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

摘要： 在心力衰竭(HF)的发展过程中，特别是在缺血损伤后，心肌β-1的紊乱。 肾上腺素能受体(βAR)信号在发病机制中起中心作用，包括信号解偶联和 受体脱敏导致心肌细胞死亡和收缩功能缺陷。心脏信号转导功能的减弱 β受体和其他G蛋白偶联受体(GPCRs)经典地通过受体磷酸化和 GPCRKs(GRKs)和β-arrestins(β-ARRs)介导的内化。这一点意义重大，因为活动 GRK2在损伤/应激后在心肌中升高，在心力衰竭时是病理性的，其抑制具有治疗作用。 通过长期的合作，科赫和斯塔姆勒实验室发现GPCR是受监管的 由一氧化氮(NO)，通过S-半胱氨酸亚硝化生成蛋白质S-亚硝硫醇(SNO)，包括 调节GRK2和β-Arr2。由于心脏β受体，包括所有三个GPCRs，都可以激活一氧化氮合酶 (NOS)酶，有必要发现这如何促进SNO介导的心脏保护，特别是 β2-和β3AR下游。我们先前的工作表明，来自内皮型一氧化氮合酶(ENOS)的NO抑制GRK2 S在Cys340上进行亚硝化反应。GRK2-C340S突变敲入(KI)小鼠SNO调节缺失导致 未受抑制和增强的GRK2活性，以及增加的缺血性损伤，以及衰老过程中的功能障碍。我们的 实验室还表明，神经元/诱导型一氧化氮合酶(n/iNOS)活性可以通过SNO-Cys253调节β-Arr2 以维持心脏中生理性的βAR信号。β-Arr2-C253S KI中这种SnO-Arr2-Arr2调控的缺失 小鼠导致β、AR脱敏和HF增加。此外，斯塔姆勒实验室最近发现，β2AR 是否S在Cys265位发生亚硝化，该修饰调控β-2AR脱敏。将这些数据放在一起 建议通过受体刺激的S-亚硝化来紧密整合调节βAR/GPC的功能，它发挥着 在控制心肌功能方面的核心作用，但在很大程度上没有被认识到。我们的数据为我们提供了对 心力衰竭时亚硝基-氧化还原失衡的后果。这一多PI方案的中心假设 心脏βAR信号转导和通过GRK2和β-Arr2的脱敏受S-亚硝酸化的调节 亚硝基-氧化还原应激可以通过改变受体、Grk和β-ARR的SNO来理解，从而显著损害 心脏对损伤的反应。具体目的是：[1]确定是否通过S-亚硝化抑制GRK2 在心脏缺血损伤期间在选择性βAR反应中起机制作用；[2]确定β2AR是否 S-亚硝酸盐在缺血心脏中的作用及其是否影响损伤和修复；[3]以确定是否调节 S的β-ARR2-硝化反应调节损伤后心功能不全期间的βAR反应，并与β2AR整合 和GRK2 SNO调节。这些研究的成功完成将阐明S-亚硝化在 心脏损伤和修复的综合肾上腺素能反应。这些根本性的发现将揭示新的 通过定义治疗干预措施来洞察健康和疾病中的心功能调节 促进心脏保护，并作为其他器官和疾病信号系统的范例。

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.