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β 1-AR信号促进凋亡性心脏细胞死亡，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-adenylyl cyclase-cAMP-PKA通路是否是必不可少的β 1-AR凋亡效应。为了避免β-AR亚型之间复杂的相互作用，我们通过使用腺病毒基因转移在β 1/β 2-AR双敲除的成年小鼠心肌细胞的零背景中表达小鼠β 1-AR来创建遗传上“纯”的β 1-AR实验设置。在这里，我们发现β 1-AR刺激显著增加心肌细胞凋亡，如通过增加TUNEL染色阳性细胞、DNA片段化（细胞死亡ELSIA和DNA梯状化）所证明的。Beat 1-AR诱导的细胞凋亡被β-AR拮抗剂普萘洛尔（1 μ M）消除。令我们惊讶的是，特异性PKA抑制剂，包括Rp-CPT-cAMP（100 μ M），H89（5 μ M）和肽抑制剂（PKI，5 μ M），虽然阻断毛喉素（0.1 μ M）诱发的心肌细胞凋亡，但不能阻止β 1-AR诱导的凋亡性细胞死亡。具有讽刺意味的是，用L-型钙通道抑制剂硝苯地平（1 μ M）阻断钙内流或用EGTA-AM（1 μ M）缓冲细胞内钙可完全保护心脏细胞免受β 1-AR介导的凋亡，这表明细胞内钙的PKA非依赖性增加是β 1-AR凋亡效应所必需的。为了描述β 1-AR/Ca 2+凋亡信号的下游事件，我们首先评估了Ca 2 +/钙调蛋白依赖性磷酸酶钙调磷酸酶的潜在作用，因为这种磷酸酶与β-AR诱导的心肌细胞凋亡有关。然而，用环孢菌素A（5 μ M）或FK 506（10 μ M）抑制钙调磷酸酶并不影响b1-AR介导的凋亡。与此形成鲜明对比的是，用KN-93（0.5 μ M）或肽抑制剂（AIP，10 μ M）抑制Ca 2 +/钙调蛋白激酶II（CaMK II）完全消除了b1-AR促进的凋亡性细胞死亡。这与β 1-AR以不依赖于PKA的方式诱导CaMKII活性稳健增加的事实一致。此外，使用腺病毒基因转移在心肌细胞中过度表达CaMKII-dC（一种主要的心脏CaMKII同种型）显著增强β 1-AR介导的心肌细胞凋亡。因此，β 1-AR凋亡效应是由CaMKII依赖性机制介导的，而不是PKA依赖性机制。这些发现阐明了我们对β 1-AR心脏有害作用的理解，也强调了治疗心力衰竭的新治疗策略。

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.