Mechanisms of Adenylyl Cyclase Effects in the Heart

描述（由申请人提供）：当前奖项的初始资助期的数据表明，增加的心脏AC 6型（AC 6），一种在哺乳动物心肌细胞中表达的主要AC亚型，对衰竭的左心室（LV）具有多种有益作用。这些作用在各种物种和病理生理学模型中如此一致，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。逻辑将决定：a）cAMP毕竟对心脏不坏-当在衰竭的心脏中cAMP水平增加时，它是导致不良结果的其他东西;或者B）增加的AC 6具有独立于cAMP的有益心脏作用，其抵消了其预期的有害作用。使用药理学抑制剂和其他方法，我们的数据表明，心脏AC 6表达增加的许多有益作用不需要增加cAMP的产生。由于使用药理学抑制的研究的固有局限性，我们通过在催化核心中的位置426处用Ala取代Asp来产生无催化活性的AC 6突变体（AC 6 mut）分子。这种AC 6 mut不产生cAMP，但保留了与AC 6相关的细胞分布模式和有利的信号传导效应。这些数据表明，AC 6的有益作用，至少部分，是独立的cAMP。我们现在建议进行一系列体外和体内实验，以建立无懈可击的有益作用机制。理想的正性肌力药将在没有2AR刺激或cAMP产生的情况下增加心脏收缩功能，改善Ca 2+处理，减少不良重塑和凋亡，并具有有利的电生理特性。除了其对cAMP生成的影响外，心脏AC 6表达的增加实现了许多这些特征，并且FDA已经批准了NHLBI资助的在患有严重CHF的患者中进行AC 6基因转移的试验（ClinicalTrials.gov NCT 00787059）。然而，有四个原因来进行cAMP缺陷型AC 6突变体的研究：1）获得关于AC 6的有益心脏作用是否与cAMP产生无关的确定性机制数据; 2）通过产生多种AC 6 mut片段分子进行AC 6 mut分子的结构-活性研究，并确定AC 6 mut片段的亚细胞靶向对Ca 2+的影响处理和Akt激活; 3）确定AC 6 mut在体内的心脏作用和4）产生编码最佳AC 6 mut片段的合适的长期调节的表达载体，并进行临床前基因转移研究以确定其在改善衰竭心脏功能中的功效和安全性。这些研究旨在揭示机制，但也具有临床意义。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剂未能延长存活。基于初步数据，AC 6 mut保留了AC 6所见的有益效果，但在不增加cAMP产生的情况下也是如此，因此实现了潜在理想的治疗性肌力药的特征。

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.