Carabin protects against cardiac hypertrophy by blocking calcineurin, Ras, and Ca2+/calmodulin-dependent protein kinase II signaling.

BACKGROUND: Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and is regulated by various signaling pathways. However, the molecular mechanisms that negatively regulate these signal transduction pathways remain poorly understood. METHODS AND RESULTS: Here, we characterized Carabin, a protein expressed in cardiomyocytes that was downregulated in cardiac hypertrophy and human heart failure. Four weeks after transverse aortic constriction, Carabin-deficient (Carabin(-/-)) mice developed exaggerated cardiac hypertrophy and displayed a strong decrease in fractional shortening (14.6±1.6% versus 27.6±1.4% in wild type plus transverse aortic constriction mice; P<0.0001). Conversely, compensation of Carabin loss through a cardiotropic adeno-associated viral vector encoding Carabin prevented transverse aortic constriction-induced cardiac hypertrophy with preserved fractional shortening (39.9±1.2% versus 25.9±2.6% in control plus transverse aortic constriction mice; P<0.0001). Carabin also conferred protection against adrenergic receptor-induced hypertrophy in isolated cardiomyocytes. Mechanistically, Carabin carries out a tripartite suppressive function. Indeed, Carabin, through its calcineurin-interacting site and Ras/Rab GTPase-activating protein domain, functions as an endogenous inhibitor of calcineurin and Ras/extracellular signal-regulated kinase prohypertrophic signaling. Moreover, Carabin reduced Ca(2+)/calmodulin-dependent protein kinase II activation and prevented nuclear export of histone deacetylase 4 after adrenergic stimulation or myocardial pressure overload. Finally, we showed that Carabin Ras-GTPase-activating protein domain and calcineurin-interacting domain were both involved in the antihypertrophic action of Carabin. CONCLUSIONS: Our study identifies Carabin as a negative regulator of key prohypertrophic signaling molecules, calcineurin, Ras, and Ca(2+)/calmodulin-dependent protein kinase II and implicates Carabin in the development of cardiac hypertrophy and failure.

Exchange protein directly activated by cAMP 1 promotes autophagy during cardiomyocyte hypertrophy.

AIMS: Stimulation of ß-adrenergic receptors (ß-AR) increases cAMP production and contributes to the pathogenesis of cardiac hypertrophy and failure through poorly understood mechanisms. We previously demonstrated that Exchange protein directly activated by cAMP 1 (Epac1)-induced hypertrophy in primary cardiomyocytes. Among the mechanisms triggered by cardiac stress, autophagy has been highlighted as a protective or harmful response. Here, we investigate whether Epac1 promotes cardiac autophagy and how altered autophagy has an impact on Epac1-induced cardiomyocyte hypertrophy. METHODS AND RESULTS: We reported that direct stimulation of Epac1 with the agonist, Sp-8-(4-chlorophenylthio)-2'-O-methyl-cAMP (Sp-8-pCPT) promoted autophagy activation in neonatal cardiomyocytes. Stimulation of ß-AR with isoprenaline (ISO) mimicked the effect of Epac1 on autophagy markers. Conversely, the induction of autophagy flux following ISO treatment was prevented in cardiomyocytes pre-treated with a selective inhibitor of Epac1, CE3F4. Importantly, we found that Epac1 deletion in mice protected against ß-AR-induced cardiac remodelling and prevented the induction of autophagy. The signalling mechanisms underlying Epac1-induced autophagy involved a Ca(2+)/calmodulin-dependent kinase kinase ß (CaMKKß)/AMP-dependent protein kinase (AMPK) pathway. Finally, we provided evidence that pharmacological inhibition of autophagy using 3-methyladenine (3-MA) or down-regulation of autophagy-related protein 5 (Atg5) significantly potentiated Epac1-promoted cardiomyocyte hypertrophy. CONCLUSION: Altogether, these findings demonstrate that autophagy is an adaptive response to antagonize Epac1-promoted cardiomyocyte hypertrophy.

Epac1 and Epac2 are differentially involved in inflammatory and remodeling processes induced by cigarette smoke.

Cigarette smoke (CS) induces inflammatory responses characterized by increase of immune cells and cytokine release. Remodeling processes, such as mucus hypersecretion and extracellular matrix protein production, are also directly or indirectly induced by CS. Recently, we showed that activation of the exchange protein directly activated by cAMP (Epac) attenuates CS extract-induced interleukin (IL)-8 release from cultured airway smooth muscle cells. Using an acute, short-term model of CS exposure, we now studied the role of Epac1, Epac2, and the Epac effector phospholipase-Cε (PLCε) in airway inflammation and remodeling in vivo. Compared to wild-type mice exposed to CS, the number of total inflammatory cells, macrophages, and neutrophils and total IL-6 release was lower in Epac2(-/-) mice, which was also the case for neutrophils and IL-6 in PLCε(-/-) mice. Taken together, Epac2, acting partly via PLCε, but not Epac1, enhances CS-induced airway inflammation in vivo. In total lung homogenates of Epac1(-/-) mice, MUC5AC and matrix remodeling parameters (transforming growth factor-ß1, collagen I, and fibronectin) were increased at baseline. Our findings suggest that Epac1 primarily is capable of inhibiting remodeling processes, whereas Epac2 primarily increases inflammatory processes in vivo.

Specific interactions between Epac1, ß-arrestin2 and PDE4D5 regulate ß-adrenergic receptor subtype differential effects on cardiac hypertrophic signaling.

ß1 and ß2 adrenergic receptors (ßARs) are highly homologous but fulfill distinct physiological and pathophysiological roles. Here we show that both ßAR subtypes activate the cAMP-binding protein Epac1, but they differentially affect its signaling. The distinct effects of ßARs on Epac1 downstream effectors, the small G proteins Rap1 and H-Ras, involve different modes of interaction of Epac1 with the scaffolding protein ß-arrestin2 and the cAMP-specific phosphodiesterase (PDE) variant PDE4D5. We found that ß-arrestin2 acts as a scaffold for Epac1 and is necessary for Epac1 coupling to H-Ras. Accordingly, knockdown of ß-arrestin2 prevented Epac1-induced histone deacetylase 4 (HDAC4) nuclear export and cardiac myocyte hypertrophy upon ß1AR activation. Moreover, Epac1 competed with PDE4D5 for interaction with ß-arrestin2 following ß2AR activation. Dissociation of the PDE4D5-ß-arrestin2 complex allowed the recruitment of Epac1 to ß2AR and induced a switch from ß2AR non-hypertrophic signaling to a ß1AR-like pro-hypertrophic signaling cascade. These findings have implications for understanding the molecular basis of cardiac myocyte remodeling and other cellular processes in which ßAR subtypes exert opposing effects.

Role of Epac in brain and heart.

Epacs (exchange proteins directly activated by cAMP) are guanine-nucleotide-exchange factors for the Ras-like small GTPases Rap1 and Rap2. Epacs were discovered in 1998 as new sensors for the second messenger cAMP acting in parallel to PKA (protein kinase A). As cAMP regulates many important physiological functions in brain and heart, the existence of Epacs raises many questions regarding their role in these tissues. The present review focuses on the biological roles and signalling pathways of Epacs in neurons and cardiac myocytes. We discuss the potential involvement of Epacs in the manifestation of cardiac and central diseases such as cardiac hypertrophy and memory disorders.

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.

New perspectives in cAMP-signaling modulation.

Cyclic adenosine 3',5'-monophosphate (cAMP) mediates the biological effects of various hormones and neurotransmitters. Stimulation of cardiac ß-adrenergic receptors (ß-AR) via catecholamines leads to activation of adenylyl cyclases and increases cAMP production to enhance myocardial function. Because many other receptors signaling through cAMP generation exist in cardiac myocytes, a central question is how different hormones induce distinct cellular responses through the same second messenger. A large body of evidence suggests that the localization and compartmentalization of ß-AR/cAMP signaling affects the net outcome of biological functions. Spatiotemporal dynamics of cAMP action is achieved by various proteins, including protein kinase A (PKA), phosphodiesterases, and scaffolding proteins such as A-kinase-anchoring proteins. In addition, the discovery of the cAMP target Epac (exchange proteins directly activated by cAMP), which functions in a PKA-independent manner, represents a novel mechanism for governing cAMP-signaling specificity. Aberrant cAMP signaling through dysregulation of ß-AR/cAMP compartmentalization may contribute to cardiac remodeling and heart failure.

Rap-linked cAMP signaling Epac proteins: compartmentation, functioning and disease implications.

Epac proteins respond to the second messenger cyclic AMP (cAMP) and are activated by Gs coupled receptors. They act as specific guanine nucleotide exchange factors (GEFs) for the small G proteins, Rap1 and Rap2 of the Ras family. A plethora of studies using 8-pCPT-2'-O-Me-cAMP, an Epac agonist, has revealed the importance of these multi-domain proteins in the control of key cellular functions such as cell division, migration, growth and secretion. Epac and protein kinase A (PKA) may act independently but are often associated with the same biological process, in which they fulfill either synergistic or opposite effects. In addition, compelling evidence is now accumulating about the formation of molecular complexes in distinct cellular compartments that influence Epac signaling and cellular function. Epac is spatially and temporally regulated by scaffold protein and its effectors are interconnected with other signaling pathways. Pathophysiological changes in Epac signaling may underlie certain diseases.

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.