HTS for Regulated Muscle Thin Filament Function.

描述(由申请人提供)：心肌病中肌瘤基因突变的一个特点是它们有能力改变心肌收缩的钙调节。一般来说，扩张型(DCM)心肌病收缩的钙敏感性降低；而在肥厚型(HCM)和限制性(RCM)心肌病中，敏感性增加。由于存在多种形式的心肌病，识别能敏化()或脱敏(-)钙敏感性的新药可能会逆转(或-)这些异常变化。因此，这项提议的目标是使用高通量筛选(HTS)来识别能够调节心肌收缩的钙敏感性的小分子。为了实现这一点，我们将使用一个由F-肌动蛋白、原肌球蛋白和肌钙蛋白(TN)组成的心肌调节细丝(RTF)组成的模型系统。RTF和肌球蛋白(粗丝)一起构成了收缩装置中的主要蛋白质。在没有肌球蛋白的情况下，RTF保留了对肌肉激活和松弛至关重要的所有钙调节功能。建议的分析将使用心脏TN(CTN)复合体，该复合体包含荧光标记的肌钙蛋白C(CTNC)，CTN复合体的钙结合亚单位。这将使我们能够监测当Ca~(2+)与cTNC调控位点结合时RTF荧光的变化。因此，检测到标记的RTF荧光强度在固定的[Ca~(2+)]和波长上的增加或减少(或-)，以响应来自HTS屏幕的化合物或“点击”，将表明cTNC的表观Ca~(2+)亲和力发生了变化(或-)。HTS的命中将使用两个生物二级筛查进行进一步验证。在此基础上，RTF系统可以提供一种可靠、稳定和生理的方法来鉴定特异性改变RTF钙敏感性的化合物，而不是通过跨桥药物相互作用来改变力的化合物。为了实现我们的目标，这项提议将追求两个具体目标。从这些研究中获得的知识可以发现潜在的新的药理药物，用于研究和治疗心肌病、高血压和其他形式的心血管疾病。 公共卫生相关性：在过去的十年里，三种主要类型的遗传性心肌病，包括扩张型、肥厚型和限制性心肌病的遗传学基础已经被仔细研究。这里提出的研究将利用现代高通量筛选技术识别新的低分子化合物，该技术可以调节在这些疾病的模型系统中观察到的关键表型。这些研究的结果最终将有益于开发治疗这些以及潜在的其他形式的心血管疾病的新的治疗方法。

DESCRIPTION (provided by applicant): A hallmark of sarcomeric gene mutations in cardiomyopathies is their ability to alter the calcium regulation of cardiac muscle contraction. In general, the Ca2+ sensitivity of contraction decreases in dilated (DCM) cardiomyopathy; whereas, in hypertrophic (HCM) and restrictive (RCM) cardiomyopathies, the sensitivity increases. Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the Ca2+ sensitivity could potentially reverse (+ or -) these aberrant changes. Therefore, the goal of this proposal is to use high throughput screening (HTS) to identify small molecules that can modulate the Ca2+ sensitivity of cardiac muscle contraction. To achieve this, we will use a model system composed of cardiac muscle regulated thin filaments (RTF) which are comprised of F-actin, tropomyosin and troponin (Tn). The RTF in combination with myosin (thick filament) make up the major proteins found in the contractile apparatus. In the absence of myosin, the RTF retains all of the Ca2+ regulated functions critical for muscle activation and relaxation. The proposed assay will use cardiac Tn (CTn) complexes that contain fluorescently labeled troponin C (CTnC), the Ca2+ binding subunit of the CTn complex. This will allow us to monitor changes in RTF fluorescence that occurs when Ca2+ binds to the CTnC regulatory site. Therefore, detecting an increase or decrease (+ or -) in the labeled RTF fluorescence intensity at a fixed [Ca2+] and wavelength in response to a compound or "hit" from the HTS screen will indicate that a change (+ or -) in the apparent Ca2+ affinity of CTnC has occurred. Hits from the HTS will be further validated using two biological secondary screens. Based on the above, the RTF system can provide a robust, stable and physiological assay to identify compounds that specifically alter the RTF Ca2+ sensitivity and not the force via cross bridge-drug interactions. To achieve our goals, this proposal will pursue two Specific Aims. Knowledge gained from these studies can uncover potentially new pharmacological agents for the investigation and treatments of cardiomyopathies, hypertension and other forms of cardiovascular diseases. PUBLIC HEALTH RELEVANCE: The genetic basis for three major types of inherited cardiomyopathies including dilated, hypertrophic and restrictive have been studied intently over the last decade. The studies proposed here will identify new low molecular weight compounds, using modern high throughput screening technology, which can modulate a key phenotype that has been observed in model systems of these diseases. Results from these studies will ultimately be beneficial in developing new therapeutic approaches for the treatment of these and potentially other forms of cardiovascular disease.