Bases fisiopatológicas y moleculares de la miocardiopatía arritmogénica en un modelo de ratón para placofilina-2 truncada

La miocardiopatía arritmogénica (MCA) es una enfermedad cardíaca degenerativa caracterizada por el remplazo de tejido cardíaco por tejido fibro-adiposo, la progresiva dilatación y disfunción del ventrículo derecho y la aparición de arritmias. Aunque es considerada una enfermedad rara, la MCA es una importante causa de muerte súbita cardíaca (MSC) entre personas jóvenes (<35 años). Además, su incidencia es hasta 5 veces mayor entre atletas, lo que sugiere que el ejercicio intensivo es un factor de riesgo importante en el desarrollo de la enfermedad. La MCA es una patología familiar en, al menos, un 50% de los casos, lo que demuestra su carácter hereditario. De hecho, mutaciones en todos los genes desmosomales que se expresan en corazón han sido asociados a la patología, lo que ha hecho acuñar el nombre de “enfermedad del desmosoma”. Entre todos los genes desmosomales implicados en la MCA destaca Placofilina-2 (PKP2), en el cual se concentran casi el 50% de todas las mutaciones encontradas en los pacientes, lo que convierte a este gen en el más importante de la patología. Gracias a los recientes avances desde el ámbito de la genética, se han podido desarrollar modelos celulares y animales para el estudio de la MCA. Ello ha permitido desvelar algunos de los mecanismos fisiopatológicos y moleculares causantes de la enfermedad. Por ejemplo, algunos modelos animales muestran defectos eléctricos antes de cualquier anomalía estructural, lo que sugiere la independencia de ambos defectos. Por otro lado, se ha comprobado que la supresión de la vía de señalización Wnt/β-cat no canónica es esencial para la formación del tejido fibro-adiposo, lo que permitió incluir este proceso como potencial diana terapéutica de la patología. Sin embargo, a pesar de su papel clave en el desarrollo de la enfermedad, pocos modelos han sido desarrollados hasta el momento para el estudio de las mutaciones en PKP2. Por tanto, el objetivo de esta tesis doctoral es estudiar en profundidad los efectos fisiopatológicos y moleculares generados por una proteína truncada de PKP2, la mutación más frecuente entre los pacientes. Para conocer el efecto de esta mutación, también se han estudiado factores que pueden ser potenciales desencadenantes de la enfermedad: i) el contenido de PKP2 truncada, ii) el ejercicio intensivo y iii) el envejecimiento. Para cumplir dichos objetivos, se ha desarrollado un modelo celular y otro animal, portadores de una versión truncada de PKP2. En el caso del modelo animal, se han generado tres líneas transgénicas diferentes, con un contenido creciente de transgén (TG-B, TG-M y TG-A, respectivamente). Además, se ha desarrollado un grupo animal sometido a un protocolo de entrenamiento intensivo en cinta andadora, para conocer el efecto del ejercicio sobre la enfermedad. Finalmente, debido al carácter degenerativo de la MCA, se ha estudiado el efecto del envejecimiento en el modelo a diferentes edades. Como resultado, se observó que ningún grupo animal sufría remplazo de tejido fibro-adiposo. Asimismo, el modelo celular mostraba una actividad normal de la vía de señalización Wnt/β-cat no canónica, en consonancia con los resultados histológicos observados en el modelo animal. A pesar de la ausencia de un fenotipo histológico, a nivel ultra-estructural se encontraron múltiples roturas de los discos intercalares de los cardiomiocitos y una reducción del número de desmosomas en el grupo de mayor contenido de transgén (TG-A). Todo ello sugiere que un alto contenido de PKP2 truncada reduce la adhesión entre los cardiomiocitos y promueve la ruptura de sus uniones. El estudio ecocardiográfico de los diferentes grupos reveló una progresiva dilatación del ventrículo derecho en relación al contenido de PKP2 truncada en cada línea animal. Además, la dilatación se exacerbaba en animales entrenados y por el envejecimiento de los animales. Curiosamente, solo el grupo TG-A mostró anomalías a nivel del ventrículo izquierdo. Por otro lado, el estudio molecular de proteínas de adhesión cardíacas mostró una reducción y remodelado de las proteínas PKP2 endógena (proteína completa), Placoglobina (PG), Desmocolina-2 (DSC2), Desmina (DES) y β-catenina (β-CAT). Ello demuestra que la degradación de las proteínas de unión celular subyace a la reducción de la adhesión entre cardiomiocitos y a la dilatación del ventrículo. El electrocardiograma (ECG) mostraba una conducción eléctrica más lenta, donde se observaba un intervalo PR y un complejo QRS prolongados. Este efecto era más pronunciado en las líneas transgénicas con mayor contenido de transgén. Sorprendentemente, estas anomalías no se agravaban por el ejercicio intensivo o el envejecimiento, como sí ocurría para los defectos estructurales. Asimismo, las arritmias eran más frecuentemente inducidas en ratones de las líneas con mayor contenido de transgén, mientras que el ejercicio intensivo no afectaba a ello. Finalmente, el estudio molecular reveló un remodelado y reducción de los canales Conexina 43 (CX43) y subunidad α del canal de sodio cardíaco (Nav1.5), lo que demuestra la asociación entre estas anomalías moleculares y los defectos eléctricos del modelo. En conclusión, esta tesis doctoral demuestra que la presencia de tejido fibro-adiposo no es indispensable para el desarrollo de la MCA. Los defectos estructurales son dependientes del contenido de PKP2 truncada, el ejercicio y el envejecimiento, mientras que las anomalías eléctricas solo dependen del contenido de PKP2 truncada. Finalmente, se han descubierto algunos de los mecanismos moleculares que subyacen a los defectos estructurales y eléctricos. Su diferente etiología molecular demuestra que las anomalías estructurales y eléctricas representan entidades fisiopatológicas independientes en este modelo.Arrhythmogenic cardiomyopathy (ACM) is a degenerative cardiac disease characterized by the replacement of cardiac tissue by fibro-fatty tissue, progressive dilatation and dysfunction of the right ventricle, and arrhythmias. Although it is considered a rare disease, ACM is an important cause of sudden cardiac death (SCD) among young people (<35 years-old). In addition, its incidence is even higher among athletes (up to 5-fold), suggesting that endurance exercise is an important risk factor in the pathology development. ACM is a familial disease, at least in 50% of the cases, what suggests its hereditary character. Indeed, mutations in all desmosomal genes expressed in heart have been associated to this pathology, what it has granted the name of “disease of the desmosome”. Among desmosomal genes involved in ACM, Plakophilin-2 (PKP2) accumulates almost 50% of total mutations found in patients, representing the most important gene in the pathology. Recent insights into the genetic basis of the disease have allowed for the generation of different cellular and animal models for the ACM study, which in turn have shed light on some pathophysiological and molecular mechanisms of the disease. For instance, some animal models show electrical defects before any structural anomaly, suggesting the independence of both abnormalities. On the other hand, it has been showed that the suppression of Wnt/β-cat signaling pathway is essential for fibro-fatty tissue formation, including this process as potential therapeutic target of the pathology. Nevertheless, despite its critical role in the pathogenesis of the disease, few models have been developed until now to study mutations in PKP2. Therefore, the aim of this doctoral thesis was to deeply study the pathophysiological and molecular effects promoted by a truncated PKP2 protein, the most frequent mutation among patients. Moreover, some potential factors which could trigger the disease had been studied: i) truncated PKP2 content; ii) endurance training and iii) ageing. In order to test such aims, a cellular and animal model carrying a truncated PKP2 version had been developed. In the animal model, three different transgenic lines were generated, expressing a distinct amount of transgene (TG-L, TG-M and TG-H, respectably). In addition, an endurance training group was including a treadmill protocol in order to characterize the influence of exercises in the disease. Finally, due to the degenerative nature of ACM, the effect of ageing in the model was also studied. As result, we did not observe fibro-fatty tissue replacement in our model. Besides, the cellular model showed normal activity of the Wnt/β-cat no canonical pathway, in concordance with the histological findings observed in the animal model. Despite absence of histological phenotype, a lot of intercalated disc ruptures as well as reduction of desmosomes were observed in the cardiomyocytes of the highest transgene expression line (TG-H) at ultra-structural level. All together, these data suggest that high truncated PKP2 expression reduces the adhesion between cardiomyocytes, promoting the breakages of their junctions. The echocardiographic study of the different groups revealed a progressive right ventricle enlargement associated to truncated PKP2 amount in each animal line. Moreover, the dilatation was exacerbated in trained and old mice. Interestingly, only the TG-H group showed left ventricle anomalies. On the other hand, the molecular study of adhesion proteins showed a reduction and remodeling of endogenous PKP2 (full protein), Placoglobin (PG), Desmocollin-2 (DSC2), Desmin (DES) and β-catenin (β-CAT). Such findings demonstrate that degradation of cellular junction proteins underlay to adhesion deficiency between cardiomyocytes and to ventricle enlargement. The electrocardiogram (ECG) showed a slower electrical conduction, due to a prolonged PR interval and QRS complex duration. This effect was deeper in lines with higher content of transgene. Strikingly, such anomalies were not affected by intense exercise or ageing, like yes happened to structural defects. Furthermore, arrhythmias occurred more often in mice of the group with higher content of transgene, whereas endurance training did not affect to that. Finally, the molecular study revealed a remodeling and reduction of the channels Connexin 43 (CX43) and α-subunit of cardiac sodium channel (Nav1.5), demonstrating the association between such molecular disturbances and electrical anomalies in the model. In conclusion, this doctoral thesis shows that presence of fibro-fatty tissue is not necessary for the ACM development. The structural defects depend on truncated PKP2 content, exercise and ageing, whereas electrical anomalies only depend on truncated PKP2 content. Finally, some molecular mechanisms were discovered, which underlay to electrical and structural anomalies. The different molecular etiology demonstrates that structural and electrical abnormalities represent independent pathophysiological entities in this model

Effect of hyperlipidic diets on normal and abnormal aortic valves in the Syrian hamster: A preliminary study

Effect of hyperlipidic diets on normal and abnormal aortic valves in the Syrian hamster: A preliminary study. MC Fernández 1,2, J Moncayo-Arlandi 1, MT Soto 1, MA López-Unzu 1, B Fernández 1,2 and AC Durán 1,2. 1 Department of Animal Biology, Faculty of Science, University of Málaga, Spain. 2 Biomedical Research Institute of Málaga (IBIMA), University of Málaga, Spain. Bicuspid aortic valve (BAV) is the most frequent human congenital cardiac malformation. It frequently becomes stenotic due to calcification by an atherosclerosis-like process. Hyperlipidic diets have been classically used to induce atherosclerosis in laboratory animals, including Syrian hamsters. The aim here is to evaluate the effect of hyperlipidic diets in hamsters having different incidence of BAVs. We used a unique inbred strain of Syrian hamsters with a high ( 40%) incidence of spontaneous BAV, morphologically similar to that in man, another inbred strain with a low ( 4%) incidence of BAV, and an outbred, second control line, acquired from Charles River Laboratories. Three experimental groups were fed with standard diet supplemented with 2% cholesterol plus 15% butter during five months. In parallel, three control groups were fed with unmodified standard diet. Hyperlipidic diets induced lesions in the aortic valve and ascending aortic wall, i.e. subendothelial lipid deposits, valve sclerosis, and neo-intima in the aorta. We performed a preliminary, qualitative, comparative study of the lesions associated with the different animal populations and valvular phenotypes. Our results indicate that (1) the type and severity of the lesions varied among the three hamster populations, suggesting that genetic factors may be involved; (2) the aortic valve morphology seems not to determine the severity of the valvular lesions. We conclude that our hamster strain with high incidence of BAV is a promising animal model for studies on human aortic stenosis. This work was supported by P10-CTS-6068.Universidad de Málaga. Campus de Excelencia Andalucía Tech. P10-CTS-6068

Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model

[EN] Background: Cardioplegic arrest is a common procedure for many types of cardiac surgery, and different formulations have been proposed to enhance its cardio-protective effect. Hydrogen sulfide is an important signaling molecule that has cardio-protective properties. We therefore studied the cardio-protective effect of hydrogen sulfide in cardiac cell culture and its potential therapeutic use in combination with cardioplegia formulations. Materials/Methods: We added hydrogen sulfide donor GYY4137 to HL-1 cells to study its protective effect in nutrient starved conditions. In addition, we tested the potential use of GYY4137 when it is added into two different cardioplegia formulations: Cardi-Braun (R) solution and del Nido solution in an ex vivo Langendorff perfused rat hearts model. Results: We observed that eight-hour pre-treatment with GYY4137 significantly suppressed apoptosis in nutrient-starved HL-1 cells (28% less compared to untreated cells; p<0.05), maintained ATP content, and reduced protein synthesis. In ex vivo experiments, Cardi-Braun (R) and del Nido cardioplegia solutions supplemented with GYY4137 significantly reduced the pro-apoptotic protein caspase-3 content and preserved ATP content. Furthermore, GYY4137 supplemented cardioplegia solutions decreased the S-(5-adenosyl)-L-methionine/S-(adenosyl)-L-homocysteine ratio, reducing the oxidative stress in cardiac tissue. Finally, heart beating analysis revealed the preservation of the inter-beat interval and the heart rate in del Nido cardioplegia solution supplemented with GYY4137. Conclusions: GYY4137 preconditioning preserved energetic state during starved conditions, attenuating the cardiomyocytes apoptosis in vitro. The addition of GYY4137 to cardioplegia solutions prevented apoptosis, ATP consumption, and oxidative stress in perfused rat hearts, restoring its electrophysiological status after cardiac arrest. These findings suggested that GYY4137 sulfide donor may improve the cardioplegia solution performance during cardiac surgery.Instituto de Salud Carlos III grants (PI10/743, PI13/0414) and RETICS RD12/0019/0025 co-funded by FEDER "Una Manera de Hacer Europa". AG. acknowledges a fellowship from Erasmus Mundus Eurotango Program. ADJ acknowledges support from the Ramon y Cajal program (RYC-2008-02378). P.S. acknowledges support from PI10/743, PI13/414 grants and the Miguel Servet I3SNS and RETICS Program (RD12/0025). We thank Dr Kenneth McCreath for helpful comments on the manuscript.Garcia, NA.; Moncayo-Arlandi, J.; Vázquez Sánchez, A.; Genoves, P.; Calvo Saiz, CJ.; Millet Roig, J.; Martí, N.... (2017). Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model. Annals of Transplantation. 22:285-295. https://doi.org/10.12659/AOT.901410S2852952

Familial Dilated Cardiomyopathy Caused by a Novel Frameshift in the BAG3 Gene.

Dilated cardiomyopathy, a major cause of chronic heart failure and cardiac transplantation, is characterized by left ventricular or biventricular heart dilatation. In nearly 50% of cases the pathology is inherited, and more than 60 genes have been reported as disease-causing. However, in 30% of familial cases the mutation remains unidentified even after comprehensive genetic analysis. This study clinically and genetically assessed a large Spanish family affected by dilated cardiomyopathy to search for novel variations.Our study included a total of 100 family members. Clinical assessment was performed in alive, and genetic analysis was also performed in alive and 1 deceased relative. Genetic screening included resequencing of 55 genes associated with sudden cardiac death, and Sanger sequencing of main disease-associated genes. Genetic analysis identified a frame-shift variation in BAG3 (p.H243Tfr*64) in 32 patients. Genotype-phenotype correlation identified substantial heterogeneity in disease expression. Of 32 genetic carriers (one deceased), 21 relatives were clinically affected, and 10 were asymptomatic. Seventeen of the symptomatic genetic carriers exhibited proto-diastolic septal knock by echocardiographic assessment.We report p.H243Tfr*64_BAG3 as a novel pathogenic variation responsible for familial dilated cardiomyopathy. This variation correlates with a more severe phenotype of the disease, mainly in younger individuals. Genetic analysis in families, even asymptomatic individuals, enables early identification of individuals at risk and allows implementation of preventive measures

Familial Dilated Cardiomyopathy Caused by a Novel Frameshift in the BAG3 Gene.

Dilated cardiomyopathy, a major cause of chronic heart failure and cardiac transplantation, is characterized by left ventricular or biventricular heart dilatation. In nearly 50% of cases the pathology is inherited, and more than 60 genes have been reported as disease-causing. However, in 30% of familial cases the mutation remains unidentified even after comprehensive genetic analysis. This study clinically and genetically assessed a large Spanish family affected by dilated cardiomyopathy to search for novel variations.Our study included a total of 100 family members. Clinical assessment was performed in alive, and genetic analysis was also performed in alive and 1 deceased relative. Genetic screening included resequencing of 55 genes associated with sudden cardiac death, and Sanger sequencing of main disease-associated genes. Genetic analysis identified a frame-shift variation in BAG3 (p.H243Tfr*64) in 32 patients. Genotype-phenotype correlation identified substantial heterogeneity in disease expression. Of 32 genetic carriers (one deceased), 21 relatives were clinically affected, and 10 were asymptomatic. Seventeen of the symptomatic genetic carriers exhibited proto-diastolic septal knock by echocardiographic assessment.We report p.H243Tfr*64_BAG3 as a novel pathogenic variation responsible for familial dilated cardiomyopathy. This variation correlates with a more severe phenotype of the disease, mainly in younger individuals. Genetic analysis in families, even asymptomatic individuals, enables early identification of individuals at risk and allows implementation of preventive measures

Familial Dilated Cardiomyopathy Caused by a Novel Frameshift in the BAG3 Gene

Dilated cardiomyopathy, a major cause of chronic heart failure and cardiac transplantation, is characterized by left ventricular or biventricular heart dilatation. In nearly 50% of cases the pathology is inherited, and more than 60 genes have been reported as disease-causing. However, in 30% of familial cases the mutation remains unidentified even after comprehensive genetic analysis. This study clinically and genetically assessed a large Spanish family affected by dilated cardiomyopathy to search for novel variations. Methods and Results Our study included a total of 100 family members. Clinical assessment was performed in alive, and genetic analysis was also performed in alive and 1 deceased relative. Genetic screening included resequencing of 55 genes associated with sudden cardiac death, and Sanger sequencing of main disease-associated genes. Genetic analysis identified a frameshift variation in BAG3 (p.H243Tfr*64) in 32 patients. Genotype-phenotype correlation identified substantial heterogeneity in disease expression. Of 32 genetic carriers (one deceased), 21 relatives were clinically affected, and 10 were asymptomatic. Seventeen of the symptomatic genetic carriers exhibited proto-diastolic septal knock by echocardiographic assessment. Conclusions We report p.H243Tfr*64_BAG3 as a novel pathogenic variation responsible for familial dilated cardiomyopathy. This variation correlates with a more severe phenotype of the disease, mainly in younger individuals. Genetic analysis in families, even asymptomatic individuals, enables early identification of individuals at risk and allows implementation of preventive measure

Unusual anatomical origins of the coronary arteries in C57BL/6 mice. Are they strain-specific?

A previous manuscript [Fernández B, et al. (2008) J Anat 212, 12] reported on the unusual coronary artery patterns in mice belonging to the C57BL/6 strain. The aim here was to elucidate whether this pattern is unique to C57BL/6 mice or appears in other laboratory mouse strains and in wild-living mice. Stereomicroscopy, scanning electron microscopy, light microscopy and a corrosion cast technique were used to examine 597 adult mice belonging to three inbred strains (C57BL/6, Balb/c, DBA/2), three outbred stocks (CD1, OF1, NMRI) two hybrid lines (129sv × BL/6, CD2F1) and wild mice. It was shown that lock-like ostium is an exclusive trait of C57BL/6 mice, whereas left septal artery, accessory ostium, high take-off, intramural course and solitary ostium in aorta are all present in the different laboratory strains and wild mice included in the present study. However, each mouse population shows a specific incidence of these coronary conditions. Several clinically relevant human coronary artery anomalies are present in healthy mice from different strains that may serve as animal models for humans. These results should be taken into consideration in research concerning the murine coronary system, especially in coronary artery occlusion experiments and in studies on cardiovascular developmental biology using murine mutant lines

Molecular disturbance underlies to arrhythmogenic cardiomyopathy induced by transgene content, age and exercise in a truncated PKP2 mouse model

13 páginas, 9 tablas, 2 figuras. Contiene material suplementario.Arrhythmogenic cardiomyopathy (ACM) is a disorder characterized by a progressive ventricular myocardial replacement by fat and fibrosis, which lead to ventricular arrhythmias and sudden cardiac death. Mutations in the desmosomal gene Plakophilin-2 (PKP2) accounts for >40% of all known mutations, generally causing a truncated protein. In a PKP2-truncated mouse model, we hypothesize that content of transgene, endurance training and aging will be determinant in disease progression. In addition, we investigated the molecular defects associated with the phenotype in this model. We developed a transgenic mouse model containing a truncated PKP2 (PKP2-Ser329) and generated three transgenic lines expressing increasing transgene content. The pathophysiological features of ACM in this model were assessed. While we did not observe fibro-fatty replacement, ultrastructural defects were exhibited. Moreover, we observed transgene content-dependent development of structural (ventricle dilatation and dysfunction) and electrophysiological anomalies in mice (PR interval and QRS prolongation and arrhythmia induction). In concordance with pathological defects, we detected a content reduction and remodeling of the structural proteins Desmocollin-2, Plakoglobin, native Plakophilin-2, Desmin and β-Catenin as well as the electrical coupling proteins Connexin 43 and cardiac sodium channel (Nav1.5). Surprisingly, we observed structural but not electrophysiological abnormalities only in trained and old mice. We demonstrated that truncated PKP2 provokes ACM in the absence of fibro-fatty replacement in the mouse. Transgene dose is essential to reveal the pathology, whereas aging and endurance training trigger limited phenotype. Molecular abnormalities underlay the structural and electrophysiological defects.This work was supported by grants from “Fundació la Caixa”, Red de Investigación Cardiovascular from Instituto Salud Carlos III (RD12/0042/0044 and RD12/0042/0019), MINECO (SAF2012-39732), and Fundació Marató TV3 2014, Spain.Peer reviewe

Echocardiographic findings in patients carrying the genetic variant associated with DCM.

<p>Gender: M, male; F, female. Symptoms: P, palpitations; T, transplanted; p-T, Pre-transplant stage; p-S, Presyncope; S, Syncope; SHF, sudden heart failure; (-) means no dyspnea. New York Heart Association Functional Classification is indicated by a rank between 1 and 4. LVEF indicates left ventricle ejection fraction; TDLVD, telediastolic left ventricle diameter; LAA1, left atrial volume; E/A ratio, relation between E-wave and A-wave in Doppler. Knock in myocardium is indicated by presence (1) or absence (0).</p