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This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2015-65722-R to Dr. Lara-Pezzi and SAF2014-59594-R to Dr. Serratosa), Autonomous Community of Madrid (2010-BMD2321, FIBROTEAM Consortium), European Union's FP7 (CardioNeT-ITN-289600, CardioNext-ITN-608027), the Spanish Carlos III Institute of Health (CPII14/00027 to Dr. Lara-Pezzi, PI13/00865 to Dr. Sanchez and RD12/0042/066 to Drs. Garcia-Pavia and Lara-Pezzi), and the National Institute of Neurological Disorders And Stroke of the National Institutes of Health (P01NS097197 to Dr. Sanchez). This work was also supported by the Plan Estatal de I+D+I 2013-2016-European Regional Development Fund (FEDER) "A way of making Europe," Spain. The Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505)., Ministerio de Economía y Competitividad (España), Comunidad de Madrid (España), Unión Europea. Comisión Europea, Instituto de Salud Carlos III, National Institutes of Health (Estados Unidos), Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF), Fundación ProCNIC, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Alternative splicing (AS) plays a major role in the generation of transcript diversity. In the heart, roles have been described for some AS variants, but the global impact and regulation of AS patterns are poorly understood. Here, we studied the AS profiles in heart disease, their relationship with heart development, and the regulatory mechanisms controlling AS dynamics in the mouse heart. We found that AS profiles characterized the different groups and that AS and gene expression changes affected independent genes and biological functions. Moreover, AS changes, specifically in heart disease, were associated with potential protein–protein interaction changes. While developmental transitions were mainly driven by the upregulation of MBNL1, AS changes in disease were driven by a complex regulatory network, where PTBP1 played a central role. Indeed, PTBP1 over-expression was sufficient to induce cardiac hypertrophy and diastolic dysfunction, potentially by perturbing AS patterns., Unión Europea, MINECO, Comunidad de Madrid, Instituto de Salud Carlos III, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

This study was supported by grants from the Spanish Ministerio de Economia y Competitividad (SAF2015-65722-R), Comunidad Autonoma de Madrid (2010-BMD2321, FIBROTEAM Consortium), European Union's FP7 (CardioNeT-ITN-289600, CardioNext-ITN-608027) and the Spanish Instituto de Salud Carlos III (CPII14/00027 to E.L-P, RD12/0042/0054 to B.I. and RD12/0042/066 to P.G.-P. and E.L-P). This work was also supported by the Plan Estatal de I+D+I 2013-2016 - European Regional Development Fund (FEDER) "A way of making Europe", Spain. The CNIC is supported by the Ministry of Economy, Industry and Competitiveness (MINECO) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505)., Aims Heart failure (HF) has become an epidemic and constitutes a major medical, social, and economic problem worldwide. Despite advances in medical treatment, HF prognosis remains poor. The development of efficient therapies is hampered by the lack of appropriate animal models in which HF can be reliably determined, particularly in mice. The development of HF in mice is often assumed based on the presence of cardiac dysfunction, but HF itself is seldom proved. Lung ultrasound (LUS) has become a helpful tool for lung congestion assessment in patients at all stages of HF. We aimed to apply this non-invasive imaging tool to evaluate HF in mouse models of both systolic and diastolic dysfunction. Methods and results We used LUS to study HF in a mouse model of systolic dysfunction, dilated cardiomyopathy, and in a mouse model of diastolic dysfunction, diabetic cardiomyopathy. LUS proved to be a reliable and reproducible tool to detect pulmonary congestion in mice. The combination of LUS and echocardiography allowed discriminating those mice that develop HF from those that do not, even in the presence of evident cardiac dysfunction. The study showed that LUS can be used to identify the onset of HF decompensation and to evaluate the efficacy of therapies for this syndrome. Conclusions This novel approach in mouse models of cardiac disease enables for the first time to adequately diagnose HF non-invasively in mice with preserved or reduced ejection fraction, and will pave the way to a better understanding of HF and to the development of new therapeutic approaches., Ministerio de Economía y Competitividad (España), Comunidad de Madrid (España), Unión Europea. Comisión Europea, Instituto de Salud Carlos III, Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF), Fundación ProCNIC, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

This study was supported by grants from the European Union (CardioNeT-ITN-289600 and CardioNext-ITN-608027 to E. Lara-Pezzi), from the Spanish Ministerio de Economía y Competitividad (RTI2018-096961-B-I00, SAF2015-65722-R, and SAF2012-31451 to E. Lara-Pezzi; BIO2015-67580-P and PGC2018-097019-B-I00 to J. Vázquez), the Spanish Carlos III Institute of Health (CPII14/00027 to E. Lara-Pezzi, RD12/0042/066 to P. García-Pavía and E. Lara-Pezzi, and RD12/0042/0056, PRB2-IPT13/0001-ISCIII-SGEFI/FEDER, ProteoRed to J. Vázquez), the Madrid Regional Government (2010-BMD-2321 Fibroteam to E. Lara-Pezzi). This study was also supported by the Plan Estatal de I+D+I 2013–2016—European Regional Development Fund (ERDF) A way of making Europe, Spain. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505)., Rationale: RBPs (RNA binding proteins) play critical roles in the cell by regulating mRNA transport, splicing, editing, and stability. The RBP SRSF3 (serine/arginine-rich splicing factor 3) is essential for blastocyst formation and for proper liver development and function. However, its role in the heart has not been explored. Objective: To investigate the role of SRSF3 in cardiac function. Methods and Results: Cardiac SRSF3 expression was high at mid gestation and decreased during late embryonic development. Mice lacking SRSF3 in the embryonic heart showed impaired cardiomyocyte proliferation and died in utero. In the adult heart, SRSF3 expression was reduced after myocardial infarction, suggesting a possible role in cardiac homeostasis. To determine the role of this RBP in the adult heart, we used an inducible, cardiomyocyte-specific SRSF3 knockout mouse model. After SRSF3 depletion in cardiomyocytes, mice developed severe systolic dysfunction that resulted in death within 8 days. RNA-Seq analysis revealed downregulation of mRNAs encoding sarcomeric and calcium handling proteins. Cardiomyocyte-specific SRSF3 knockout mice also showed evidence of alternative splicing of mTOR (mammalian target of rapamycin) mRNA, generating a shorter protein isoform lacking catalytic activity. This was associated with decreased phosphorylation of 4E-BP1 (eIF4E-binding protein 1), a protein that binds to eIF4E (eukaryotic translation initiation factor 4E) and prevents mRNA decapping. Consequently, we found increased decapping of mRNAs encoding proteins involved in cardiac contraction. Decapping was partially reversed by mTOR activation. Conclusions: We show that cardiomyocyte-specific loss of SRSF3 expression results in decapping of critical mRNAs involved in cardiac contraction. The molecular mechanism underlying this effect likely involves the generation of a short mTOR isoform by alternative splicing, resulting in reduced 4E-BP1 phosphorylation. The identification of mRNA decappin, Unión Europea, Ministerio de Economía y Competitividad, de la Comunidad de Madrid, Plan Estatal de I+D+I 2013–2016 European Regional Development Fund (ERDF), “A way to build Europe” de la European Regional Development Fund (ERDF)., The Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence, Ministerio de Ciencia, Innovación y Universidades (MCNU), Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Background: Heart failure (HF) with preserved ejection fraction (HFpEF) prevalence is increasing, and large clinical trials have failed to reduce mortality. A major reason for this outcome is the failure to translate results from basic research to the clinics. Evaluation of HFpEF in mouse models requires assessing three major key features defining this complex syndrome: the presence of a preserved left ventricular ejection fraction (LVEF), diastolic dysfunction, and the development of HF. In addition, HFpEF is associated with multiple comorbidities such as systemic arterial hypertension, chronic obstructive pulmonary disease, sleep apnea, diabetes, and obesity; thus, non-cardiac disorders assessment is crucial for a complete phenotype characterization. Non-invasive procedures present unquestionable advantages to maintain animal welfare and enable longitudinal analyses. However, unequivocally determining the presence of HFpEF using these methods remains challenging. Main text: Transthoracic echocardiography (TTE) represents an invaluable tool in HFpEF diagnosis, allowing evaluation of LVEF, diastolic dysfunction, and lung congestion in mice. Since conventional parameters used to evaluate an abnormal diastole like E/A ratio, isovolumic relaxation time, and E/e′ may pose limitations in mice, including advanced TTE techniques to characterize cardiac motion, including an assessment under stress, will improve diagnosis. Patients with HFpEF also show electrical cardiac remodelling and therefore electrocardiography may add valuable information in mouse models to assess chronotropic incompetence and sinoatrial node dysfunction, which are major contributors to exercise intolerance. To complete the non-invasive diagnosis of HF, low aerobic exercise capacity and fatigue using exercise tests, impaired oxygen exchange using metabolic cages, and determination of blood biomarkers can be determined. Finally, since HFpEF patients commonly present non-cardiac pathological conditions, Plan Estatal de I+D+I, Pro CNIC Foundation, Spanish Ministerio de Ciencia e Innovacion, Instituto de Salud Carlos III Spain, Ministerio de Ciencia e Innovación Spain, European Regional Development Fund European Union, Agencia Estatal de Investigación Spain, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Follistatins are extracellular inhibitors of the TGF-beta family ligands including activin A, myostatin and bone morphogenetic proteins. Follistatin-like 3 (FSTL3) is a potent inhibitor of activin signalling and antagonises the cardioprotective role of activin A in the heart. FSTL3 expression is elevated in patients with heart failure and is upregulated in cardiomyocytes by hypertrophic stimuli, but its role in cardiac remodelling is largely unknown. Here, we show that the production of FSTL3 by cardiomyocytes contributes to the paracrine activation of cardiac fibroblasts, inducing changes in cell adhesion, promoting proliferation and increasing collagen production. We found that FSTL3 is necessary for this response and for the induction of cardiac fibrosis. However, full activation requires additional factors, and we identify connective tissue growth factor as a FSTL3 binding partner in this process. Together, our data unveil a novel mechanism of paracrine communication between cardiomyocytes and fibroblasts that may provide potential as a therapeutic target in heart remodelling., British Heart Foundation, Unión Europea, Ministerio de Ciencia e Innovación, Comunidad de Madrid, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2015-65722-R to E.L-P), Autonomous Community of Madrid (2010-BMD2321, FIBROTEAM Consortium), European Union's FP7 (CardioNeT-ITN-289600, CardioNext-ITN-608027 to E.L-P), the Spanish Carlos III Institute of Health (CPII14/00027 and RD12/0042/066 to E.L-P). This work was also supported by the Plan Estatal de I+D+I 2013-2016-European Regional Development Fund (FEDER) "A way of making Europe," Spain. The CNIC is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505)., Heart failure (HF) is a major cause of death and hospitalization worldwide. Despite advances in reducing mortality, prognosis remains poor and prevalence has reached epidemic proportions. The limitations of available preclinical models represent a major hurdle in the development of new therapies. Myocardial infarction (MI) is a main cause of HF in humans, and mouse models of MI are often used to study HF mechanisms and experimental treatments. We investigated whether MI in mice constitutes an appropriate model of HF. Permanent ligation of the left coronary artery induced severe and persistent systolic dysfunction and ventricular dilatation. Mouse follow-up for 10 months showed no significant evidence of lung congestion or other pulmonary defects associated with HF. No difference was observed in the capacity of infarcted mice to exercise compared to control animals. These results indicate that severe cardiac dysfunction in mice is not sufficient to demonstrate the presence of HF, Ministerio de Economía y Competitividad (España), Unión Europea. Comisión Europea, Instituto de Salud Carlos III, Comunidad de Madrid (España), Fundación ProCNIC, Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF), Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Cardiac cine magnetic resonance imaging (MRI) can be considered the optimal criterion for measuring cardiac function. This imaging technique can provide us with detailed information about cardiac structure, tissue composition and even blood flow, which makes it highly used in medical science. But due to the image time acquisition and several other factors the MRI sequences can easily get corrupted, causing radiologists to misdiagnose 40 million people worldwide each and every single year. Hence, the urge to decrease these numbers, researchers from different fields have been introducing novel tools and methods in the medical field. Aiming to the same target, we consider in this work the application of the higher order dynamic mode decomposition (HODMD) technique. The HODMD algorithm is a linear method, which was originally introduced in the fluid dynamics domain, for the analysis of complex systems. Nevertheless, the proposed method has extended its applicability to numerous domains, including medicine. In this work, HODMD in used to analyze sets of MR images of a heart, with the ultimate goal of identifying the main patterns and frequencies driving the heart dynamics. Furthermore, a novel interpolation algorithm based on singular value decomposition combined with HODMD is introduced, providing a three-dimensional reconstruction of the heart. This algorithm is applied (i) to reconstruct corrupted or missing images, and (ii) to build a reduced order model of the heart dynamics., SIMOPAIR, Spain, The European Union’s Horizon 2020, The Marie Skłodowska-Curie, Centro Nacional de Investigaciones Cardiovasculares Carlos III. (CNIC), Madrid, Spain, Ministerio de Ciencia e Innovación (MCIN). Madrid, Spain, The Pro CNIC Foundation. Severo Ochoa Center of Excellence, The European Union, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Objective: To investigate the role of SRSF4 in the heart. Methods and Results: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long noncoding RNAs, including GAS5 (growth arrest-specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotide-resolution cross-linking and immuno-precipitation. GAS5 is a repressor of the GR (glucocorticoid receptor) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. Conclusions: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in patients with Cushing syndrome., European Union, Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, Comunidad de Madrid, Plan Estatal I+D+I 2013-2016, Ministerio de Ciencia, Innovación y Universidades, Fundación Pro-CNIC, Severo Ochoa Center de Excellence, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

This study has been funded by Instituto de Salud Carlos III through the project PI18/00462 to M.V.G.G., co‐funded 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.). Tis 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. Te 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)., Heart diseases are associated with changes in the biomechanical properties of the myocardial wall. However, there is no modality available to assess myocardial stiffness directly. Brillouin microspectroscopy (mBS) is a consolidated mechanical characterization technique, applied to the study of the viscoelastic and elastic behavior of biological samples and may be a valuable tool for assessing the viscoelastic properties of the cardiac tissue. In this work, viscosity and elasticity were assessed using mBS in heart samples obtained from healthy and unhealthy mice (n = 6 per group). Speckle-tracking echocardiography (STE) was performed to evaluate heart deformation. We found that mBS was able to detect changes in stiffness in the ventricles in healthy myocardium. The right ventricle showed reduced stiffness, in agreement with its increased compliance. mBS measurements correlated strongly with STE data, highlighting the association between displacement and stiffness in myocardial regions. This correlation was lost in pathological conditions studied. The scar region in the infarcted heart presented changes in stiffness when compared to the rest of the heart, and the hypertrophied left ventricle showed increased stiffness following aortic stenosis, compared to the right ventricle. We demonstrate that mBS can be applied to determine myocardial stiffness, that measurements correlate with functional parameters and that they change with disease., Instituto de Salud Carlos III, Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF), Ministerio de Ciencia. (España), Ministerio de Ciencia e Innovación (España), Fundación ProCNIC, Ministerio de Ciencia e Innovación. Centro de Excelencia Severo Ochoa (España), Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Introduction: Removal of poorly perfused capillaries by pruning contributes to remodeling the microvasculature to optimize oxygen and nutrient delivery. Blood flow drives this process by promoting the intravascular migration of endothelial cells in developing networks, such as in the yolk sac, zebrafish brain or postnatal mouse retina., Methods: In this study, we have implemented innovative tools to recognize capillary pruning in the complex 3D coronary microvasculature of the postnatal mouse heart. We have also experimentally tested the impact of decreasing pruning on the structure and function of this network by altering blood flow with two different vasodilators: losartan and prazosin., Results: Although both drugs reduced capillary pruning, a combination of experiments based on ex vivo imaging, proteomics, electron microscopy and in vivo functional approaches showed that losartan treatment resulted in an inefficient coronary network, reduced myocardial oxygenation and metabolic changes that delayed the arrest of cardiomyocyte proliferation, in contrast to the effects of prazosin, probably due to its concomitant promotion of capillary expansion., Discussion: Our work demonstrates that capillary pruning contributes to proper maturation and function of the heart and that manipulation of blood flow may be a novel strategy to refine the microvasculature and improve tissue perfusion after damage.

Introduction: Removal of poorly perfused capillaries by pruning contributes to remodeling the microvasculature to optimize oxygen and nutrient delivery. Blood flow drives this process by promoting the intravascular migration of endothelial cells in developing networks, such as in the yolk sac, zebrafish brain or postnatal mouse retina. Methods: In this study, we have implemented innovative tools to recognize capillary pruning in the complex 3D coronary microvasculature of the postnatal mouse heart. We have also experimentally tested the impact of decreasing pruning on the structure and function of this network by altering blood flow with two different vasodilators: losartan and prazosin. Results: Although both drugs reduced capillary pruning, a combination of experiments based on ex vivo imaging, proteomics, electron microscopy and in vivo functional approaches showed that losartan treatment resulted in an inefficient coronary network, reduced myocardial oxygenation and metabolic changes that delayed the arrest of cardiomyocyte proliferation, in contrast to the effects of prazosin, probably due to its concomitant promotion of capillary expansion. Discussion: Our work demonstrates that capillary pruning contributes to proper maturation and function of the heart and that manipulation of blood flow may be a novel strategy to refine the microvasculature and improve tissue perfusion after damage., European Regional Development Fund, Deutsche Forschungsgemeinschaft, MICINN, Comunidad de Madrid, Fundación “La Caixa”, FPI Severo Ochoa, Severo Ochoa Center of Excellence, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

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.