br Conclusion Our study demonstrates that the effects of
Conclusion Our study demonstrates that the effects of Epac activation are strongly conditioned by the steady-state Ca levels at which the myocyte is functioning. As intracellular Ca increases, the activity of CaMKII enhances and the phosphorylation of its SR substrates acquires different relevance in the control of SR Ca handling. The Ca uptake stimulated by the CaMKII-dependent PLN phosphorylation is gradually overcome by the Ca leak facilitated by the CaMKII-dependent RyR2 phosphorylation. In this scenario, stimulation of Epac through the Epac/PKC/CaMKII exacerbates the prevailing mechanism. Therefore, under different physiological or pathological situations, this cAMP-induced signaling pathway may produce beneficial (increased contractility) or detrimental (impaired contractility and triggered arrhythmias) effects depending on the myocyte intracellular Ca availability, dynamically regulated by the balance between SR Ca uptake and leak.
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Introduction β-adrenergic receptor (β-AR) mediated myocyte Ca mishandling is commonly described in heart failure (HF) and arrhythmia, which are increasing rapidly . Therefore understanding fundamental mechanisms of β-AR effects on Ca handling is critical. β-AR activation is an integral part of the cardiac fight-or-flight response, but its chronic activation (e.g. in HF) contributes to pathological hypertrophic remodeling, contractile dysfunction and arrhythmia. Acute β-AR activation enhances cardiac contraction (ionotropy) and relaxation (lusitropy), in large part by increasing myocyte Ca transients and accelerating [Ca]i reuptake by the sarcoplasmic reticulum (SR). These effects are mainly produced by PKA-dependent phosphorylation of L-type Ca a83 and which increases Ca current (ICa), and phospholamban (PLB) which enhances SR Ca uptake and content [Ca]SRT (making more Ca available for release; Fig. 1, right). The higher Ca transient causes stronger contraction, functionally offsetting myofilament Ca desensitization by PKA (that otherwise participates in lusitropy) . Studies have also shown that β-AR can also sensitizes ryanodine receptors (RyR) gating and SR Ca leak, but while PKA can phosphorylate RyR, the functional effects on RyR are mediated via CaMKII activation and phosphorylation of RyR , , , , , , , , , , , , , , , . Chronic CaMKII activation and consequent increase of SR Ca is now generally accepted as part of the HF syndrome , , . Indeed, SR Ca leak can contribute directly to both diastolic and systolic dysfunction in HF as well as β-AR triggered arrhythmias, via Ca wave-induced inward Na+/Ca exchange current (INCX) that causes delayed afterdepolarizations (DADs), triggered action potentials and premature ventricular contractions (PVCs) , , , . Therefore, inhibition of SR Ca leak is a valid therapeutic strategy in HF , ,  which could improve systolic and diastolic function and limit β-AR-induced arrhythmias. Two pathways have been independently implicated to mediate β-AR activation of CaMKII and SR Ca leak. One pathway involves exchange protein directly activated by cAMP (Epac), a cAMP target parallel to PKA, which may involve some downstream Epac targets leading to CaMKII autophosphorylation and RyR phosphorylation RyR (Fig. 1, blue) , , , , , , , , . Indeed, we have shown that this pathway specifically requires β1-AR, Epac2 (which localizes at myocyte T-tubules), CaMKIIδ and RyR2 phosphorylation at S2814 , . For this pathway, it is clear that cAMP is involved and that PKA only contributes indirectly (via PLB-dependent increase in SR Ca load), but the details of the pathway from the Epac-Rap1 level to CaMKII are not well resolved (with several mediators implicated, Fig. 1) , , , , , , , . The other β-AR to CaMKII-RyR pathway involving nitric oxide synthase 1 (NOS1), seemed cAMP-independent, but involving protein kinase B (PKB or Akt) as upstream activators of NOS1dependent CaMKII activation via S-nitrosylation (Fig. 1, red) , , , , , , . But in this case, the steps upstream from the β-AR to Akt were less clearly defined. This pathway was thought to be independent of cAMP and Epac because neither forskolin (direct adenylyl cyclase activator) nor 8-CPT (selective Epac agonist) mimicked β-AR effects , , . This raised the idea of β-arrestin mediated signaling to CaMKII , as a parallel pathway from (β-AR to Akt and NOS1; Fig. 1). Recent studies that have revealed the molecular mechanism by which S-nitrosylation occurs and mediates CaMKII activation , , localization of NOS1 at the junctional SR domain , and robust evidence for cardiac CaMKIIδ in regulating RyR2 have solidified our understanding of the bottom part of the NOS1-RyR pathway.