Kenji Suita

Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses

PKA phosphorylates multiple molecules involved in calcium (Ca2+) handling in cardiac myocytes and is considered to be the predominant regulator of β-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca2+ storage and the magnitude of Ca2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition.

Background Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition. Methodology We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB. Results and Conclusion Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy.

Oscillation of cAMP and Ca 2+ in cardiac myocytes: a systems biology approach

Cyclic adenosine monophosphate (cAMP) and Ca2+ levels may oscillate in harmony within excitable cells; a mathematical oscillation loop model, the Cooper model, of these oscillations was developed two decades ago. However, in that model all adenylyl cyclase (AC) isoforms were assumed to be inhibited by Ca2+, and it is now known that the heart expresses multiple AC isoforms, among which the type 5/6 isoforms are Ca2+-inhibitable whereas the other five (AC2, 3, 4, 7, and 9) are not. We used a computational systems biology approach with CellDesigner simulation software to develop a comprehensive graphical map and oscillation loop model for cAMP and Ca2+. This model indicated that Ca2+-mediated inhibition of AC is essential to create oscillations of Ca2+ and cAMP, and the oscillations were not altered by incorporation of phosphodiesterase-mediated cAMP hydrolysis or PKA-mediated inhibition of AC into the model. More importantly, they were created but faded out immediately in the co-presence of Ca2+-noninhibitable AC isoforms. Because the subcellular locations of AC isoforms are different, spontaneous cAMP and Ca2+ oscillations may occur within microdomains containing only Ca2+-inhibitable isoforms in cardiac myocytes, which might be necessary for fine tuning of excitation–contraction coupling.

Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2‐adrenoceptor stimulation

Epac (exchange protein directly activated by cyclic AMP (cAMP)), a PKA‐independent cAMP sensor, plays important roles in multiple cellular processes, but its role in the pathogenesis of skeletal muscle hypertrophy and myosin heavy chain (MHC) transition is poorly understood. Chronic stimulation of β2‐adrenoceptor (β2‐AR) with clenbuterol (CB), a selective β2‐AR agonist, induced masseter muscle hypertrophy in wild‐type (WT) mice, but not in Epac1‐null mice (Epac1KO), even if slow‐to‐fast MHC isoform transition was similarly induced by CB treatment in both WT and Epac1KO. Disruption of Epac1 inhibited development of masseter muscle hypertrophy concomitantly with decreased phosphorylation of Akt and its downstream molecules 70 kDa ribosomal S6 kinase 1 and eukaryotic initiation factor 4E‐binding protein 1, and also, in parallel, glycogen synthase kinase‐3β. Disruption of Epac1 decreased histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy. We conclude that Epac1 induces β2‐AR‐mediated masseter muscle hypertrophy without influencing slow‐to‐fast MHC isoform transition, probably via activation of Akt and its downstream molecules and increase of CaMKII‐mediated HDAC4 phosphorylation.

Norepinephrine-Induced Adrenergic Activation Strikingly Increased the Atrial Fibrillation Duration through β1- and α1-Adrenergic Receptor-Mediated Signaling in Mice

Background Atrial fibrillation (AF) is the most common arrhythmias among old people. It causes serious long-term health problems affecting the quality of life. It has been suggested that the autonomic nervous system is involved in the onset and maintenance of AF in human. However, investigation of its pathogenesis and potential treatment has been hampered by the lack of suitable AF models in experimental animals. Objectives Our aim was to establish a long-lasting AF model in mice. We also investigated the role of adrenergic receptor (AR) subtypes, which may be involved in the onset and duration of AF. Methods and Results Trans-esophageal atrial burst pacing in mice could induce AF, as previously shown, but with only a short duration (29.0±8.1 sec). We found that adrenergic activation by intraperitoneal norepinephrine (NE) injection strikingly increased the AF duration. It increased the duration to more than 10 minutes, i.e., by more than 20-fold (656.2±104.8 sec; P<0.001). In this model, a prior injection of a specific β1-AR blocker metoprolol and an α1-AR blocker prazosin both significantly attenuated NE-induced elongation of AF. To further explore the mechanisms underlying these receptors’ effects on AF, we assessed the SR Ca2+ leak, a major trigger of AF, and consequent spontaneous SR Ca2+ release (SCR) in atrial myocytes. Consistent with the results of our in-vivo experiments, both metoprolol and prazosin significantly inhibited the NE-induced SR Ca2+ leak and SCR. These findings suggest that both β1-AR　and α1-AR may play important roles in the development of AF. Conclusions We have established a long-lasting AF model in mice induced by adrenergic activation, which will be valuable in future AF study using experimental animals, such as transgenic mice. We also revealed the important role of β1- and α1-AR-mediated signaling in the development of AF through in-vivo and in-vitro experiments.

Disruption of Epac1 protects the heart from adenylyl cyclase type 5-mediated cardiac dysfunction

Type 5 adenylyl cyclase (AC5) plays an important role in the development of chronic catecholamine stress-induced heart failure and arrhythmia in mice. Epac (exchange protein activated by cAMP), which is directly activated by cAMP independent of protein kinase A, has been recently identified as a novel mediator of cAMP signaling in the heart. However, the role of Epac in AC5-mediated cardiac dysfunction and arrhythmias remains poorly understood. We therefore generated AC5 transgenic mice (AC5TG) with selective disruption of the Epac1 gene (AC5TG-Epac1KO), and compared their phenotypes with those of AC5TG after chronic isoproterenol (ISO) infusion. Decreased cardiac function as well as increased susceptibility to pacing-induced atrial fibrillation (AF) in response to ISO were significantly attenuated in AC5TG-Epac1KO mice, compared to AC5TG mice. Increased cardiac apoptosis and cardiac fibrosis were also concomitantly attenuated in AC5TG-Epac1KO mice compared to AC5TG mice. These findings indicate that Epac1 plays an important role in AC5-mediated cardiac dysfunction and AF susceptibility.

Cardiac overexpression of Epac1 in transgenic mice rescues lipopolysaccharide-induced cardiac dysfunction and inhibits Jak-STAT pathway

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic stimulation leads to coupling of the β-adrenergic receptor/Gs/adenylyl cyclase, a membrane-bound enzyme that catalyzes the conversion of ATP to cAMP, thereby stimulating protein kinase A (PKA) and ultimately compensating for cardiac dysfunction. The mechanism of such compensation by catecholamine has been traditionally understood as PKA-mediated enforcement of cardiac contractility. We hypothesized that exchange protein activated by cyclic AMP (Epac), a new target of cAMP signaling that functions independently of protein kinase A, also plays a key role in protection against acute stresses or changes in hemodynamic overload. Lipopolysaccharide injection induced cytokine release and severe cardiac dysfunction in mouse. In mouse overexpressing Epac1 in the heart, however, the magnitude of such dysfunction was significantly smaller. Epac1 overexpression inhibited the Jak-STAT pathway, as indicated by decreased phosphorylation of STAT3 and increased SOCS3 expression, with subsequent inhibition of iNOS expression. In cultured cardiomyocytes treated with isoproterenol or forskolin, the increase of SOCS3 expression was blunted when Epac1 or PKCα was silenced with siRNA. Activation of the cAMP/Epac/PKCα pathway protected the heart against cytokine-induced cardiac dysfunction, suggesting a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac1 and its downstream pathways may be novel targets for treating cardiac dysfunction in endotoxemia.

Role of phosphodiesterase 4 expression in the Epac1 signaling‐dependent skeletal muscle hypertrophic action of clenbuterol

Clenbuterol (CB), a selective β2‐adrenergic receptor (AR) agonist, induces muscle hypertrophy and counteracts muscle atrophy. However, it is paradoxically less effective in slow‐twitch muscle than in fast‐twitch muscle, though slow‐twitch muscle has a greater density of β‐AR. We recently demonstrated that Epac1 (exchange protein activated by cyclic AMP [cAMP]1) plays a pivotal role in β2‐AR‐mediated masseter muscle hypertrophy through activation of the Akt and calmodulin kinase II (CaMKII)/histone deacetylase 4 (HDAC4) signaling pathways. Here, we investigated the role of Epac1 in the differential hypertrophic effect of CB using tibialis anterior muscle (TA; typical fast‐twitch muscle) and soleus muscle (SOL; typical slow‐twitch muscle) of wild‐type (WT) and Epac1‐null mice (Epac1KO). The TA mass to tibial length (TL) ratio was similar in WT and Epac1KO at baseline and was significantly increased after CB infusion in WT, but not in Epac1KO. The SOL mass to TL ratio was also similar in WT and Epac1KO at baseline, but CB‐induced hypertrophy was suppressed in both mice. In order to understand the mechanism involved, we measured the protein expression levels of β‐AR signaling‐related molecules, and found that phosphodiesterase 4 (PDE4) expression was 12‐fold greater in SOL than in TA. These results are consistent with the idea that increased PDE4‐mediated cAMP hydrolysis occurs in SOL compared to TA, resulting in a reduced cAMP concentration that is insufficient to activate Epac1 and its downstream Akt and CaMKII/HDAC4 hypertrophic signaling pathways in SOL of WT. This scenario can account for the differential effects of CB on fast‐ and slow‐twitch muscles.

Epac activation inhibits IL-6-induced cardiac myocyte dysfunction.

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic and thus catecholamine signaling is activated thereafter to compensate for cardiac dysfunction. The mechanism of such compensation by catecholamine signaling has been traditionally understood to be cyclic AMP-dependent protein kinase (PKA)-mediated enforcement of cardiac contractility. We hypothesized that the exchange protein activated by cAMP (Epac), a newly identified target of cAMP signaling that functions independently of PKA, also plays a key role in this mechanism. In cultured cardiac myocytes, activation of Epac attenuated the inhibitory effect of interleukin-6 on the increase of intracellular Ca2+ concentration and contractility in response to isoproterenol, most likely through inhibition of the Jak-STAT pathway via SOCS3, with subsequent changes in inducible nitric oxide synthase expression. These findings suggest a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac and its downstream pathway may be a novel target for treating cardiac dysfunction in endotoxemia.

Vidarabine, an anti-herpesvirus agent, prevents catecholamine-induced arrhythmias without adverse effect on heart function in mice

Sympathetic activation causes clinically important arrhythmias including atrial fibrillation (AF) and ventricular tachyarrhythmia. Although the usefulness of β-adrenergic receptor blockade therapy is widely accepted, its multiple critical side effects often prevent its initiation or continuation. The aim of this study is to determine the advantages of vidarabine, an adenylyl cyclase (AC)-targeted anti-sympathetic agent, as an alternative treatment for arrhythmia. We found that vidarabine, which we identified as a cardiac AC inhibitor, consistently shortens AF duration and reduces the incidence of sympathetic activation-induced ventricular arrhythmias. In atrial and ventricular myocytes, vidarabine inhibits adrenergic receptor stimulation-induced RyR2 phosphorylation, sarcoplasmic reticulum (SR) Ca2+ leakage, and spontaneous Ca2+ release from SR, the last of which has been considered as a potential arrhythmogenic trigger. Moreover, vidarabine also inhibits sympathetic activation-induced reactive oxygen species (ROS) production in cardiac myocytes. The pivotal role of vidarabine’s inhibitory effect on ROS production with regard to its anti-arrhythmic property has also been implied in animal studies. In addition, as expected, vidarabine exerts an inhibitory effect on AC function, which is more potent in the heart than elsewhere. Indexes of cardiac function including ejection fraction and heart rate were not affected by a dosage of vidarabine sufficient to exert an anti-arrhythmic effect. These findings suggest that vidarabine inhibits catecholamine-induced AF or ventricular arrhythmia without deteriorating cardiac function in mice.