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

Negative screening of Fabry disease in patients with conduction disorders requiring a pacemaker.

Identification of Fabry disease (FD) in cardiac patients has been restricted so far to patients with left ventricular hypertrophy. Conduction problems are frequent in FD and could precede other manifestations, offering a possible earlier diagnosis. We studied the prevalence of FD in 188 patients < 70 years with conduction problems requiring pacemaker implantation. Although classical manifestations of FD were not rare, no patient with FD was identified. Screening efforts should not be conducted in this population.post-print523 K

Assessment of myocardial viscoelasticity with Brillouin spectroscopy in myocardial infarction and aortic stenosis models.

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.post-print6652 K

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

Early preventive treatment with Enalapril improves cardiac function and delays mortality in mice with arrhythmogenic right ventricular cardiomyopathy type 5.

Background: Arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5) is an inherited cardiac disease with complete penetrance and an aggressive clinical course caused by mutations in TMEM43 (transmembrane protein 43). There is no cure for ARVC5 and palliative treatment is started once the phenotype is present. A transgenic mouse model of ARVC5 expressing human TMEM43-S358L (TMEM43mut) recapitulates the human disease, enabling the exploration of preventive treatments. The aim of this study is to determine whether preventive treatment with heart failure drugs (β-blockers, ACE [angiotensin-converting enzyme] inhibitors, mineralocorticoid-receptor antagonists) improves the disease course of ARVC5 in TMEM43mut mice. Methods: TMEM43mut male/female mice were treated with metoprolol (β-blockers), enalapril (ACE inhibitor), spironolactone (mineralocorticoid-receptor antagonist), ACE inhibitor + mineralocorticoid-receptor antagonist, ACE inhibitor + mineralocorticoid-receptor antagonist + β-blockers or left untreated. Drugs were initiated at 3 weeks of age, before ARVC5 phenotype, and serial ECG and echocardiograms were performed. Results: TMEM43mut mice treated with enalapril showed a significantly increased median survival compared with untreated mice (26 versus 21 weeks; P=0.003). Enalapril-treated mice also exhibited increased left ventricular ejection fraction at 4 months compared with controls (37.0% versus 24.9%; P=0.004), shorter QRS duration and reduced left ventricle fibrosis. Combined regimens including enalapril also showed positive effects. Metoprolol decreased QRS voltage prematurely and resulted in a nonsignificant decrease in left ventricular ejection fraction compared with untreated TMEM43mut mice. Conclusions: Preventive enalapril-based regimens reduced fibrosis, improved ECG, echocardiographic parameters and survival of ARVC5 mice. Early metoprolol did not show positive effects and caused premature ECG abnormalities. Our findings pave the way to consider prophylactic enalapril in asymptomatic ARVC5 genetic carriers.pre-print326 K

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

The SRSF4–GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy.

RATIONALE: RBPs (RNA-binding proteins) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP SRSF4 (serine/arginine-rich splicing factor 4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. 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-nucleotideresolution 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.post-print2695 K

The SRSF4–GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy.

RATIONALE: RBPs (RNA-binding proteins) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP SRSF4 (serine/arginine-rich splicing factor 4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. 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-nucleotideresolution 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.post-print2695 K

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