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标志是它们能够改变心肌收缩的钙调节。一般来说，扩张型（DCM）心肌病的Ca2+收缩敏感性降低；而在肥厚性（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，对Ca2+敏感性增敏（+）或脱敏（-）的新药的鉴定可能潜在地逆转（+或-）这些异常变化。因此，本提案的目标是使用高通量筛选（HTS）来识别可以调节心肌收缩Ca2+敏感性的小分子。为了实现这一目标，我们将使用由心肌调节细丝（RTF）组成的模型系统，RTF由f -肌动蛋白、原肌球蛋白和肌钙蛋白（Tn）组成。RTF与肌凝蛋白（粗纤维）结合构成了在收缩装置中发现的主要蛋白质。在没有肌凝蛋白的情况下，RTF保留了所有对肌肉激活和放松至关重要的Ca2+调节功能。拟议的分析将使用心脏Tn （CTn）复合物，其中含有荧光标记的肌钙蛋白C (CTnC)， CTn复合物的Ca2+结合亚基。这将使我们能够监测Ca2+结合到CTnC调控位点时RTF荧光的变化。因此，检测到在固定的[Ca2+]和波长下标记的RTF荧光强度的增加或减少（+或-），以响应来自HTS屏幕的化合物或“命中”，将表明CTnC的表观Ca2+亲和力发生了变化（+或-）。HTS的命中将使用两个生物二级筛选进一步验证。基于上述，RTF系统可以提供一个强大的，稳定的和生理分析，以确定化合物特异性地改变RTF Ca2+的敏感性，而不是通过交叉桥药物相互作用的力。为了实现我们的目标，本建议将追求两个具体目标。从这些研究中获得的知识可以为心肌病、高血压和其他形式的心血管疾病的调查和治疗发现潜在的新药理学药物。

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.