北京大学分子医学研究所（IMM）与美国杜克大学医学院合作，首次建立果蝇心脏钙信号活体成像技术,并将这一新技术应用于心脏疾病致病基因研究。 合作论文“A method to measure myocardial calcium handling in adult Drosophila”于4月15日在线发表于  Circulation Research 。

心脏疾病尤其是心力衰竭中的很多致病基因并不清楚，在哺乳动物中难以进行大规模的筛选。果蝇较短的繁殖周期、低廉的培养成本、丰富的突变体库使其成为用遗传学高通量筛选致病基因的理想模式动物。论文作者构建了表达钙荧光蛋白探针的转基因果蝇，采用高速显微成像系统采集果蝇管状心脏搏动时钙兴奋波（见图示）,研究了野生型果蝇心脏钙信号动态及其离子机制。同时，与一种有类似哺乳动物扩张性心肌病的肌钙蛋白I突变体果蝇进行了对比，发现后者心脏钙信号上升和下降速率都变慢，时程变长,与哺乳动物扩张性心肌病的钙调控异常十分类似。新方法及实验结果表明果蝇模型可用于高通量筛选哺乳动物心脏中进化上高度保守的一类致病基因。

分子医学研究所博士研究生林娜为论文第一作者，课题由杜克大学医学院Matthew Wolf，Howard Rockman，Nenad Bursac和北京大学分子医学研究所程和平教授共同指导完成。

图示:果蝇管状心脏紧靠腹背侧，长约1毫米，钙兴奋波以自后向前(a), 自前向后(b), 前后相向(c)三种方式传播，持续时间约200毫秒。

原文出处：

Circulation Research. 2011  doi: 10.1161/CIRCRESAHA.110.238105

A Method to Measure Myocardial Calcium Handling in Adult Drosophila

Na Lin, Nima Badie, Lin Yu, Dennis Abraham, Heping Cheng, Nenad Bursac, Howard A. Rockman Matthew J. Wolf

From the Institute of Molecular Medicine, Peking University, Beijing, China (N.L., H.C.); Department of Biomedical Engineering, Duke University, Durham, NC (N.Badie, N.Bursac); Department of Medicine, Duke University, Durham, NC (L.Y., D.A., H.A.R., M.J.W.); Departments of Cell Biology and Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC (H.A.R.).

Correspondence to Matthew J. Wolf, MD, PhD, Assistant Professor of Medicine, Division of Cardiology, Duke University Medical Center, Durham, NC 27710. E-mail wolf0008@mc.duke.edu

Abstract

Rationale:Normal cardiac physiology requires highly regulated cytosolic Ca2+ concentrations, and abnormalities in Ca2+ handling are associated with heart failure. The majority of approaches to identifying the components that regulate intracellular Ca2+ dynamics rely on cells in culture, mouse models, and human samples. However, a genetically robust system for unbiased screens of mutations that affect Ca2+ handling remains a challenge.

Objective:We sought to develop a new method to measure myocardial Ca2+ cycling in adult Drosophila and determine whether cardiomyopathic fly hearts recapitulate aspects of diseased mammalian myocardium.

Methods and Results:Using engineered transgenic Drosophila that have cardiac-specific expression of Ca2+-sensing fluorescent protein, GCaMP2, we developed methods to measure parameters associated with myocardial Ca2+ handling. The following key observations were identified: (1) Control w1118 Drosophila hearts have readily measureable Ca2+-dependent fluorescent signals that are dependent on L-type Ca2+ channels and SR Ca2+ stores and originate from rostral and caudal pacemakers. (2) A fly mutant, held-up2 (hdp2), that has a point mutation in troponin I and has a dilated cardiomyopathic phenotype demonstrates abnormalities in myocardial Ca2+ handling that include increases in the duration of the 50% rise in intensity to peak intensity, the half-time of fluorescence decline from peak, the full duration at half-maximal intensity, and decreases in the linear slope of decay from 80% to 20% intensity decay. (3) Hearts from hdp2 mutants had reductions in caffeine-induced Ca2+ increases and reductions in ryanodine receptor (RyR) without changes in L-type Ca2+ channel transcripts in comparison with w1118.

Conclusions:Our results show that the cardiac-specific expression of GCaMP2 provides a means of characterizing propagating Ca2+ transients in adult fly hearts. Moreover, the adult fruit fly heart recapitulates several aspects of Ca2+ regulation observed in mammalian myocardium. A mutation in Drosophila that causes an enlarged cardiac chamber and impaired contractile function is associated with abnormalities in the cytosolic Ca2+ transient as well as changes in transcript levels of proteins associated with Ca2+ handling. This new methodology has the potential to permit an examination of evolutionarily conserved myocardial Ca2+-handing mechanisms by applying the vast resources available in the fly genomics community to conduct genetic screens to identify new genes involved in generated Ca2+ transients and arrhythmias.

Key Words: Drosophila heart myocardial calcium  GCaMP2