Effect of chronic respiratory loading on the subunit composition of cytochrome c oxidase in the diaphragm

To elucidate in the diaphragm, 1) whether chronic inspiratory loading increases the amount of cytochrome c oxidase (COX) subunit proteins, and 2) how well the regulation of mitochondrially and nuclearly coded COX subunits is coordinated, we have trained six adult sheep with inspiratory flow-resistive loads for 3 h/day for 3 wk. Six other sheep served as controls. Proteins from crude muscle homogenates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotted, and reacted with polyclonal rabbit anti-bovine COX antibodies. A mitochondrially coded subunit (II) and nuclearly coded subunits (IV and VII) reacted with anti-COX antibodies and were quantified with laser densitometry using purified COX as a standard. In the costal diaphragm and for the equivalent amount of muscle homogenate protein, the integrated optical densities (IOD) for subunits II, IV, and VII were significantly greater in the trained sheep than in the controls. Similarly, the IOD for subunits II and VII were significantly greater in the trained than in the controls in the crural diaphragm. There were no differences between the two groups in the quadriceps, a muscle that was used as an untrained, internal control muscle. The ratios of the IOD for each of the two nuclearly coded subunits to that for mitochondrially coded subunit II were not different between the two groups. These data suggest that chronic inspiratory loading increases both mitochondrially and nuclearly coded COX subunits in the diaphragm and that the subunits coded by the two genetic systems are coordinately regulated.