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Transthyretin amyloid cardiomyopathy (ATTR-CM) is increasingly recognized as a cause of heart failure in the elderly. Although wild-type transthyretin amyloidosis is the most frequent form of ATTR-CM found in the elderly, hereditary transthyretin amyloidosis (ATTRv) can also occur. We sought to determine the prevalence of ATTRv among elderly ATTR-CM patients, identify predictors of ATTRv and evaluate the clinical consequences of positive genetic testing in this population. Prevalence of ATTRv in elderly ATTR-CM patients (≥70 years) was assessed in a cohort of 300 consecutive ATTR-CM patients (median age 78 years at diagnosis, 82% ≥70 years, 16% female, 99% Caucasian). ATTRv was diagnosed in 35 (12%; 95% confidence interval [CI] 3.1–8.8) and 13 (5.3%; 95% CI 5.6–26.7) patients in the overall cohort and in those ≥70 years, respectively. Prevalence of ATTRv among elderly female patients with ATTR-CM was 13% (95% CI 2.1–23.5). Univariate analysis identified female sex (odds ratio [OR] 3.66; 95% CI 1.13–11.85; p = 0.03), black ancestry (OR 46.31; 95% CI 3.52–Inf; p = 0.005), eye symptoms (OR 6.64; 95% CI 1.20–36.73; p = 0.03) and polyneuropathy (OR 10.05; 95% CI 3.09–32.64; p, This study has been funded by Instituto de Salud Carlos III (ISCIII) through the projects ‘PI18/0765 & PI20/01379’ (co-funded by European Regional Development Fund/European Social Fund ‘A way to make Europe’/‘Investing in your future’). AMB receives grant support by ISCIII (CM20/002209). The CNIC is supported by the ISCIII, MCIN, the Pro-CNIC Foundation, and the Severo Ochoa grant (CEX2020-001041-S)

Original article: Assessment of myocardial viscoelasticity with Brillouin spectroscopy in myocardial infarction and aortic stenosis models. In Scientific Reports (Vol. 11, Issue 1). Springer Science and Business Media LLC. https://doi.org/10.1038/s41598-021-00661-4 This study has been funded by Instituto de Salud Carlos III through the project PI18/00462 to M.V.G.G., cofunded by European Regional Development Fund "A way to make Europe, (CB16/11/00432 to P.G.-P. and E.L-P, and RD12/0042/005) and the Spanish Ministerio de Ciencia (RTI2018-096961-B-I00 to E.L-P. and RTI2018- 096918-B-C41 to R.J.J.R.). This study was also supported by the Plan Estatal de I+D+I 2013-2016, with funding from the European Regional Development Fund (ERDF) “A way to build Europe” initiative. The CNIC is supported by Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare genetically-determined cardiac heart muscle disorder characterized by fibro-fatty replacement of the myocardium that causes heart failure and sudden cardiac death (SCD), predominantly in young males. The disease is often caused by mutations in genes encoding proteins of the desmosomal (cell-to-cell adhesion) complex, with a significant minority caused by mutations in non-desmosomal proteins. Existing treatment options are based on SCD prevention with the implantable cardioverter defibrillator and anti-heart failure medication. Heart transplantation may also be required and there is currently no cure. Several transgenic animal models have been developed to characterize the disease, assess its progression and determine the influence of potential environmental factors. Transgenic models are also very valuable for translational therapeutic approaches, to screen new treatment options that prevent and/or regress the disease. Here we review the available ARVC animal models reported to date, highlighting the most important pathophysiological findings and discussing the effect of treatments tested so far in this setting. We also describe gaps in current disease knowledge with the goal of facilitating research and improving patient outcomes. pre-print 396 KB

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFβ1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFβ1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFβ1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFβ1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 13-01-2021, Introns populate eukaryotic genes to a variable extent across species, being widespread in vertebrates and mammals. While the evolutionary advantages, if any, of introns, remain unclear, their expansion has provided the opportunity to splice genes in more than a single way, allowing the production of diferent mRNAs from a single gene through Alternative splicing (AS). AS patterns change during the development of complex organisms and diverge across diferent tissues and experimental conditions. These highly reproducible changes evidences the existence of a regulatory network that ensures repeatable responses to certain stimuli and suggest that, at least some of them, play a role in the overall physiological response or adaptation. Not surprisingly, perturbation of some elements of this network is often associated with pathological conditions. However, not only we are far from a complete characterization of the molecular mechanisms that drive AS changes in most pathologies like those afecting the heart, but the computational tools that are currently used to study these regulatory networks are limiting our ability to extract all the information that is hidden in the data. It has been long hypothesized that AS contributes to a great expansion of the proteome and facilitates the evolution of new functions from pre-existing ones without gene duplication. While there are very well known examples of how AS enables the production of diferent functional proteins or mRNAs, the proportion of AS isoforms that are actually functional remains large unknown. Indeed, recent studies from diferent perspectives, including both transcriptomic, proteomics and sequence evolutionary analysis suggest that this percentage may be rather small and that much of the observed transcriptomic diversity is driven by non-functional noise in the splicing process. In this thesis, we have studied global AS patterns through computational analysis of large RNA-seq datasets to characterize the causes and consequences of AS changes from diferent perspectives. First, we have analyzed how AS global patterns change during heart development and disease using data from a variety of mouse models. We found that AS changes modulate diferent biological processes than gene expression ones and are associated to isoform speci c protein-protein interactions. Disease patterns partially recapitulate developmental patterns probably through the upregulation of PTBP1, which is suficient to induce pathological changes in the heart. Second, in an attempt to improve computational tools for identi cation of regulatory elements, we have developed dSreg. This tool leverages the power of bayesian inference and hierarchical models to pool information across the whole transcriptome to infer, not only the changes in the activities of the underlying regulatory elements, but also the changes in inclusion rates, outperforming competing methods and tools made for both purposes separately. Finally, we have studied the evolutionary process driving AS divergence during mammalian evolution using models of phenotypic evolution in a phylogenetic framework. We found that AS patterns have evolved under weak stabilizing selection that allows widespread variability in AS patterns across species, with only about 5% of the genes probably encoding AS isoforms with dif erent functions. Rates of neutral evolution are high, preventing the identi cation of adaptive changes at this long evolutionary scale. In summary, this thesis provides new computational tools and knowledge about the evolution and regulation of AS in diferent biological conditions and helps to better understand its relevance from diferent persepectives.

16 p.-5 fig.-3 tab.-1 il. cent., BACKGROUND: The mechanisms underlying early atherosclerotic plaque formation are not completely understood. Moreover, plasma biomarkers of subclinical atherosclerosis are lacking., OBJECTIVES: The purpose of this study was to analyze the temporal and topologically resolved protein changes taking place in human aortas with early atherosclerosis to find new potential diagnostic and/or therapeutic targets., METHODS: The protein composition of healthy aortas (media layer) or with early atheroma (fatty streak and fibrolipidic, media and intima layers) was analyzed by deep quantitative multiplexed proteomics. Further analysis was performed by Western blot, immunohistochemistry, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. Plasma levels of complement C5 were analyzed in relation to the presence of generalized (>2 plaques) or incipient (0 to 2 plaques) subclinical atherosclerosis in 2 independent clinical cohorts (PESA [Progression of Early Subclinical Atherosclerosis] [n=360] and NEFRONA [National Observatory of Atherosclerosis in Nephrology] [n=394])., RESULTS: Proteins involved in lipid transport, complement system, immunoglobulin superfamily, and hemostasis are increased in early plaques. Components from the complement activation pathway were predominantly increased in the intima of fibrolipidic plaques. Among them, increased C5 protein levels were further confirmed by Western blot, enzyme-linked immunosorbent assay and immunohistochemistry, and associated with in situ complement activation. Plasma C5 was significantly increased in individuals with generalized subclinical atherosclerosis in both PESA and NEFRONA cohorts, independently of risk factors. Moreover, in the PESA study, C5 plasma levels positively correlated with global plaque volume and coronary calcification., CONCLUSIONS: Activation of the complement system is a major alteration in early atherosclerotic plaques and is reflected by increased C5 plasma levels, which have promising value as a novel circulating biomarker of subclinicalatherosclerosis., This study was funded by the Spanish MINECO(SAF2016-80843-R, BIO2015-67580-P, and PGC2018-097019-B-I00), CAM (Complemento II-CM, S2017/BMD-3673),Fondo de Investigaciones Sanitarias ISCiii-FEDER (Biobancos RD09/0076/00101) and PRB3 (IPT17/0019, ProteoRed), and FEDER “Una manera de hacer Europa.” CIBERCV is an Instituto de Salud Carlos III project. The work leading to these results has also received funding from “la Caixa” Banking Foundation under the project code HR17-00247. The PESA study is cofunded equally by the Centro Nacional de Investigaciones Cardiovasculares (CNIC), and Banco Santander, Madrid, Spain. The study also receives funding from the Instituto de Salud Carlos III (PI15/02019) and the European Regional Development Fund (ERDF) “Una manera de hacer Europa.”

BACKGROUND: Filamin C (encoded by the FLNC gene) is essential for sarcomere attachment to the plasmatic membrane. FLNC mutations have been associated with myofibrillar myopathies, and cardiac involvement has been reported in some carriers. Accordingly, since 2012, the authors have included FLNC in the genetic screening of patients with inherited cardiomyopathies and sudden death. OBJECTIVES: The aim of this study was to demonstrate the association between truncating mutations in FLNC and the development of high-risk dilated and arrhythmogenic cardiomyopathies. METHODS: FLNC was studied using next-generation sequencing in 2,877 patients with inherited cardiovascular diseases. A characteristic phenotype was identified in probands with truncating mutations in FLNC. Clinical and genetic evaluation of 28 affected families was performed. Localization of filamin C in cardiac tissue was analyzed in patients with truncating FLNC mutations using immunohistochemistry. RESULTS: Twenty-three truncating mutations were identified in 28 probands previously diagnosed with dilated, arrhythmogenic, or restrictive cardiomyopathies. Truncating FLNC mutations were absent in patients with other phenotypes, including 1,078 patients with hypertrophic cardiomyopathy. Fifty-four mutation carriers were identified among 121 screened relatives. The phenotype consisted of left ventricular dilation (68%), systolic dysfunction (46%), and myocardial fibrosis (67%); inferolateral negative T waves and low QRS voltages on electrocardiography (33%); ventricular arrhythmias (82%); and frequent sudden cardiac death (40 cases in 21 of 28 families). Clinical skeletal myopathy was not observed. Penetrance was >97% in carriers older than 40 years. Truncating mutations in FLNC cosegregated with this phenotype with a dominant inheritance pattern (combined logarithm of the odds score: 9.5). Immunohistochemical staining of myocardial tissue showed no abnormal filamin C aggregates in patients with truncating FLNC mutations. CONCLUSIONS: Truncating mutations in FLNC caused an overlapping phenotype of dilated and left-dominant arrhythmogenic cardiomyopathies complicated by frequent premature sudden death. Prompt implantation of a cardiac defibrillator should be considered in affected patients harboring truncating mutations in FLNC. Instituto de Salud Carlos III [PI11/0699, PI14/0967, PI14/01477, RD012/0042/0029, RD012/0042/0049, RD012/0042/0066, RD12/0042/0069]; Spanish Ministry of Economy and Competitiveness [SAF2015-71863-REDT]; Plan Nacional de I+D+I; Plan Estatalde I+D+I, European Regional Development Fund; Health in Code SL Sí

Las enfermedades cardiovasculares constituyen la mayor causa de muerte a nivel mundial, sin embargo la regulación de los mecanismos post-transcripcionales y el papel de las proteínas de unión a RNA (RBPs) en estas enfermedades continúa siendo un campo por descubrir. Estudios recientes muestran que la expresión aberrante de RBPs puede afectar a varios puntos del procesamiento del RNA, alterando la función de sus genes diana. La RBP serine-arginine rich factor 4 (SRSF4) está implicada en el desarrollo de patologías neurológicas y en cáncer. Sin embargo, su papel en el corazón es completamente desconocido. Nuestros análisis ecocardiográficos mostraron que los ratones con pérdida cardio-específica de SRSF4 (SRSF4 KO) desarrollan hipertrofia ventricular izquierda por un incremento del área de los cardiomiocitos, y disfunción diastólica en edades más avanzadas. Además, los ratones SRSF4 KO presentaron una actividad electrofisiológica alterada bajo condiciones de estrés cardiaco inducido por isoproterenol, incluyendo una depresión después del QRS y un QT largo, indicando un elevado riesgo de muerte súbita. El análisis por RNA-seq mostró cambios de expresión en un gran número de RNAs largos no codificantes (lncRNAs), entre los que se encontraba el lncRNA-GAS5 (Growth Arrest Specific 5), el cual hemos identificado como diana directa de SRSF4 en cardiomiocitos mediante individual nucleotide-resolution Cross-Linking and ImmunoPrecipitation (iCLIP). GAS5 es un inhibidor del receptor de glucocorticoides (GR), y su expresión se encontraba disminuida en los corazones SRSF4 KO. Consecuentemente, encontramos una elevación de la actividad transcripcional del GR que conducía a un incremento de marcadores de hipertrofia y del área celular. Además la sobreexpresión de GAS5 reducía la hipertrofia de los cardiomiocitos.