No Calpain, No Gain: Newly Developed Procedures for the Separation and Characterization of The Calpain Family of Proteins in Human Dystrophic and Non-dystophic Muscle

Abstract

Muscular dystrophy is a disease which gradually deteriorates skeletal muscle cells, leading to the eventual death of such cells and the surrounding tissue. Calpains are Ca2+- dependent proteases and together with the Ca2+-dependent specific inhibitor of calpains, calpastatin, are widely distributed in eukaryotic cells. It has been suggested that part of the enhanced deterioration in the dystrophic state is due to enhanced calpain activity; therefore analysis of normal and dystrophic muscle was essential. Conventional techniques for the isolation and characterization of calpain and calpastatin utilize relatively large muscle samples (>100g), whereas biopsy or post-mortem samples are considerably less than this. Thus, the initial and main objective of this project was to develop methods suitable for purification and analysis of the calpain family from limited muscle samples. With these restrictions in mind, techniques were developed for samples ranging from 0.2-1g, a realistic biopsy extraction. The hypothesis to be further evaluated is that some dystrophies are characterized by increased calpain activity, caused either by an increased expression of m- or μ- calpain or decreased inhibition by calpastatin, or both. The procedures are now in place to test this hypothesis further and extensive analyses are required using defined dystrophic types and increased sampling numbers.