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F. H. Andrade, S. P. Patel, J. Gamboa, C. A. McMullen, A. G. Rabchevsky; Unexpected Constraints of Extraocular Muscle Mitochondrial Function: Lower Respiration Rates and Enzymatic Activity. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4490.
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© ARVO (1962-2015); The Authors (2016-present)
The fast and constant activity of the extraocular muscles (EOMs) requires ATP from mitochondria. Given the metabolic load, it seemed likely that EOM mitochondria would require adaptive changes in their composition and function in order to match aerobic capacity to energy demand. Therefore, the purpose of this study was to test the hypothesis that EOM mitochondria respire at faster rates than those from limb muscles because of higher content of the protein complexes that integrate the electron transport chain (ETC).
We obtained EOMs and gastrocnemius (Gast, a mixed-fiber limb muscle) from adult male Sprague-Dawley rats (300-350 g) and isolated mitochondria by differential centrifugation. We measured mitochondrial respiration rates with a miniature Clark-type electrode and then determined the activity (by spectrophotometry) and content (by western blotting) of ETC complexes.
EOM mitochondria had lower state 4 (substrates but no ADP), state 3 (substrates+ADP) and state 5 (substrates+ADP+uncoupler) respiration rates. State 4 was 7.4±0.3 vs. 12.7±0.8 and state 3 was 53.7±1.7 vs. 114.9±6.2 (nmoles O2/min/mg protein, EOM vs. Gast respectively, P<0.001 for both comparisons). The respiratory control ratios (state 3/state 4) were consistently high for EOM and Gast mitochondria: 7.3±0.3 and 9.1±0.3, evidence that the isolation procedure did not preferentially alter EOM mitochondria. State 5 respiration was 56.4±1.3 for EOM and 138.8±14.2 for Gast (nmoles O2/min/mg protein, P<0.001). ETC complex I content in EOM mitochondria was 116±4.1% of Gast mitochondria, but activity was 55.1±4.5% (P<0.05 for both). ETC complex II content and activity were not different between EOM and Gast mitochondria. ETC complex IV content in EOM mitochondria was 142.4±10.5 of Gast mitochondria and activity was only 47.9±4.9% (P<0.01 for both).
Based on these results, we reject the original hypothesis that EOM mitochondria respire at faster rates. Instead, our data support the conclusion that EOM mitochondria have a lower respiratory capacity, apparently due to a combination of lower activity and content of ETC complexes. We propose that these intrinsic differences render EOMs susceptible to certain mitochondrial myopathies.
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