Laboratory of Dr. Robert Brown, Day Neuromuscular Research Laboratory, Massachusetts General Hospital
Development of high-throughput drug screening assay for membrane repair
David N Reshef1,2, Eduardo Gallardo1,3, Stuart Gibb1, Louise E Glover1, John E Landers1, Isabel Illa3, Robert H. Brown Jr.1
1CB Day Neuromuscular Research Laboratory, MA General Hospital, Harvard Medical School, Boston, MA 02129; 2Massachusetts Institute of Technology; 3Department of Neurology, Neuromuscular Division, Hospital Santa Creu i St Pau, Universitat Autonoma de Barcelona, Barcelona, Spain
Miyoshi myopathy (MM) and limb girdle dystrophy 2B are recessively inherited disorders caused by deficiencies of the membrane protein dysferlin. Both disorders demonstrate early adult onset and markedly elevated serum creatine kinase levels. While LGMD 2B presents with typical limb-girdle distribution of weakness, in MM the initial weakness initially affects the gastrocnemius muscles. We and others have reported that dysferlin is
important in accelerating calcium-triggered muscle membrane repair. To facilitate the analysis of this repair process, and ultimately devise an approach to screen potential therapies for dysferlin-deficiency, we have devised a muscle membrane repair assay that characterizes the time course and extent of membrane resealing in muscle cells after injury. In a 6-well format, this assay clearly distinguishes wild-type (WT) and MM myotubes; repair after a physical injury is slowed in the latter. We have now reduced this assay to a 96 well format, initially studying the injury repair process in myotubes formed from C2C12 myoblasts. Briefly, to achieve simultaneous injury in multiple wells, we injure the myotubes with a short application of detergent, combined in some experiments with glass beads. Following the detergent pulse, injury is quantified at different time points by the addition of a cell impermeant fluorescent dye (propridium iodide, typically applied at 0, 3, 6, 12, 15 and 20 minutes after injury). The level of dye uptake is inversely proportional to the structural integrity of the myotube membrane. The time course of membrane injury and repair in this micro-titer assay reproduces that reported previously in larger in vitro systems. To facilitate uniform delivery of the key reagents to multiple wells in the 96 well plates, we employ a robot to accelerate fluid handling. Critical for this miniaturized assay is the ability to quantify the uptake of propridium iodide as the marker for membrane permeability. Toward this end, we use a quantitative, high-content image analysis system. After the assays are run, the plates of cells are fixed and individual images of each well in the 96 well plate are acquired using a General Electric InCell plate reader, which requires about 5 minutes per 96 well plate. Gross observation of each well using low magnification microscopy clearly indicates the presence or absence of PI-labeled nuclei in myotubes. However, while manual counting of the myotubes clearly quantifies the degree of membrane injury, it is laborious and prohibitive for rapid screening. We have therefore developed software to perform pixel-by-pixel analysis of each well, using edge-detection, cluster recognition, and optimized recursive algorithms to analyze the 3 million+ images generated in each assay. The software has been devised to include a simple graphical user interface to provide flexibility in searching and identifying almost any type of object within an image by simply changing a few mathematical parameters. Finally, the user interface highlights all identified instances of the object found within each image, and allows the user to immediately validate results via inspection, if desired. This system should allow us to screen rapidly for compounds that will modify the timing and amplitude of membrane repair in both WT and dysferlin-deficient cells.