Cardiac β-adrenergic receptors (β-AR) and Ca2+-Calmodulin dependent protein kinase (CaMKII) regulate both physiological and pathophysiological Ca2+ signaling. Elevated diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) contributes to contractile dysfunction in heart failure and to arrhythmogenesis. β-AR activation is known to increase SR Ca2+ leak via CaMKII-dependent phosphorylation of the ryanodine receptor. Two independent and reportedly parallel pathways have been implicated in this β-AR-CaMKII cascade, one involving exchange protein directly activated by cAMP (Epac2) and another involving nitric oxide synthase 1 (NOS1). Here we tested whether Epac and NOS function in a single series pathway to increase β-AR induced and CaMKII-dependent SR Ca2+ leak. Leak was measured as both Ca2+ spark frequency and tetracaine-induced shifts in SR Ca2+, in mouse and rabbit ventricular myocytes. Direct Epac activation by 8-CPT (8-(4-chlorophenylthio)-2'-O-methyl-cAMP) mimicked β-AR-induced SR Ca2+ leak, and both were blocked by NOS inhibition. The same was true for myocyte CaMKII activation (assessed via a FRET-based reporter) and ryanodine receptor phosphorylation. Inhibitor and phosphorylation studies also implicated phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) downstream of Epac and above NOS activation in this pathway. We conclude that these two independently characterized parallel pathways function mainly via a single series arrangement (β-AR-cAMP-Epac-PI3K-Akt-NOS1-CaMKII) to mediate increased SR Ca2+ leak. Thus, for β-AR activation the cAMP-PKA branch effects inotropy and lusitropy (by effects on Ca2+ current and SR Ca2+-ATPase), this cAMP-Epac-NOS pathway increases pathological diastolic SR Ca2+leak. This pathway distinction may allow novel SR Ca2+ leak therapeutic targeting in treatment of arrhythmias in heart failure that spare the inotropic and lusitropic effects of the PKA branch.

中文翻译：

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β-肾上腺素诱导的SR Ca2 +泄漏是由Epac-NOS途径介导的。

心脏β-肾上腺素受体（β-AR）和Ca2 +-钙调蛋白依赖性蛋白激酶（CaMKII）调节生理和病理生理上的Ca2 +信号传导。肌浆网（SR）舒张期Ca2 +泄漏的增加导致心力衰竭的收缩功能障碍和心律失常。已知β-AR激活通过赖氨酸的受体的CaMKII依赖性磷酸化增加SR Ca2 +的泄漏。β-AR-CaMKII级联涉及两个独立且据报道的平行途径，一个途径涉及被cAMP直接激活的交换蛋白（Epac2），另一个涉及一氧化氮合酶1（NOS1）。在这里，我们测试了Epac和NOS是否在单个串联途径中起作用，以增加β-AR诱导的CaMKII依赖性SR Ca2 +泄漏。用Ca2 +火花频率和丁卡因诱导的SR Ca2 +位移来测量泄漏，在小鼠和兔子的心室肌细胞中。8-CPT（8-（4-氯苯硫基）-2'-O-甲基-cAMP）的直接Epac激活模仿了β-AR诱导的SR Ca2 +泄漏，两者均被NOS抑制所阻断。肌细胞CaMKII激活（通过基于FRET的报告基因评估）和ryanodine受体磷酸化也是如此。抑制剂和磷酸化研究还暗示了Epac下游的磷酸肌醇3激酶（PI3K）和蛋白激酶B（Akt）在该途径中高于NOS活化。我们得出的结论是，这两个独立表征的平行途径主要通过单个串联排列（β-AR-cAMP-Epac-PI3K-Akt-NOS1-CaMKII）起作用，以介导增加的SR Ca2 +泄漏。因此，对于β-AR激活，cAMP-PKA分支会影响正性和异性（通过对Ca2 +电流和SR Ca2 + -ATPase的影响），此cAMP-Epac-NOS途径会增加病理舒张性SR Ca2 +泄漏。这种途径的区别可能允许新的SR Ca2 +泄漏治疗靶向治疗心力衰竭的心律失常，而无需使用PKA支的正性和正性作用。