PKA phosphorylates multiple molecules involved in calcium (Ca 2+ ) 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 Ca 2+ storage and the magnitude of Ca 2+ 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.

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Key pointsr 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.r Chronic stimulation of β 2 -adrenoceptor (β 2 -AR) with clenbuterol (CB), a selective β 2 -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.r 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.r 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.r We conclude that Epac1 induces β 2 -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.Abstract The predominant isoform of β-adrenoceptor (β-AR) in skeletal muscle is β 2 -AR and that in the cardiac muscle is β 1 -AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A-independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic β 2 -AR stimulation with clenbuterol (CB) in Epac1-null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild-type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB-induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB-mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB-induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in β 2 -AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling.

In skeletal muscle, the major isoform of β-adrenergic receptor (β-AR) is β 2 -AR and the minor isoform is β 1 -AR, which is opposite to the situation in cardiac muscle. Despite extensive studies in cardiac muscle, the physiological roles of the β-AR subtypes in skeletal muscle are not fully understood. Therefore, in this work, we compared the effects of chronic β 1 - or β 2 -AR activation with a specific β 1 -AR agonist, dobutamine (DOB), or a specific β 2 -AR agonist, clenbuterol (CB), on masseter and cardiac muscles in mice. In cardiac muscle, chronic β 1 -AR stimulation induced cardiac hypertrophy, fibrosis and myocyte apoptosis, whereas chronic β 2 -AR stimulation induced cardiac hypertrophy without histological abnormalities. In masseter muscle, however, chronic β 1 -AR stimulation did not induce muscle hypertrophy, but did induce fibrosis and apoptosis concomitantly with increased levels of p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204), calmodulin kinase II (Thr-286) and mammalian target of rapamycin (mTOR) (Ser-2481) phosphorylation. On the other hand, chronic β 2 -AR stimulation in masseter muscle induced muscle hypertrophy without histological abnormalities, as in the case of cardiac muscle, concomitantly with phosphorylation of Akt (Ser-473) and mTOR (Ser-2448) and increased expression of microtubule-associated protein light chain 3-II, an autophagosome marker. These results suggest that the β 1 -AR pathway is deleterious and the β 2 -AR is protective in masseter muscle. These data should be helpful in developing pharmacological approaches for the treatment of skeletal muscle wasting and weakness.