Targeting Gbg-GRK2 signaling in fibrotic remodeling

 描述（由申请人提供）：心力衰竭（HF）是一种预后不良的毁灭性疾病。病理性心脏重构的特征在于进行性肥大、凋亡和纤维化。纤维化加剧心脏重塑、心律失常、心源性猝死和心室顺应性降低;新出现的证据表明，心脏成纤维细胞（CF）在这些过程中发挥关键作用。到目前为止，还没有针对纤维化的治疗方法。阐明CF在病理性重塑中的作用可能具有巨大的治疗潜力。CF研究受到缺乏体内遗传试剂的限制。我们正在合作表征两种新型小鼠模型（PeriostinMerCreMer和Tcf 21 MerCreMer敲入小鼠），其允许对CF功能/活力进行靶向和诱导性操纵，以直接确定心脏纤维化的机制基础。HF中的慢性G蛋白偶联受体（GPCR）刺激诱发GPCR激酶2（GRK 2）的病理性上调，GRK 2被募集到膜Gβγ亚基以使激动剂占据的GPCR脱敏。我们和其他人已经证明了在HF中抑制Gβγ-GRK 2相互作用的治疗潜力。我们的新型小分子Gβγ-GRK 2抑制剂gallein可减弱病理性心脏重塑，使GPCR信号正常化，并减少HF中的间质性心肌纤维化。为了探索GRK 2在心肌纤维化中的成纤维细胞限制作用，GRK 2fl/fl小鼠与POSTNMerCreMer和Tcf-21 MerCreMer一起繁殖。我们令人兴奋的初步数据表明，GRK 2fl/fl-POSTNMerCreMer小鼠在心脏缺血/再灌注（I/R）损伤后受到显著保护，而没有由gallein赋予的进一步保护。有趣的是，GRK 2fl/fl-POSTNMerCreMer小鼠似乎比GRK 2fl/fl-αMHCMerCreMer更具保护性，这表明CF可能比以前认为的提供更大的心脏保护。肾功能恶化和纤维化通常伴随HF进展;它是晚期HF患者死亡率的强预测因子（心肾综合征2，CRS 2），但仍知之甚少。在我们的研究中，TAC或I/R损伤导致大量的肾纤维化和功能障碍;全身性gallein减弱了CRS 2以及急性肾I/R损伤后的肾功能障碍和纤维化。我们的假设是，Gβγ-GRK 2在HF进展和肾功能不全的病理性CF激活中起重要作用，并且其抑制对HF和CRS 2具有治疗前景。为了解决我们的假设，我们提出了以下目的：目的1：在野生型和诱导型CF或CM GRK 2-/-小鼠（+/- Gallein）的临床相关HF模型中确定Gβγ-GRK 2抑制或GRK 2消融的治疗功效和特异性。目标二：阐明成纤维细胞中Gβγ-GRK 2抑制的细胞机制，并验证Gβγ-GRK 2抑制作为小鼠和人类心脏纤维化和HF的治疗靶点。目的3：确定Gβγ-GRK 2信号在肾纤维化和CRS 2中的作用。我们相信，在CF限制性GRK 2-/-小鼠中验证的Gβγ-GRK 2抑制化合物的这一提议，具有治疗HF的前景。该原理也可以扩展到CRS 2和其他纤维化疾病。

 DESCRIPTION (provided by applicant): Heart failure (HF) is a devastating disease with poor prognosis. Pathologic cardiac remodeling is characterized by progressive hypertrophy, apoptosis and fibrosis. Fibrosis exacerbates cardiac remodeling, arrhythmias, sudden cardiac death, and decreased ventricular compliance; emerging evidence suggests that cardiac fibroblasts (CFs) play a key role in these processes. To date, there are no therapies that target fibrosis. Elucidating the role(s) of CF in pathologic remodeling may hold substantial therapeutic potential. CF studies have been limited by the lack of genetic reagents in vivo. We are collaboratively characterizing two novel mouse models (PeriostinMerCreMer and Tcf21MerCreMer knock-in mice) that permit targeted and inducible manipulation of CF function/viability to directly determine the mechanistic basis of cardiac fibrosis. Chronic G- protein coupled receptor (GPCR) stimulation in HF elicits pathologic upregulation of GPCR kinase 2 (GRK2) that is recruited to membrane Gβγ subunits to desensitize agonist-occupied GPCRs. We and others have demonstrated therapeutic potential for inhibiting Gβγ-GRK2 interaction in HF. Our novel, small molecule Gβγ- GRK2 inhibitor, gallein, attenuates pathologic cardiac remodeling, normalizes GPCR signaling and reduces interstitial myocardial fibrosis in HF. To explore a fibroblast-restricted role for GRK2 in myocardial fibrosis, GRK2fl/fl mice have been bred with POSTNMerCreMer and Tcf-21MerCreMer. Our exciting preliminary data suggest that the GRK2fl/fl-POSTNMerCreMer mice are significantly protected following cardiac ischemia/reperfusion (I/R) injury, with no further protection conferred by gallein. Interestingly, GRK2fl/fl-POSTNMerCreMer mice appeared to be more protective than GRK2fl/fl-αMHCMerCreMer, suggesting that greater cardioprotection may be provided via the CF than previously thought. Worsening renal function and fibrosis often accompanies HF progression; it is a strong predictor of mortality in advanced HF patients (cardio-renal syndrome 2, CRS2) but remains poorly understood. TAC or I/R injury resulted in substantial kidney fibrosis and dysfunction in our studies; systemic gallein attenuated CRS2 as well as renal dysfunction and fibrosis following acute renal I/R injury. Our hypothesis is that Gβγ-GRK2 plays an important role in pathologic CF activation in HF progression and kidney dysfunction and that its inhibition holds therapeutic promise for HF and CRS2. To address our hypothesis, we propose the following Aims: Aim 1: Determine the therapeutic efficacy and specificity of Gβγ-GRK2 inhibition or GRK2 ablation in a clinically-relevant HF model in wild type and inducible CF or CM GRK2-/- mice (+/- Gallein). Aim 2: Elucidate the cellular mechanisms underlying Gβγ-GRK2 inhibition in fibroblasts and validate Gβγ-GRK2 inhibition as a therapeutic target in both mouse and human cardiac fibrosis and HF. Aim 3: Determine the role of Gβγ-GRK2 signaling in renal fibrosis and CRS2. We believe this proposal, with Gβγ-GRK2 inhibitory compounds, validated in CF-restricted GRK2-/- mice, holds therapeutic promise for HF. The principle may also extend to CRS2 and other fibrotic diseases.