Measuring Muscle Myofibril Fragmentation

As an indirect measure of calpain activity, myofibril fragmentation (MFI) in muscle samples was assessed. A total of 4 g of minced muscles, free of visible connective tissue and external fat, were homogenized for 30 s in a blender (Ultra Turrax; IKA-Werke, Staufen, Germany) with 40 ml of cold MFI buffer at 2°C. Following several washes, suspension aliquots were diluted in MFI buffer to a final protein concentration of 0.5 mg/ml and poured into a cuvette for immediate absorbance measurement at 540 nm with a spectrophotometer (HACH DR/3000 Spectrophotometer, USA). Each sample's MFI was multiplied by 200.