. This study not only presents a transferable methodology for accurately classifying KCNQ2 missense variants, but also provides valuable insights for clinical counseling and aids in the determination of variant severity. The research context emphasizes the necessity of precise variant classification, especially for genes like KCNQ2, contributing to the broader understanding of gene-specific challenges in the field of genomic research. The MLe-KCNQ2 model stands as a promising tool for enhancing clinical decision making and prognosis in the realm of KCNQ2-related pathologies.
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Epilepsia Neonatal Benigna , Epilepsia Generalizada , Recém-Nascido , Humanos , Inteligência Artificial , Mutação de Sentido Incorreto , Mutação , Epilepsia Neonatal Benigna/genética , Canal de Potássio KCNQ2/genéticaRESUMO
Background: Aging correlates with increased frailty, multi-morbidity, and chronic diseases. Furthermore, treating the aged often entails polypharmacy to achieve optimal disease management, augmenting medication-related problems (MRPs). Few guidelines and tools address the problem of polypharmacy and MRPs, mainly within the institutionalized elderly population. Routine pharmacological review is needed among institutionalized patients. This pharmacological review may improve with a multidisciplinary approach of a collaboration of multiple health professionals. This study aimed to describe institutionalized patients, systematically review their medication plans, and then give recommendations and identify MRPs. Methods: A cross-sectional study was performed using data obtained from patients living in five nursing homes in the northern area of Barcelona, Spain. The inclusion criteria comprised institutionalized patients with public health coverage provided by the Health Department of Catalonia. A detailed description of the clinical characteristics, chronic diseases, pharmacological treatments, recommendations, incomplete data, and MRPs, such as potential drug-drug interactions, therapeutic duplications, contraindications, and drugs deemed inappropriate or of doubtful efficacy, was made. The clinical pharmacologist was the medical doctor specialist who acted as the coordinator of the multidisciplinary team and actively reviewed all the prescribed medications to make recommendations and detect MRPs. Results: A total of 483 patients were included. Patients had a mean age of 86.3 (SD 8.8) years, and 72.0% were female individuals. All patients had at least three health-related problems, with a mean of 17.4 (SD 5.6). All patients, except one, had a minimum of one prescription, with a mean of 8.22 drugs prescribed (SD 3.5) per patient. Recommendations were made for 82.4% of the patients. Of these recommendations, verification of adequate use was made for 69.3% and withdrawal of a drug for 49.5%. Conclusion: This study demonstrates a high prevalence of health-related problems and several prescribed drugs in nursing homes in Catalonia. Many recommendations were made, confirming the increased proportion of polypharmacy, MRPs, and the need for standardized interventions. A multidisciplinary team approach, including general practitioners, geriatric assessments, a clinical pharmacist, and a clinical pharmacologist, should address this problem.
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Hypothyroidism is the most frequent endocrine pathology. Although clinical or overt hypothyroidism has been traditionally associated to low T3 / T4 and high thyrotropin (TSH) circulating levels, other forms exist such as subclinical hypothyroidism, characterized by normal blood T3 / T4 and high TSH. In its different forms is estimated to affect approximately 10% of the population, especially women, in a 5:1 ratio with respect to men. Among its consequences are alterations in cardiac electrical activity, especially in the repolarization phase, which is accompanied by an increased susceptibility to cardiac arrhythmias. Although these alterations have traditionally been attributed to thyroid hormone deficiency, recent studies, both clinical trials and experimental models, demonstrate a fundamental role of TSH in cardiac electrical remodeling. Thus, both metabolic thyroid hormones and TSH regulate cardiac ion channel expression in many and varied ways. This means that the different combinations of hormones that predominate in different types of hypothyroidism (overt, subclinic, primary, central) can generate different forms of cardiac electrical remodeling. These new findings are raising the relevant question of whether serum TSH reference ranges should be redefined. (AU)
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Hipotireoidismo , Eletrofisiologia Cardíaca , Tri-Iodotironina , Tireotropina , Arritmias CardíacasRESUMO
Hypothyroidism is the most frequent endocrine pathology. Although clinical or overt hypothyroidism has been traditionally associated to low T3 / T4 and high thyrotropin (TSH) circulating levels, other forms exist such as subclinical hypothyroidism, characterized by normal blood T3 / T4 and high TSH. In its different forms is estimated to affect approximately 10% of the population, especially women, in a 5:1 ratio with respect to men. Among its consequences are alterations in cardiac electrical activity, especially in the repolarization phase, which is accompanied by an increased susceptibility to cardiac arrhythmias. Although these alterations have traditionally been attributed to thyroid hormone deficiency, recent studies, both clinical trials and experimental models, demonstrate a fundamental role of TSH in cardiac electrical remodeling. Thus, both metabolic thyroid hormones and TSH regulate cardiac ion channel expression in many and varied ways. This means that the different combinations of hormones that predominate in different types of hypothyroidism (overt, subclinic, primary, central) can generate different forms of cardiac electrical remodeling. These new findings are raising the relevant question of whether serum TSH reference ranges should be redefined. (AU)
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Hipotireoidismo , Eletrofisiologia Cardíaca , Tri-Iodotironina , Tireotropina , Arritmias CardíacasRESUMO
Hypothyroidism is the most frequent endocrine pathology. Although clinical or overt hypothyroidism has been traditionally associated to low T3 / T4 and high thyrotropin (TSH) circulating levels, other forms exist such as subclinical hypothyroidism, characterized by normal blood T3 / T4 and high TSH. In its different forms is estimated to affect approximately 10% of the population, especially women, in a 5:1 ratio with respect to men. Among its consequences are alterations in cardiac electrical activity, especially in the repolarization phase, which is accompanied by an increased susceptibility to cardiac arrhythmias. Although these alterations have traditionally been attributed to thyroid hormone deficiency, recent studies, both clinical trials and experimental models, demonstrate a fundamental role of TSH in cardiac electrical remodeling. Thus, both metabolic thyroid hormones and TSH regulate cardiac ion channel expression in many and varied ways. This means that the different combinations of hormones that predominate in different types of hypothyroidism (overt, subclinic, primary, central) can generate different forms of cardiac electrical remodeling. These new findings are raising the relevant question of whether serum TSH reference ranges should be redefined.
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Remodelamento Atrial , Hipotireoidismo , Masculino , Feminino , Humanos , Tri-Iodotironina/uso terapêutico , Tireotropina/uso terapêutico , Tiroxina/uso terapêuticoRESUMO
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 TNFα 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, IFNγ, and TNFα. 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.
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Remodelamento Atrial , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Resistência à Insulina , Metformina , Camundongos , Ratos , Animais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fator de Necrose Tumoral alfa , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Ratos Sprague-Dawley , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/prevenção & controle , CitocinasRESUMO
Background: Data related to adverse drug reactions (ADRs), specifically immune-related adverse events (irAEs), in long-term treatment with immunotherapy in real-world practice is scarce, as is general information regarding the management of ADRs. Objectives: To characterize and describe the incidence of ADRs in patients who began immunotherapy treatment in clinical practice. Methods: In a prospective observational study cancer patients ≥18 years of age who were treated with a monotherapy regime of PD-1/PD-L1 inhibitors were evaluated. The study period was from November 2017 to June 2019 and patients were followed up until June 2021. Patients were contacted monthly by telephone and their electronic health records were reviewed. Each ADR was graded according to the Common Terminology Criteria for Adverse Events (CTCAE 5.0). Results: Out of 99 patients, 86 met the inclusion criteria. Most were male (67.4%), with a median age of 66 (interquartile range, IQR: 59-76). The most frequent cancer was non-small cellular lung cancer (46 cases, 53.5%), followed by melanoma (22, 25.6%). A total of 74 patients (86%) were treated with anti-PD-1 drugs and 12 (14%) were treated with anti-PD-L1 drugs. The median treatment durations were 4.9 (IQR: 1.9-17.0) and 5.9 months (IQR: 1.2-12.3), respectively. Sixty-three patients (73%) developed from a total of 156 (44% of the total number of ADR) irADRs, wherein the most frequent were skin disorders (50 cases, 32%, incidence = 30.5 irADRs/100 patients per year [p-y]), gastrointestinal disorders (29, 19%, 17.7 irADRs/100 p-y), musculoskeletal disorders (17, 11%, 10.4 irADRs/100 p-y), and endocrine disorders (14, 9%, 8.6 irADRs/100 p-y). A total of 22 irADRs (14%) had a latency period of ≥12 months. Twelve irADRs (7.7%) were categorized as grade 3-4, and while 2 (1.3%) were categorized as grade 5 (death). Sixty-one irADRs (39.1%) in 36 patients required pharmacological treatment and 47 irADRs (30.1%) in 22 patients required treatment with corticosteriods. Conclusion: The majority of patients treated with anti-PD1/PDL1-based immunotherapy experienced adverse reactions. Although most of these reactions were mild, 11.5% were categorized as grade 3 or above. A high percentage of the reactions were immune-related and occurred throughout the treatment, thereby indicating that early identification and close monitoring is essential.
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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.
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Diabetes Mellitus Tipo 2 , Células-Tronco Pluripotentes Induzidas , Metformina , Potenciais de Ação , Animais , Arritmias Cardíacas/tratamento farmacológico , Células HEK293 , Humanos , Metformina/farmacologia , Miócitos Cardíacos , Potássio/farmacologia , RatosRESUMO
Resumen Introducción: El primer capítulo dará cuenta de las percepciones sobre educación inicial y su relación con los ámbitos familiar y escolar y cómo inciden en el desempeño escolar en la infancia. El segundo capítulo referencia los estudios relacionados con las responsabilidades y obligaciones de la familia y la escuela en el acompañamiento educativo de los niños y finalmente se desarrolla un apartado que indaga las estrategias de acompañamiento educativo en el contexto familiar y escolar. Objetivo: Realizar una revisión bibliográfica sobre las estrategias de acompañamiento educativo y familiar y su incidencia en el proceso formativo en la infancia. Materiales y Métodos: El enfoque privilegiado, es cualitativo a partir de una estrategia de revisión documental. Se presenta un estudio analítico de artículos de investigación, lo cual permite evidenciar el grado de profundización que ha tenido el tema en los últimos tiempos. Resultado: Carencia de estrategias de acompañamiento en el entorno familiar que sean exitosas y favorezcan el proceso educativo de los niños. Conclusión: Las instituciones gubernamentales continúan apostándole a la vinculación familia y escuela, pero la realidad muestra una brecha entre ellas que afecta de manera indirecta el proceso educativo en la infancia.
Abstract Introduction: The first will give an account of the perceptions about initial education and its relationship with the family and school environments and how they affect school performance in childhood. The second chapter refers to the studies related to the responsibilities and obligations of the family and the school in the educational accompaniment of children and finally a section is developed that investigates the educational accompaniment strategies in the family and school context. Objective: To carry out a bibliographic review on the strategies of educational and family support and their impact on the formative process in childhood. Methodology: The privileged approach is qualitative based on a document review strategy. An analytical study of research articles is presented, which shows the degree of deepening that the subject has had in recent times. Result: Lack of support strategies in the family environment that are successful and favor the educational process of children. Conclusion: Government institutions continue to focus on family and school ties, but reality shows a gap between them that indirectly affects the educational process in childhood.
Resumo Introdução: O acompanhamento educativo a nível escolar e familiar é o tema que pretendemos explorar em profundidade através da revisão documental das principais categorias deste estudo. O tema surge da necessidade observada nos centros educativos relativamente às estratégias utilizadas para acompanhar as curianas e à forma como as famílias são assertivamente envolvidas nos mesmos processos para benefício da formação académica dos seus filhos. O Objectivo deste artigo é realizar uma revisão bibliográfica das estratégias educativas e de acompanhamento familiar e do seu impacto no processo formativo na infância. Materiais e métodos: O enfoque é qualitativo, com base numa estratégia de revisão documental, é apresentado um estudo analítico de artigos de investigação, o que permite demonstrar o grau de estudo aprofundado do tema nos últimos tempos. Os resultados indicam uma falta de estratégias de apoio bem sucedidas no ambiente familiar que favorecem o processo educativo das crianças. Conclusões que as instituições governamentais continuam a concentrar-se na ligação entre a família e a escola, mas a realidade mostra um fosso entre elas que afecta indirectamente o processo educativo na infância.
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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.
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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.
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Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Biomarcadores/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Suscetibilidade a Doenças , Mediadores da Inflamação/sangue , Animais , Arritmias Cardíacas/diagnóstico , Citocinas/sangue , Diabetes Mellitus Tipo 2/etiologia , Modelos Animais de Doenças , Insulina/metabolismo , Resistência à Insulina , Canais de Potássio/metabolismo , Ratos , Remodelação VentricularRESUMO
Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5GFP reporter cardiac fibroblasts. We first established a new NKX2.5GFP reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5GFP hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5GFP cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.
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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.
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Arritmias Cardíacas/mortalidade , Arritmias Cardíacas/fisiopatologia , Remodelamento Atrial/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Compostos de Metilmercúrio/intoxicação , Potenciais de Ação , Animais , Canais de Cálcio/metabolismo , Simulação por Computador , Suscetibilidade a Doenças , Células HEK293 , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Humanos , Masculino , Modelos Cardiovasculares , Canais de Potássio/metabolismo , Ratos Wistar , Redução de PesoRESUMO
BACKGROUND/AIMS: To test whether the physiological regulation of the cardiac Kv4 channels by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is restricted to lipid rafts and whether the interactions observed in rat cardiomyocytes also occur in the human ventricle. METHODS: Ventricular myocytes were freshly isolated from Sprague-Dawley rats. Ito was recorded by the whole-cell Patch-Clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. The presence of the proteins of interest was analysed by western blot. Immunogold staining and electron microscopy of heart vibrosections was performed to localize Kv4.2/Kv4.3 and CaMKII proteins. Protein-protein interactions were determined by co-immunoprecipitation experiments in rat and human ventricular mycoytes. RESULTS: Patch-Clamp recordings in control conditions and after lipid raft or caveolae disruption show that the CaMKII-Kv4 channel complex must associate in non-caveolar lipid rafts to be functional. Separation in density gradients, co-immunoprecipitation and electron microscopy show that there are two Kv4 channel populations: one located in caveolae, that is CaMKII independent, and another one located in planar membrane rafts, which is bound to CaMKII. CONCLUSION: CaMKII regulates only the Kv4 channel population located in non-caveolar lipid rafts. Thus, the regulation of cardiac Kv4 channels in rat and human ventricle depends on their subcellular localization.
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Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Microdomínios da Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio Shal/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/análise , Cavéolas/metabolismo , Células Cultivadas , Humanos , Transporte de Íons , Potássio/metabolismo , Mapas de Interação de Proteínas , Ratos Sprague-Dawley , Canais de Potássio Shal/análiseRESUMO
BACKGROUND: There is an urgent need to develop better materials to provide anatomical support to the pelvic floor without compromising its function. OBJECTIVE: Our aim was to assess outcomes after simulated vaginal prolapse repair in a sheep model using three different materials: (1) ultra-lightweight polypropylene (PP) non-degradable textile (Restorelle) mesh, (2) electrospun biodegradable ureidopyrimidinone-polycarbonate (UPy-PC), and (3) electrospun non-degradable polyurethane (PU) mesh in comparison with simulated native tissue repair (NTR). These implants may reduce implant-related complications and avoid vaginal function loss. DESIGN, SETTING, AND PARTICIPANTS: A controlled trial was performed involving 48 ewes that underwent NTR or mesh repair with PP, UPy-PC, or PU meshes (n=12/group). Explants were examined 60 and 180 d (six per group) post-implantation. INTERVENTION: Posterior rectovaginal dissection, NTR, or mesh repair. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Implant-related complications, vaginal contractility, compliance, and host response were assessed. Power calculation and analysis of variance testing were used to enable comparison between the four groups. RESULTS: There were no visible implant-related complications. None of the implants compromised vaginal wall contractility, and passive biomechanical properties were similar to those after NTR. Shrinkage over the surgery area was around 35% for NTR and all mesh-augmented repairs. All materials were integrated well with similar connective tissue composition, vascularization, and innervation. The inflammatory response was mild with electrospun implants, inducing both more macrophages yet with relatively more type 2 macrophages present at an early stage than the PP mesh. CONCLUSIONS: Three very different materials were all well tolerated in the sheep vagina. Biomechanical findings were similar for all mesh-augmented repair and NTR. Constructs induced slightly different mid-term inflammatory profiles. PATIENT SUMMARY: Product innovation is needed to reduce implant-related complications. We tested two novel implants, electrospun and an ultra-lightweight polypropylene textile mesh, in a physiologically relevant model for vaginal surgery. All gave encouraging outcomes.
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Polipropilenos , Telas Cirúrgicas , Prolapso Uterino/cirurgia , Animais , Materiais Biocompatíveis , Modelos Animais de Doenças , Feminino , Procedimentos Cirúrgicos em Ginecologia , Teste de Materiais , Modelos Animais , Desenho de Prótese , Pirimidinonas , Ovinos , Têxteis , Resultado do TratamentoRESUMO
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 function.
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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 ß-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 TSH-receptor/protein kinase A pathway in the heart is responsible for the cardiac electrical remodeling and arrhythmia generation seen in hypothyroidism.
Assuntos
Arritmias Cardíacas/metabolismo , Remodelamento Atrial/fisiologia , Hipotireoidismo/metabolismo , Miócitos Cardíacos/metabolismo , Tireotropina/metabolismo , Potenciais de Ação , Animais , Antitireóideos/toxicidade , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Bexaroteno/toxicidade , Cálcio/metabolismo , Simulação por Computador , Modelos Animais de Doenças , Suscetibilidade a Doenças , Eletrocardiografia , Humanos , Hipotireoidismo/complicações , Hipotireoidismo/fisiopatologia , Isoproterenol/farmacologia , Canal de Potássio KCNQ1/efeitos dos fármacos , Canal de Potássio KCNQ1/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Propiltiouracila/toxicidade , RNA Mensageiro/metabolismo , Ratos , Canais de Potássio Shal/efeitos dos fármacos , Canais de Potássio Shal/genética , Tireotropina/farmacologiaRESUMO
PURPOSE: Electrospun meshes may be considered as substitutes to textile polypropylene implants. We compared the host response and biomechanical properties of the rat abdominal wall following reinforcement with either polycaprolactone (PCL) modified with ureidopyrimidinone-motifs (UPy) or polypropylene mesh. METHODS: First we measured the response to cyclic uniaxial load within the physiological range both dry (room temperature) and wet (body temperature). 36 rats underwent primary repair of a full-thickness abdominal wall defect with a polypropylene suture (native tissue repair), or reinforced with either UPy-PCL or ultra-light weight polypropylene mesh (n = 12/group). Sacrifice was at 7 and 42 days. Outcomes were compliance of explants, mesh dimensions, graft related complications and semi-quantitative assessment of inflammatory cell (sub) types, neovascularization and remodeling. RESULTS: Dry UPy-PCL implants are less stiff than polypropylene, both are more compliant in wet conditions. Polypropylene loses stiffness on cyclic loading. Both implant types were well incorporated without clinically obvious degradation or herniation. Exposure rates were similar (n = 2/12) as well as mesh contraction. There was no reinforcement at low loads, while, at higher tension, polypropylene explants were much stiffer than UPy-PCL. The latter was initially weaker yet by 42 days it had a compliance similar to native abdominal wall. There were eventually more foreign body giant cells around UPy-PCL fibers yet the amount of M1 subtype macrophages was higher than in polypropylene explants. There were less neovascularization and collagen deposition. CONCLUSION: Abdominal wall reconstruction with electrospun UPy-PCL mesh does not compromise physiologic tissue biomechanical properties, yet provokes a vivid inflammatory reaction.