Deficiency of -sarcoglycan differently affects fast- and slow-twitch skeletal muscles

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1 Am J Physiol Regul Integr Comp Physiol 289: R1328 R1337, First published July 7, 2005; doi: /ajpregu Deficiency of -sarcoglycan differently affects fast- and slow-twitch skeletal muscles Daniela Danieli-Betto, 1 Alessandra Esposito, 1 Elena Germinario, 1 Dorianna Sandonà, 2 Tiziana Martinello, 2 Anna Jakubiec-Puka, 3 Donatella Biral, 4 and Romeo Betto 4 Departments of 1 Human Anatomy and Physiology and 2 Biomedical Sciences, University of Padova, Padova; 3 Department of Cell Biochemistry, Nencki Institute of Experimental Biology, Warszawa, Poland; and 4 Neuromuscular Biology and Physiopathology Unit, Consiglio Nazionale delle Ricerca Institute of Neuroscience, Padova, Italy Submitted 30 September 2004; accepted in final form 29 June 2005 Danieli-Betto, Daniela, Alessandra Esposito, Elena Germinario, Dorianna Sandonà, Tiziana Martinello, Anna Jakubiec- Puka, Donatella Biral, and Romeo Betto. Deficiency of -sarcoglycan differently affects fast- and slow-twitch skeletal muscles. Am J Physiol Regul Integr Comp Physiol 289: R1328 R1337, First published July 7, 2005; doi: /ajpregu Sarcoglycan (Sgca) is a transmembrane glycoprotein of the dystrophin complex located at skeletal and cardiac muscle sarcolemma. Defects in the -sarcoglycan gene (Sgca) cause the severe human-type 2D limb girdle muscular dystrophy. Because Sgca-null mice develop progressive muscular dystrophy similar to human disorder they are a valuable animal model for investigating the physiopathology of the disorder. In this study, biochemical and functional properties of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of the Sgca-null mice were analyzed. EDL muscle of Sgcanull mice showed twitch and tetanic kinetics comparable with those of wild-type controls. In contrast, soleus muscle showed reduction of twitch half-relaxation time, prolongation of tetanic half-relaxation time, and increase of maximal rate of rise of tetanus. EDL muscle of Sgca-null mice demonstrated a marked reduction of specific twitch and tetanic tensions and a higher resistance to fatigue compared with controls, changes that were not evident in dystrophic soleus. Contrary to EDL fibers, soleus muscle fibers of Sgca-null mice distinctively showed right shift of the pca-tension (pca is the negative log of Ca 2 concentration) relationships and reduced sensitivity to caffeine of sarcoplasmic reticulum. Both EDL and soleus muscles showed striking changes in myosin heavy-chain (MHC) isoform composition, whereas EDL showed a larger number of hybrid fibers than soleus. In contrast to the EDL, soleus muscle of Sgca-null mice contained a higher number of regenerating fibers and thus higher levels of embryonic MHC. In conclusion, this study revealed profound distinctive biochemical and physiological modifications in fast- and slow-twitch muscles resulting from -sarcoglycan deficiency. limb girdle muscular dystrophy 2D; skeletal muscle fiber types; skeletal muscle contractile properties; caffeine sensitivity of sarcoplasmic reticulum; calcium sensitivity of myofibrillar proteins THE SARCOGLYCAN COMPLEX IS a group of four single-pass transmembrane proteins ( -, -, -, and -sarcoglycan) associated with the major dystrophin-based membrane cytoskeletal complex (4, 23, 29). Although the exact function of the sarcoglycan complex is not well defined, it is well established that mutations in any of the sarcoglycan genes result in distinct forms of limb-girdle muscular dystrophy, collectively named sarcoglycanopathies (5, 29, 36, 37, 45). A clear sign that the four Address for reprint requests and other correspondence: R. Betto, Muscle Biology and Physiopathology Unit, Consiglio Nazionale delle Ricerca Neuroscience Institute. Viale G. Colombo 3, Padova, Italy ( romeo.betto@bio.unipd.it). sarcoglycans act as a complex is the fact that mutations in any one of the sarcoglycan genes cause the loss of or defects in cognate proteins and typically produces deficiencies of other sarcoglycan proteins (23, 50). Besides the sarcoglycans, the multimeric complex formed by dystrophin includes also dystroglycan ( and ), syntrophins, dystrobrevin, and sarcospan (4). Dystrophin and dystrophin-associated proteins (DAP) provide muscle cell membrane for a mechanical connection between myofibrils and the extracellular matrix. Particularly, dystrophin links in the intracellular side actin cytoskeleton with the transmembrane anchor point of -dystroglycan, whereas, on the extracellular side, -dystroglycan connects to laminin of the extracellular matrix (4, 32). The DAP complex is regularly distributed all over muscle cell membrane and forms a supportive cytoskeletal structure devoted to protect muscle membrane from lateral solicitation due to contractile activity (38, 41). As a consequence, the lack of dystrophin as it occurs in Duchenne muscular dystrophy (DMD), in causing full dismantling of the entire structure makes the muscle fiber more prone to membrane damage (4, 50). The sarcoglycans, with sarcospan, form a distinct subcomplex that stabilize the association of dystroglycans to dystrophin and thus reinforce the overall structure (9). Therefore, deficiencies in the sarcoglycans also determine destabilization of dystrophin complex leading to membrane instability and higher sensibility to membrane injury (14, 24, 25, 51). It is worth reporting that additional sarcoglycans functions have been described that could help revealing additional lossof-function mechanisms in the pathogenesis of the dystrophy (23). It has been shown that sarcoglycans, within the dystrophin complex, represent an additional anchor point for neuronal nitric oxide (NO) synthase (nnos) via -syntrophin and -dystrobrevin (62). Consistently, the loss of sarcoglycans determines the loss of nnos both in sarcoglycan-null mice and in patients of sarcoglycanopathies (8). Moreover, through and -sarcoglycan, the sarcoglycan complex associates filamin-2, a protein normally involved in transduction signaling pathways (55). Also, because all sarcoglycan can be phosphorylated (61) and -, - and -sarcoglycan possess an extracellular epidermal growth factor-like domain, the complex has been proposed as an independent signaling module (23). Finally, we have demonstrated that -sarcoglycan is an ecto- ATPase, indicating that -sarcoglycan might control the ATP The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. R /05 $8.00 Copyright 2005 the American Physiological Society

2 level and the signaling action of the nucleotide at the surface of muscle cells (3, 49). Thus the combined loss of mechanical and signaling functions associated with sarcoglycan deficiency might contribute to the pathological phenotype of dystrophic muscle. Identification of animal models of muscular dystrophy has been helpful in research of the pathogenesis, physiopathology, and treatment of these disorders (15, 57). The most widely used model of muscle dystrophy is the mdx mouse, the dystrophinnull animal model for DMD. In general, it is reported (17, 31, 39, 41, 44) that the absence of dystrophin does not alter twitch kinetics of muscles from mdx mice, whereas specific twitch and tetanic forces are significantly reduced. Because it has been demonstrated that fast-twitch fibers are preferentially affected in DMD patients (58), studies were undertaken to reveal any possible difference in the contractile characteristics of fast- and slow-twitch muscles. Although not all studies of hind-limb muscles from mdx mice were performed at the same age, it emerged that specific tensions of dystrophic extensor digitorum longus (EDL) and soleus are equally reduced compared with wild-type controls (1, 17, 31, 39). On the other hand, studies at the single-fiber level revealed that EDL and soleus muscles of mdx mice are affected differently by the absence of dystrophin (13, 29). The different dysfunction of the mdx dystrophic muscles has been correlated with changes of myosin heavy-chain (MHC) composition and enzymatic kinetic of myofibrillar proteins Ca 2 -sensitivity as well as of excitation-contraction coupling (1, 12, 13, 28, 35, 41, 43). Murine models of the diverse sarcoglycanopathies have also been generated and, with the naturally occurring -sarcoglycan-null Bio 14.6 hamster, have been used to investigate the functional consequence of sarcoglycan-deficiency. All sarcoglycan-null animals show progressive muscular dystrophy of variable severity (2, 14, 16, 24, 30). Functionally, fast-twitch muscles from Bio 14.6 dystrophic hamster showed either no changes (34) or the significant reduction (53, 60) of absolute and specific twitch and tetanic forces compared with wild-type control. Soleus muscle of dystrophic hamster equally showed the dramatic decrease of specific tensions (7). In contrast to the Bio 14.6 dystrophic hamster, mice lacking -sarcoglycan showed no changes in the absolute tetanic force produced by EDL (23). Mice lacking -sarcoglycan develop progressive muscular dystrophy similar to human muscular dystrophy (24). However, fast EDL muscle from -sarcoglycan-null and wild-type mice showed no significant differences in the absolute and specific twitch and tetanic tensions (P 0 ) and, surprisingly, in the resistance to mechanical strain (22). Unfortunately, it is not possible to compare force production of fast- and slow-twitch muscles from Sgcd- and Sgcg-null mice, because, so far, contractile properties of slow muscles have not been analyzed. From the physiological point of view, Sgca-null mice show the significant reduction of specific force of hind-limb and diaphragm muscles, compared with wild-type controls (14, 40, 48). However, it is evident that fast EDL muscle reacts differently than slow-twitch soleus muscle to the absence of -sarcoglycan, particularly in the ability to generate absolute force (14, 48). An additional difference between fast and slow muscles is that EDL muscle of Sgca-null mice has a very high resistance to passive stretch compared with control muscle, whereas soleus is not different from control (14). In contrast, both fast and slow muscles of Sgca-null mice are susceptible to contraction-induced injury after exercise, a sensitivity evident also in fibers from Sgcb- and Sgcd-null mice but not in fibers from -sarcoglycan-deficient muscles (22). In summary, functional studies have revealed that consequences of primary gene defects are more complex and variable than predicted (57). Defects in individual sarcoglycans seem to cause either similar or opposing responses to fast- and slow-twitch muscles. In particular, Sgca-null muscles show a dramatic difference in the adaptive response of fast- and slow-twitch muscles, an evidence that evoked the need for additional investigation (14). In the present study, contractile properties and fatigue characteristics of fast-twitch EDL and slow-twitch soleus muscles from Sgca-null mice were investigated. These data were complemented with analyses at the single-fiber level of Ca 2 -sensitivity of myofibrillar proteins, of sarcoplasmic reticulum caffeine sensitivity, and of MHC isoforms expression. Results make evident distinctive functional and contractile differences between EDL and soleus muscles from Sgca-null mice. MATERIALS AND METHODS R1329 Animals. This study was approved by the Ethics Committee of the Medical Faculty of the University of Padova. The protocol utilized in the study has been authorized by the Public Veterinary Health Department of the Italian Ministry of Health. The experiments were carried out on the Sgca-null mice previously characterized (30) by using the congenic C57BL/6J strain (Charles River) as control. All experiments were performed on male mice of the same age (12 13 wk). The animals were first weighed and then killed by cervical dislocation. Soleus and EDL muscles were excised, weighed, and used for the experimental analyses. Histological and immunofluorescence analysis. Muscles were frozen in liquid nitrogen in a slightly stretched position. Serial cross sections (8- m thick) were cut in a cryostat microtome set at 24 2 C (Slee Pearson). For the histochemical analysis, hematoxylin and eosin staining was performed on muscle sections to examine the general morphology and to determine the cross-sectional area (CSA) of individual fibers. Muscle cryostat sections were also stained for the SDH activity. Digital photographs were taken of each muscle section and analyzed by the ImageJ NIH imaging software. MHC expression in muscle fibers was detected by immunofluorescence by using specific antibodies. Serial sections from control and Sgca-null soleus and EDL muscles were probed with the following antibodies: BA-D5 (1:400) for type 1 MHC, A4 74 (1:50) for type 2A, BF-F3 (1:400) for type 2B, RT-D9 (1:400) for type 2B/2X, and F1.652 (1:5) for the embryonic isoform. A4 74 and F1.652 were from Developmental Studies Hybridoma Bank, (University of Iowa, Iowa City, Iowa). BA-D5, BF-F3, and RT-D9 antibodies were from S. Schiaffino (University of Padova, Padova, Italy). All primary antibodies were diluted in PBS and incubated at room temperature for 45 min. Tetramethylrhodamine isothiocyanate-conjugated rabbit antimouse immunoglobulins (Dako, Denmark, 1:100), incubated at room temperature for 30 min, were used as secondary antibodies. Whole muscle mechanical properties. Contractile properties were investigated in vitro at 30 1 C. Muscle bathing, stimulation conditions, and tension recordings were performed as previously described (33). Twitches were obtained by applying single supramaximal stimuli (0.5 ms duration), whereas tetani were obtained by applying trains of stimuli at frequency of 100 and 140 Hz for soleus and EDL, respectively. The following parameters were measured: contraction time, half-relaxation time of the twitch, and twitch (P t) and P 0. The specific force (i.e., force per CSA) produced by each muscle was calculated from the absolute force, muscle weight, and

3 R1330 muscle length, assuming a density of g/ml (25). The maximum rate of rise of tetanus was also measured (expressed both as N/ms and N ms 1 mm 2 ). Muscle responses were recorded via an AT-MIO 16 AD card and data were analyzed by the LabView computer program (National Instruments). Force-frequency curves were determined by stimulating soleus muscle at 20, 40, 60, 80, and 100 Hz and EDL muscle at 30, 60, 100, 140, and 160 Hz. Fatigue was produced, as previously reported (11, 19), by submitting muscles to a 10-min protocol consisting of a train of short tetani at low frequency (40 Hz for 300 ms, 0.5/s, in soleus and 60 Hz for 300 ms, 0.3/s in EDL). Force recorded during the fatigue protocol was normalized to the initial tension. At the end of each experiment, the muscles were weighed. Preparation of single fibers. Muscle fibers were chemically skinned as previously described (10, 20, 33). Briefly, muscles were tied to a wooden stick and quickly immersed into an ice-cold skinning solution containing (in mm): 170 K-propionate, 2.5 Mg-propionate, 2.5 Na 2K 2ATP, 5 K 2EGTA, and 10 imidazole buffer, ph 7.0. Skinned fibers were stored at 20 C for no more than 4 wk in a skinning solution supplemented with 50% (vol/vol) glycerol. Single fiber functional studies-ca 2 sensitivity of tension development. pca-tension curves (pca is the negative log of Ca 2 concentration) were obtained by exposing the fiber sequentially to solutions with different free Ca 2 concentration (10, 20). Single-fiber segments ( 1.5-mm length) were inserted between two clamps, one fixed and the other connected to a tension transducer (model 801; AK Sensonor, Horten, Norway). Fibers were immersed in a relaxing solution at room temperature (22 24 C) with the following composition (in mm): 170 K-propionate, 2.5 Mg-propionate, 5 Na 2K 2ATP, 5 K 2EGTA, and 10 imidazole buffer, ph 7.0. Fibers were stretched manually to a sarcomere length of 3 m as measured by light diffraction with a helium neon laser lamp (33). The tension generated in each pca solution was continuously recorded, and the baseline tension was established as the steady-state voltage output recorded with the fiber in the relaxing solution. Specific tension for each single fiber was calculated by normalizing the maximum tension measured at pca 5 to the fiber CSA, as calculated by three different diameter determinations along the fiber length considering the fiber immersed in solution as a cylinder (11). Functional studies of single fiber-sarcoplasmic reticulum caffeine response. The threshold for sarcoplasmic reticulum Ca 2 -release in response to caffeine was determined as previously described (11, 20, 33). Fibers were first incubated for 30 s in a Ca 2 -loading solution (pca 7.0 solution) and then in a relaxing solution deprived of EGTA. The fibers were stepwise challenged with increasing concentrations of caffeine until tension was recorded (11, 20, 33). To completely deplete sarcoplasmic reticulum cisterns of Ca 2, 20 mm caffeine was used. The tensions developed at caffeine threshold (P th) and by 20 mm caffeine (P 20), were measured. Caffeine threshold was defined as the lowest concentration (usually at least 3 5% P 20) of caffeine that was able to induce an appreciable tension (11, 47). SDS-PAGE of single fibers and whole muscle extracts. Analysis of MHC isoforms of the whole muscle and single fibers was performed by the method described by Talmadge and Roy (54). Small muscle fragments from control and Sgca-null muscles were weighed, ground with a ceramic pestle in liquid nitrogen, and extracted at 2 mg/ml in SDS-PAGE sample buffer (62.5 mm Tris, ph 6.8, 2.3% SDS, 5% 2-mercaptoethanol, 10% glycerol). Chemically skinned single fibers were inserted in a microcapillary tube and proteins dissolved in 20 l of the SDS-PAGE buffer. Single fibers or 40 g of each muscle sample were electrophoresed on 8% SDS-PAGE slab gels in SDS (54). MHC protein bands from whole muscles or single fibers were revealed by Coomassie brilliant blue or silver staining, respectively. MHC isoform percentage composition was determined by densitometry of gels by using a Bio-Rad Imaging Densitometer (model GS-670). Statistical analysis. Means SE were calculated according to standard procedures. The statistical significance of differences between means of dystrophic muscles and cognate controls was determined with the Student s unpaired t-test, setting the level of significance at P 0.05 or as indicated. pca-tension data were fitted by a least squares method using the Table Curve fitting program (Jandel Scientific) according to the following equation: y max x N /(x N k N ), where max is the maximal value of pca-tension curve normalized to 1, k is pca 50 (the pca at 50% of maximum tension), and N is the Hill coefficient. RESULTS Histological analysis of Sgca-null muscles. Mice lacking -sarcoglycan develop progressive muscular dystrophy similar to human muscular dystrophy (14, 27, 48). At 3 mo of age, EDL and soleus muscles from Sgca-null mice showed a significantly larger mass compared with wild-type controls (Table 1). Moreover, both muscles showed an ample variability of Table 1. Contractile properties of Sgca-null muscles EDL Soleus control Sgca-null control Sgca-null Muscle mass, mg Muscle/body mass * Fiber CSA, m Twitch P t,mn P t, N/cm CT, ms HRT, ms * Tetanus P 0,mN * * P 0, N/cm MRRT, mn/ms * HRTT, ms * P t/p Values are means SE. The number of muscles utilized for the analysis of contractile properties was 13 wild-type and 14 Sgca-null extendor digitorum longus (EDL) and 9 wild-type and Sgca-null soleus. P t, maximal twitch tension; CT, contraction time; HRT, half-relaxation time; P 0, maximal tetanic tension; MRRT, maximal rate of rise of tetanus; HRTT, half relaxation tetanic tension; P t/p 0, twitch tension-to-tetanic tension ratio; fiber CSA, muscle fiber cross-sectional area, as determined in fibers from Sgca-null and wild-type EDL (667 and 673, respectively) and soleus (844 and 887, respectively). The muscle-to-body mass ratio was calculated taking into account that the body mass of control and Sgca-null mice were (n 6) and (n 6) g, respectively, with the difference being significant (P 0.005). Table data significant differences: *P 0.05; P

4 Fig. 1. Histological analysis of Sgca-null muscles. Hematoxylin and eosin (A and B) and succinate dehydrogenase (C and D) staining of cryostat sections of extensor digitorum longus (EDL) and soleus muscles from Sgca-null mice. Hematoxylin and eosin staining performed in EDL (A) and soleus (B) muscles from Sgca-null mice reveals very large fibers with small regenerating ones whose number is higher in soleus than in EDL muscle. Some large fibers of EDL muscle from Sgca-null mice show longitudinal splitting (arrows). *Numerous fibers of both EDL and soleus muscles from Sgca-null mice show internally-located nuclei, with some fibers containing 2 or 3 nuclei. Succinate staining performed in EDL muscle sections from wild-type (C) and Sgca-null mice (D) show an overall higher staining in the dystrophic muscle. R1331 muscle fiber size, with the presence of very small fibers with very large ones. EDL muscle fibers from Sgca-null mice showed a mean CSA 43% larger compared with wild-type control fibers (Table 1), whereas a minor, still significant, CSA increase (24%) was shown in soleus muscle fibers (Table 1). A significant number of the large fibers of EDL muscles from Sgca-null mice showed partial or complete splitting, a rare occurrence in soleus fibers (Fig. 1). Moreover, EDL and soleus muscle fibers from Sgca-null mice showed a high number of internally located nuclei ( %, n 1,243, and %, n 975, respectively), with some large fibers containing two or three internal nuclei, particularly in EDL muscles (Fig. 1). In contrast, the number of fibers with internally-located nuclei was very low ( 0.2%) in both EDL and soleus wildtype controls. Figure 1 also shows that soleus muscles from Sgca-null mice contain a larger number of small regenerating fibers ( 18%) compared with dystrophic EDL muscles ( 2%). EDL muscle fibers from Sgca-null mice showed an overall more intense succinate dehydrogenase (SDH) staining intensity, an estimate of mitochondria number, compared with wild-type controls (Fig. 1). In contrast, soleus muscle fibers from Sgca-null mice showed no significant difference in SDH reaction compared with controls (not shown). Contractile properties of Sgca-null muscles. Figure 2 shows typical examples of twitches and tetani produced in EDL and soleus muscles from Sgca-null and wild-type mice. EDL muscles from Sgca-null and wild-type mice showed no significant differences in the average absolute twitch tension (Table 1). In contrast, soleus muscles from Sgca-null mice produced a higher absolute twitch tension than wild-type controls (Fig. 2 and Table 1). EDL muscles from Sgca-null and wild-type mice showed no significant differences in the contraction and relaxation times, whereas the latter was shorter in Sgca-null soleus compared with wild-type controls (Fig. 2C and Table 1). In EDL muscles from Sgca-null mice, the absolute P 0 was significantly lower than in wild-type controls (Table 1). In contrast, in soleus muscles from Sgca-null mice, the absolute P 0 was slightly higher (Table 1). When normalized to the CSA, on the other hand, the average specific twitch tension was significantly lower in Sgca-null EDL compared with controls, whereas that of soleus muscles was not different (Table 1).The muscle twitch-to-tetanus ratio was significantly higher in Sgcanull EDL mice compared with controls, whereas that of soleus was not different in Sgca-null and wild-type mice (Table 1). EDL muscles from Sgca-null mice showed no significant differences in the absolute maximum rate of rise of tetanus, whereas in soleus muscles from Sgca-null mice the rate was significantly higher than in wild-type controls (Table 1). Similarly, the half-relaxation P 0 time was higher only in soleus muscles from Sgca-null mice compared with controls (Table 1). Force-frequency relationship of Sgca-null muscles. Figure 3 shows the force-frequency relationship in EDL and soleus muscles from Sgca-null and wild-type mice. EDL muscles from Sgca-null mice produced a higher relative force at all frequencies up to 100 Hz compared with controls, thus showing a clear leftward shift of the force-frequency relationship. In fact, the frequency giving 50% force was 45 Hz in Sgca-null EDL muscles and 60 Hz in controls. Soleus muscles from Sgca-null mice produced a higher relative force at frequencies above 40 Hz compared with controls (Fig. 3B). Soleus muscles from Sgca-null and wild-type mice showed no significant differences in the frequency giving 50% force ( 22 Hz). Fatigue of Sgca-null muscles. Force production during fatigue produced by low-frequency tetanic stimulation of muscles is shown in Fig. 3, C and D. EDL muscles from Sgca-null mice were more fatigue resistant than controls (Fig. 3C). In fact, EDL muscles from Sgca-null mice produced significantly higher mean relative forces during the whole fatiguing protocol, which includes an initial transient potentiation phase. In EDL muscles from Sgca-null mice, relative force at the end of the 10-min stimulation was and % in controls. In contrast, there was no significant difference in the fatigue resistance in soleus muscles compared with wild-type controls (Fig. 3D). MHC composition of Sgca-null muscles. Figure 4 shows MHC isoform composition of EDL and soleus muscles from Sgca-null mice as determined by SDS-PAGE analysis (Fig. 4A) performed with the method described by Talmadge and Roy (54). EDL muscles from Sgca-null and wild-type mice

5 R1332 Fig. 2. Twitch and tetanic tensions of EDL and soleus muscles from Sgca-null mice. Typical force records from an EDL (A and B) and a soleus (C and D) muscle of control (black lines) and Sgca-null (grey lines) mouse. Records are from twitches (A and C) and maximal tetani [150 Hz for EDL (B) and 100 Hz for soleus (D)]. showed no significant difference in the overall MHC isoforms composition, both muscles showing a comparable net prevalence of type 2B MHC isoform (Fig. 4B). On the other hand, in soleus muscles from Sgca-null mice, expression level of type 1 MHC was significantly lower, and that of type 2X MHC was significantly higher compared with wild-type controls. Moreover, the protein band comprising both type 2A and embryonic MHC isoforms (the electrophoretic method does not permit the Fig. 3. Force-frequency relationships and low-frequency fatigue of Sgca-null muscles. Force-frequency relationships (A and B) and low-frequency fatigue (C and D) of EDL (A and C) and soleus (B and D) muscles from Sgca-null mice. Open symbols indicate controls (3 animals) and closed symbols the dystrophic muscles (3 animals). Values are means SE. *Statistical significance is P 0.05.

6 R1333 Fig. 4. Myosin heavy-chain (MHC) isoform composition in EDL and soleus Sgca-null muscles. Composition of MHC isoforms was estimated by SDS-PAGE of muscle fragment lysates. A: Coomassie blue staining of 2 SDS-PAGE slab gels loaded with mixed EDL and soleus muscle lysates (mix) from wild-type mice, muscles from newborn wildtype mice (nb), EDL and soleus muscle lysates from wildtype (Ec and Sc, respectively), and from Sgca-null mice (E and S, respectively). The 4 MHC isoforms expressed by adult muscles are shown in the mix lane, namely, in the migration order, type 2A, 2X, 2B, and 1 MHC (54). The 2 developmental MHC isoforms, embryonic (emb) and neonatal (neo) are shown in the nb lane. Note that embryonic and 2A MHC isoforms comigrate under this electrophoretic condition. B: %composition of MHC isoforms expression in wild-type (open bars) and Sgca-null muscle lysates (filled bars); MHC isoforms were identified by SDS-PAGE by analyzing 5 muscles each. Because of the comigration of embryonic and 2A MHC isoform (2A-e), it was not possible to indicate the relative content of these isoforms. Values are means SE; *P 0.05 and **P separation of these two bands) was significantly reduced in soleus muscles from Sgca-null mice compared with controls (Fig. 4B). pca-tension relationship of Sgca-null muscle fibers. Ca 2 sensitivity of myofibrillar proteins was measured in chemically skinned fibers isolated from wild-type and Sgca-null muscles. EDL muscle fibers isolated from Sgca-null and wild-type mice showed no difference in the pca/tension relationships (Fig. 5 and Table 2). In contrast, soleus muscle fibers isolated from Sgca-null mice showed a significant right shift of the pcatension curve compared with controls (Fig. 5). In fact, the pca threshold for tension development and the pca 50 were significantly lower in soleus fibers of Sgca-null mice compared with controls (Table 2). The Hill coefficient, an estimate of cooperative interactions between myofibrillar elements, was not different between soleus muscle fibers of Sgca-null and control mice (Table 2). Sarcoplasmic reticulum caffeine sensitivity of Sgca-null muscle fibers. Figure 6 shows caffeine sensitivity of Ca 2 release from sarcoplasmic reticulum of muscle fibers isolated from wild-type and Sgca-null muscles. In wild-type mice, the mean threshold caffeine concentration able to stimulate the release of enough Ca 2 to cause a detectable fiber contraction (11, 20) was significantly lower in soleus compared with EDL fibers (Table 3 and Fig. 6A). In soleus muscle fibers from Sgca-null mice, the mean caffeine threshold was significantly higher compared with control fibers, but it was not different compared with wild-type EDL fibers. Moreover, sarcoplasmic reticulum of soleus fibers isolated from Sgca-null mice released less calcium than control fibers (Table 3). In fact, the ratio between tension developed at threshold caffeine concentration (P th /P 20 ) and that developed at 20 mm caffeine (P 20,a concentration able to completely empty sarcoplasmic reticulum) was significantly reduced (Table 3). Contracture amplitude produced by 20 mm caffeine, corresponding to the amount of Ca 2 accumulated by sarcoplasmic reticulum during 30 s of incubation, was also significantly lower in soleus fibers isolated from Sgca-null mice compared with wild-type controls (Table 3). MHC composition of single fibers from Sgca-null muscles. Figure 7 shows SDS-PAGE analysis of MHC isoforms expressed by the chemically skinned fibers used for the physiological analysis described in Figs. 5 and 6. Seventy-four percent of fibers isolated from EDL muscles of wild-type mice (n 34) contained type 2B MHC isoform, whereas the remaining fibers (26%) contained both type 2X and 2B MHC. In contrast, 67% of fibers isolated from EDL muscles of Sgca-null mice (n 42) were hybrid fibers coexpressing 2B and 2X MHC isoforms, whereas the remaining fibers contained the type 2B MHC only. Single fibers isolated from soleus muscles (n 38) presented a more variable composition of MHC isoforms with a large number of hybrid fibers (Fig. 7). Approximately 37% of the single soleus fibers isolated from wild-type muscles ex- Fig. 5. pca-tension relationships of single fibers isolated from EDL and soleus muscles from wild-type (open symbols) and Sgca-null (solid symbols) muscles. Tension developed at each pca value (P) was reported relative to the maximal tension produced by the fiber (P 0). The total number of fibers examined was 23 in wild-type and 25 in Sgca-null EDL and 23 and 24 in wild-type and Sgca-null soleus, respectively. Values are means SE. *P 0.05.

7 R1334 Table 2. pca-tension relationships of single fibers pressed MHC 1, either alone or with other MHC isoforms (usually 2A and 2X); 45% of fibers contained MHC 2A, either alone or with 1 and/or MHC 2X; 58% contained the MHC 2X, alone or with (2A and 2B), and 18% contained the MHC 2B. Usually, 50% of all soleus fibers from wild-type controls contained more than one MHC isoform. In soleus muscle fibers from Sgca-null mice, the number of fibers expressing type 1 MHC decreased to 33%, with respect to wild-type controls, whereas fibers with 2A and 2X MHC increased to 51 and 72%, respectively. The number of hybrid fibers isolated from Sgcanull muscle also augmented to 59% of total fibers. DISCUSSION EDL Soleus control Sgca-null control Sgca-null n pca th * pca N Values are means SE; n is number fibers, pca th is the lowest log Ca 2 concentration giving a detectable tension; pca 50 is the pca value corresponding to 50% of maximum tension; N, the Hill coefficient. Significant differences between control and Sgca-null mice are indicated. *P 0.01; P The present study shows that the absence of -sarcoglycan in the Sgca-null mice produces profound modifications of contractile properties that are largely muscle-type specific, in that changes noted in the fast-twitch EDL muscle were often either not present in dystrophic soleus muscles or changed in the opposite direction. Limb-girdle muscular dystrophy 2D is an autosomal recessive disorder caused by mutations in the Sgca gene (45). The Sgca-null mice was developed to determine how the sarcoglycan deficiency causes muscular dystrophy (14, 30). It was found that Sgca-null mice develop progressive muscular dystrophy similar to human Sgca-deficient limb-girdle muscular Table 3. Sarcoplasmic reticulum caffeine sensitivity of single fibers EDL Soleus control Sgca-null control Sgca-null n Threshold (mm) * P th/p P 20 (mn mm 2 ) Values are means SE, n is number of fibers, P th/p 20, ratio between the tension developed at caffeine threshold level and 20 mm caffeine. Significant differences between control and Sgca-null mice are indicated. *P 0.05; P dystrophy-type 2D. The main histological changes of Sgca-null muscles include extensive central nucleation, increased variability of muscle fiber diameter, and the persisted presence of degeneration and regeneration (14, 27, 30, 48). Immunofluorescence and immunoblot analyses demonstrated that the absence of -sarcoglycan causes complete loss of the other sarcoglycans and of sarcospan (9, 14, 27). Moreover, the absence of -sarcoglycan, and thus of the sarcoglycan-sarcospan complex, destabilizes the dystrophin complex by greatly reducing the amount of bound -dystroglycan (9, 14). This results in the structural instability of sarcolemma, as indicated by the uptake of Evans blue dye into muscle fibers and the elevated serum levels of cytoplasmic enzymes (14). Functionally, Sgca-null muscles show a dramatic difference in the adaptive response of fast- and slow-twitch muscles, an evidence that evoked additional investigation (14). Likewise in mdx mice (17, 39), EDL and soleus muscles from 3-mo-old Sgca-null mice display a larger mass (27 and 36%, respectively) and mean CSA than age-matched controls. Hypertrophy of EDL and soleus muscles in younger (8-wk-old) Sgca-null mice was much larger, 40 and 62%, respectively(14), than that reported here in older animals, suggesting that the phenomenon could be progressively attenuated with age. Fig. 6. Sarcoplasmic reticulum caffeine sensitivity of single fibers isolated from EDL and soleus muscles of wild-type (open bars) and Sgca-null (filled bars) muscles. Single fibers, whose sarcoplasmic reticulum was filled with calcium (see MATERIALS AND METHODS), were probed with increasing concentrations of caffeine, and the first one producing significant tension was reported as the threshold concentration. The total number of fibers examined was 11 in wild-type, 17 in Sgcanull EDL, 15 in wild-type, and Sgca-null soleus.

8 Fig. 7. MHC isoforms expressed by chemically skinned single fibers from soleus and EDL Sgca-null muscles. MHC isoform expression was analyzed in chemically skinned single fibers isolated from EDL and soleus muscles of wild-type and Sgca-null mice. A: gel electrophoresis analysis of muscle fiber lysates according to Talmadge and Roy (54); lane 1, MHC isoforms present in mixed soleus and EDL muscles; lanes 2 and 3, MHC isoforms of single fibers from wild-type EDL; lanes 4-6, MHC isoforms of single fibers from Sgca-null EDL; lanes 7-9, MHC isoforms of single fibers from wild-type soleus; lanes 10-12, MHC isoforms of single fibers from Sgca-null soleus. B: graphs showing the composition of fibers isolated from wild-type (control) and Sgca-null muscles grouped according to the MHC isoforms. The total number of fibers examined of wild-type and Sgca-null EDL was 34 and 42, respectively, whereas that of wild-type and Sgca-null soleus was 38 and 39, respectively. A first important functional difference between fast- and slow-twitch Sgca-null hindlimb muscles is that, despite a similar level of hypertrophy, absolute tetanic force of 3-mo-old EDL was reduced by 39%, whereas that of soleus was increased by 38%, compared with age-matched controls. As a consequence, specific P 0 of Sgca-null EDL was about half that of control ( 46%), whereas that of dystrophic soleus was not different. In contrast, it was reported that the absolute tetanic force of 8-wk-old Sgca-null EDL was not different from that of controls, whereas absolute force of the 8-wk-old dystrophic soleus increased in the same way as in the 3-mo-old animals analyzed in the present study (14). Thus from these data, it results that also specific P 0 of 8-wk-old Sgca-null EDL is reduced by 31%. Interestingly, another fast-twitch muscle, R1335 the tibialis anterior of 9-wk-old Sgca-null mice, showed a comparable reduction of specific P 0 ( 35%) than that of age-matched control (27). Moreover, specific tension of singletype 2B fast-twitch fibers isolated from 6-mo-old Sgca-null gastrocnemius was 32% lower than that of control fibers (48). Therefore, our data and those so far present in the literature suggest that the inability to generate force of fast-twitch muscles of Sgca-null mice is maintained with age. On the other hand, the concurrent increase of muscle mass and of absolute P 0 demonstrated by Sgca-null soleus, independently of age, results in specific P 0 values equivalent to those of control, indicating that the slow-twitch muscle maintains the capacity to generate force independently of age and progression of dystrophy. To ascertain whether force generation capacity of both EDL and soleus of Sgca-null mice is maintained at an older age will require future investigation. Thus even though both Sgca-null EDL and soleus become hypertrophic, only the soleus appears to be able to produce force with some efficiency during hypertrophy. The more evident functional deficiency in EDL compared with the dystrophic soleus is attributable to the presence of numerous large fibers undergoing longitudinal splitting in the Sgca-null EDL, a finding which was not present in the dystrophic soleus muscle. Since fiber splitting is known to contribute to the weakening of pathological muscles (26, 46, 56), the observed increase in EDL fiber splitting may contribute to the lower force generation of Sgca-null EDL. Fiber splitting was also reported in -sarcoglycan-null mice (16) and in the diaphragm of Sgca-null mice (14); however, physiological data of these muscles were not presented. Fiber splitting was not reported for -sarcoglycan-null muscles and, consistent with our hypothesis, specific P 0 of EDL muscle from -sarcoglycannull mice at wk of age was identical to that of control muscle (22). An additional possible explanation for the reduced force production of Sgca-null EDL could be found in the large increase of hybrid fibers, i.e., fibers contemporarily expressing type 2X and 2B myosin at detriment of the most powerful type 2B fibers. Because 2X-2B hybrid fibers are less efficient in generating force than are 2B fibers (6), the prominence of hybrid fibers in the dystrophic EDL may also contribute to the lower contractile efficiency of the Sgca-null fast-twitch muscle. It is worth noting that the increase of hybrid fibers is also a characteristic of mdx muscles (39, 41), suggesting that the transition between fiber types could explain the lower force production also of dystrophin-null mice. An additional functional difference between fast- and slowtwitch muscles of Sgca-null mice relates to the calcium sensitivity of myofibrillar proteins. The pca/tension curve of Sgcanull soleus fibers demonstrated a rightward shift with respect to control fibers, a modification that indicates a transformation of muscle fibers toward faster characteristics, whereas no difference was evident in EDL. SDS-PAGE analysis of the fibers used for the pca-tension measurements shows in Sgca-null soleus the overall increase of fibers expressing type 2X MHC, from 60% in control to 80% in dystrophic soleus. Thus it is possible that the changes noted in the mean myofibrillar Ca 2 sensitivity of Sgca-null soleus fibers could be attributed to the contribution of changes in the myofibrillar proteins of the fast-type 2X phenotype. These changes could be correlated with the expression of either

9 R1336 regulative proteins (troponin and tropomyosin) and/or changes in the phosphorylation state and processes of regulative myosin light chains. Moreover, the higher content of fibers expressing type 2X MHC could also explain the higher maximum rate of rise of tetanus and capacity to generate force of dystrophic soleus. Because the number of hybrid 2X/2B fibers was much higher in Sgca-null EDL (83%) than in the control muscle (61%), the lack of any difference in the pca sensitivity of myofibrillar proteins of the two muscles could be explained by the possibility that pca sensitivity of type 2B fibers is similar to that of hybrid-type 2X/2B fibers. Sgca-null EDL muscle showed a higher resistance to fatigue than control, whereas soleus muscle was not different than control. Several factors may be responsible for the lower fatigability of dystrophic EDL. It has been reported that hindlimb muscles of mdx mice are less fatigable than those of control animals (44, 46), a property that was attributed to the lower relative force produced by the dystrophic muscles and thus to the lower energy demand. The same explanation could be applied to the Sgca-null EDL, which has a relative maximal tension half that of normal EDL and thus uses less energy and develops a slower rate of fatigue. An additional explanation could be found in the observed disproportion of MHC isoform composition where the reduction of glycolytic fibers expressing the fast-fatiguing 2B MHC is compensated by the large increase of hybrid fibers, resulting in a more oxidative phenotype of dystrophic EDL. Consistently, the overall SDH staining of Sgca-null EDL, an estimate of mitochondria number, was higher than that of control muscle. An additional difference associated with the absence of -sarcoglycan was found in the excitation-contraction coupling mechanism of dystrophic muscle fibers. Caffeine sensitivity of the sarcoplasmic reticulum (an indication of how easily the opening of the Ca 2 -release channel) of soleus fibers was significantly decreased with respect to control fibers, whereas that of EDL fibers was unaffected. This alteration of sarcoplasmic reticulum properties of the slow-twitch Sgca-null soleus fibers would make them similar to fast fibers. It is in fact known in several species that sarcoplasmic reticulum of slow muscle fibers is more sensitive to caffeine than that of fast fibers (18, 47). Whereas in mouse, the different caffeine sensitivity of sarcoplasmic reticulum from fast and slow muscles was demonstrated at the muscle level (52), our data represent the first confirmation at the fiber level. Mdx muscles also display profound modifications of Ca 2 handling to the extent that this characteristic is considered a critical pathogenetic feature of muscle dystrophy (21). A recent study, performed in chemically skinned fibers isolated from soleus and EDL mdx muscles (13), showed that sarcoplasmic reticulum properties of these muscles are differently affected by the downstream effects associated with the absence of dystrophin. In the present study, we show that also the absence of -sarcoglycan produces distinctive effects on fast and slow muscles, and we hypothesize that caffeine sensitivity changes of Sgcanull soleus could be attributed to the shift of the muscle toward the type 2X phenotype. In conclusion, the present work demonstrated that substantial modifications occur to muscles as a consequence of -sarcoglycan deficiency characterized by changes that are muscle fiber-type dependent. In DMD patients, the absence of dystrophin seems to affect more fast- than slow-twitch fibers (58). In Sgca-null mice, the absence of -sarcoglycan also perturbs more fast-twitch than slow-twitch fibers. Therefore, we speculate that within the dystrophin complex, probably in the sarcoglycan-sarcospan subcomplex, reside structural/signaling properties that distinctively affect the design of muscle fiber phenotype, perhaps also under physiological conditions. REFERENCES 1. Anderson JE, Bressler BH, and Ovalle WK. Functional regeneration in the hindlimb skeletal muscle of the mdx mouse. J Muscle Res Cell Motil 9: , Araishi K, Sasaoka T, Imamura M, Noguchi S, Hama H, Wakabayashi E, Yoshida M, Hori T, and Ozawa E. Loss of the sarcoglycan complex and sarcospan leads to muscular dystrophy in -sarcoglycandeficient mice. Hum Mol Genet 8: , Betto R, Senter L, Ceoldo S, Tarricone E, Biral D, and Salviati G. Ecto-ATPase activity of -sarcoglycan (adhalin). J Biol Chem 274: , Blake DJ, Weir A, Newey SE, and Davies KE. Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev 82: , Bönnemann CG, Modi R, Noguchi S, Mizuno Y, Yoshida M, Gussoni E, McNally EM, Duggan DJ, Angelini C, and Hoffman EP. -Sarcoglycan (A3b) mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex. Nat Genet 11: , Bottinelli R, Betto R, Schiaffino S, and Reggiani C. Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle fibers. J Physiol 478: , Burbach JA, Schlenker EH, and Johnson JL. Morphometry, histochemistry, and contractility of dystrophic hamster diaphragm. Am J Physiol Regul Integr Comp Physiol 253: R275 R284, Crosbie RH, Barresi R, and Campbell KP. Loss of sarcolemma nnos in sarcoglycan-deficient muscle. FASEB J 16: , Crosbie RH, Lebakken CS, Holt KH, Venzke DP, Straub V, Lee JC, Grady RM, Chamberlain JS, Sanes JR, and Campbell KP. Membrane targeting and stabilization of sarcospan is mediated by the sarcoglycan subcomplex. J Cell Biol 145: , Danieli-Betto D, Betto R, and Midrio M. Calcium sensitivity and myofibrillar protein isoforms of rat skinned skeletal muscle fibers. Pflügers Arch 417: , Danieli-Betto D, Germinario E, Esposito A, Biral D, and Betto R. Effects of fatigue on sarcoplasmic reticulum and myofibrillar properties of rat slow- and fast-twitch muscle fibers. J Appl Physiol 89: , De Luca A, Pierno S, Liantonio A, Cetrone M, Camerino C, Simonetti S, Papadia F, and Camerino DC. Alteration of excitation-contraction coupling mechanism in extensor digitorum longus muscle fibers of dystrophic mdx mouse and potential efficacy of taurine. Br J Pharmacol 132: , Divet A and Huchet-Cadiou C. Sarcoplasmic reticulum function in slowand fast-twitch skeletal muscles from mdx mice. Pflügers Arch 444: , Duclos F, Straub V, Moore SA, Venzke DP, Hrstka RF, Crosbie RH, Durbeej M, Lebakken CS, Ettinger AJ, van der Meulen J, Holt KH, Lim LE, Sanes JR, Davidson BL, Faulkner JA, Williamson R, and Campbell KP. Progressive muscular dystrophy in -sarcoglycan-deficient mice. J Cell Biol 142: , Durbeej M and Campbell KP. Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models. Curr Opin Genet Dev 12: , Durbeej M, Cohn RD, Hrstka RF, Moore SA, Allamand V, Davidson BL, Williamson RA, and Campbell KP. Disruption of the -sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E. Mol Cell 5: , Faulkner JA, Brooks SV, Dennis RG, and Lynch GS. The functional status of dystrophic muscles and functional recovery by skeletal muscles following myoblast transfer. Basic Appl Myol 7: , Fryer MW and Neering IR. Actions of caffeine on fast- and slow-twitch muscles of the rat. J Physiol 416: , Germinario E, Esposito A, Megighian A, Midrio M, Betto R, and Danieli-Betto D. Effects of modulators of sarcoplasmic Ca 2 release on

10 R1337 the development of skeletal muscle fatigue. J Appl Physiol 96: , Germinario E, Esposito A, Megighian A, Midrio M, Biral D, Betto R, and Danieli-Betto D. Early changes of sarcoplasmic reticulum and myofibrillar properties of type 2B fibers after denervation of rat EDL skeletal muscle. J Appl Physiol 92: , Gillis JM. Membrane abnormalities and Ca 2 homeostasis in muscles of the mdx mouse, an animal model of the Duchenne muscular dystrophy: a review. Acta Physiol Scand 156: , Hack AA, Cordier L, Shoturma DI, Lam MY, Sweeney HL, and McNally EM. Muscle degeneration without mechanical injury in sarcoglycan deficiency. Proc Natl Acad Sci USA 96: , Hack AA, Groh ME, and McNally EM. Sarcoglycans in muscular dystrophy. Microsc Res Tech 48: , Hack AA, Ly CT, Jiang F, Clendenin CJ, Sigrist KS, Wollmann RL, and McNally EM. -Sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin. J Cell Biol 142: , Holt KH, Lim LE, Straub V, Venzke DP, Duclos F, Anderson RD, Davidson BL, and Campbell KP. Functional rescue of the sarcoglycan complex in the BIO 14.6 hamster using delta-sarcoglycan gene transfer. Mol Cell 1: , Hosaka Y, Yokota T, Miyagoe-Suzuki Y, Yuasa K, Imamura M, Matsuda R, Ikemoto T, Kameya S, and Takeda S. 1-Syntrophindeficient skeletal muscle exhibits hypertrophy and aberrant formation of neuromuscular junctions during regeneration. J Cell Biol 158: , Imamura M, Mochizuki Y, Engvall E, and Takeda S. Epsilon-sarcoglycan compensates for lack of -sarcoglycan in a mouse model of limb-girdle muscular dystrophy. Hum Mol Genet 14: , Khammari A, Pereon Y, Baudet S, and Noireaud J. In situ study of the sarcoplasmic reticulum function in control and mdx mouse diaphragm muscle. Can J Physiol Pharmacol 76: , Lim LE and Campbell KP. The sarcoglycan complex in limb-girdle muscular dystrophy. Curr Opin Neurol 11: , Liu LA and Engvall E. Sarcoglycan isoforms in skeletal muscles. J Biol Chem 274: , Lynch GS, Hinkle RT, Chamberlain JS, Brooks SV, and Faulkner JA. Force and power output of fast and slow skeletal muscles from mdx mice 6 28 months old. J Physiol 535: , Michele DE and Campbell KP. Dystrophin-glycoprotein complex: posttranslational processing and dystroglycan function. J Biol Chem 278: , Midrio M, Danieli-Betto D, Megighian A, and Betto R. Early effects of denervation on sarcoplasmic reticulum properties of slow-twitch rat muscle fibers. Pflügers Arch 434: , Montgomery A. The isometric responses of fast and slow twitch muscles from normal and genetically dystrophic hamsters. Exp Neurol 46: , Nicolas-Metral V, Raddatz E, Kucera P, and Ruegg UT. Mdx myotubes have normal excitability but show reduced contraction-relaxation dynamics. J Muscle Res Cell Motil 22: 69 75, Nigro V, Moreira E, Piluso G, Vainzof M, Belsito A, Politano L, Puca AA, Passo-Bueno MR, and Zatz M. Autosomal recessive limb-girdle muscular dystrophy, LGMD 2F, is caused by a mutation in the -sarcoglycan gene. Nat Genet 14: , Noguchi S, McNally EM, Ben Othmane K, Hagiwara Y, Mizuno Y, Yoshida M, Yamamoto H, Bonnemann CG, Gussoni E, Denton PH, Kyriakides T, Middleton L, Hentati F, Ben Hamida M, Nonaka I, Vance JM, Kunkel LM, and Ozawa E. Mutations in the dystrophinassociated protein -sarcoglycan in chromosome 13 muscular dystrophy. Science 270: , Pasternak C, Wong S, and Elson EL. Mechanical function of dystrophin in muscle cells. J Cell Biol 128: , Pastoret C and Sebille A. Time course study of the isometric contractile properties of mdx mouse striated muscles. J Muscle Res Cell Motil 14: , Patel ND, Jannapureddy SR, Hwang W, Chaudhry I, and Boriek AM. Altered muscle force and stiffness of skeletal muscles in -sarcoglycandeficient mice. Am J Physiol Cell Physiol 284: C962 C968, Petrof BJ, Shrager JB, Stedman HH, Kelly AM, and Sweeney HL. Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci USA 90: , Petrof BJ, Stedman HH, Shrager JB, Eby J, Sweeney HL, and Kelly AM. Adaptations in myosin heavy chain expression and contractile function in dystrophic mouse diaphragm. Am J Physiol Cell Physiol 265: C834 C841, Quinlan JG, Johnson SR, McKee MK, and Lyden SP. Twitch and tetanus in mdx mouse muscle. Muscle Nerve 15: , Rezvani M, Cafarelli E, and Hood DA. Performance and excitability of mdx mouse muscle at 2, 5, and 13 wk of age. J Appl Physiol 78: , Roberds SL, Leturcq F, Allamand V, Piccolo F, Jeanpierre M, Anderson RD, Lim LE, Lee JC, Tomé FM, Romero NB, and Campbell KP. Missense mutations in the adhalin gene linked to autosomal recessive muscular dystrophy. Cell 78: , Sacco P, Jones DA, Dick JR, and Vrbova G. Contractile properties and susceptibility to exercise-induced damage of normal and mdx mouse tibialis anterior muscle. Clin Sci (Lond) 82: , Salviati G, Betto R, Ceoldo S, Tegazzin V, and Della Puppa A. Caffeine sensitivity of sarcoplasmic reticulum of fast and slow fibers from normal and malignant hyperthermia human muscle. Muscle Nerve 12: , Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D Antona G, Pellegrino MA, Barresi R, Bresolin N, De Angelis MG, Campbell KP, Bottinelli R, and Cossu G. Cell therapy of -sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 301: , Sandonà D, Gastaldello S, Martinello T, and Betto R. Characterization of the ATP-hydrolyzing activity of -sarcoglycan. Biochem J 381: , Straub V and Campbell KP. Muscular dystrophies and the dystrophinglycoprotein complex. Curr Opin Neurol 10: , Straub V, Rafael JA, Chamberlain JS, and Campbell KP. Animal models for muscular dystrophy show different patterns of sarcolemmal disruption. J Cell Biol 139: , Singh YN and Dryden WF. Isometric contractile properties and caffeine sensitivity of the diaphragm, EDL and soleus in the mouse. Clin Exp Pharmacol Physiol 16: , Singh YN, Schlenker EH, Singh BN, and Burbach JA. Consequences of thyroxine treatment on diaphragm and EDL of normal and dystrophic hamsters. Can J Physiol Pharmacol 82: , Talmadge RJ and Roy RR. Electrophoretic separation of rat skeletal muscle myosin heavy-chain isoforms. J Appl Physiol 75: , Thompson TG, Chan YM, Hack AA, Brosius M, Rajala M, Lidov HG, McNally EM, Watkins S, and Kunkel LM. Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein. J Cell Biol 148: , Walton JN. Clinical examination, differential diagnosis and classification. In: Disorder of voluntary muscle (5th ed.). Edited by Walton JN. New York: Churchill Livingstone, 1988, p Watchko JF, O Day TL, and Hoffman EP. Functional characteristics of dystrophic skeletal muscle: insights from animal models. J Appl Physiol 93: , Webster C, Silberstein L, Hays AP, and Blau HM. Fast muscles are preferentially affected in Duchenne muscular dystrophy. Cell 52: , Williams DA, Head SI, Lynch GS, and Stephenson DG. Contractile properties of skinned muscle fibres from young and adult normal and dystrophic (mdx) mice. J Physiol 460: 51 67, Xiao X, Li J, Tsao YP, Dressman D, Hoffman EP, and Watchko JF. Full functional rescue of a complete muscle (TA) in dystrophic hamsters by adeno-associated virus vector-directed gene therapy. J Virol 74: , Yamamoto H, Hagiwara Y, Mizuno Y, Yoshida M, and Ozawa E. Heterogeneity of dystrophin-associated proteins. J Biochem (Tokyo) 114: , Yoshida M, Hama H, Ishikawa-Sakurai M, Imamura M, Mizuno Y, Araishi K, Wakabaiashi-Takai E, Noguchi S, Sasaoka T, and Ozawa E. Biochemical evidence for association of dystrobrevin to sarcoglycansarcospan complex as a basis for understanding sarcoglycanopathy. Hum Mol Genet 9: , 2000.