Embryonic echocardiography for assessment of congenital and functional cardiac defects.

Cardiac function and morphology by mouse fetal echocardiography can be assessed by scanning the uterus extracted from the abdominal cavity (trans-uterine ultrasound) or the womb (trans-abdominal ultrasound). Advantages of transabdominal ultrasound include (1) non-invasive longitudinal analysis at different stages, reducing animal use; and (2) maintenance of natural environment, diminishing perturbations on functional parameters, which are more frequent in transuterine conditions. Here we describe both approaches, explaining how to identify congenital cardiac defects and defining the correlation between echocardiography findings and histological analysis.post-print5184 K

Cardiorespiratory, Sedative and Antinociceptive Effects of a Medetomidine Constant Rate Infusion with Morphine, Ketamine or Both

Standing surgery under sedation reduces anesthetic-related mortality in horses. Medetomidine, alone and combined with morphine in a constant rate infusion (CRI), has been described for standing surgery but their cardiorespiratory, sedative and antinociceptive effects have never been compared. The addition of ketamine could improve analgesia in these procedures with minimal cardiorespiratory consequences. The objectives were to compare the cardiorespiratory effects, quality of sedation, antinociception and ataxia produced by administration of a medetomidine-based CRI with morphine, ketamine or both, in standing horses. A prospective, blind, randomized crossover, experimental design with six healthy adult horses was performed, in which four treatments were administered to all horses with at least two weeks of washout period: medetomidine (M); medetomidine and ketamine (MK); medetomidine and morphine (MMo); and medetomidine, morphine and ketamine (MMoK). Dosages were the same in all treatment groups: medetomidine at 5 µg/kg bwt followed by 5 µg/kg bwt/h, ketamine at 0.4 mg/kg/h and morphine at 50 µg/kg bwt, followed by morphine 30 µg/kg bwt/h. Drug infusions were maintained for 120 min. Cardiorespiratory variables, sedation degree and antinociceptive effects were evaluated during the procedure. All combinations produced similar sedation and antinociceptive effects and no clinically relevant alterations in cardiorespiratory variables occurred. Medetomidine CRI combined with morphine, ketamine or both are suitable and safe protocols for standing sedation in horses and the addition of morphine and/or ketamine did not cause any negative effect but no improving effect on sedation and antinociception was detected

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

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

Elaboración de material docente para la enseñanza de la exploración física del caballo

El objetivo principal del presente proyecto fue incorporar una serie de recursos educativos virtuales que facilitaran e implementaran la enseñanza de la exploración física del caballo

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

γ-Linolenic acid in maternal milk drives cardiac metabolic maturation.

Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA-RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism.S