To determine whether expression of a nuclear gene encoding a mitochondrial fatty acid oxidation enzyme is regulated in parallel with skeletal muscle fibre-type-specific energy substrate preference, expression of the gene encoding medium-chain acyl-CoA dehydrogenase (MCAD) was delineated in canine latissimus dorsi muscle subjected to chronic motor nerve stimulation. In predominantly fast-twitch canine latissimus dorsi muscle, MCAD mRNA levels were regulated by chronic stimulation in a biphasic pattern. During the 1st wk of stimulation, steady-state MCAD mRNA levels decreased to 50% of unstimulated levels. MCAD mRNA levels began to increase during the 3rd wk of stimulation to reach a level 3.0-fold higher than levels in unstimulated contralateral control muscle by day 70. Immunodetectable MCAD mRNA levels throughout the stimulation period. The temporal pattern and magnitude of MCAD mRNA accumulation in response to muscle stimulation was distinct from that of mRNAs encoding other enzymes known to be regulated by this stimulus, including glyceraldehyde phosphate dehydrogenase, citrate synthase, and sarcoplasmic reticulum Ca-ATPase, but paralleled the protein levels of the peroxisome proliferator-activated receptor (PPAR), an orphan member of the nuclear hormone receptor superfamily known to regulate genes encoding fatty acid oxidation enzymes in liver. The skeletal muscle expression pattern of PPAR was also similar to that of MCAD in unstimulated rat skeletal muscles with distinct fiber-type compositions. These results demonstrate that a nuclear gene encoding a mitochondrial beta-oxidation enzyme is dynamically regulated in a pattern that parallels skeletal muscle fiber-type-specific energy substrate utilization and implicate an orphan nuclear receptor transcription factor as a candidate transducer of this response.

1. Salbutamol, a fl2-agonist, increased the weight of the canine latissimus dorsi muscle. It also increased fusion frequency, and decreased time-to-peak tension, half-relaxation time, and total contraction time. These changes in twitch times and fusion frequency were associated with changes in the levels of proteins expressed in slow-and fast-twitch fibres. Salbutamol decreased the levels of the slow-twitch cardiac isoform of sarco-/endoplasmic reticulum Ca2P-ATPase (SERCA2a) and phospholamban proteins, and increased the level of the fast-twitch isoform of sarco-/endoplasmic reticulum Ca2+-ATPase (SERCAla).2. Changes in the levels of SERCA proteins, particularly SERCAla, could account for most of the increases in calcium uptake rate observed in homogenates of muscles from the salbutamol-treated animals and could partially account for the changes in half-relaxation rates and other twitch times.3. Changes in the levels of SERCAla, SERCA2a and phospholamban protein did not always follow changes in the levels of their corresponding mRNAs. Divergence depended upon the SERCA isoform and muscle. The muscles studied were latissimus dorsi and vastus intermedius. 4. Salbutamol did not change the level of myosin heavy chain (HC)-I isoforms in either muscle, suggesting that it did not increase the proportion of slow-twitch fibres in these muscles. It did increase the level of HC-IIx and decrease the level of HC-IIa isoforms in the latissimus dorsi. Salbutamol did not produce these effects in the vastus intermedius. It is of particular interest that salbutamol changed the relative levels of SERCA proteins in the latissimus dorsi muscle without producing significant changes in the level of HC-I isoform.

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Using an immunohistochemical double-labeling technique, we observed that different isoforms of sarcoplasmic reticulum Ca-ATPase are co-expressed in single fibers of canine fast-twitch skeletal muscles stimulated chronically at low frequency. By 7 days of neuromuscular stimulation, the population of hybrid fibers expressing both SERCA1 and SERCA2a [fast- and slow-twitch isoforms of sarco(endo)plasmic reticulum Ca(2+)-ATPase] had increased from 1.5% to 9.2% of fibers. By 14 days of stimulation 90% of the pure fast-twitch fibers (expressing only SERCA1) were replaced by hybrid fibers. An additional 28 days of stimulation caused all fast-twitch fibers to express SERCA2a at the same level as found in nonstimulated slow-twitch fibers (expressing only SERCA2a). At this time, one-half of the previously hybrid fibers had become pure-slow-twitch fibers. The remaining one-half of the hybrid fibers expressed SERCA1 at a very low level. Extending stimulation to 70 days did not further change the percentage of fibers that were slow-twitch or hybrid. Immunoblot studies at the whole-muscle level confirmed that changes in SERCA expression at 42 days of neuromuscular stimulation were complete. Immunohistochemical analysis of longitudinal sections of muscle showed that the changes in SERCA protein were uniform along the length of the muscle fiber, indicating that nuclei along its length responded equally to chronic stimulation.