The 31-amino acid proteolipid, sarcolipin (SLN), is associated with the fast-twitch skeletal muscle sarcoplasmic reticulum Ca 2؉ -ATPase (SERCA1). Constructs of human and rabbit SLN and of rabbit SLN with the FLAG epitope at its N terminus (NF-SLN) or its C terminus (SLN-FC) were coexpressed with SERCA1 in HEK-293 T-cells. Immunohistochemistry was used to demonstrate colocalization of NF-SLN and SERCA1 in the endoplasmic reticulum membrane and to demonstrate the cytosolic orientation of the N terminus of SLN. Coexpression of native rabbit SLN or NF-SLN with SERCA1 decreased the apparent affinity of SERCA1 for Ca 2؉ but stimulated maximal Ca 2؉ uptake rates (V max ). The N terminus of SLN is not well conserved among species, and the addition of an N-terminal FLAG epitope did not alter SLN function. Anti-FLAG antibody reversed both the inhibition of Ca 2؉ uptake by NF-SLN at low Ca 2؉ concentrations and the stimulatory effect of NF-SLN on V max . Addition of the FLAG epitope to the highly conserved C terminus decreased the apparent affinity of SERCA1 for Ca 2؉ relative to native SLN and decreased V max significantly. Mutations in the C-terminal domain showed that this sequence is critical for SLN function. Mutational analysis of the transmembrane helix, together with the additive regulatory effects of coexpression of both SLN and phospholamban (PLN) with SERCA1, provided evidence for different mechanisms of interaction of SLN and PLN with SERCA molecules. Ca 2؉ uptake rates in sarcoplasmic reticulum vesicles, isolated from rabbit fast-twitch muscle (tibialis anterior) subjected to chronic low frequency stimulation, were reduced by approximately 40% in 3-and 4-day stimulated muscle, with a marginal increase in apparent affinity of SERCA1 for Ca 2؉ . SERCA1 mRNA and protein levels were unaltered after stimulation. In contrast, SLN mRNA was decreased by 15%, and SLN protein was reduced by 40%. Reduced SLN expression could explain the decrease in SERCA1 activity observed in these muscles and might represent an early functional adaptation to chronic low frequency stimulation.

Fast-twitch tibialis anterior and extensor digitorum longus rabbit muscles were subjected to long-term intermittent (8 h daily) or continuous (24 h daily) indirect stimulation with a frequency pattern resembling that of a slow motoneuron. Increases in time to peak of isometric twitch contraction were observed without parallel changes in the pattern of myosin light chains or alterations in the distribution of slow and fast fibres as discernible by the histochemical ATPase reaction. However, changes in the fibre population and in the myosin light chain pattern were observed after intermittent stimulation periods exceeding 40 days or continuous stimulation periods longer than 20 days. Under these conditions even higher increases were found in contraction time. In one animal a complete change in fbire population was observed. In this case myosin light chains of the slow (LCS1, LCS2) and of the fast type (LCf1) were obviously synthetized simultaneously within the same fibre. Early changes in the enzyme activity pattern of energy metabolism indicated a conversion of the fibres including their mitochondrial population. These changes and the earlier reported changes in the sarcoplasmic reticulum are probably responsible for the early changes in contractile properties which occur before the transformation of the myosin.

The present study investigates the role of two major proteolytic systems in transforming rabbit and rat muscles. The fast-to-slow transformation of rabbit muscle by chronic low-frequency stimulation (CLFS) induces fast-to-slow transitions of intact, mature fibers and replacement of degenerating fibers by newly formed slow fibers. Ubiquitination, an indicator of the ATP-dependent proteasome system, and calpain activity were measured in homogenates of control and stimulated extensor digitorum longus muscles. Calpain activity increased similarly (approximately 2-fold) in stimulated rat and rabbit muscles. CLFS had no effect on protein ubiquitination in rat muscle but led to elevations in ubiquitin protein conjugates in rabbit muscle. Immunohistochemistry was used to study the distribution of micro-calpain and m-calpain and of ubiquitinated proteins in myosin heavy chain-based fiber types. The findings suggest that both proteolytic systems are involved in fiber transformation and replacement. Transforming mature fibers displayed increases in micro-calpain and accumulation of ubiquitin protein conjugates. The majority of these fibers were identified as type IIA. Enhanced ubiquitination was also observed in degenerating and necrotic fibers. Such fibers additionally displayed elevated m-calpain levels. Conversely, p94, the skeletal muscle-specific calpain, decayed rapidly after stimulation onset and was hardly detectable after 4 days of CLFS.

Specific antisera against glycogen phosphorylase, phosphofructokinase, aldolase, glyceraldehyde-phosphate dehydrogenase, enolase, lactate dehydrogenase, cytosolic and mitochondrial malate dehydrogenase from rabbit muscle were obtained from sheep. The gamma-globulins were used for indirect immunofluorescent localization of the respective enzymes in rabbit skeletal muscle and heart. In stretched skeletal muscle a cross-striation like distribution was observed for all enzymes studied. In the case of mitochondrial malate dehydrogenase this pattern is due to the staining of I-band mitochondria. In cross-sections, an intense staining of the sarcolemma and of subsarcolemmal mitochondria was observed. Comparative analyses with polarized light revealed that the cytosolic enzymes under study are distributed in the relaxed muscle fibre predominantly within the isotropic zones. The same distribution holds also for heart. In contracting muscle a decrease in cross-striated fluorescence and a faint staining of the interfibrillar spaces suggests a location also within the interfibrillar space.

After chronic, low-frequency stimulation, a rapid decline in Ca(2+) pump activity is observed during the early stages of skeletal muscle transformation. However, this variation in enzymatic activity does not coincide with a drastic reduction in the amount of sarcoplasmic reticulum Ca(2+)-ATPases. To investigate whether changes in subunit interactions within Ca(2+) pump complexes contribute to this phenomena, we performed a chemical cross-linking analysis of 4 days continuously, and 4 days discontinuously, electrostimulated fast muscle fibers. The abundance of the slow and fast Ca(2+)-ATPase isoforms sarco(endo)plasmic reticulum Ca(2+)- ATPase types 1 and 2 was affected during the fast-to-slow transition process, demonstrating that, even after short-term stimulation, distinct changes in the isoform expression pattern of muscle proteins occur. However, the oligomeric status of both ion pump species did not change. Hence, chemical modifications of critical enzyme domains must be responsible for the rapid stimulation-induced activity changes, not variations in protein-protein interactions within Ca(2+)-ATPase units. Oligomerization appears to be of central importance to the proper physiological functioning of the Ca(2+)-ATPase and does not undergo changes during skeletal muscle conditioning.