QUANTITATIVE STUDIES ON ENZYMES IN STRUCTURES IN STRIATED MUSCLES BY LABELED INHIBITOR METHODS : II. Confirmation of Radioautographic Measurement by Liquid-Scintillation Counting

Fragments of mouse diaphragm and sternomastoid muscles were incubated in diisopropyl-fluorophosphate (DFP)-³H in conditions known to saturate all the available DFP-sensitive reaction sites. After being extensively washed, the enzyme acetylcholinesterase (AChase) was specifically reactivated by treatment with pyridine-2-aldoxime methiodide (2-PAM). The radioactive DP-groups released into solution by 2-PAM were measured by liquid scintillation counting, and related to the known number of motor endplates present. Considerable difficulty was encountered in reducing the excess, adsorbed radioactivity to acceptable levels: long washing routines, extraction with organic solvents, and removing excess muscle fiber by microdissection were necessary. Six experiments gave a mean value of 2.4 x 10⁷molecules AChase per sternomastoid endplate, in reasonable agreement with the previously reported measurements by radioautography.     

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography

The distribution of acetylcholinesterase (ACHe) in the twitch fibers of the extraocular muscles of the mouse was examined by light and electron microscope autoradiography after labeling with radioactive diisopropyl fluorophosphate (DFP) with, and without, 2-pyridine aldoxime methiodide (2-PAM) reactivation. The values obtained were compared with those previously reported for the diaphragm and sternomastoid muscles. The extraocular muscles were studied because they differ from the other two muscles in that they are among the fastest of the mammalian muscles, yet their endplates have sparse junctional folds. They could thus provide information on the extent to which ACHe concentration is an invariant feature of endplate morphology and what, if any aspects may be related to their fast speed of response. We found, using light microscope autoradiography, that in the twitch fibers of the extraocular muscle, there is n average of 6.4 +/- 2.1 X 10(7) DFP- binding sites per endplate, of which 29% (1.8 X 10(7)) are reactivated by 2-PAM and are thus AChe. The morphology of the extraocular endplates allowed us to conclude, on statistical grounds, that the AChe site are probably localized not only along the surface area of the postjunctional membrane (PJM) but also along the surface of the presynaptic axonal membrane. Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites/micron 2 of surface area of pre-and postjunctional membrane. This stacking density of DFP- binding sites per surface area of membrane ( probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.     

ELECTRON MICROSCOPE RADIOAUTOGRAPHY AS A QUANTITATIVE TOOL IN ENZYME CYTOCHEMISTRY : II. The Distribution of DFP-Reactive Sites at Motor Endplates of A Vertebrate Twitch Muscle

The distribution of diisopropylfluorophosphate (DFP)-sensitive enzyme sites at the neuromuscular junction was determined quantitatively by electron microscope radioautography after incubation of muscle fragments in DFP-³H. Most of the sensitive sites were located in the subneural apparatus at a concentration of 90,000 sites per µ³ of cleft tissue or 12,000 sites per µ² of postjunctional membrane surface area. A considerable concentration is also present in the teloglial cap. It has previously been demonstrated (Rogers et al., 1966) that one-third of the DFP-sensitive sites at the endplate can be reactivated by pyridine-2-aldoxime methiodide (2-PAM)—a compound which selectively reactivates phosphorylated acetylcholinesterase. In the present study, it was found that this ratio of 1:2 holds also on a fine-structural level. Muscle mast cells were found to have a heavy concentration of bound DFP.     

Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor end plates of a vertebrate twitch muscle

Tritiated diisopropylfluorophosphate (DFP) was used to phosphorylate acetylcholinesterase (AChase) in the motor end plate of mouse sternomastoid muscle, and its distribution within the end plate was evaluated quantitatively by electron microscope radioautography. With the use of emulsion layers whose sensitivity to tritium had been calibrated, the density of AChase in different components of the end plate was calculated. The AChase was primarily localized (85%) in the junctional fold region. The concentration of AChase there was more than 20,000 active sites per cubic micron of tissue. The resolution of the technique was not sufficient to determine whether there was some AChase in the nerve end bulb; however, if there is any there, the concentration must be less than 10% of that at the junctional fold region.     

Obidoxime antagonizes the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate via different mechanisms

The efficacies and mechanisms of obidoxime in antagonizing the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate (DFP) were studied in mouse phrenic nerve/diaphragm preparations. Obidoxime antagonized neostigmine-induced tetanic fade (EC₅₀: 300 µM) by inhibiting the regenerative and sustained depolarization during repetitive stimulation. The antagonism was associated with a depression and shortening of single endplate potentials (EPPs) and miniature EPPs (MEPPs). In contrast, the neuromuscular failure induced irreversibly after treatment with DFP and followed by washout was restored by obidoxime at concentrations (EC₅₀: 0.6 µM) 500-fold lower than that against neostigmine. The regenerative depolarization was abolished with no depression of single EPPs and MEPPs, and the antagonistic action persisted after washout of obidoxime. The EC₅₀ of obidoxime was proportionately increased in the presence of increasing concentrations of DFP. Nevertheless, the EC₅₀ against DFP, at a concentration (30 µM) 15-fold in excess of that which caused tetanic fade, was still 10-fold lower than that which antagonized neostigmine. In both cases, the amplitudes of train EPPs were increased. It is concluded that obidoxime antagonizes neostigmine-induced neuromuscular failure by a curare-like action but antagonizes DFP by an enzyme reactivation.     

The ultrastructural localization and quantitation of cholinergic receptors at the mouse motor endplate

Summary The snake venom toxin, -bungarotoxin, is known to bind specifically to the acetylcholine receptor at skeletal muscle endplates. In this study, tritiated -bungarotoxin has been used in conjunction with electron-microscope autoradiography to visualize and enumerate acetylcholine receptor sites at the neuromuscular junctions of the mouse diaphragm. From an analysis of the grain distribution, the receptor sites appear to be located specifically on the postjunctional membrane. The density there is about 8,500/² of membrane surface. For comparison purposes, cholinesterases and related active centers were labeled using [³H] diisopropylfluorophosphate; they were shown to be at this same concentration over the synaptic membranes (or along the cleft). The 11 relationship of the receptors to the cholinesterase type of site, found previously to hold in studies on whole endplates, is also true at the ultrastructural level in this case. In fact, this 11 relationship is believed to be a characteristic of the postsynaptic membranes of endplates in other muscles and other vertebrates.Based on the constant density value thus arrived at, the total surface areas of postsynaptic and of presynaptic membranes are at once obtained from the known total numbers of these sites per endplate, available from previous studies in this laboratory. Examples of such synaptic surface area values are given. These values are only reliable for a given muscle type if the approximate fiber size is defined.     

Sequence of Age-Associated Changes to the Mouse Neuromuscular Junction and the Protective Effects of Voluntary Exercise

Loss of connections between motor neurons and skeletal muscle fibers contribute to motor impairment in old age, but the sequence of age-associated changes that precede loss of the neuromuscular synapse remains uncertain. Here we determine changes in the size of neuromuscular synapses within the tibialis anterior muscle across the life span of C57BL/6J mice. Immunofluorescence, confocal microscopy and morphometry were used to measure the area occupied by nerve terminal synaptophysin staining and postsynaptic acetylcholine receptors at motor endplates of 2, 14, 19, 22, 25 and 28month old mice. The key findings were: 1) At middle age (14-months) endplate acetylcholine receptors occupied 238±11 µm² and nerve terminal synaptophysin 168±14 µm² (mean ± SEM). 2) Between 14-months and 19-months (onset of old age) the area occupied by postsynaptic acetylcholine receptors declined 30%. At many endplates the large acetylcholine receptor plaque became fragmented into multiple smaller acetylcholine receptor clusters. 3) Between 19- and 25-months, the fraction of endplate acetylcholine receptors covered by synaptophysin fell 21%. By 28-months, half of the endplates imaged retained ≤50 µm² area of synaptophysin staining. 4) Within aged muscles, the degree to which an endplate remained covered by synaptophysin did not depend upon the total area of acetylcholine receptors, nor upon the number of discrete receptor clusters. 5) Voluntary wheel-running exercise, beginning late in middle-age, prevented much of the age-associated loss of nerve terminal synaptophysin. In summary, a decline in the area of endplate acetylcholine receptor clusters at the onset of old age was followed by loss of nerve terminal synaptophysin from the endplate. Voluntary running exercise, begun late in middle age, substantially inhibited the loss of nerve terminal from aging motor endplates.     

The number of acetylcholinesterase molecules in the rat megakaryocyte.

A megakaryocyte cell series from rat bone marrow has been examined by the isotopic di-isopropyl fluorophosphate (DFP) method for esterases. After complete reaction with 32P-DFP, the numbers of DFP-reacted molecules inindividual cells havebeen determined by beta trackauto-radiography. Previous work has shown the percentage of organophosphate-sensitive sites in these cells which can be taken as active centers of acetylcholinesterase (AChase). Combining these data, the absolute numbers of organophosphate-sensitive esterase molecules and AChase molecules per cell were determined. Histograms show a narrow spread of values within each of four size classes from megakaryoblast to fully mature megakaryocyte, but, with means increasing 4-fold through this series, approximately in proportion to cell volume. A rat megakaryoblast has 2 X 10(6) AChase molecules, and a megakaryocyte (of 48-micro diameter) has 7.6 X 10(6) molecules. The apparent turnover number of the enzyme for intracellular reaction with substrate is calculated and compared with turnover numbers available for other AChases.     

CHOLINESTERASE ACTIVITY OF THE MOTOR ENDPLATE IN ISOLATED MUSCLE MEMBRANE

Abstract— The cholinesterase activity of motor endplates in tibialis anterior muscle of rats accounted for about 20 per cent of the total cholinesterase activity of the muscle. In the isolated muscle membrane preparation of rat intercostal muscle, the cholinesterase activity was localized solely in the motor endplate, as shown by cholinesterase staining. The cholinesterase activity of the membrane per unit of nitrogen was 26·9 times that of the muscle homogenate. The membrane (endplate) cholinesterase had an optimal pH of 8, K_m value of 3·1 m m , and was stable at 4° for at least 13 days. Cholinesterase of a motor endplate hydrolysed 2·69 × 10⁸ acetylcholine molecules in 1 msec. Since it is estimated that 10⁸ cholinesterase active sites are present in a motor endplate, the turnover time (time necessary for one enzyme site to hydrolyse one acetylcholine molecule) is calculated to be 372 μ sec, and the turnover number (molecules of acetylcholine hydrolysed by one enzyme site/min) to be 1·61 × 10⁵. From studies with cholinesterase inhibitors, cholinesterase activity was estimated to be due mostly to acetylcholinesterase, and only a minor part to pseudocholinesterase. The muscle membrane preparation seems to be useful for the study of other properties of the motor endplate.     

Pectoralis muscle morphology in the little brown bat,Myotis lucifugus: A non-convergence with birds

Recent studies of muscle architecture demonstrate that many mammalian muscles are composed of short, interdigitating fibers. In addition, the avian pectoralis, a muscle capable of producing high frequency oscillations has been shown to possess a serially arranged pattern of muscle endplate in all sizes of birds studied. The pectoralis muscle of the little brown bat, Myotis lucifugus (Chiroptera: Vespertilionidae), is composed of fairly uniform fibers that span the length of the muscle and is characterized by a zone of motor endplates within the middle third of the muscle. The homogeneous fiber architecture of the bat pectoralis muscle is in contrast to the serial arrangement of endplates (and presumably muscle muscle fibers) in the avian pectoralis in species equivalent in size to Myotis. The short fiber organization and motor endplate pattern observed in most birds is thus not a requisite design for flying vertebrates. © 1994 Wiley-Liss, Inc.     

Experimental autoimmune myasthenia gravis: can pretreatment with 125I-labeled receptor prevent functional damage at the neuromuscular junction?

Rats were immunized with purified receptor from electric fish to induce experimental autoimmune myasthenia gravis (EAMG). It is implied by the clonal selection theory that antigens react only with receptors on specific immunocompetent cell subpopulations. In an attempt to damage these specific cells with the aid of highly radioactive antigen, one group of rats was pretreated with an additional injection of radiolabeled receptor of high specific activity 3 days before the basic immunization. The success of the immunization was monitored by measuring changes in the following three parameters: antibody titers against nicotinic acetylcholine receptor; number of alpha-bungarotoxin-binding sites at endplates; and number of acetylcholine-operated ionic endplate channels, using quantitative electrophysiologic methods. Conventionally immunized animals showed the classical signs of EAMG: elevated antibody titers against nicotinic acetylcholine receptor and a reduction of the number of alpha-bungarotoxin-binding sites, as well as reduction of the number of acetylcholine-operated ionic channels. The same symptoms were found in animals pretreated with unlabeled receptor and in animals pretreated with radioactive albumin. Animals pretreated with radioactively labeled receptor showed far less reduction of functional nicotinic acetylcholine receptor and only slightly raised antibody titers. This study suggests that preimmunization with radioactive antigen selectively eliminates immunocompetent cells, thus precluding the production of antibodies by a subsequent immunization procedure. The same protective effect cannot be obtained by either preimmunization with unlabeled antigen or by radioactively labeled unspecific antigen.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Isolation of a tripeptide (Ala-Gly-Ser) exhibiting weak acetylthiocholine hydrolyzing activity from a high-salt soluble form of monkey diaphragm acetylcholinesterase

A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammoniumchloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [³H]Diisopropyl fluorophosphate ([³H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight 300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [³H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme.A synthetic peptide of sequence Ala-Gly-Ser was also found to exhibit acetylthiocholine hydrolyzing activity. The kinetics and inhibition characteristics of the synthetic peptide was similar to those of the peptide derived from the purified enzyme, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 29480 times less than that of the purified AChE.Abbreviations AChE Acetylcholinesterase - BW284C51 1,5-bis(4-allyl dimethylammonium phenyl) pentan 3-one-dibromide - DFP Diisobropyl fluorophosphate - TIPP Tetra isopropyl pyrophosphoramide - TPCK N-Tosyl-L-phenylalanylchloromethyl ketone - MAP m-Aminophenyl trimethylammonium chloride - RCA₁ Ricinus communis agglutinin 120 - TEAB Tetraethylammonium bromide - DTT Dithiothreitol     

Inseminatory behavior of Philophthalmus megalurus and Philophthalmus hegeneri in concurrent infections of chicks

Sperm labeled with ³H-thymidine and³H-tyrosine detected by autoradiography were used to determine inseminatory patterns in concurrent infections of Philophthalmus megalurus and Philophthalmus hegeneri. In infections of one labeled P. megalurus and 1 to 3 unlabeled P. hegeneri self-insemination but not cross-insemination was detected. When P. hegeneri was labeled and transplanted with 1 to 3 unlabeled P. megalurus no insemination was found. Transplantation of one labeled P. megalurus with various numbers of unlabeled adults of both species resulted in exclusive cross-insemination ofP. megalurus. When one labeled P. hegeneri was in the presence of unlabeled adults of both species, only cross-insemination of P. hegeneri was detected. Thus no insemination between species was found precluding hybrid production. This may be due to a separation of feeding sites within the microenvironment of the conjunctival sac of the chick's eye. Both species carried on normal inseminatory behavior in the presence of the other species. Recovery rates of both species from concurrent infections were not greatly different than those from single species infections. However, the recovery rate forP. megalurus (61·2 %) was much greater than for P. hegeneri (44·7 %).     

Hereditary motor endplate disease (med) of the mouse: Observations on dissociated myogenic cells and their development in culture

Summary Myogenic cells from mice homozygous for the lethal mutation motor endplate disease (med/med) were grown in culture. Like muscle cells taken from wild type (+/?) litter mates they fused to form myotubes which contracted, developed cross striations, and exposed acetylcholine receptors (AChR) on their surface. However, a decrease of 30% in the number of mononucleated cells per unit fresh weight of muscle was observed as early as 2–3 days postnatal, i.e., at least one week prior to the onset of physiological symptoms. Hence, in addition to influencing the functional maintenance of motor endplates, the med gene seems to control early events in muscle development.     

Activity and Distribution of Tissue Transglutaminase in Association with Nerve-Muscle Synapses

Abstract: We have measured, characterized, and localized calcium-dependent protein cross-linking activity in rat skeletal muscle, and in myotubes cultured independently or In coculture with spinal neurones, catalyzed by the enzyme tissue transglutaminase (tTG). The enzyme activity was present in both motor endplate and endplate-free zones of rat diaphragm muscle. tTG in the endplate zone was more tightly associated with the tissue. This form of association was absent in extracts of peripheral nerve. Cross-linking of endogenous proteins, as measured by the content of ɛ-(γ-glutamyl)lysine isppeptide, was higher in the endplate than in the nonendplate zone. Cytosolic (C) and paniculate (B) forms of tTG were separated by ion-exchange chromatography from both regions of the muscle. In the motor endplate zone, a higher proportion of tightly bound tTG was recovered as a separate (B₁) particulate form. K _m values for calcium activation of the three forms of tTG were in the range of 5–15 μM. Immunocytochemistry with polyclonal and monoclonal antibodies revealed the enzyme at motor endplates and at contacts between neurites of rat embryo spinal neurones and myotubes in primary cocultures. Appearance of the B₁ transglutaminase could be induced by coculturing myotubes of the mouse C2C12 cell line with neurones. The results suggest that tTG is most concentrated and active at the motor endplate.     

Density and apparent location of the sodium pump in frog sartorius muscle

Summary The binding of the cardiosteroid³H-ouabain to frog skeletal muscle was determined by studying the kinetics of its uptake and release.The amount of ouabain bound as a function of drug concentration in the external medium follows a hyperbolic relationship with a maximum binding (B _max) of the order of 2500 molecules per square micrometer of surface membrane and an affinity constant (K) of 2.2×10^–7 m. The data do not suggest a drug-receptor (Na pump site) relation other than one-to-one.Ouabain molecules are released from whole muscle into ouabain-free media very slowly. The release is a single exponential function of time (25 hr). When re-binding is prevented by the presence of unlabeled ouabain in the external medium, the loss of labeled ouabain is increased (15 hr). Increasing [K⁺]₀ from 2.5 to 10mm slows the time course of binding without any significant change in binding capacity of the muscle fibers.Experiments on detubulated muscles indicate that the density of pump sites is considerably higher in the surface than in the T-tubular membrane. These findings agree with the report by Narahara et al. [Narahara, H.T., Vogrin, V.G., Green, J.D., Kent, R.A., Gould, M.K. (1979)Biochim. Biophys. Acta 552:247] on the distribution of (Na⁺+K⁺)-ATPase among different cell membrane fractions from frog skeletal muscle.     

The clustering of acetylcholine receptors and formation of neuromuscular junctions in regenerating mammalian muscle grafts

The present investigation was undertaken to study the relationship between acetylcholine receptor (AchR) clustering and endplate formation within regenerating skeletal muscle grafts. Silver staining of nerves was combined with rhodamine-alpha-bungarotoxin labeling of AchR clusters in heterotopic grafts of the rat soleus muscle. Two major graft procedures were used: whole muscle grafts and grafts which lacked the zone of original motor endplates (MEP-less grafts). These categories were subdivided into standard grafts, where subsequent innervation was allowed, and noninnervated grafts, which were experimentally deprived of innervation. Grafting brought about the death and removal of muscle fibers, followed by regeneration of myotubes within surviving basal lamina sheaths. A transient population of small extra-junctional AchR clusters spontaneously appears shortly after myotube formation in all four muscle graft types. Early myotubes of whole muscle grafts (both innervated and standard grafts, prior to the time of innervation) also develop presumptive secondary synaptic clefts and large, organized aggregations of AchRs at original synaptic sites. At later times, nerves regenerating into standard whole muscle and MEP-less grafts lead to the formation of numerous ectopic endplates. In whole muscle grafts, endplates may also form at original synaptic sites. Functional graft innervation is achieved in whole muscle and MEP-less grafts as early as 20 days postgrafting. The results of this study support the existence of still-unknown factors associated with the original synaptic site which can direct postsynaptic differentiation independent of innervation. They also demonstrate that functional endplates may form in mammalian muscle grafts at both original synaptic sites and ectopic locations, thus indicating that the zone of original synaptic sites is not necessary for the establishment of numerous functional and morphologically well-differentiated endplates.     

INCREASE IN THE APPARENT AChE ACTIVITY IN THE MOUSE DIAPHRAGM INDUCED BY PROTEOLYTIC TREATMENT

Abstract— Isolated endplate regions from the mouse diaphragm were treated with different agents before or after homogenization in order to solubilize junctional AChE and study the effect of solubilization on its apparent activity. Total AChE activity (solubilized + nonsolubilized) of samples treated with collagenase or papain before homogenization was nearly twice as high as in control samples. If collagenase was added after homogenization no increase in apparent activity was observed although in both cases about 70–80% of AChE activity was solubilized. The access of ACh to the membrane-bound enzyme is probably not a limiting factor in the AChE assay as is the case in the electric organ homogenates. Both 1 m -NaCl and Triton X-100 were quite ineffective as solubilizers when applied before homogenization and had an insignificant effect on the apparent AChE activity.
The increase in apparent AChE activity cannot be explained either by a de novo synthesis or by the change in kinetic properties of different species of AChE, or by the release of AChE possibly sequestrated in the membrane vesicles. The possibility is discussed that a part of junctional AChE is inactivated at the beginning of homogenization while it can be preserved by previous solubilization, or that proteolytic treatment may activate some 'silent' AChE sites in motor endplates.
However, the mere fact that the difference does exist suggests that all AChE activity present in intact motor endplates may not be measurable after homogenization.     

Diaphragm Muscle Remodeling in a Rat Model of Chronic Intermittent Hypoxia

Respiratory muscle remodeling occurs in human sleep apnea—a common respiratory disorder characterized by chronic intermittent hypoxia (CIH) due to recurrent apnea during sleep. We sought to determine if CIH causes remodeling in rat sternohyoid (upper airway dilator) and diaphragm muscles. Adult male Wistar rats were exposed to CIH (n=8), consisting of 90 sec of hypoxia (5% at the nadir; SaO₂ ~80%)/90 sec of normoxia, 8 hr per day, for 7 consecutive days. Sham animals (n=8) were exposed to alternating air/air cycles in parallel. The effect of CIH on myosin heavy-chain (MHC) isoform (1, 2a, 2x, 2b) distribution, sarcoplasmic reticulum calcium ATPase (SERCA) isoform distribution, succinate dehydrogenase activity, glycerol phosphate dehydrogenase activity, and Na⁺/K⁺ ATPase pump content was determined. Sternohyoid muscle structure was unaffected by CIH treatment. CIH did not alter oxidative/glycolytic capacity or the Na⁺/K⁺-ATPase pump content of the diaphragm. CIH significantly increased the areal density of MHC 2b fibers in the rat diaphragm, and this was associated with a shift in SERCA proteins from SERCA2 to SERCA1. We conclude that CIH causes a slow-to-fast fiber transition in the rat diaphragm after just 7 days of treatment. Respiratory muscle functional remodeling may drive aberrant functional plasticity such as decreased muscle endurance, which is a feature of human sleep apnea.