Beta1-adrenergic Apoptotic Signal Delivered By CaMKII

β-肾上腺素能受体(AR)是心脏功能的关键调节因子。然而，增强的β1-AR信号促进了心肌细胞的凋亡性死亡，这与心肌重塑和心力衰竭有关。本研究的目的是确定经典的Gs-腺苷环化酶-cAMP-PKA通路是否在β1-AR的凋亡效应中起重要作用。为了避免β-AR亚型之间复杂的相互作用，我们通过腺病毒基因转移在Beta1/Beta2-AR双基因敲除的成年小鼠心肌细胞的零背景中表达小鼠Beta1-AR，从而创建了一个遗传上“纯”的Beta1-AR实验环境。在此我们发现，β1-AR的刺激显著增加了心肌细胞的凋亡率，表现为TUNEL阳性细胞增多，DNA片段化(细胞死亡ELSIA和DNA梯状条带)。Beat1-AR诱导的细胞凋亡可被β-AR拮抗剂心得安(1um)阻断。令我们惊讶的是，特定的PKA抑制剂，包括RP-CPT-cAMP(100um)、H89(5um)和多肽抑制剂(PKI，5um)，在阻断Forsklin(0.1um)诱导的心肌细胞凋亡的同时，不能阻止β1-AR诱导的细胞凋亡。具有讽刺意味的是，用L类钙通道阻滞剂硝非地平(1um)阻断细胞内钙内流或用EGTA-AM(1um)缓冲细胞内钙离子完全保护心肌细胞不受β1-AR介导的细胞凋亡的影响，提示非依赖于PKA的细胞内钙离子增加是β1-AR凋亡效应的必由之路。为了描述β1-AR/Ca~(2+)凋亡信号的下游事件，我们首先评估了钙/钙调素依赖的磷酸酶钙调神经磷酸酶的潜在作用，因为这种磷酸酶参与了β-AR诱导的心肌细胞的凋亡。然而，用环孢素A(5um)或FK506(10um)抑制钙调神经磷酸酶并不影响b1-AR介导的细胞凋亡。与之形成鲜明对比的是，用KN-93(0.5um)或多肽抑制剂(AIP，10uM)抑制钙/钙调蛋白激酶II(CaMKII)可完全消除b1-AR诱导的细胞死亡。这与β1-AR以非PKA依赖的方式诱导CaMKII活性显著增加的事实一致。此外，通过腺病毒基因转移在心肌细胞中过表达CaMKII-DC，显著增强了β1-AR介导的心肌细胞凋亡。因此，β1-AR的凋亡效应是由CaMKII-而不是PKA-依赖的机制介导的。这些发现有助于我们了解β1-AR对心脏的有害影响，也强调了治疗心力衰竭的新的治疗策略。

Beta-adrenergic receptor (AR) is a pivotal regulator of cardiac function. However, enhanced beta1-AR signaling promotes apoptotic heart cell death, which is implicated in myocardial remodeling and heart failure. The goal of this study is to determine whether the classic Gs-adenylyl cyclase-cAMP-PKA pathway is essential to beta1-AR apoptotic effect. To avoid complicated interactions between beta-AR subtypes, we created a genetically "pure" beta1-AR experimental setting by expressing the mouse beta1-AR in the null background of beta1/beta2-AR double knockout adult mouse cardiomyocytes using adenoviral gene transfer. Here we show that beta1-AR stimulation is markedly increases myocyte apoptosis, as evidenced by increased TUNEL staining positive cells, DNA fragmentation (cell death ELSIA and DNA laddering). Beat1-AR-induced apoptosis is abolished by a beta-AR antagonist, propranolol (1 uM). To our surprise, specific PKA inhibitors, including Rp-CPT-cAMP (100 uM), H89 (5 uM) and a peptide inhibitor (PKI, 5uM), while blocking forskolin (0.1 uM)-evoked myocyte apoptosis, cannot prevent beta1-AR-induced apoptotic cell death. Ironically, blocking Ca2+ influx by a L-type Ca2+ channel inhibitor nifidipine (1 uM) or buffering intracellular Ca2+ with EGTA-AM (1 uM) fully protects heart cells against beta1-AR-mediatd apoptosis, suggesting that a PKA-independent increase in intracellular Ca2+ is obligatory to beta1-AR apoptotic effect. To delineate the downstream events of beta1-AR/Ca2+ apoptotic signaling, we first evaluated the potential role of a Ca2+/ calmodulin-dependent phosphatase, calcineurin, since this phosphatase has been implicated in beta-AR-induced apoptosis in cardiomyocytes. However, inhibition of calcineurin with cyclosporin A (5 uM) or FK506 (10 uM) does not affect b1-AR-mediated apoptosis. In sharp contrast, inhibition of Ca2+/calmodulin kinase II (CaMKII) with KN-93 (0.5 uM) or a peptide inhibitor (AIP, 10uM) fully abolishes b1-AR-promoted apoptotic cell death. This is consistent with the fact that beta1-AR induces a robust increase in CaMKII activity in a PKA-independent manner. Furthermore, overexpression of CaMKII-dC, a predominant cardiac CaMKII isoform, in cardiac myocytes using adenoviral gene transfer markedly enhances beta1-AR mediated myocyte apoptosis. Thus, beta1-AR apoptotic effect is mediated by a CaMKII-, rather then PKA-, dependent mechanism. These findings shed light on our understanding of beta1-AR cardiac detrimental effects, also underscore novel therapeutic strategies for the treatment of heart failure.