The Notch pathway controls fibrotic and regenerative repair in the adult heart.

AIMS: In the adult heart, Notch signalling regulates the response to injury. Notch inhibition leads to increased cardiomyocyte apoptosis, and exacerbates the development of cardiac hypertrophy and fibrosis. The role of Notch in the mesenchymal stromal cell fraction, which contains cardiac fibroblasts and cardiac precursor cells, is, however, largely unknown. In the present study, we evaluate, therefore, whether forced activation of the Notch pathway in mesenchymal stromal cells regulates pathological cardiac remodelling. METHODS AND RESULTS: We generated transgenic mice overexpressing the Notch ligand Jagged1 on the surface of cardiomyocytes to activate Notch signalling in adjacent myocyte and non-myocyte cells. In neonatal transgenic mice, activated Notch sustained cardiac precursor and myocyte proliferation after birth, and led to increased numbers of cardiac myocytes in adult mice. In the adult heart under pressure overload, Notch inhibited the development of cardiomyocyte hypertrophy and transforming growth factor-ß/connective tissue growth factor-mediated cardiac fibrosis. Most importantly, Notch activation in the stressed adult heart reduced the proliferation of myofibroblasts and stimulated the expansion of stem cell antigen-1-positive cells, and in particular of Nkx2.5-positive cardiac precursor cells. CONCLUSIONS: We conclude that Notch is pivotal in the healing process of the injured heart. Specifically, Notch regulates key cellular mechanisms in the mesenchymal stromal cell population, and thereby controls the balance between fibrotic and regenerative repair in the adult heart. Altogether, these findings indicate that Notch represents a unique therapeutic target for inducing regeneration in the adult heart via mobilization of cardiac precursor cells.

Epac enhances excitation-transcription coupling in cardiac myocytes.

Epac is a guanine nucleotide exchange protein that is directly activated by cAMP, but whose cardiac cellular functions remain unclear. It is important to understand cardiac Epac signaling, because it is activated in parallel to classical cAMP-dependent signaling via protein kinase A. In addition to activating contraction, Ca(2+) is a key cardiac transcription regulator (excitation-transcription coupling). It is unknown how myocyte Ca(2+) signals are decoded in cardiac myocytes to control nuclear transcription. We examine Epac actions on cytosolic ([Ca(2+)](i)) and intranuclear ([Ca(2+)](n)) Ca(2+) homeostasis, focusing on whether Epac alters [Ca(2+)](n) and activates a prohypertrophic program in cardiomyocytes. Adult rat cardiomyocytes, loaded with fluo-3 were viewed by confocal microscopy during electrical field stimulation at 1Hz. Acute Epac activation by 8-pCPT increased Ca(2+) sparks and diastolic [Ca(2+)](i), but decreased systolic [Ca(2+)](i). The effects on diastolic [Ca(2+)](i) and Ca(2+) spark frequency were dependent on phospholipase C (PLC), inositol 1,4,5 triphosphate receptor (IP(3)R) and CaMKII activation. Interestingly, Epac preferentially increased [Ca(2+)](n) during both diastole and systole, correlating with the perinuclear expression pattern of Epac. Moreover, Epac activation induced histone deacetylase 5 (HDAC5) nuclear export, with consequent activation of the prohypertrophic transcription factor MEF2. These data provide the first evidence that the cAMP-binding protein Epac modulates cardiac nuclear Ca(2+) signaling by increasing [Ca(2+)](n) through PLC, IP(3)R and CaMKII activation, and initiates a prohypertrophic program via HDAC5 nuclear export and subsequent activation of the transcription factor MEF2.

Role of the cAMP-binding protein Epac in cardiovascular physiology and pathophysiology.

Exchange proteins directly activated by cyclic AMP (Epac) were discovered 10 years ago as new sensors for the second messenger cyclic AMP (cAMP). Epac family, including Epac1 and Epac2, are guanine nucleotide exchange factors for the Ras-like small GTPases Rap1 and Rap2 and function independently of protein kinase A. Given the importance of cAMP in the cardiovascular system, numerous molecular and cellular studies using specific Epac agonists have analyzed the role and the regulation of Epac proteins in cardiovascular physiology and pathophysiology. The specific functions of Epac proteins may depend upon their microcellular environments as well as their expression and localization. This review discusses recent data showing the involvement of Epac in vascular cell migration, endothelial permeability, and inflammation through specific signaling pathways. In addition, we present evidence that Epac regulates the activity of various cellular compartments of the cardiac myocyte and influences calcium handling and excitation-contraction coupling. The potential role of Epac in cardiovascular disorders such as cardiac hypertrophy and remodeling is also discussed.

Epac stimulation induces rapid increases in connexin43 phosphorylation and function without preconditioning effect.

It has been recently shown that beta-adrenergic receptors are able to activate phospholipase C via the cyclic adenosine monophosphate-binding protein Epac. This new interconnection may participate in isoproterenol (Iso)-induced preconditioning. We evaluated here whether Epac could induce PKCepsilon activation and could play a role in ischemic preconditioning through the phosphorylation of connexin43 (Cx43) and changes in gap junctional intercellular communication (GJIC). In cultured rat neonatal cardiomyocytes, we showed that in response to Iso and 8-CPT, a specific Epac activator, PKCepsilon content was increased in particulate fractions of cell lysates independently of protein kinase A (PKA). This was associated with an increased Cx43 phosphorylation. Both Iso and 8-CPT induced an increase in GJIC that was blocked by the PKC inhibitor bisindolylmaleimide. Interestingly, inhibition of PKA partly suppressed both Iso-induced increases in Cx43 phosphorylation and in GJIC. The same PKCepsilon-dependent Cx43 phosphorylation by beta-adrenergic stimulation via Epac was found in adult rat hearts. However, in contrast with Iso that induced a preconditioning effect, perfusion of isolated hearts with 8-CPT prior to ischemia failed to improve the post-ischemia functional recovery. In conclusion, Epac stimulation induces PKCepsilon activation and Cx43 phosphorylation with an increase in GJIC, but Epac activation does not induce preconditioning to ischemia in contrast with beta-adrenergic stimulation.

Epac mediates beta-adrenergic receptor-induced cardiomyocyte hypertrophy.

Cardiac hypertrophy is promoted by adrenergic overactivation and can progress to heart failure, a leading cause of mortality worldwide. Although cAMP is among the most well-known signaling molecules produced by beta-adrenergic receptor stimulation, its mechanism of action in cardiac hypertrophy is not fully understood. The identification of Epac (exchange protein directly activated by cAMP) proteins as novel sensors for cAMP has broken the dogma surrounding cAMP and protein kinase A. However, their role and regulation in the mature heart remain to be defined. Here, we show that cardiac hypertrophy induced by thoracic aortic constriction increases Epac1 expression in rat myocardium. Adult ventricular myocytes isolated from banded animals display an exaggerated cellular growth in response to Epac activation. At the molecular level, Epac1 hypertrophic effects are independent of its classic effector, Rap1, but rather involve the small GTPase Ras, the phosphatase calcineurin, and Ca(2+)/calmodulin-dependent protein kinase II. Importantly, we find that in response to beta-adrenergic receptor stimulation, Epac1 activates Ras and induces adult cardiomyocyte hypertrophy in a cAMP-dependent but protein kinase A-independent manner. Knockdown of Epac1 strongly reduces beta-adrenergic receptor-induced hypertrophic program. Finally, we report for the first time that Epac1 is mainly expressed in human heart as compared with Epac2 isoform and is increased in heart failure. Taken together, our data demonstrate that the guanine nucleotide exchange factor Epac1 contributes to the hypertrophic effect of beta-adrenergic receptor in a protein kinase A-independent fashion and may, therefore, represent a novel therapeutic target for the treatment of cardiac disorders.

Functional characterization of the cAMP-binding proteins Epac in cardiac myocytes.

The cyclic AMP (cAMP)-binding proteins, Epac, are guanine nucleotide exchange factors for the Ras-like small GTPases. Since their discovery in 1998 and with the development of specific Epac agonists, many data in the literature have illustrated their critical role in multiple cellular events mediated by the second messenger cAMP. Given the importance of cAMP in cardiovascular physiology and physiopathology, there is a growing interest to delineate the role of these multi-domain Epac in the cardiovascular system. This review will focus on recent pharmacological and biochemical studies aiming at understanding the role of Epac in cardiomyocyte signaling and hypertrophy.

Small GTP-binding proteins and their regulators in cardiac hypertrophy.

Small GTP-binding proteins (small G proteins) act as GDP-GTP-regulated molecular switches and are activated by guanine nucleotide exchange factors (GEFs) in response to diverse extracellular stimuli. During this last decade, numerous molecular and cellular studies, as well as genetically-modified animal models, have highlighted the role of small G proteins in the regulation of cardiac hypertrophy. The growing interest in small G protein signalling comes from the fact that chronic hypertrophic response is considered maladaptive and predisposes individuals to heart failure. Although some of the hypertrophic signalling pathways involving small G proteins have now been identified, a central question deals with the identity of the GEFs that modulate small G protein activation in the context of cardiac hypertrophy. Here, we discuss the precise regulation of Ras and Rho subfamilies of GTPases by GEFs and other regulatory proteins during cardiac hypertrophy. In addition, we summarize recent published data, mainly those describing the role of small G proteins in the development of myocardial hypertrophy and we further present the importance of their downstream effectors in myocardial remodelling.

Diverging effects of enalapril or eplerenone in primary prevention against doxorubicin-induced cardiotoxicity.

Aims: Clinical studies suggest beneficial effects of renin-angiotensin system blockade for prevention of left ventricular (LV) dysfunction after chemotherapy. However, the efficacy of this strategy as primary prevention has been poorly studied. This study aimed at identifying the pathophysiological mechanisms by which mineralocorticoid receptor antagonism (MRA) or angiotensin converting enzyme inhibition (ACEi) provide protection against doxorubicin-induced cardiotoxicity (DIC) in mouse models of acute and chronic toxicity. Methods and results: Acute DIC was induced by a single injection of Dox at 15 mg/kg and chronic DIC applied 5 injections of Dox at 4 mg/kg/week. MRA was achieved using eplerenone or cardiomyocyte-specific ablation of the MR gene in transgenic mice and ACEi using enalapril. Drugs were provided with the first dose of Dox and applied until the end of the study. In both model of DIC, Dox induced cardiac atrophy with decreased LV volume, reduced cardiomyocyte cell size, and cardiac dysfunction. In the acute model, neither MRA nor ACEi protected against these manifestations of DIC. In the chronic model, concomitant treatment with eplerenone did not protect against DIC and drastically increased plasma aldosterone levels and cardiac levels of angiotensin II type 1 receptor and of connective tissue growth factor (CTGF), as observed in acute DIC. Enalapril treatment in the chronic model, however, protected against cardiac dysfunction and cardiomyocyte atrophy and was associated with increased activation of the PI3K/AKT/mTOR pathway along with normal levels of CTGF. Conclusion: Enalapril and eplerenone disparately impact on cellular signalling in DIC. Eplerenone, on top of Dox treatment was not protective and associated with increased levels of plasma aldosterone and of cardiac CTGF. In contrast, we show that primary prevention with enalapril preserves LV morphology and function in a clinically relevant model of chronic DIC, with increased stimulation of the PI3K/AKT/mTOR axis and normal CTGF levels suggesting potential therapeutic implications.

Adrenergic Receptor Polymorphism and Maximal Exercise Capacity after Orthotopic Heart Transplantation.

BACKGROUND: Maximal exercise capacity after heart transplantion (HTx) is reduced to the 50-70% level of healthy controls when assessed by cardiopulmonary exercise testing (CPET) despite of normal left ventricular function of the donor heart. This study investigates the role of donor heart ß1 and ß2- adrenergic receptor (AR) polymorphisms for maximal exercise capacity after orthotopic HTx. METHODS: CPET measured peak VO2 as outcome parameter for maximal exercise in HTx recipients ≥9 months and ≤4 years post-transplant (n = 41; mean peak VO2: 57±15% of predicted value). Donor hearts were genotyped for polymorphisms of the ß1-AR (Ser49Gly, Arg389Gly) and the ß2-AR (Arg16Gly, Gln27Glu). Circumferential shortening of the left ventricle was measured using magnetic resonance based CSPAMM tagging. RESULTS: Peak VO2 was higher in donor hearts expressing the ß1-Ser49Ser alleles when compared with ß1-Gly49 carriers (60±15% vs. 47±10% of the predicted value; p = 0.015), and by trend in cardiac allografts with the ß1-AR Gly389Gly vs. ß1-Arg389 (61±15% vs. 54±14%, p = 0.093). Peak VO2 was highest for the haplotype Ser49Ser-Gly389, and decreased progressively for Ser49Ser-Arg389Arg > 49Gly-389Gly > 49Gly-Arg389Arg (adjusted R2 = 0.56, p = 0.003). Peak VO2 was not different for the tested ß2-AR polymorphisms. Independent predictors of peak VO2 (adjusted R2 = 0.55) were ß1-AR Ser49Gly SNP (p = 0.005), heart rate increase (p = 0.016), and peak systolic blood pressure (p = 0.031). Left ventricular (LV) motion kinetics as measured by cardiac MRI CSPAMM tagging at rest was not different between carriers and non-carriers of the ß1-AR Gly49allele. CONCLUSION: Similar LV cardiac motion kinetics at rest in donor hearts carrying either ß1-AR Gly49 or ß1-Ser49Ser variant suggests exercise-induced desensitization and down-regulation of the ß1-AR Gly49 variant as relevant pathomechanism for reduced peak VO2 in ß1-AR Gly49 carriers.

Red cell distribution width and mortality in acute heart failure patients with preserved and reduced ejection fraction.

BACKGROUND: Elevated red blood cell distribution width (RDW) is a valid predictor of outcome in acute heart failure (AHF). It is unknown whether elevated RDW remains predictive in AHF patients with either preserved left ventricular ejection fraction (LVEF) ≥50% or reduced LVEF (<50%). METHODS AND RESULTS: Prospective local registry including 402 consecutive hospitalized AHF patients without acute coronary syndrome or need of intensive care. The primary outcome was all-cause mortality (ACM) at 1 year after admission. Demographic and clinical data derive from admission, echocardiographic examinations (n = 269; 67%) from hospitalization. The Cox proportional hazard model including all patients (P < 0.001) was adjusted for age, gender, and RDW quartiles. Independent predictors of 1-year ACM were cardiogenic shock (HR 2.86; CI: 1.3-6.4), male sex (HR 1.9; CI: 1.2-2.9), high RDW quartile (HR 1.66; CI: 1.02-2.8), chronic HF (HR 1.61; CI: 1.05-2.5), valvular heart disease (HR 1.61; CI: 1.09-2.4), increased diastolic blood pressure (HR 1.02 per mmHg; CI: 1.01-1.03), increasing age (HR 1.04 by year; CI: 1.02-1.07), platelet count (HR 1.002 per G/l; CI: 1.0-1.004), systolic blood pressure (HR 0.99 per mmHg; CI: 0.98-0.99), and weight (HR 0.98 per kg; CI: 0.97-0.99). A total of 114 patients (28.4%) died within the first year; ACM of all patients increased with quartiles of rising RDW (χ2 18; P < 0.001). ACM was not different between RDW quartiles of patients with reduced LVEF (n = 153; χ2 6.6; P = 0.084). In AHF with LVEF ≥50% the probability of ACM increased with rising RDW (n = 116; χ2 9.9; P = 0.0195). CONCLUSIONS: High RDW is associated with increased ACM in AHF patients with preserved but not with reduced LVEF in this study population.

Jagged1 intracellular domain-mediated inhibition of Notch1 signalling regulates cardiac homeostasis in the postnatal heart.

AIMS: Notch1 signalling in the heart is mainly activated via expression of Jagged1 on the surface of cardiomyocytes. Notch controls cardiomyocyte proliferation and differentiation in the developing heart and regulates cardiac remodelling in the stressed adult heart. Besides canonical Notch receptor activation in signal-receiving cells, Notch ligands can also activate Notch receptor-independent responses in signal-sending cells via release of their intracellular domain. We evaluated therefore the importance of Jagged1 (J1) intracellular domain (ICD)-mediated pathways in the postnatal heart. METHODS AND RESULTS: In cardiomyocytes, Jagged1 releases J1ICD, which then translocates into the nucleus and down-regulates Notch transcriptional activity. To study the importance of J1ICD in cardiac homeostasis, we generated transgenic mice expressing a tamoxifen-inducible form of J1ICD, specifically in cardiomyocytes. Using this model, we demonstrate that J1ICD-mediated Notch inhibition diminishes proliferation in the neonatal cardiomyocyte population and promotes maturation. In the neonatal heart, a response via Wnt and Akt pathway activation is elicited as an attempt to compensate for the deficit in cardiomyocyte number resulting from J1ICD activation. In the stressed adult heart, J1ICD activation results in a dramatic reduction of the number of Notch signalling cardiomyocytes, blunts the hypertrophic response, and reduces the number of apoptotic cardiomyocytes. Consistently, this occurs concomitantly with a significant down-regulation of the phosphorylation of the Akt effectors ribosomal S6 protein (S6) and eukaryotic initiation factor 4E binding protein1 (4EBP1) controlling protein synthesis. CONCLUSIONS: Altogether, these data demonstrate the importance of J1ICD in the modulation of physiological and pathological hypertrophy, and reveal the existence of a novel pathway regulating cardiac homeostasis.

Epac activation induces histone deacetylase nuclear export via a Ras-dependent signalling pathway.

Epac (Exchange protein directly activated by cAMP) is a sensor for cAMP and represents a novel mechanism for governing cAMP signalling. Epac is a guanine nucleotide exchange factor (GEF) for the Ras family of small GTPases, Rap. Previous studies demonstrated that, in response to a prolonged beta-adrenergic stimulation Epac induced cardiac myocyte hypertrophy. The aim of our study was to further characterize Epac downstream effectors involved in cardiac myocyte growth. Here, we found that Epac led to the activation of the small G protein H-Ras in primary neonatal cardiac myocytes. A Rap GTPase activating protein (RapGAP) partially inhibited Epac-induced H-Ras activation. Interestingly, we found that H-Ras activation involved the GEF domain of Epac. However, Epac did not directly induce exchange activity on this small GTPase protein. Instead, the effect of Epac on H-Ras activation was dependent on a signalling cascade involving phospholipase C (PLC)/inositol 1,3,5 triphosphate receptor (IP3R) and an increase intracellular calcium. In addition, we found that Epac activation induced histone deacetylase type 4 (HDAC4) translocation. Whereas HDAC5 alone was unresponsive to Epac, it became responsive to Epac in the presence of HDAC4 in COS cells. Consistent with its effect on HDAC cytoplasmic shuttle, Epac activation also increased the prohypertrophic transcription factor MEF2 in a CaMKII dependent manner in primary cardiac myocytes. Thus, our data show that Epac activates a prohypertrophic signalling pathway which involves PLC, H-Ras, CaMKII and HDAC nuclear export.

Functional effects of PTPN11 (SHP2) mutations causing LEOPARD syndrome on epidermal growth factor-induced phosphoinositide 3-kinase/AKT/glycogen synthase kinase 3beta signaling.

LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3beta (GSK-3beta) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3beta hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3beta stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.

The cAMP binding protein Epac modulates Ca2+ sparks by a Ca2+/calmodulin kinase signalling pathway in rat cardiac myocytes.

cAMP is a powerful second messenger whose known general effector is protein kinase A (PKA). The identification of a cAMP binding protein, Epac, raises the question of its role in Ca(2+) signalling in cardiac myocytes. In this study, we analysed the effects of Epac activation on Ca(2+) handling by using confocal microscopy in isolated adult rat cardiomyocytes. [Ca(2+)](i) transients were evoked by electrical stimulation and Ca(2+) sparks were measured in quiescent myocytes. Epac was selectively activated by the cAMP analogue 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT). Patch-clamp was used to record the L-type calcium current (I(Ca)), and Western blot to evaluate phosphorylated ryanodine receptor (RyR). [Ca(2+)](i) transients were slightly reduced by 10 microm 8-CPT (F/F(0): decreased from 4.7 +/- 0.5 to 3.8 +/- 0.4, P < 0.05), an effect that was boosted when cells were previously infected with an adenovirus encoding human Epac. I(Ca) was unaltered by Epac activation, so this cannot explain the decreased [Ca(2+)](i) transients. Instead, a decrease in the sarcoplasmic reticulum (SR) Ca(2+) load underlies the decrease in the [Ca(2+)](i) transients. This decrease in the SR Ca(2+) load was provoked by the increase in the SR Ca(2+) leak induced by Epac activation. 8-CPT significantly increased Ca(2+) spark frequency (Ca(2+) sparks s(-1) (100 microm)(-1): from 2.4 +/- 0.6 to 6.9 +/- 1.5, P < 0.01) while reducing their amplitude (F/F(0): 1.8 +/- 0.02 versus 1.6 +/- 0.01, P < 0.001) in a Ca(2+)/calmodulin kinase II (CaMKII)-dependent and PKA-independent manner. Accordingly, we found that Epac increased RyR phosphorylation at the CaMKII site. Altogether, our data reveal a new signalling pathway by which cAMP governs Ca(2+) release and signalling in cardiac myocytes.