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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.

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.

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.

Background It has been suggested that protein directly activated by cAMP (Epac), one of the downstream signaling molecules of β-adrenergic receptor (β-AR), may be an effective target for the treatment of arrhythmia. However, there have been no reports on the anti-arrhythmic effects or cardiac side-effects of Epac1 inhibitors in vivo. Methods and Results: In this study, the roles of Epac1 in the development of atrial and ventricular arrhythmias are examined. In addition, we examined the usefulness of CE3F4, an Epac1-selective inhibitor, in the treatment of the arrhythmias in mice. In Epac1 knockout (Epac1-KO) mice, the duration of atrial fibrillation (AF) was shorter than in wild-type mice. In calsequestrin2 knockout mice, Epac1 deficiency resulted in a reduction of ventricular arrhythmia. In both atrial and ventricular myocytes, sarcoplasmic reticulum (SR) Ca2+ leak, a major trigger of arrhythmias, and spontaneous SR Ca2+ release (SCR) were attenuated in Epac1-KO mice. Consistently, CE3F4 treatment significantly prevented AF and ventricular arrhythmia in mice. In addition, the SR Ca2+ leak and SCR were significantly inhibited by CE3F4 treatment in both atrial and ventricular myocytes. Importantly, cardiac function was not significantly affected by a dosage of CE3F4 sufficient to exert anti-arrhythmic effects. Conclusions These findings indicated that Epac1 is involved in the development of atrial and ventricular arrhythmias. CE3F4, an Epac1-selective inhibitor, prevented atrial and ventricular arrhythmias in mice.

Cyclic adenosine monophosphate (cAMP) and Ca(2+) 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 Ca(2+), and it is now known that the heart expresses multiple AC isoforms, among which the type 5/6 isoforms are Ca(2+)-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 Ca(2+). This model indicated that Ca(2+)-mediated inhibition of AC is essential to create oscillations of Ca(2+) 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 Ca(2+)-noninhibitable AC isoforms. Because the subcellular locations of AC isoforms are different, spontaneous cAMP and Ca(2+) oscillations may occur within microdomains containing only Ca(2+)-inhibitable isoforms in cardiac myocytes, which might be necessary for fine tuning of excitation-contraction coupling.

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.

Hashimoto's thyroiditis (HT) has long been epidemiologically associated with excess iodine levels. However, the underlying immunological mechanisms still remain largely unexplored. Th17 cells are commonly recognized as playing vital roles in various autoimmune diseases. Here we show that intra-thyroid infiltrating Th17 cells and serum IL-17 levels were significantly increased in HT patients. However, the concentration of serum IL-17 was inversely correlated with patients' residual thyroid function while the heterogeneously expressed thyroid IL-17 was directly correlated with local fibrosis. Administration of moderate high levels of iodine was found to facilitate the polarization of murine splenic naïve T cells into Th17 cells, whereas extreme high levels of iodine favored Th1 polarization and inhibited Treg development. These findings suggest that both Th1 and Th17 cells may be involved in the pathogenesis of HT and high levels of iodine may play a critical role in this process by modulating T cell differentiation.

Despite various suspected causes, ranging from endocrine and genetic to infectious and immunological aspects, the molecular mechanisms of miscarriage still remain enigmatic. This work provides evidence that downregulation of 11β-hydroxysteroid dehydrogenase (HSD) type 2, the key enzyme inactivating glucocorticoid activities, insults the pregnant inflammatory milieu by inhibiting the biosynthesis of lipoxin A4 (LXA4), a metabolite of arachidonic acid, leading to an early loss of the pregnancy. Both LXA4 and its biosynthetic enzymes were found to be decreased in women with spontaneous miscarriages and in the murine miscarriage model. Replenishing LXA4 reversed LPS-induced miscarriages in mouse models, whereas blocking LXA4 signaling resulted in miscarriages in the pregnant mice. The protective effect of LXA4 might be explained by LXA4’s role in regulating uterine and placental inflammatory factors and mast cells. The underlying molecular mechanism involved miscarriage-inducing infections or stresses that downregulate the expression of 11β-HSD2, but not 11β-HSD1, resulting in increases in glucocorticoid activity and decreases in LXA4. Together, these findings suggest that the stress/glucocorticoid/LXA4 axis might be a common pathway through which miscarriages occur.

Chronic administration of clenbuterol (CB), a lipophilic β2-adrenoceptor (β2-AR) agonist, induces skeletal muscle hypertrophy and slow-to-fast fiber–type transitions in mammalian species, but the mechanism and pathophysiological roles of these changes have not been explored. Here, we examined the effects of CB not only on masseter muscle mass, fiber diameter, and myosin heavy chain (MHC) composition, but also on daily muscle activity, a factor influencing muscle phenotype, by means of electromyogram analysis in rats. MHC transition towards faster isoforms was induced by 2-week CB treatment. In addition, daily duty time was increased at 1 day, 1 week, and 2 weeks after the start of CB treatment and its increase was greater at high activity level (6-fold) than at low activity level (2-fold). In order to examine whether these effects of CB were mediated through muscle or CNS β2-AR stimulation, we compared these effects of CB with those of salbutamol (SB), a hydrophilic β2-AR agonist. SB treatment induced masseter hypertrophy and MHC transition, like CB, but did not increase daily activity. These results suggest that CB-mediated slow-to-fast MHC transition with hypertrophy was induced through direct muscle β2-AR stimulation, but the increase of daily duty time was mediated through the CNS. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.12271FP] Keywords:: β2-adrenergic signaling, masseter muscle, electromyogram, myosin heavy chain, hypertrophy

Background In heart failure patients, chronic hyperactivation of sympathetic signaling is known to exacerbate cardiac dysfunction. In this study, the cardioprotective effect of vidarabine, an anti-herpes virus agent, which we identified as a cardiac adenylyl cyclase inhibitor, in dogs with pacing-induced dilated cardiomyopathy (DCM) was evaluated. In addition, the adverse effects of vidarabine on basal cardiac function was compared to those of the β-blocker, carvedilol.Methods and Results:Vidarabine and carvedilol attenuated the development of pacing-induced systolic dysfunction significantly and with equal effectiveness. Both agents also inhibited the development of cardiac apoptosis and fibrosis and reduced the Na+-Ca2+exchanger-1 protein level in the heart. Importantly, carvedilol significantly enlarged the left ventricle and atrium; vidarabine, in contrast, did not. Vidarabine-treated dogs maintained cardiac response to β-AR stimulation better than carvedilol-treated dogs did. Conclusions Vidarabine may protect against pacing-induced DCM with less suppression of basal cardiac function than carvedilol in a dog model. (Circ J 2016; 80: 2496-2505).

Gluconeogenesis is a fundamental feature of hepatocytes. Whether this gluconeogenic activity is also present in malignant hepatocytes remains unexplored. A better understanding of this biological process may lead to novel therapeutic strategies. Here we show that gluconeogenesis is not present in mouse or human malignant hepatocytes. We find that two critical enzymes 11β-HSD1 and 11β-HSD2 that regulate glucocorticoid activities are expressed inversely in malignant hepatocytes, resulting in the inactivation of endogenous glucocorticoids and the loss of gluconeogenesis. In patients' hepatocarcinoma, the expression of 11β-HSD1 and 11β-HSD2 is closely linked to prognosis and survival. Dexamethasone, an active form of synthesized glucocorticoids, is capable of restoring gluconeogenesis in malignant cells by bypassing the abnormal regulation of 11β-HSD enzymes, leading to therapeutic efficacy against hepatocarcinoma. These findings clarify the molecular basis of malignant hepatocyte loss of gluconeogenesis and suggest new therapeutic strategies.

Clenbuterol (CB), a selective β 2‐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 β 2‐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.

We previously demonstrated that type 5 adenylyl cyclase (AC5) functions in autonomic regulation in the heart. Based on that work, we hypothesized that pharmacological modulation of AC5 activity could regulate the autonomic control of the heart rate under micro- and hypergravity. To test this hypothesis, we selected the approach of activating AC5 activity in mice with a selective AC5 activator (NKH477) or inhibitor (vidarabine) and examining heart rate variability during parabolic flight. The standard deviation of normal R-R intervals, a marker of total autonomic variability, was significantly greater under micro- and hypergravity in the vidarabine group, while there were no significant changes in the NKH477 group, suggesting that autonomic regulation was unstable in the vidarabine group. The ratio of low frequency and high frequency (HF) in heart rate variability analysis, a marker of sympathetic activity, became significantly decreased under micro- and hypergravity in the NKH477 group, while there was no such decrease in the vidarabine group. Normalized HF, a marker of parasympathetic activity, became significantly greater under micro- and hypergravity in the NKH477 group. In contrast, there was no such increase in the vidarabine group. This study is the first to indicate that pharmacological modulation of AC5 activity under micro- and hypergravity could be useful to regulate the autonomic control of the heart rate. Keywords:: adenylyl cyclase isoform, autonomic nerve activity, parabolic flight, heart rate variability, microgravity

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