Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin

Background: Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome). Results: Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate 1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region. Conclusions: Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs

Glycogen synthase kinases 3α and 3β in cardiac myocytes: regulation and consequences of their inhibition

Inhibition of glycogen synthase kinase 3β (GSK3β) as a consequence of its phosphorylation by protein kinase B/Akt (PKB/Akt) has been implicated in cardiac myocyte hypertrophy in response to endothelin-1 or phenylephrine. We examined the regulation of GSK3α (which we show to constitute a significant proportion of the myocyte GSK3 pool) and GSK3β in cardiac myocytes. Although endothelin increases phosphorylation of GSK3 and decreases its activity, the response is less than that induced by insulin (which does not promote cardiac myocyte hypertrophy). GSK3 phosphorylation induced by endothelin requires signalling through the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade and not the PKB/Akt pathway, whereas the reverse is true for insulin. Cardiac myocyte hypertrophy involves changes in morphology, and in gene and protein expression. The potent GSK3 inhibitor 1-azakenpaullone increases myocyte area as a consequence of increased cell length whereas phenylephrine increases both length and width. Azakenpaullone or insulin promotes AP1 transcription factor binding to an AP1 consensus oligonucleotide, but this was significantly less than that induced by endothelin and derived principally from increased binding of JunB protein, the expression of which was increased. Azakenpaullone promotes significant changes in gene expression (assessed by Affymetrix microarrays), but the overall response is less than with endothelin and there is little overlap between the genes identified. Thus, although GSK3 may contribute to cardiac myocyte hypertrophy in some respects (and presumably plays an important role in myocyte metabolism), it does not appear to contribute as significantly to the response induced by endothelin as has been maintained

Cardiomyocyte BRAF is a key signalling intermediate in cardiac hypertrophy in mice

Cardiac hypertrophy is necessary for the heart to accommodate an increase in workload. Physiological, compensated hypertrophy (e.g. with exercise) is reversible and largely due to cardiomyocyte hypertrophy. Pathological hypertrophy (e.g. with hypertension) is associated with additional features including increased fibrosis, and can lead to heart failure. RAF kinases (ARAF/BRAF/RAF1) integrate signals into the ERK1/2 cascade, a pathway implicated in cardiac hypertrophy, and activation of BRAF in cardiomyocytes promotes compensated hypertrophy. Here, we used mice with tamoxifen-inducible cardiomyocyte-specific BRAF knockout (CM-BRAFKO) to assess the role of BRAF in hypertension-associated cardiac hypertrophy induced by angiotensin II (AngII; 0.8 mg/kg/d, 7 d) and physiological hypertrophy induced by phenylephrine (40 mg/kg/d, 7 d). Cardiac dimensions/function were measured by echocardiography with histological assessment of cellular changes. AngII promoted cardiomyocyte hypertrophy and increased fibrosis within the myocardium (interstitial) and around the arterioles (perivascular) in male mice; cardiomyocyte hypertrophy and interstitial (but not perivascular) fibrosis were inhibited in mice with CM-BRAFKO. Phenylephrine had a limited effect on fibrosis, but promoted cardiomyocyte hypertrophy and increased contractility in male mice; cardiomyocyte hypertrophy was unaffected in mice with CM-BRAFKO, but the increase in contractility was suppressed and fibrosis increased. Phenylephrine induced a modest hypertrophic response in female mice and, in contrast to the males, tamoxifen-induced loss of cardiomyocyte BRAF reduced cardiomyocyte size, had no effect on fibrosis and increased contractility. The data identify BRAF as a key signalling intermediate in both physiological and pathological hypertrophy in male mice, and highlight the need for independent assessment of gene function in females

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

Phosphorylation and activation of mitogen- and stress-activated protein kinase-1 in adult rat cardiac myocytes by G-protein-coupled receptor agonists requires both extracellular-signal-regulated kinase and p38 mitogen-activated protein kinase.

G-protein-coupled receptor agonists are powerful stimulators of mitogen-activated protein kinase (MAPK) cascades in cardiac myocytes. However, little is known regarding the physiological activation of enzymes downstream of MAPKs. We examined the activation of mitogen- and stress-activated protein kinase-1 (MSK1), a downstream target of MAPKs, in adult rat cardiac myocytes by phenylephrine and endothelin-1. Both agonists induced the phosphorylation of MSK1 at Thr-581 and Ser-376 but not at Ser-360. Maximal phosphorylation was observed at 10-15 min after stimulation and it correlated with increased activity. Maximal activation of MSK1 in adult cardiomyocytes temporally coincided with maximal p38 MAPK activation while activation of the extracellular-signal-regulated kinase (ERK) cascade was more rapid. Phosphorylation and activation of MSK1 was completely inhibited by either PD98059 (ERK1/2 pathway inhibitor) or SB203580 (p38 MAPK inhibitor) alone. These data demonstrate that MSK1 activation in adult rat cardiac myocytes by G-protein-coupled receptor agonists requires the simultaneous activation of both the ERK and p38 MAPK pathways. However, the lack of phosphorylation at Ser-360, an identified phosphorylation site targeted by MAPKs, may indicate that MSK1 is not a direct substrate of ERK1/2 and p38 MAPK in adult rat cardiomyocytes

Indrocellular signaling mechanisms mediated by G protein coupled receptors in rat heart

In cardiac myocytes, GPCRs (G-protein coupled receptors) are involved in rapid changes in contractility, metabolic responses and electrophysiological properties. Stimulation of GPCR may also lead to long-term maintenance of cardiac function through regulation of gene expression and cell growth. The purpose of this study was to investigate the pathways leading to MAPK activation in response to GPCR agonists and to identify the molecular downstream targets of these kinases in adult rat ventricular myocytes. As a first step, we investigated the activation of MAPKs in response to hypertrophic agonists, such as phenylephrine and endothelin-1. Both agonists induced activation of ERK1/2 and p38 MAPK, whereas JNK1/2 phosphorylation was modest under these conditions. Using selective inhibitors, activation of ERK1/2 was shown to involve MEK1/2, PKC protein tyrosine kinase and PI3K. In contrast, p38 MAPK phosphorylation was not mediated through MEK1/2 or PKC. Mitogen- and stress-activated protein kinase-1 (MSK1) is a new MAPK target protein that is present in various mammalian cell types. MSK1 is activated by a variety of extracellular signals that include growth factors, phorbol esters, and cell damaging stimuli. In the present study, we demonstrate that MSK1 is present in adult rat cardiac myocytes and it is activated in response to hypertrophic GPCR agonists, such as phenylephrine and endothelin. Both agonist induced phosphorylation of MSK1 at Thr581 and Ser376, but not Ser360. Maximal phosphorylation was observed at 10-15 min after stimulation and it correlated with increased activity. Phosphorylation and activation of MSK1 was completely inhibited by either PD98059, or SB203580, suggesting that MSK1 activation by GPCR agonists in adult rat ventricular myocytes requires the simultaneous activation of both p38 MAPK and ERKs pathways. In contrast the PKA inhibitor, RpcAMP, had no effect on MSK1 activation and phosphorylation. MSK1 has been implicated in the regulation of transcriptional activation and one potential substrate is the cAMP responsive element (CRE) binding protein (CREB), a basic leucine zipper (bZip) transcription factor. Several studies suggested CREB as an important regulator of gene expression in cardiomyocytes with possible relevance for the pathophysiology of heart. However, the signaling events involved in the activation of CREB by hypertrophic stimuli have not yet been demonstrated in cardiomyocytes. In the present study, we characterized the phosphorylation of CREB by the hypertrophic G-protein-coupled receptor agonist, PE, in adult rat ventricular myocytes. Based on our reports that MSK1 is activated in response to PE, we used the inhibitors Ro318220 and H89 to determine the role of MSK1 in the activation ofΟι συζευγμένοι με G πρωτεΐνες υποδοχείς διαδραματίζουν σημαντικό ρόλο στη φυσιολογία της καρδιάς επηρεάζοντας τις συσταλτικές, ηλεκτροφυσιολογικές και μεταβολικές ιδιότητες της και Επιπλέον συμμετέχουν σε διαδικασίες κυτταρικής αύξησης ρυθμίζοντας την έκφραση των γονιδίων. Σκοπός της παρούσας εργασίας ήταν η διερεύνηση των σηματοδοτικών που οδηγούν στην ενεργοποίηση των MAPKs από τους αγωνιστές των συζευγμένων με G πρωτεΐνες υποδοχέων και η ταυτοποίηση των πρωτεϊνών-στόχων των κινασών στα καρδιακά μυοκύτταρα ενήλικου αρουραίου. Στο πρώτο στάδιο της εργασίας διερευνήθηκε η ενεργοποίηση των MAPKs μετά από τη διέγερση υποδοχέων που εμπλέκονται στο φαινόμενο της υπερτροφίας της καρδιάς, όπως είναι οι α1-αδρενεργικοί υποδοχείς και οι υποδοχείς της ενδοθηλίνης- 1. Διαπιστώθηκε ότι οι αγωνιστές των συγκεκριμένων υποδοχέων, φαινυλεφρίνη και ενδοθηλίνη-1, επάγουν την ενεργοποίηση των ERK1/2 και p38 MAPK, ενώ δεν αυξάνουν σημαντικά τη φωσφορυλίωση των JNK1/2. Η χρήση αναστολέων διαφόρων κινασών αποκάλυψε ότι η ενεργοποίηση των ERK1/2 στις συγκεκριμένες πειραματικές συνθήκες επιτυγχάνεται με τη διαμεσολάβηση των ΜΕΚ1/2, PKC πρωτεϊνικών κινασών τυροσίνης και PI3K. Αντίθετα, η επαγόμενη από τη φαινυλεφρίνη και την ενδοθηλίνη-1 φωσφορυλίωση της p38 MAPK εξαρτάται από τη ΡΙ3Κ και τις πρωτεϊνικές κινάσες τυροσίνης, ενώ δεν εμπλέκονται οι ΜΕΚ1/2 και η PKC. Η MSK1 είναι μια κινάση που αναγνωρίστηκε πρόσφατα σε διάφορους κυτταρικούς τύπους και θεωρείται ότι είναι υπόστρωμα των MAPKs. Τα ερεθίσματα που την ενεργοποιούν περιλαμβάνουν αυξητικούς παράγοντες, φορβολεστέρες και διάφορες μορφές κυτταρικού στρες. Στην παρούσα μελέτη αποδείχτηκε ότι η MSK1 εκφράζεται στα καρδιακά μυοκύτταρα ενήλικου αρουραίου και ότι ενεργοποιείται από τη φαινυλεφρίνη και ενδοθηλίνη. Οι συγκεκριμένοι αγωνιστές επάγουν τη φωσφορυλίωση της κινάσης στα κατάλοιπα Thr581 και Ser376, όχι όμως στη Ser360. Η μέγιστη τιμή της φωσφορυλίωσης της κινάσης εντοπίζεται στα 10-15 λεπτά επώασης με τους αγωνιστές και συνοδεύεται από αύξηση της ενζυμικής της δραστικότητας. Η ενεργοποίηση και η φωσφορυλίωση της MSK1 αναστέλλεται πλήρως από τον SB203580 ή το PD98059, γεγονός που υποδηλώνει ότι η ενεργοποίηση της κινάσης στα καρδιακά μυοκύτταρα προϋποθέτει τη συντονισμένη οδό των ERK1/2 και p38 MAPK. Αντίθετα, ο αναστολέας της ΡΚΑ, RpcAMP, δεν επηρέασε την ενεργοποίησητης MSK1. Η MSK1 θεωρείται ότι συμμετέχει στη ρύθμιση της μεταγραφής και ένα πιθανό υπόστρωμα της είναι ο bZip μεταγραφικός παράγοντας CREB. Αρκετές μελέτε

Lung injury after simulated cardiopulmonary bypass in an isolated perfused rat lung preparation:Role of mitogen-activated protein kinase/Akt signaling and the effects of theophylline

ObjectivesLung deflation and inflation during cardiac surgery with cardiopulmonary bypass contributes to pulmonary dysfunction postoperatively. Theophylline treatment for lung diseases has traditionally been thought to act by phosphodiesterase inhibition; however, increasing evidence has suggested other plausible mechanisms. We investigated the effects of deflation and reinflation on signaling pathways (p38-mitogen-activated protein kinase [MAPK], extracellular signal-regulated kinase 1 and 2 [ERK1/2], and Akt) and whether theophylline influences the deflation-induced lung injury and associated signaling.MethodsIsolated rat lungs were perfused (15 mL/min) with deoxygenated rat blood in bicarbonate buffer and ventilated. After 20 minutes' equilibration, the lungs were deflated (60 minutes, aerobic perfusion 1.5 mL/min), followed by reinflation (60 minutes, anaerobic reperfusion 15 mL/min). Compliance, vascular resistance, and kinase phosphorylation were assessed during deflation and reinflation. The effects of SB203580 (50 μM), a p38-MAPK inhibitor, and theophylline (0.083 mM [therapeutic] or 3 mM [supratherapeutic]) on physiology and signaling were studied.ResultsDeflation reduced compliance by 44% compared with continuously ventilated lungs. p38-MAPK and Akt phosphorylation increased (three to fivefold) during deflation and reinflation, and ERK1/2 phosphorylation increased (approximately twofold) during reinflation. SB203580 had no effect on lung physiology or ERK1/2 and Akt activation. Both theophylline doses increased cyclic adenosine monophosphate, but only 3 mM theophylline improved compliance. p38-MAPK phosphorylation was not affected by theophylline; 0.083 mM theophylline inhibited reinflation-induced ERK1/2 phosphorylation (72% ± 3%); and 3 mM theophylline inhibited Akt phosphorylation during deflation (75% ± 5%) and reinflation (87% ± 4%).ConclusionsLung deflation and reinflation stimulates differential p38-MAPK, ERK1/2, and Akt activation, suggesting a role in lung injury during cardiopulmonary bypass. However, p38-MAPK was not involved in the compromised compliance. A supratherapeutic theophylline dose protected lungs against deflation-induced injury and was associated with inhibition of phosphoinositide 3-kinase/Akt rather than phosphodiesterase

Signaling pathways mediating cardiac myocyte gene expression in physiological and stress responses

The contractile cells in the heart (the cardiac myocytes) are terminally differentiated. In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis, responses associated with the development of cardiac pathologies. There has been much effort expended in gaining an understanding of the stimuli which promote these responses, and in identifying the intracellular signaling pathways which are activated and potentially involved. These signaling pathways presumably modulate gene and protein expression to elicit the end-stage response. For the regulation of gene expression, the signal may traverse the cytoplasm to modulate nuclear-localized transcription factors as occurs with the mitogen-activated protein kinase or protein kinase B/Akt cascades. Alternatively, the signal may promote translocation of transcription factors from the cytoplasm to the nucleus as is seen with the calcineurin/NFAT and JAK/STAT systems. We present an overview of the principal signaling pathways implicated in the regulation of gene expression in cardiac myocyte pathophysiology, and summarize the current understanding of these pathways, the transcription factors they regulate and the changes in gene expression associated with the development of cardiac pathologies. Finally, we discuss how intracellular signaling and gene expression may be integrated to elicit the overall change in cellular phenotype