Mechanisms of Adenylyl Cyclase Effects in the Heart

描述（由申请人提供）：来自当前奖项初始资助期的数据表明，增加的心脏AC6型（AC6），一种在哺乳动物心肌细胞中表达的显性AC异构体，对衰竭的左心室（LV）具有多种有益作用。这些影响在各种物种和病理生理模型中是一致的，必须与2-肾上腺素能受体刺激和细胞内cAMP升高对心脏的可怕后果相协调。从逻辑上讲，要么是：a)毕竟cAMP对心脏没有坏处——当衰竭的心脏中cAMP水平升高时，它是导致不良结果的其他东西；或b)增加的AC6具有独立于cAMP的有益心脏作用，从而抵消其预期的有害作用。使用药物抑制剂和其他方法，我们的数据表明，心脏AC6表达增加的许多有益效果不需要增加cAMP的产生。由于使用药物抑制研究的固有局限性，我们通过在催化核心的426位用Ala取代Asp生成了一个催化无活性的AC6突变体（AC6mut）分子。该AC6mut不产生cAMP，但保留了与AC6相关的细胞分布模式和有利的信号传导作用。这些数据表明，AC6的有益作用，至少在一定程度上是独立于cAMP的。我们现在建议进行一系列的体外和体内实验，以建立无懈可击的有益作用机制。理想的肌力剂应在没有2AR刺激或cAMP生成的情况下增加心脏收缩功能，改善Ca2+处理，减少不良重构和凋亡，并具有良好的电生理特性。除了对cAMP产生的影响外，心脏AC6表达的增加完成了许多这些特征，FDA已经批准了nhlbi资助的AC6基因转移在严重CHF患者中的试验（ClinicalTrials.gov NCT00787059）。然而，对cAMP功能不全的AC6突变体进行研究有四个原因：1)获得AC6对心脏的有益作用是否与cAMP生成无关的确切机制数据；2)通过生成多种AC6mut片段分子，对AC6mut分子进行结构活性研究，确定AC6mut片段亚细胞靶向对Ca2+处理和Akt活化的影响；3)确定AC6mut在体内的心脏作用；4)生成合适的长期调控表达载体，编码最佳AC6mut片段，并进行临床前基因转移研究，确定其改善衰竭心脏功能的有效性和安全性。这些研究旨在揭示机制，但也具有临床相关性。例如，在临床心力衰竭中，通过增加cAMP水平来增加心脏收缩功能的药物未能延长生存期。根据初步数据，AC6mut保留了AC6的有益作用，但没有增加cAMP的产生，因此实现了潜在理想治疗性肌力的特征。

DESCRIPTION (provided by applicant): Data from the initial funding period of the current award indicate that increased cardiac AC type 6 (AC6), a dominant AC isoform expressed in mammalian cardiac myocytes, has protean beneficial effects on the failing left ventricle (LV). These effects, so consistent in a variety of species and pathophysiological models, must be reconciled with the dire consequences on the heart of 2-adrenergic receptor stimulation and elevations in intracellular cAMP. Logic would dictate that either: a) cAMP is not bad for the heart after all-that it is something else which leads to poor outcomes when cAMP levels are increased in the failing heart; or b) increased AC6 has beneficial cardiac effects independent of cAMP, which counterbalance its expected deleterious effects. Using pharmacological inhibitors and other approaches, our data suggest that many of the beneficial effects of increased cardiac AC6 expression do not require increased cAMP generation. Because of the inherent limitations of studies using pharmacological inhibition, we generated a catalytically inactive AC6 mutant (AC6mut) molecule by substitution of Ala for Asp at position 426 in the catalytic core. This AC6mut does not generate cAMP, but retains the cellular distribution pattern and favorable signaling effects associated with AC6. These data indicate that the beneficial effects of AC6, at least in part, are independent of cAMP. We propose now to conduct a series of experiments, both in vitro and in vivo, to establish unassailable mechanisms for the beneficial effects. The ideal inotrope would increase cardiac contractile function in the absence of 2AR stimulation or cAMP generation, improve Ca2+ handling, reduce adverse remodeling and apoptosis, and have favorable electrophysiological properties. With the exception of its effects on cAMP generation, increased cardiac AC6 expression accomplishes many of these features, and the FDA has approved an NHLBI-funded trial of AC6 gene transfer in patients with severe CHF (ClinicalTrials.gov NCT00787059). However, there are four reasons to conduct studies of the cAMP-incompetent AC6 mutant: 1) to obtain definitive mechanistic data on whether AC6's beneficial cardiac effects are unrelated to cAMP generation; 2) to conduct structure-activity studies of the AC6mut molecule by generating a variety of AC6mut fragment molecules and to determine the effects of subcellular targeting of the AC6mut fragments on Ca2+ handling and Akt activation; 3) to determine the cardiac effects of AC6mut in vivo and 4) to generate a suitable long term regulated expression vector encoding the optimal AC6mut fragment, and conduct preclinical gene transfer studies to determine its efficacy and safety in improving function of the failing heart. The studies are designed to uncover mechanisms, but also to have clinical relevance. For example, in clinical heart failure, agents that increase cardiac contractile function by increasing cAMP levels have failed to prolong survival. The AC6mut, based on preliminary data, preserves the beneficial effects seen with AC6, but does so in the absence of increased cAMP production, thus fulfilling features of a potentially ideal therapeutic inotrope. PUBLIC HEALTH RELEVANCE: We have modified adenylyl cyclase type 6 (AC6), a molecule that regulates cardiac function. This modified AC6 (AC6mut) preserves the beneficial effects on the heart that are seen with AC6, but does so in the absence of increased cAMP production. In patients with heart failure, drugs that improve heart function by increasing cAMP levels have failed to reduce mortality. Our studies will determine if this modified AC6 molecule will be a suitable heart failure treatment.