Extracellular cysteine disulfide bond break at Cys122 disrupts PIP2-dependent Kir2.1 channel function and leads to arrhythmias in Andersen-Tawil Syndrome

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Extracellular Kir2.1C122Y Mutant Upsets Kir2.1-PIP2 Bonds and Is Arrhythmogenic in Andersen-Tawil Syndrome.

BACKGROUND Andersen-Tawil syndrome type 1 is a rare heritable disease caused by mutations in the gene coding the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys (cysteine)122-to-Cys154 disulfide bond in the channel structure is crucial for proper folding but has not been associated with correct channel function at the membrane. We evaluated whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state. METHODS We identified a Kir2.1 loss-of-function mutation (c.366 A>T; p.Cys122Tyr) in an ATS1 family. To investigate its pathophysiological implications, we generated an AAV9-mediated cardiac-specific mouse model expressing the Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments. RESULTS Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking. Molecular dynamics predicted that the C122Y mutation provoked a conformational change over the 2000-ns simulation, characterized by a greater loss of hydrogen bonds between Kir2.1 and phosphatidylinositol 4,5-bisphosphate than wild type (WT). Therefore, the phosphatidylinositol 4,5-bisphosphate-binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch clamping, the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing phosphatidylinositol 4,5-bisphosphate concentrations. In addition, the Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins. CONCLUSIONS The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential for the channel function. We demonstrate that breaking disulfide bonds in the extracellular domain disrupts phosphatidylinositol 4,5-bisphosphate-dependent regulation, leading to channel dysfunction and defects in Kir2.1 energetic stability. The mutation also alters functional expression of the NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.The authors thank the Centro Nacional de Investigaciones Cardiovasculares (CNIC) Viral Vectors Unit for producing the adeno-associated virus serotype 9. Confocal experiments were conducted at the CNIC Microscopy and Dynamic Imaging Unit. The authors thank the CNIC Bioinformatics Unit for generating the in silico homology modeling simulations, F-function analysis, and helpful discussions. The authors also thank the Centro de Supercomputación de Galicia for the use of the Finis Terrae III supercomputer to perform molecular dynamics studies. The CNIC was supported by the Instituto de Salud Carlos III, the Ministerio de Ciencia, Innovación y Universidades, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIU/AEI/10.13039/501100011033). This work was supported by the National heart, Lung and Blood Institute under National Institutes of Health (NIH) grant R01HL163943; the La Caixa Banking Foundation project code HR18-00304 (grant LCF/PR/HR19/52160013); grants PI-FIS-2020, PI20/01220, PI-FIS-2023, and PI23/01039 from the Instituto de Salud Carlos III and cofunded by the Fondo Europeo de Desarrollo Regional (FEDER) and the European Union, respectively; grants PID2020-116935RB-I00 and BFU2016-75144-R funded by MICIU/AEI/10.13039/501100011033; the Fundación La Marató de TV3 (736/C/2020) amb el suport de la Fundació La Marató de TV3; the CIBER (Centro de Investigación Biomédica en Red) de Enfermedades Cardiovasculares (grant CB16/11/00458); the European Union’s Horizon 2020 grant agreement GA-965286; and the Program S2022/BMD7229-CM ARCADIACM funded by the Comunidad de Madrid to J. Jalife; grant PID2021-126423OB-C22 (to M. Martín-Martínez) funded by MICIU/AEI/10.13039/501100011033; and European Regional Development Fund (ERDF) grant PID2022-137214OB-C22 (to M. Gutierrez-Rodríguez) funded by MICIU/AEI/10.13039/501100011033. The imaging studies were performed in the TRIMA@CNIC (Infraestructura de Imagen Traslacional Avanzada del CNIC) node of the ICTS ReDIB (Infraestructuras Científicas y Técnicas Singulares: Red Distribuida de Imagen Biomédica) grant ICTS-2018- 04-CNIC-16 funded by MICIU/AEI/10.13039/501100011033 and ERDF, and project EQC2018-005070-P funded by MICIU/AEI/10.13039/501100011033 and FEDER. A.I. Moreno-Manuel holds an formación profesional universitaria (FPU) contract (FPU20/01569) from the Ministerio de Universidades. J.M. Ruiz Robles holds an FPU contract (FPU22/03253) from the Ministerio de Universidades. L.K. Gutiérrez holds an FPI contract (PRE2018-083530) from the Ministerio de Economía y Competitividad de España cofunded by the Fondo Social Europeo, attached to project SEV-2015-0505-18-2. I. Martínez-Carrascoso holds a PFIS (Contratos predoctorales de formación en investigación en salud) contract (FI21/00243) funded by Instituto de Salud Carlos III and the Fondo Social Europeo Plus cofunded by the European Union. M.L. Vera-Pedrosa held contract PEJD-2019-PRE/BMD15982 funded by the Consejería de Educación e Investigación de la Comunidad de Madrid y Fondo Social Europeo.S

Predictores de riesgo en una cohorte española con cardiolaminopatías. Registro REDLAMINA

[Abstract] Introduction and objectives. According to sudden cardiac death guidelines, an implantable cardioverter-defibrillator (ICD) should be considered in patients with LMNA-related dilated cardiomyopathy (DCM) and ≥ 2 risk factors: male sex, left ventricular ejection fraction (LVEF) < 45%, nonsustained ventricular tachycardia (NSVT), and nonmissense genetic variants. In this study we aimed to describe the clinical characteristics of carriers of LMNA genetic variants among individuals from a Spanish cardiac-laminopathies cohort (REDLAMINA registry) and to assess previously reported risk criteria. Methods. The relationship between risk factors and cardiovascular events was evaluated in a cohort of 140 carriers (age ≥ 16 years) of pathogenic LMNA variants (54 probands, 86 relatives). We considered: a) major arrhythmic events (MAE) if there was appropriate ICD discharge or sudden cardiac death; b) heart failure death if there was heart transplant or death due to heart failure. Results. We identified 11 novel and 21 previously reported LMNA-related DCM variants. LVEF < 45% (P = .001) and NSVT (P < .001) were related to MAE, but not sex or type of genetic variant. The only factor independently related to heart failure death was LVEF < 45% (P < .001). Conclusions. In the REDLAMINA registry cohort, the only predictors independently associated with MAE were NSVT and LVEF < 45%. Therefore, female carriers of missense variants with either NSVT or LVEF < 45% should not be considered a low-risk group. It is important to individualize risk stratification in carriers of LMNA missense variants, because not all have the same prognosis.[Resumen] Introducción y objetivos. Según las guías de muerte súbita, se debe considerar un desfibrilador automático implantable (DAI) para los pacientes con miocardiopatía dilatada debida a variantes en el gen de la lamina (LMNA) con al menos 2 factores: varones, fracción de eyección del ventrículo izquierdo (FEVI) < 45%, taquicardia ventricular no sostenida (TVNS) y variantes no missense. Nuestro objetivo es describir las características clínicas de una cohorte española de pacientes con cardiolaminopatías (registro REDLAMINA) y evaluar los criterios de riesgo vigentes. Métodos. Se evaluó la relación entre factores de riesgo y eventos cardiovasculares en una cohorte de 140 portadores de variantes en LMNA (54 probandos, 86 familiares, edad ≥ 16 años). Se consideró: a) evento arrítmico mayor (EAM) si hubo descarga apropiada del DAI o muerte súbita, y b) muerte por insuficiencia cardiaca, incluidos los trasplantes. Resultados. Se identificaron 11 variantes nuevas y 21 previamente publicadas. La FEVI < 45% (p = 0,001) y la TVNS (p < 0,001) se relacionaron con los EAM, pero no el sexo o el tipo de variante (missense frente a no missense). La FEVI < 45% (p < 0,001) fue el único factor relacionado con la muerte por insuficiencia cardiaca. Conclusiones. En el registro REDLAMINA, los únicos 2 predictores asociados con EAM fueron la TVNS y la FEVI < 45%. No se debería considerar grupo de bajo riesgo a las portadoras de variantes missense con TVNS o FEVI < 45%. Es importante individualizar la estratificación del riesgo de los portadores de variantes missense en LMNA, porque no todas tienen el mismo pronóstico.This study received a grant from the Proyecto de investigación de la Sección de Insuficiencia Cardiaca 2017 from the Spanish Society of Cardiology and grants from the Instituto de Salud Carlos III (ISCIII) [PI14/0967, PI15/01551, AC16/0014] and ERA-CVD Joint Transnational Call 2016 (Genprovic). Grants from the ISCIII and the Ministerio de Economía y Competitividad de España (Spanish Department of Economy and Competitiveness) are supported by the Plan Estatal de I+D+i 2013-2016: Fondo Europeo de Desarrollo Regional (FEDER) “Una forma de hacer Europa”

Clinical characteristics and determinants of the phenotype in TMEM43 arrhythmogenic right ventricular cardiomyopathy type 5.

Arrhythmogenic right ventricular cardiomyopathy type V (ARVC-5) is the most aggressive heterozygous form of ARVC. It is predominantly caused by a fully penetrant mutation (p.S358L) in the nondesmosomal gene TMEM43-endemic to Newfoundland, Canada. To date, all familial cases reported worldwide share a common ancestral haplotype. It is unknown whether the p.S358L mutation by itself causes ARVC-5 or whether the disease is influenced by genetic or environmental factors. The purpose of this study was to examine the phenotype, clinical course, and the impact of exercise on patients with p.S358L ARVC-5 without the Newfoundland genetic background. We studied 62 affected individuals and 73 noncarriers from 3 TMEM43-p.S358L Spanish families. The impact of physical activity on the phenotype was also evaluated. Haplotype analysis revealed that the 3 Spanish families were unrelated to patients with ARVC-5 with the Newfoundland genetic background. Two families shared 10 microsatellite markers in a 4.9 cM region surrounding TMEM43; the third family had a distinct haplotype. The affected individuals showed a 38.7% incidence of sudden cardiac death, which was higher in men. Left ventricular involvement was common, with 40% of mutation carriers showing a left ventricular ejection fraction of <50%. Compared with noncarriers, the R-wave voltage in lead V3 was lower (3.2 ± 2.8 mV vs 7.5 ± 3.6 mV; P < .001) and QRS complex in right precordial leads wider (104.7 ± 24.0 ms vs 88.2 ± 7.7 ms; P = .001). A history of vigorous exercise showed a trend toward more ventricular arrhythmias only in women (P = .053). ARVC-5 is associated with a high risk of sudden cardiac death and characteristic clinical and electrocardiographic features irrespective of geographical origin and genetic background. Our data suggest that, as in desmosomal ARVC, vigorous physical activity could aggravate the phenotype of TMEM43 mutation carriers.This work was supported by grants from the Instituto de Salud Carlos III (PI14/0967 and PI17/1941, CPII14/00027, PI14/01477, PI18/0158 and La Fe Biobank PT17/0015/0043), the Isabel Gemio Foundation, the Spanish Society of Cardiology (2014 Basic Research Grant), the European Union (CardioNeTITN-289600 and CardioNext-608027), and from the Spanish Ministry of Economy and Competitiveness (RTI2018-096961-B-I00, SAF2015-65722-R, and SAF2012-31451). This work was also supported by the Plan Estatal de I1D1I 2013-2016 – European Regional Development Fund (FEDER) “AWay ofMaking Europe,” Spain. The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU), and the ProCNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505)S

Efecto de la Pentoxifilina en hepatitis colestásica aguda: reporte de dos casos

Introduction. Cholestatic hepatitis is a rare entity characterized by a bile flow obstruction,and it has a multi-factorial etiology. It can be acute or chronic and occasionally triggers fibrosis, cirrhosis and severe hepatitis. The current pharmacological management is based on corticosteroids and azathioprine or other drugs, such as, methotrexate, cyclosporine, budesonide, ursodeoxycholic acid, mefenamic acid, and pentoxifylline (PTX). We report two patients with autoimmune cholestatic hepatitis treated with pentoxifylline. Clinical cases. Two cases with diagnosis of cholestatic hepatitis confirmed by ultrasound and serological and histological studies. In both cases, corticosteroids were used as a pharmacological treatment without a favorable response. The therapy was replaced by pentoxifylline plus symptomatic treatment at base of ursodeoxycholic acid, vitamin E and cholestyramine. Both patients showed a remarkable improvement after two weeks of treatment. One of the patients showed a total bilirubin of 22.30 mg/dL with a decreased of direct bilirubin from 22.23 mg/dL to 2.10 mg/ dL; meanwhile, the second patient showed a total bilirubin of 63.7 mg/dL with a decreased of direct bilirubin levels from 60.2 mg/dL to 2.33 mg/ dL. In both patients the levels of transaminases decreased and an improvement of their clinical conditions were observed. Discussion. Our results showed that there was a favorable response in two patients treated with pentoxifylline. This drug has anti-inflammatory, anti-oxidant and anti-fibrotic effects as well as inhibitors effects of the transcription factor NF- κβ, so that it could be considered as an alternative treatment in patients with cholestatic hepatitis after validating its actual effectiveness.Introducción. La hepatitis colestásica es una entidad poco frecuente que se caracteriza por obstrucción del flujo biliar y presentar etiología multifactorial; puede ser aguda o crónica y, ocasionalmente, desencadena fibrosis, cirrosis y hepatitis grave. El manejo farmacológico habitual es a base de corticoides y azatioprina, así como otros medicamentos como el metotrexate, la ciclosporina, la budesonida, el ácido ursodesoxicólico, el ácido micofenólico y la pentoxifilina (PTX); dentro de los medicamentos citados, utilizamos pentoxifilina en dos pacientes con hepatitis colestásica autoinmune. Casos clínicos. Se reportan dos casos con diagnóstico de hepatitis colestásica, corroborada a través de estudios ecosonográfico, serológicos e histológicos. Se utilizaron corticoides para el tratamiento farmacológico en ambos casos; sin embargo, al no obtener resultados favorables, la terapia fue sustituida por pentoxifilina, más tratamiento sintomático a base de ácido ursodesoxicólico, vitamina E y colestiramina. Ambos pacientes mostraron una mejoría después de dos semanas de tratamiento; una paciente con bilirrubina total (BT) de 22.30 mg/dL disminuyó bilirrubina directa (BD) de 22.23 mg/dL a 2.10 mg/dL; en el segundo caso con BT de 63.7 mg/ dL disminuyó sus cifras de BD de 60.2 mg/dL a 2.33 mg/dL; además, redujo transaminasemia y las condiciones de ambas pacientes presentaron mejoría. Discusión. Nuestros resultados mostraron que hubo respuesta favorable en los dos pacientes tratados con pentoxifilina; considerando que este fármaco ha demostrado efectos antiinflamatorios, antioxidantes, antifibróticos e inhibidores del factor de transcripción NF-κβ, podría ser utilizado como un tratamiento alternativo en los pacientes con hepatitis colestásica, aclarando que se necesitan más estudios clínicos para validar su real eficacia