Comparison of skeletal muscle mitochondrial properties isolated by protease digestion and mechanical homogenization

Skeletal muscle contains two heterogeneous mitochondrial subfractions located in the subsarcolemmal (SS) and intermyofibrillar (IMF) regions. Traditionally, the isolation of IMF mitochondria has required the use of limited protease (Nagarse) digestion. The purpose of this investigation was to compare the properties of SS with those of IMF mitochondria isolated by protease (PROT; 0.25mg/g tissue) or mechanical (MECH; Teflon‐glass, ~900 rpm) protocols. Protein yield was similar among IMF mitochondria obtained by PROT and MECH digestion compared to the SS subfraction, as were the respiratory control ratios (6–10), indicating the isolation of well‐coupled mitochondria independent of the procedure used. State 4 and 3 respiration rates in IMF PROT and IMF MECH mitochondria were 3‐5‐ and 2‐2.5‐fold greater than SS mitochondria, respectively. Membrane potential of IMF PROT and IMF MECH mitochondria were 55% lower and 12% higher compared to SS mitochondria. ROS production was 60–80% lower in IMF PROT and 30–45% lower in IMF MECH mitochondria during state 4 and state 3 respiration compared to the SS subfraction. Electron microscopy revealed that the MECH protocol produced IMF mitochondria which were less pure than with the PROT method. These data indicate that properties of SS and IMF subfractions remain distinct following IMF PROT and MECH isolation procedures. However, these established disparities between SS and IMF mitochondria are attenuated with MECH fractionation, which may be due in part to contamination by non‐mitochondrial constituents. Support from NSERC and HSFC.