Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the “RAF paradox”) may have the same effect. BRAF was upregulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a “RAF paradox” effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the “RAF paradox”. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function

Nitric oxide signaling in cardiac development, function and disease

A variety of roles have been attributed to Nitric Oxide (NO) in the cardiovascular system. During development, exposure of mouse embryonic stem cells to high levels of NO has been shown to promote cardiomyocyte differentiation through activation of transcription factors such as Nkx2.5. In adults, NO has been implicated in control of blood pressure and regulation of cardiac contractility. In disease, it is evident that elevated levels of NO can have a protective effect during myocardial infarction. ADMA and L-NMMA regulate NO biosynthesis by endogenously inhibiting Nitric Oxide Synthases (NOS). DDAH (1 and 2) enzymes metabolise ADMA in order to regulate the production of NO. The aims of this PhD thesis are (1) to identify the role of NO levels during development of the cardiovascular system and (2) to identify the role of the Ddah2 gene during an ischemia/reperfusion injury to the myocardium. Treatment of mouse embryonic stem cells during differentiation with ADMA reduced NO biosynthesis and showed significantly lower expression of cardiac associated markers and endothelial cell markers. This decrease in cardiac differentiation was accompanied by an increase in Runx1, an important hematopoietic marker. Our findings suggest that reduced NO levels guide differentiating cells towards haematopoiesis by parallel reduction of endothelial and cardiac cell formation. Global Ddah2-/- mouse hearts that underwent a global ischemia/reperfusion injury showed significantly higher tissue survival when compared to WT hearts. Gene expression of iNOS and eNOS genes was elevated in Ddah2-/- hearts along with other cellular survival markers. In an attempt to repeat our observations, WT and Ddah2-/- adult cardiomyocytes were isolated and underwent a hypoxia/normoxia treatment. Analysis of cardiomyocytes, showed no increase in hypoxia associated markers such as Glut1 and Ddah1 gene suggesting that either Ddah2-/- cardiomyocytes do not sense hypoxia or that they do not need to induce a hypoxic response to low oxygen levels. This work provides novel insights into the role of NO in cardiac differentiation and Ddah2 gene as a potential therapeutic target for ischemia/reperfusion injuries of the myocardium.Open Acces

Zebrafish sex determination and sex differentiation. Genetic and environmental influences

1st Meeting on Emerging Technologies in Zebrafish, 25-26 Noviembre de 2009, BilbaoPeer Reviewe

Epigenetic mechanisms mediating environmental influences on fish basic functions.

I Jornada de Cromatina i Epigenètica Organitzada per la Secció de Biologia Molecular de la Societat Catalana de Biologia. Institut d'Estudis Catalans, 5 de març 2010 BarcelonaIt is well established that the phenotype is the result of the interaction of the genotype and the environment. However, the mechanisms by which the environment affects and directs the expression of a given genotype into a particular phenotype are still not completely understood. The mechanisms that integrate environmental cues and result in lasting phenotypic effects that can be heritable without changes into the nucleotide sequences of DNA belong to the realm of epigenetics. Recent findings in our lab evidence the presence of an epigenetic mechanism directing environmental influences on the sex phenotype that explains the altered sex ratios commonly found in fish exposed to thermal contamination. In this regard, farmed fish experience an early environment that is different from that to which wild conspecifics are exposed to and thus provide an excellent model where to study epigenetic effects. We thus argue that the early life environment that fish experience under farming conditions influences the pathways regulating epigenetic programming. This may result in long-lasting differences in the expression of genes critical for key biological functions, including behavior, stress responsivity, reproduction, and susceptibility to disease. We discuss our ongoing research on both production (sea bass) and model fish (zebrafish) to decipher epigenetic mechanisms mainly controlling reproductionPeer Reviewe

An epigenetic mechanism mediating temperature effects on fish sex ratios

I Jornada de Cromatina i Epigenètica Organitzada per la Secció de Biologia Molecular de la Societat Catalana de Biologia. Institut d'Estudis Catalans, 5 de març 2010, BarcelonaMany reptiles and fish have temperature-dependent sex determination (TSD), where the temperature experienced during early development determines gonadal sex. However, the underlying mechanism has remained elusive. In the developing gonads of non-mammalian vertebrates, expression of cyp19a, the gene coding for aromatase, is essential for female development. In ectothermic vertebrates, exposure to female-promoting temperatures is associated with cyp19a upregulation, whereas exposure to male-producing temperatures is associated with cyp19a suppression. The European sea bass has a sex determination mechanism influenced by temperature. Water temperatures 3–4°C higher than normal masculine about 50% of genetic females. The period of highest temperature sensitivity, 0–15 days post fertilization (dpf), is located not only before sex differentiation (150 dpf) but also before the formation of the gonadal ridges (35 dpf), and even before the migration of the primordial germ cells (25 dpf). How, then, temperature can determine the fate of an organ that is still not formed, not even in a rudimentary manner? This suggested the possibility of an epigenetic mechanism by which increased temperature could silence the expression of genes essential for female development. Analysis of a ~500 bp region of the sea bass cyp19a promoter showed the presence of seven CpGs. Males had double DNA methylation levels than females. Further, we also found an inverse relationship between cyp19a promoter DNA methylation and cyp19a gene expression levels. Induced methylation of the cyp19a promoter suppressed transcription in vitro. Finally, a CpG differentially methylated by temperature was found to be conserved across species. We have suggested that DNA methylation of the aromatase promoter most likely is the long-sought after mechanism connecting environmental temperature and sex ratios in species with TSD, including fish and reptiles. Currently, we are investigating the appearance of the sex-related differences in cyp19a promoter methylation and testing the effects of DNA methylation inhibitorsPeer Reviewe

Cardiomyocyte-Restricted Expression of IL11 Causes Cardiac Fibrosis, Inflammation, and Dysfunction

Cardiac fibrosis is a common pathological process in heart disease, representing a therapeutic target. Transforming growth factor β (TGFβ) is the canonical driver of cardiac fibrosis and was recently shown to be dependent on interleukin 11 (IL11) for its profibrotic effects in fibroblasts. In the opposite direction, recombinant human IL11 has been reported as anti-fibrotic and anti-inflammatory in the mouse heart. In this study, we determined the effects of IL11 expression in cardiomyocytes on cardiac pathobiology and function. We used the Cre-loxP system to generate a tamoxifen-inducible mouse with cardiomyocyte-restricted murine Il11 expression. Using protein assays, bulk RNA-sequencing, and in vivo imaging, we analyzed the effects of IL11 on myocardial fibrosis, inflammation, and cardiac function, challenging previous reports suggesting the cardioprotective potential of IL11. TGFβ stimulation of cardiomyocytes caused Il11 upregulation. Compared to wild-type controls, Il11-expressing hearts demonstrated severe cardiac fibrosis and inflammation that was associated with the upregulation of cytokines, chemokines, complement factors, and increased inflammatory cells. IL11 expression also activated a program of endothelial-to-mesenchymal transition and resulted in left ventricular dysfunction. Our data define species-matched IL11 as strongly profibrotic and proinflammatory when secreted from cardiomyocytes and further establish IL11 as a disease factor

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors

10.1016/j.celrep.2019.02.083CELL REPORTS26123231-