Here two labs that helped characterize these two pathways
Here, two labs that helped characterize these two pathways have worked together to further test whether these apparently parallel pathways from β-AR to CaMKII-RyR might be related in series. Much of the work leading to the Epac pathway involved measurement of Ca sparks as an index of SR Ca leak, often used 8-CPT as an Epac agonist and did not explore NOS involvement. The studies leading to NOS1 involvement in β-AR-induced SR Ca leak, more often used the Shannon-Bers method of tetracaine-induced Δ[Ca]i and Δ[Ca]SRT shifts to measure SR Ca leak. Here, we find similar results regardless of SR Ca leak method, that 8-CPT (when freshly prepared) mimics the β-AR effects on tetracaine-sensitive SR Ca leak, and that NOS mediates Epac-dependent increase of Ca sparks, and that Akt is involved. We conclude that these pathways are largely in series (Fig. 1).
Materials and methods
Discussion Prior work had demonstrated two apparently independent parallel pathways by which cardiac β-ARs activate CaMKII-dependent diastolic SR Ca leak involving Epac2 (Fig. 1, blue) , , , , , , , ,  and NOS1 (Fig. 1, red) , , , , , . Here, we have demonstrated that these are part of a single series signaling cascade involving both Epac and NOS (Fig. 1). Key novel findings here demonstrate such a link. Indeed, direct Epac activation by 8-CPT: 1) activates Ca sparks, an effect prevented by inhibition of NOS or PI3K (as seen for ISO), 2) increases tetracaine-sensitive SR Ca leak in rabbit (overturning prior conclusions ) and again this was NOS-dependent, 3) activates myocyte CaMKII in a NOS- and PI3K-dependent manner (as seen for ISO ), and 4) promotes NOS-dependent RyR S2815 phosphorylation. We conclude that β1-AR activation triggers a series cascade via cAMP-Epac2-PI3K-Akt-NOS1-CaMKIIδ to cause s1p receptor of RyR at S2815 to increase pathological diastolic SR Ca leak.
Acknowledgements We thank Kayvon Jabbari for his technical assistance. This work was supported by grants from the National Institutes of Health (R01-HL030077 and P01-HL080101 to D.M.B) and American Heart Association (14GRNT20380907 to TRS).
cAMP signalling as a therapeutic target Synthesis of cAMP (see Glossary) in cells is regulated by G protein-coupled receptors (GPCRs), which can either activate or inhibit adenylate cyclase (AC) through the actions of stimulatory (Gs) or inhibitory (Gi) heterotrimeric G proteins. Active AC catalyses the conversion of ATP into cAMP and pyrophosphate, a process that is terminated through the actions of the cAMP phosphodiesterase (PDE) family, which catalyse the hydrolysis of cAMP into 5′-AMP. This ensures that the cAMP signal is transient, thereby allowing precise control over the localisation, intensity, and duration of the cAMP signal. Elevations in intracellular cAMP lead to the activation of a select range of intracellular effector proteins containing cyclic nucleotide-binding domains (CNBDs), including EPAC enzymes, 1 and 2 1, 2, PKA isoforms , cAMP-responsive ion channels , and Popeye domain-containing proteins . Drugs that target the cAMP system are currently prescribed for a range of medical conditions, including β2-adrenoceptor agonists such as salbutamol and formoterol, which form the basis of bronchodilators for the treatment of asthma 6, 7, and selective PDE4 inhibitors such as roflumilast , which have shown promise in the treatment of inflammatory diseases such as chronic obstructive pulmonary disorder. The challenge now is to specifically target cAMP signalling in a pathway-specific manner to reduce the side effects associated with these treatments. For example, PDE4 inhibitor treatment is associated with nausea and emesis and cAMP elevation in the heart produces cardiac inotropy and chronotropy. Recent research has therefore been directed at limiting off-target effects by specifically regulating the actions of the EPAC enzymes independently of PKA and cyclic nucleotide-gated ion channels. This review focuses on the cellular actions of EPAC enzymes in health and disease and the various strategies being used to identify EPAC-directed small-molecule regulators. We discuss whether the development of EPAC agonists or antagonists is the best way forward for the development of EPAC-centred pharmaceuticals with true clinical efficacy.