Purpose: The ultrafast extraocular muscles necessitate tight regulation of free cytosolic Ca2+ concentration ([Ca2+]i). Mitochondrial Ca2+ influx may be fast enough for this role. In the present study, three hypotheses were tested: (1) Mitochondrial Ca2+ uptake regulates [Ca2+]i and production of force in extraocular muscle; (2) mitochondrial content correlates with their use as Ca2+ sinks; and (3) mitochondrial content in extraocular muscle is determined by the transcription factors and coactivators that initiate muscle adaptation to aerobic exercise.
Methods: Extraocular and extensor digitorum longus (EDL) muscles from adult Sprague-Dawley rats were used to examine how the Ca2+ release agonists caffeine and 4-chloro-3-ethylphenol (CEP), calcimycin (a Ca2+ ionophore) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP; a mitochondrial uncoupler) alter [Ca2+]i and force transients. Mitochondrial volume density and capillary density were analyzed by stereology and citrate synthase and cytochrome c oxidase by biochemical assays. Real-time PCR measured mRNAs of genes involved in mitochondrial biogenesis.
Results: Caffeine, CEP, and calcimycin increased resting [Ca2+]i to a greater extent in EDL. Peak tetanic [Ca2+]i increased in extraocular muscle with caffeine and CEP. CCCP augmented peak tetanic and submaximum [Ca2+]i and force significantly more in extraocular muscles. Mitochondrial volume density and capillary density were three times greater, and citrate synthase and cytochrome c oxidase were only approximately 2-fold higher in extraocular muscle. Calcineurin Aalpha, calcineurin B, and peroxisome proliferator activated receptor (PPAR)gamma were more abundant in extraocular muscle.
Conclusions: These data support the hypothesis that mitochondria serve as Ca2+ sinks in extraocular muscles. The high mitochondrial content of these muscles may partly reflect this additional function. It is likely that mitochondrial Ca2+ influx increases the dynamic response range of the extraocular muscles and matches metabolic demand to supply.