Role of S-Nitrosylation on Beta-Adrenergic Signaling in Cardiac Injury and Repair

总结： 在心力衰竭（HF）的发展过程中，特别是缺血性损伤后，心肌β- 肾上腺素能受体（βAR）信号传导在发病机制中起着重要作用，包括信号解偶联和 受体脱敏导致肌细胞死亡和收缩性缺陷。通过心脏的信号传导减弱 β AR和其他G蛋白偶联受体（GPCR）通常通过受体磷酸化进行调节， 由GPCR激酶（GRKs）和β-抑制蛋白（β-Arrs）介导的内化。这一点很重要，因为活动 GRK 2在损伤/应激后在心肌中升高，在HF中是病理性的，其抑制是治疗性的。 通过长期的合作，科赫和斯塔姆勒实验室发现GPCR受到调控， 通过一氧化氮（NO），通过半胱氨酸的S-亚硝基化形成蛋白质S-亚硝基硫醇（SNO），包括深刻的 GRK 2和β-Arr 2的调节。由于心脏GPCR，包括所有三种β AR，可以激活NO合酶， (NOS)酶，有必要发现这是如何促进SNO介导的心脏保护，特别是 β2-和β 3AR的下游。我们先前的工作表明，来自内皮型一氧化氮合酶（eNOS）的一氧化氮抑制GRK 2 通过Cys 340处的S-亚硝基化。GRK 2-C340 S突变敲入（KI）小鼠中基于SNO的调节的丧失导致 未检查和增强的GRK 2活性，并增加缺血性损伤，以及衰老过程中的功能障碍。我们 实验室还表明，神经元/诱导型NOS（n/iNOS）活性可以通过SNO-Cys 253调节β-Arr 2， 来维持心脏中的生理βAR信号。β-Arr 2-C253 S KI中SNO-β-Arr 2调节的丧失 小鼠导致βAR脱敏和HF增加。此外，Stamler实验室最近发现β2AR 在Cys 265处被S-亚硝基化，并且这种修饰调节β2AR脱敏。这些数据一起 表明βAR/GPCR功能通过受体刺激的S-亚硝基化进行紧密整合调节， 在控制心肌功能中的核心作用，但在很大程度上未被重视。我们的数据提供了新的见解， 亚硝基氧化还原失衡的后果在衰竭的心脏。此Multi-PI提案的中心假设 心脏βAR信号传导和通过GRK 2和β-Arr 2的脱敏受S-亚硝基化调节， 亚硝基氧化还原应激可通过改变受体、GRK和β-Arr的SNO来理解， 心脏对损伤的反应具体目的是：[1]确定GRK 2是否通过S-亚硝基化抑制 在心脏缺血性损伤时，β 2 AR在选择性βAR反应中起机制作用; [2]为了确定β 2 AR是否是 缺血心脏中的S-亚硝基化以及这是否会影响损伤和修复; [3]为了确定是否调节 β-Arr 2通过S-亚硝基化调节损伤后心功能不全时βAR的反应并与β2AR整合 和GRK 2 SNO调节。这些研究的成功完成将阐明S-亚硝基化在 对心脏损伤和修复的综合肾上腺素能反应。这些基本发现将揭示新的 通过定义治疗干预措施， 促进心脏保护，并作为其他器官和疾病中信号系统的范例。

SUMMARY: During the development of heart failure (HF), especially after ischemic injury, derangements in myocardial β- adrenergic receptor (βAR) signaling contribute centrally to pathogenesis, including signal uncoupling and receptor desensitization leading to myocyte death and contractility defects. Waning of signaling through cardiac βARs and other G protein-coupled receptors (GPCRs) is classically regulated via receptor phosphorylation and internalization mediated by GPCR kinases (GRKs) and β-arrestins (β-Arrs). This is significant since the activity of GRK2, which is elevated in myocardium after injury/stress, is pathologic in HF and its inhibition is therapeutic. Through a long-standing collaboration, the Koch and Stamler laboratories have found that GPCRs are regulated by nitric oxide (NO), through S-nitrosylation of cysteine to form protein S-nitrosothiol (SNO), including profound regulation of GRK2 and of β-Arr2. Since cardiac GPCRs, including all three βARs, can activate NO synthase (NOS) enzymes, there is a need to discover how this can promote SNO-mediated cardioprotection, especially downstream of β2- and β3ARs. Our prior work has shown that NO from endothelial NOS (eNOS) inhibits GRK2 by S-nitrosylation at Cys340. Loss of SNO-based regulation in GRK2-C340S mutant knock-in (KI) mice leads to un-checked and enhanced GRK2 activity, and to increased ischemic injury, and to dysfunction during aging. Our labs have also shown that neuronal/inducible NOS (n/iNOS) activity can regulate β-Arr2 through SNO-Cys253 to maintain physiological βAR signaling in the heart. The loss of this SNO-β-Arr2 regulation in β-Arr2-C253S KI mice leads to increased βAR desensitization and HF. Additionally, the Stamler lab recently discovered that β2AR is S-nitrosylated at Cys265 and that this modification regulates β2AR desensitization. Together these data suggest tightly integrated regulation of βAR/GPCR function via receptor-stimulated S-nitrosylation, which plays a central but largely unappreciated role in controlling myocardial function. Our data provides novel insight into consequences of the nitroso-redox imbalance in failing heart. The Central Hypothesis of this Multi-PI proposal is that cardiac βAR signaling and desensitization via GRK2 and β-Arr2 are regulated by S-nitrosylation and that nitroso-redox stress can be understood in terms of altered SNO of receptor, GRK and β-Arr to significantly impair the heart’s response to injury. Specific Aims are: [1] To determine whether GRK2 inhibition via S-nitrosylation plays a mechanistic role in selective βAR responses during cardiac ischemic injury; [2] To determine if β2AR is S-nitrosylated in the ischemic heart and whether this impacts injury and repair; [3] To determine if regulation of β-Arr2 by S-nitrosylation tunes βAR responses during cardiac dysfunction after injury and is integrated with β2AR and GRK2 SNO regulation. Successful completion of these studies will illuminate the role of S-nitrosylation in the integrated adrenergic response to cardiac injury and repair. These fundamental discoveries will reveal new insights into the regulation of cardiac function in health and disease by defining therapeutic interventions to promote cardioprotection, and to serve as a paradigm for signaling systems in other organs and diseases.