Systolic Dysfunction in Infarcted Mice Does Not Necessarily Lead to Heart Failure: Need to Refine Preclinical Models

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.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).S

Functional Impact and Regulation of Alternative Splicing in Mouse Heart Development and Disease.

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.S

Lafora Disease Is an Inherited Metabolic Cardiomyopathy

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).S

Induction of the calcineurin variant CnAβ1 after myocardial infarction reduces post-infarction ventricular remodelling by promoting infarct vascularization

AIMS: Ventricular remodelling following myocardial infarction progressively leads to loss of contractile capacity and heart failure. Although calcineurin promotes maladaptive cardiac hypertrophy, we recently showed that the calcineurin splicing variant, CnAβ1, has beneficial effects on the infarcted heart. However, whether this variant limits necrosis or improves remodelling is still unknown, precluding translation to the clinical arena. Here, we explored the effects and therapeutic potential of CnAβ1 overexpression post-infarction. METHODS AND RESULTS: Double transgenic mice with inducible cardiomyocyte-specific overexpression of CnAβ1 underwent left coronary artery ligation followed by reperfusion. Echocardiographic analysis showed depressed cardiac function in all infarcted mice 3 days post-infarction. Induction of CnAβ1 overexpression 1 week after infarction improved function and reduced ventricular dilatation. CnAβ1-overexpressing mice showed shorter, thicker scars, and reduced infarct expansion, accompanied by reduced myocardial remodelling. CnAβ1 induced vascular endothelial growth factor (VEGF) expression in cardiomyocytes, which resulted in increased infarct vascularization. This paracrine angiogenic effect of CnAβ1 was mediated by activation of the Akt/mammalian target of rapamycin pathway and VEGF. CONCLUSIONS: Our results indicate that CnAβ1 exerts beneficial effects on the infarcted heart by promoting infarct vascularization and preventing infarct expansion. These findings emphasize the translational potential of CnAβ1 for gene-based therapies.European Union [ERG-239158, CardioNeT-ITN-289600]; Spanish Ministry of Science and Innovation [BFU2009-10016, SAF2012-31451]; Regional Government of Madrid [2010-BMD-2321]; Fondo de Investigaciones Sanitarias [RD12/0042/0066]; Spanish Ministry of Economy and Competitiveness; Pro-CNIC FoundationS

Lung ultrasound as a translational approach for non-invasive assessment of heart failure with reduced or preserved ejection fraction in mice

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.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).S

Calcineurin splicing variant calcineurin Aβ1 improves cardiac function after myocardial infarction without inducing hypertrophy

BACKGROUND: Calcineurin is a calcium-regulated phosphatase that plays a major role in cardiac hypertrophy. We previously described that alternative splicing of the calcineurin Aβ (CnAβ) gene generates the CnAβ1 isoform, with a unique C-terminal region that is different from the autoinhibitory domain present in all other CnA isoforms. In skeletal muscle, CnAβ1 is necessary for myoblast proliferation and stimulates regeneration, reducing fibrosis and accelerating the resolution of inflammation. Its role in the heart is currently unknown. METHODS AND RESULTS: We generated transgenic mice overexpressing CnAβ1 in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter. In contrast to previous studies using an artificially truncated calcineurin, CnAβ1 overexpression did not induce cardiac hypertrophy. Moreover, transgenic mice showed improved cardiac function and reduced scar formation after myocardial infarction, with reduced neutrophil and macrophage infiltration and decreased expression of proinflammatory cytokines. Immunoprecipitation and Western blot analysis showed interaction of CnAβ1 with the mTOR complex 2 and activation of the Akt/SGK cardioprotective pathway in a PI3K-independent manner. In addition, gene expression profiling revealed that CnAβ1 activated the transcription factor ATF4 downstream of the Akt/mTOR pathway to promote the amino acid biosynthesis program, to reduce protein catabolism, and to induce the antifibrotic and antiinflammatory factor growth differentiation factor 15, which protects the heart through Akt activation. CONCLUSIONS: Calcineurin Aβ1 shows a unique mode of action that improves cardiac function after myocardial infarction, activating different cardioprotective pathways without inducing maladaptive hypertrophy. These features make CnAβ1 an attractive candidate for the development of future therapeutic approaches.British Heart Foundation [PG/07/020/22503]; Spanish Ministry of Science and Innovation [BFU2009-10016]; National Institutes of Health Research, Cardiovascular Biomedical Research Unit at the Royal Brompton; Hare-field NHS Foundation Trust; Imperial College; Spanish Fondo Nacional de Investigaciones Sanitarias [EIF-040545, ERG-239158, CP08/00144

Follistatin-like 3 mediates paracrine fibroblast activation by cardiomyocytes

Follistatins are extracellular inhibitors of the TGF-β 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 [PG/08/084/25827]; Heart Research UK; National Institute for Health Research Cardiovascular Biomedical Research Unit at the Royal Brompton; Harefield NHS Foundation Trust; Imperial College; European Union [ERG-239158, ITN-289600]; Spanish Ministry of Science and Innovation [BFU2009-10016, CP08/00144]; Regional Government of Madrid [S2010/BMD-2321 'Fibroteam']S

Activation of Serine One-Carbon Metabolism by Calcineurin A beta 1 Reduces Myocardial Hypertrophy and Improves Ventricular Function

BACKGROUND In response to pressure overload, the heart develops ventricular hypertrophy that progressively decompensates and leads to heart failure. This pathological hypertrophy is mediated, among others, by the phosphatase calcineurin and is characterized by metabolic changes that impair energy production by mitochondria. OBJECTIVES The authors aimed to determine the role of the calcineurin splicing variant CnA beta 1 in the context of cardiac hypertrophy and its mechanism of action. METHODS Transgenic mice overexpressing CnAb1 specifically in cardiomyocytes and mice lacking the unique C-terminal domain in CnA beta 1 (CnA beta 1(Delta i12) mice) were used. Pressure overload hypertrophy was induced by transaortic constriction. Cardiac function was measured by echocardiography. Mice were characterized using various molecular analyses. RESULTS In contrast to other calcineurin isoforms, the authors show here that cardiac-specific overexpression of CnA beta 1 in transgenic mice reduces cardiac hypertrophy and improves cardiac function. This effect is mediated by activation of serine and one-carbon metabolism, and the production of antioxidant mediators that prevent mitochondrial protein oxidation and preserve ATP production. The induction of enzymes involved in this metabolic pathway by CnAb1 is dependent on mTOR activity. Inhibition of serine and one-carbon metabolism blocks the beneficial effects of CnA beta 1. CnA beta 1(Delta i12) mice show increased cardiac hypertrophy and declined contractility. CONCLUSIONS The metabolic reprogramming induced by CnAb1 redefines the role of calcineurin in the heart and shows for the first time that activation of the serine and one-carbon pathway has beneficial effects on cardiac hypertrophy and function, paving the way for new therapeutic approaches. (J Am Coll Cardiol 2018; 71: 654-67) (C) 2018 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/).This work was supported by grants from the European Union (CardioNeT-ITN-289600 and CardioNext-608027 to Dr. Lara-Pezzi; Meet-ITN-317433 to Dr. Enriquez; UE0/MCA1108 to Dr. Acin-Perez), from the Spanish Ministry of Economy and Competitiveness (SAF2015-65722-R and SAF2012-31451 to Dr. Lara-Pezzi; SAF2015-71521-REDC, BFU2013-50448, and SAF2012-32776 to Dr. Enriquez; RyC-2011-07826 to Dr. Acin-Perez; BIO2012-37926 and BIO2015-67580-P to Dr. Vazquez), from the Spanish Carlos III Institute of Health (CPII14/00027 to Dr. Lara-Pezzi; RD12/0042/066 to Drs. Garcia-Pavia and Lara-Pezzi), from the Regional Government of Madrid (2010-BMD-2321 ``Fibroteam´´ to Dr. Lara-Pezzi; 2011-BMD-2402 ``Mitolab´´ to Dr. Enriquez) and the FIS-ISCIII (PRB2-IPT13/0001 and RD12/0042/0056-RIC-RETICS to Dr. Vazquez). This work was also supported by the Plan Estatal de IthornDthornI 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 and by the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). Drs. Vazquez and Garcia-Pavia have served as consultants for VL39. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Padron-Barthe, Villalba-Orero, and Gomez-Salinero contributed equally to this work and are joint first authors. Robyn Shaw, MD, PhD, served as Guest Editor for this paper.S

Loss of SRSF3 in Cardiomyocytes Leads to Decapping of Contraction-Related mRNAs and Severe Systolic Dysfunction

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 decapping as a mechanism of systolic heart failure may open the way to the development of urgently needed therapeutic tools.This study was supported by grants from the European Union (CardioNeT-ITN-289600 and CardioNext-ITN-608027 to E.L-P.), from the Spanish Ministerio de Economía y Competitividad (RTI2018-096961-BI00, SAF2015-65722-R and SAF2012-31451 to E.L-P.; BIO2015-67580-P and PGC2018-097019-B-I00 to J.V.), the Spanish Carlos III Institute of Health (CPII14/00027 to E.L-P, RD12/0042/066 to P.G.-P. and E.L-P, and RD12/0042/0056, PRB2-IPT13/0001-ISCIII-SGEFI/FEDER, ProteoRed to J.V.), the Madrid Regional Government (2010-BMD-2321 “Fibroteam” to E.L-P.). 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).S

The Calcineurin Variant CnAβ1 Controls Mouse Embryonic Stem Cell Differentiation by Directing mTORC2 Membrane Localization and Activation

Embryonic stem cells (ESC) have the potential to generate all the cell lineages that form the body. However, the molecular mechanisms underlying ESC differentiation and especially the role of alternative splicing in this process remain poorly understood. Here, we show that the alternative splicing regulator MBNL1 promotes generation of the atypical calcineurin Aβ variant CnAβ1 in mouse ESCs (mESC). CnAβ1 has a unique C-terminal domain that drives its localization mainly to the Golgi apparatus by interacting with Cog8. CnAβ1 regulates the intracellular localization and activation of the mTORC2 complex. CnAβ1 knockdown results in delocalization of mTORC2 from the membrane to the cytoplasm, inactivation of the AKT/GSK3β/β-catenin signaling pathway, and defective mesoderm specification. In summary, here we unveil the structural basis for the mechanism of action of CnAβ1 and its role in the differentiation of mESCs to the mesodermal lineage.European Union's FP7 [CardioNext-ITN-608027, Cardio-NeT-ITN-289600]; Spanish Ministry of Science and Innovation [SAF2012-31451, CP08/00144]; Regional Government of Madrid [2010-BMD-2321]; Spanish Ministry of Economy and Competitiveness; Pro-CNIC Foundation; Severo Ochoa Center of Excellence (MINECO award) [SEV-2015-0505]S