Mitsugumin 29 Research Articles

Acute Knockdown of MG29 in Mouse Muscle Cells Reveals Signaling Mechanisms Associated with Polyunsaturated Fatty Acid (PUFA) – Implications for Sarcopenia

Estimates of humans with sarcopenia (i.e., the age related loss of muscle loss and strength) ranges from 30% to over 70% depending on the age group, and specific population studied. Mitsugumin 29 (MG29), a muscle-specific protein, is a member of the synaptophysin-like protein family (SYPL2) that contain MAL and related proteins for vesicle trafficking and membrane link (MARVEL) domains. MG29 is important for the formation of the transverse-tubule (TT) system in skeletal muscles, efficient signaling between RyR1 and the store-operated Ca2+ entry (SOCE) machinery, and maintenance of lipid content of the sarcolemmal membrane. Our previous in vivo studies using a global knockout model of MG29 suggested a role of MG29 in the aging decline in muscle function. To avoid potential adaptive effects of the global absence of MG29, we also tested the siRNA-mediated acute knockdown of MG29 in mouse primary skeletal muscle cells, which led to extensive myogenic changes characterized by increased myogenic differentiation that led to abnormally thinner and longer myotubes. To examine the mechanistic basis of these changes, we measured changes in lipid signaling following knockdown of MG29. We collected both cell culture medium and cell homogenates 72 h after siRNA transfection, and applied a custom-targeted LC/MS lipidomics approach to quantify specific Lipid Signaling Mediators (LMs) in cell culture media and we used our customized skeletal muscle-specific RT-qPCR gene array to investigate specific genes showing alteration expression following acute MG29 knockdown. Compared with negative control siRNA treated cells, in MG29 siRNA treated cells, the cell culture media levels of arachidonoylethanolamine (AEA), oleoylethanolamide (OEA) increased significantly (p < 0.05) and the levels of prostaglandin D2 (PGD2), 9,10-DiHOME, 9-HOTrE, 9-HODE decreased significantly (p < 0.05). In addition, the PCR gene array revealed that in MG29 siRNA treated cells the expression of Cacna1s, RyR3, Btk and Sod3 increased 2.6 ± 0.48, 4.5 ± 0.89, 2.7 ± 0.51 and 6.5 ± 1.06 fold, respectively, while Fkbp1b and Ccl2 decreased 2.8 ± 0.38 and 7.3 ± 1.74 fold, respectively. These genes have function in calcium signaling and homeostasis and oxidative stress as well as inflammation and immune-regulation. We conclude that MG29 modulation of myogenic differentiation occurs via a complex signaling mechanism related to changes in LMs derived from polyunsaturated fatty acid (PUFA). This study further positions MG29 as an important regulator of signaling pathways associated with intracellular calcium homeostasis, oxidative stress, and inflammation, which emphasizes the role of MG29 as an orchestrator of alterations in skeletal muscle during sarcopenia.

Acute Knockdown of MG29 Alters Skeletal Muscle Cells Differentiation and Leads to Cellular Atrophy

Sarcopenia is defined as the progressive loss of skeletal muscle mass and development of force/power that occurs during aging. It is estimated that more than 30% of humans over 60 years of age suffer from sarcopenia and age‐related loss of muscle function that contributes to more than half of all chronic conditions in people over 50 and accounts for 1.5% of total U.S. healthcare expenditures (&gt;$40 billion). Mitsugumin 29 (MG29), is a muscle‐specific protein belonging to the synaptophysin and MAL and related proteins for vesicle trafficking and membrane link (MARVEL) families of proteins which are involved in cholesterol binding and formation of oligomers of these proteins. MG29 is essential for the proper formation of the transverse‐tubule (TT) system, maintenance of lipid content of the sarcolemmal membrane, and efficient signaling between RyR1 and the store‐operated Ca2+ entry (SOCE) machinery. MG29 null (mg29−/−) mouse muscle displays abnormalities in the membrane ultrastructure of the triad junction where the TT are swollen and the sarcoplasmic reticulum (SR) networks show a vacuolated and fragmented structure. Clear phenotypic changes are seen in mg29−/− skeletal muscle at young ages (3–6 months) that resemble those seen in aged wildtype muscle. Our previous work clearly demonstrated that SOCE is compromised in an MG29‐associated manner in aged skeletal muscle and that reduced SOCE associates with a drastic decrease in MG29 content (~50%), altered intracellular Ca2+ homeostasis and decreased specific contractile force. But the function of MG29 on muscle differentiation remains unclear. In this study, we knocked down MG29 expression in mouse primary skeletal muscle cells by using MG29 siRNA. Our data shows that 48h after MG29 siRNA (10nM) transfection, the expression of MG29 decreased by ~ 80%, compared to negative control siRNA. After MG29 siRNA treatment, at day 3 of differentiation, thinner, longer myotubes were observed compared to negative controls (diameter of myotubes: negative control = 24.9 ± 2.08μm; MG29 siRNA = 19.1 ± 0.65μm, p &lt; 0.05; Length of myotubes: negative control = 424.9 ± 50.2μm; MG29 siRNA = 580.4 ± 39.9 μm, p &lt; 0.05). Surprisingly, fusion index (FI) was significantly increased compared to control (FI values: negative control= 53.2% ± 6.8; MG29 siRNA = 70.3% ± 7.9, p &lt; 0.05), in agreement with increased mRNA expression of myogenenic markers MyoG and MyoD detected by real time PCR. These results suggest that the acute knock down of MG29 leads in just three days to complex myogenic changes characterized by increased myogenic differentiation but with abnormally thinner and longer muscle cells. These studies further indicate specific roles of MG29 in maintaining normal muscle mass and function, and its importance for cellular homeostasis during aging.Support or Funding InformationThis work was supported by NIH‐National Institutes of Aging R01AG056504 (NW, MB), and the George W. and Hazel M. Jay and Evanston Research Endowments (MB).

MG29 Interacts with Bin‐1 to Maintain T‐Tubule Structure in Skeletal Muscle Physiology and Regeneration

Mitsugumin 29 (MG29), a member of the synaptophysin‐like family proteins, is a transmembrane protein primarily expressed in the t‐tubule membranes of skeletal muscle. In the current study we have found that t‐tubules in skeletal muscle of mg29−/− have defective morphology, indicating importance of MG29 in maintenance of muscle structure and function. To elucidate the role of MG29 in muscle physiology, we performed two‐color STORM super‐resolution imaging analysis confirmed co‐localization of MG29 and Bin‐1 on t‐tubules of skeletal muscle. We also performed co‐immunoprecipitation and found that MG29 can bind to Bin‐1, another t‐tubular protein. Recombinant MG29 protein domain fragments tagged with GST were expressed and Bin‐1 was found to bind to the cytosolic domains of MG29. Furthermore, organized distribution of Bin‐1 is severely disrupted in mg29−/− muscle. To test the function of MG29 in muscle regeneration, we injured gastrocnemius muscle with cardiotoxin (CTX) and tracked muscle repair and regeneration. Western blot showed that following CTX injury, MG29 protein levels were transiently reduced from day 1 to day 3, followed by recovery associated with muscle regeneration by day 5. Protein level of Bin‐1 in wild type muscle was also reduced upon CTX‐injury and increased during the recovery process. Compared with wild type muscle, the mg29−/− muscle displayed delayed regeneration and increased fibrosis following CTX‐induced injury. Together our data suggest that functional interaction between MG29 and Bin‐1 contribute to maintenance and formation of t‐tubule network while playing a role in muscle regeneration and regeneration. Targeting this MG29‐Bin‐1 interaction might provide a potential effective approach for treatment of muscle diseasesSupport or Funding InformationThis work was supported by grants from the National Institutes of Health (NIH) (AR067766 and HL124122)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MG29 Interacts with Bin1 for Maintaining T-Tubule Structure in Skeletal Muscle Physiology and Regeneration

Mitsugumin 29 (MG29), a member of the synaptophysin-like family proteins, is a transmembrane protein mainly expressed in the t-tubule membranes of skeletal muscle. Electron microscopy analysis of skeletal muscle derived from mg29-/- mice revealed morphological defects of t-tubules, indicating importance of MG29 in maintenance of muscle structure and function. To determine the function of MG29 in muscle physiology and regeneration, we performed co-immunoprecipitation and found that MG29 can bind to Bin1, another t-tubular protein.

Interaction between mitsugumin 29 and TRPC3 participates in regulating Ca2+ transients in skeletal muscle

Mitsugumin 29 (MG29) is related to the fatigue and aging processes of skeletal muscle. To examine the roles of MG29 in conjunction with its binding protein, the canonical-type transient receptor potential cation channel 3 (TRPC3), in skeletal muscle, the binding region of MG29 to TRPC3 was studied along with the functional relevance of the binding in mouse primary skeletal myotubes using co-immunoprecipitation assays and Ca2+ imaging experiments. The N-terminus and the I–II loop of MG29 constitute the binding region for TRPC3. The myotubes that expressed the MG29 mutant missing the entire TRPC3-binding region showed a disrupted binding between endogenous MG29 and TRPC3 and a reduction in Ca2+ transients in response to membrane depolarization without affecting ryanodine receptor 1 activity, the resting cytosolic Ca2+ level, and the amount of releasable Ca2+ from the sarcoplasmic reticulum. Among the proteins mediating Ca2+ movements in skeletal muscle, TRPC4 expression was significantly decreased by the MG29 mutant. Therefore, MG29 could be a new factor for regulating Ca2+ transients during skeletal muscle contraction possibly via a correlation with TRPC3 and TRPC4.

MG29/SYPL2 Contributes to Dysregulation of Lipid Composition and Store Operated Ca2+ Entry in Aging Skeletal Muscle

We proposed over one decade ago that age-related muscle weakness and not the loss of muscle size is the principal contributor to disability. In searching for cellular and molecular explanations for this weakness that exceeds the loss of muscle mass, we found that elements of contractile force generation in skeletal muscle are dependent on store-operated Ca2+ entry (SOCE) and that this capacity is lost in aging skeletal muscle. This altered SOCE is directly associated with reduced content of mitsugumin 29 (MG29), a synaptophysin family protein (SYPL2) containing a MARVEL domain involved in cholesterol binding and formation of oligomers of these proteins. MG29 is essential for the formation of the transverse-tubule system and efficient SOCE, creating a link between reduced MG29 expression in aged muscle and the development of muscle dysfunction in sarcopenia as our previous data demonstrate that aspects of aged skeletal muscle are present in young mg29-/- muscle. Here we test how the loss of MG29 affects the phospholipid composition of skeletal muscle fibers and if producing similar changes in muscle fibers can influence SOCE activity. Lipidomic analysis of young mg29-/- skeletal muscle showed decreased cholesterol content and altered levels of various phospholipid species compared to young control muscle. To simulate the reduced cholesterol content observed in mg29-/- muscle we partially extracted it from mature C2C12 myotubes with methyl-β-cyclodextrin (MCD) and then measured SOCE in these myotubes. Reduced SOCE was observed following MCD extraction, similar to the limited SOCE we previously observed in young mg29-/- and wild-type aged muscle fibers. Further studies will determine if mutation of conserved residues in the MARVEL domain of MG29 modulate SOCE function in, and the lipid composition of, skeletal muscle cells.

Mitsugumin 29 is transcriptionally induced in senile plaque-associated astrocytes

Astrocytes associated with beta-amyloid (Aβ)-deposited senile plaques are a common neuropathological feature of Alzheimer's disease (AD). There is little doubt that the association of Aβ with the major component in the central nervous system cells significantly influences disease progression, however, the molecular mechanisms by which Aβ contributes to the astrocyte-mediated neuropathological changes have not been well established. In an effort to identify astrocyte-derived molecules that may be closely associated with exacerbation of AD, we identified a novel Aβ-induced rat gene, whose mouse counterpart, mitsugumin 29 (MG29), is known to be involved in intracellular Ca2+ homeostasis in skeletal muscle. To evaluate the roles of human MG29 in AD, we investigated its expression in AD brain. In non-AD brains, MG29 mRNA has been detected in neurons, but not in quiescent astrocytes. On the contrary, in AD brains, MG29 is expressed in activated astrocytes associated with senile plaques, but not expressed in neurons around lesions. Human MG29-transfected cells express the protein in the endoplasmic reticulum and the level of expression is involved in Ca2+-dependent exocytosis mechanisms. Increased MG29 expression in senile plaques-associated activated astrocytes suggests that intracellular Ca2+ turnovers may be changed in these astrocytes. This might be related in sustained Ca2+-dependent exocytosis of various transmitters including glutamate, leading to the acceleration of neurodegeneration in the lesion observed in AD.

Mitsugumin-29 Regulates RyR1 Activity In Mouse Skeletal Myotubes

Canonical-type transient receptor potential cation channel type 3 (TRPC3) in plasma membrane allows the entry of Ca2+ ions into various cells. In skeletal myotubes, functional interaction between TRPC3 and RyR1 (ryanodine receptor1, a Ca2+ channel in sarcoplasmic reticulum (SR) membrane) regulates the gain of skeletal excitation-contraction coupling (J. Biol. Chem., 2006). Mitsugumin-29 (MG29) is a four membrane-spanning protein and is found in both plasma and SR membrane. MG29 has been known as a TRPC3-interacting protein in skeletal myotubes (Biochem. J., 2008).To identify critical region(s) of MG29 that participate in binding to TRPC3 or the role of MG29 in skeletal muscle, N-terminus, three intervenient loops among four transmembrane regions, and C-terminus of MG29 were expressed in E. coil as N-terminal GST-fused forms, and subjected to co-immunoprecipitation assay with intact TRPC3 from rabbit skeletal muscle. Cytoplasmic N-terminus and a loop between first and second transmembrane domains of MG29 effectively bound to TRPC3. Two deletion mutants of MG29 (missing the TRPC3-binding sites: deleting the N-terminus only or longer N-terminus covering the loop region) was expressed in mouse skeletal myotubes, and the myotubes was subjected to the measurement of Ca2+ transients with Fura-2 or Fluo-4. The later mutant showed significantly decreased responsiveness of RyR1 to caffeine, suggesting that MG29 may be a mediator between the functional interaction between TRPC3 and RyR1.

Mitsugumin 29 as a TRPC3-interacting Protein

Canonical-type transient receptor potential cation channel type3 (TRPC3) allows the entry of Ca2+ into various cells. In skeletal myotubes, functional interaction between TRPC3 and RyR1 (ryanodine receptor1) regulates the gain of excitation-contraction coupling. Mitsugumin 29 (MG29) is a TRPC3-interacting protein in skeletal myotubes. To identify critical region(s) of MG29 that participate in binding to TRPC3, N-terminus, three intervenient loops among four transmembrane regions, and C-terminus of MG29 were expressed in E. coil as N-terminal GST-fused forms. The five GST-fused MG29 portions were subjected to co-immunoprecipitation assay with intact TRPC3. Cytoplasmic N-terminus and a loop between first and second transmembrane regions of MG29 effectively bound to TRPC3.

Muscle aging is associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+ release

Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation–contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.

When sparks get old

Sparks are transient local elevations of Ca ion concentration observed in different types of muscle cells. Such local Ca2+ signals can be provoked in skeletal muscle cells by altering the osmotic pressure of the extracellular solution. In this issue, Weisleder et al. (see p. 639) demonstrate that the Ca2+ response to osmotic stress is substantially altered in aged muscle. The study presents evidence for a link between this finding and a reduced expression of mitsugumin 29 (MG29), a small membrane protein of the sarcoplasmic reticulum (SR).

Enhanced resistance to fatigue and altered calcium handling properties of sarcalumenin knockout mice

Sarcalumenin is a Ca2+-binding protein located in the sarcoplasmic reticulum of striated muscle cells, the physiological function of which has not been fully determined yet. Using sarcalumenin knockout (sar(-/-)) mice, we showed that sar ablation altered store-operated Ca2+ entry (SOCE) and enhanced muscle fatigue resistance. Sar(-/-) mice fatigued less with treadmill exercise, and intact isolated soleus and extensor digitorum longus muscles from sar(-/-) mice were more resistant to intermittent fatiguing stimulation than those from wild-type mice. Enhanced SOCE was observed in the sar(-/-) muscles. Biochemical analysis revealed that sar(-/-) muscles contained significantly elevated expression of mitsugumin 29 (MG29), a synaptophysin-related membrane protein located in the triad junction of skeletal muscle. Because the ablation of mg29 has been shown to cause increased fatigability and dysfunction of SOCE, the enhanced SOCE activity seen in sar(-/-) muscle may be correlated with the increased expression of MG29. Our data suggest that systemic ablation of sarcalumenin caused enhanced resistance to muscle fatigue by compensatory changes in Ca2+ regulatory proteins that effect SOCE.

Defective maintenance of intracellular Ca2+ homeostasis is linked to increased muscle fatigability in the MG29 null mice

Mitsugumin 29 (MG29) is a transmembrane protein that is normally found in the triad junction of skeletal muscle. Our previous studies have shown that targeted deletion of mg29 from the skeletal muscle resulted in abnormality of the triad junction structure, and also increased susceptibility to muscle fatigue. To elucidate the basis of these effects, we investigated the properties of Ca2+-uptake and -release in toxin-skinned Extensor Digitorium Longus (EDL) muscle fibers from control and mg29 knockout mice. Compared with the control muscle, submaximal Ca2+-uptake into the sarcoplasmic reticulum (SR) was slower and the storage of Ca2+ inside the SR was less in the mutant muscle, due to increased leakage process of Ca2+ movement across the SR. The leakage pathway is associated with the increased sensitivity of Ca2+/caffeine -induced Ca2+ release to myoplasmic Ca2+. Therefore, the increased fatigability of mutant EDL muscles can result from a combination of a slowing of Ca2+ uptake, modification of Ca2+-induced Ca2+ release (CICR), and a reduction in total SR Ca2+ content.

Co-expression of MG29 and Ryanodine Receptor Leads to Apoptotic Cell Death: EFFECT MEDIATED BY INTRACELLULAR Ca2+ RELEASE

Perturbation of intracellular Ca2+ homeostasis has been shown to regulate the process of cell proliferation and apoptosis. Our previous studies show that mitsugumin 29 (MG29), a synaptophysin-related protein localized in the triad junction of skeletal muscle, serves an essential role in muscle Ca2+ signaling by regulating the process of store-operated Ca2+ entry. Here we report a functional interaction between MG29 and the ryanodine receptor (RyR)/Ca2+ release channel. The purified MG29 protein enhances activity of the RyR/Ca2+ release channel incorporated into the lipid bilayer membrane. Co-expression of MG29 and RyR in Chinese hamster ovary cells leads to apoptotic cell death resulting from depletion of intracellular Ca2+ stores, despite neither protein expression alone exhibits any significant effect on cell viability. In transient expression studies, the presence of RyR in the endoplasmic reticulum leads to retention of MG29 from the plasma membrane into the intracellular organelles. This functional interaction between MG29 and RyR could have important implications in the Ca2+ signaling processes of muscle cells. Our data also show that perturbation of intracellular Ca2+ homeostasis can serve as a key signal in the initiation of apoptosis.

Changes in Ca 2+ handling in adult MG29-deficient skeletal muscle

It was reported that a lack of Mitsugumin29 (MG29), a protein expressed at the triad junction, caused morphological changes in sarcoplasmic reticulum and T-tubules, reduced twitch/tetanus ratio, and increased susceptibility to fatigue in adult skeletal muscle and dysfunction of store-operated Ca 2+ entry (SOC) in embryonic and neonatal muscles. To deepen our understanding of the role of MG29 in the Ca 2+ handling in adult skeletal muscle, Ca 2+ stores of wild-type and mutant muscle fibers were depleted by repetitive high-K + treatments in a Ca 2+-free medium. Although wild-type muscle showed only minor caffeine contracture after high-K + response had disappeared, the mutant muscle showed remarkable caffeine contracture under the conditions used, suggesting functional compartmentalization of the Ca 2+-store in the mutant. Activation of SOC in adult mutant muscle was observed upon the voltage-sensitive store depletion as is true with the wild-type muscle. Thus MG29 is not involved in the SOC activation at variance with the previous conclusion with immature muscles.

Dysfunction of store-operated calcium channel in muscle cells lacking mg29.

The store-operated calcium channel (SOC) located in the plasma membrane (PM) mediates capacitative entry of extracellular calcium after depletion of intracellular calcium stores in the endoplasmic or sarcoplasmic reticulum (ER/SR). An intimate interaction between the PM and the ER/SR is essential for the operation of this calcium signalling pathway. Mitsugumin 29 (MG29) is a synaptophysin-family-related protein located in the junction between the PM and SR of skeletal muscle. Here, we identify SOC in skeletal muscle and characterise its regulation by MG29 and the ryanodine receptor (RyR) located in the SR. Targeted deletion of mg29 alters the junctional membrane structure, causes severe dysfunction of SOC and SR calcium homeostasis and increases the susceptibility of muscle to fatigue stimulation. Severe dysfunction of SOC is also identified in muscle cells lacking both type 1 and type 3 RyRs, indicating that SOC activation requires an intact interaction between the PM and the SR, and is linked to conformational changes of RyRs. Whereas defective SOC seems to be inconsequential to short-term excitation-contraction coupling, the slow cumulative calcium entry through SOC is crucial for long-term calcium homeostasis, such that reduced SOC activity exaggerates muscle fatigue under conditions of intensive exercise.

Abnormal formation of sarcoplasmic reticulum networks and triads during early development of skeletal muscle cells in mitsugumin29-deficient mice.

Recently, we detected a novel membrane protein, mitsugumin29 (MG29), in the triads in rabbit skeletal muscle cells and suggested important roles for this membrane protein in the formation of the sarcoplasmic reticulum (SR) networks and triads in muscle cells. In the present study, we examined the development of skeletal muscle cells in MG29-deficient mice to try to determine the roles played by MG29 in the formation of the SR networks and triads. Ultrastructural observations revealed some morphological abnormalities in these mice, such as incomplete formation of the SR networks, an irregular running of the transverse tubule and a partial defect in the triads at the A-I junctional region. These ultrastructural abnormalities occurred during early myogenesis and were preserved until the adult stage. The possible roles for MG29 in the formation of SR networks and triads in skeletal muscle cells are discussed in the light of these observations.

Abnormal features in skeletal muscle from mice lacking mitsugumin29.

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.

Immunolocalization of mitsugumin29 in developing skeletal muscle and effects of the protein expressed in amphibian embryonic cells.

The temporal appearance and subcellular distribution of mitsugumin29 (MG29), a 29-kDa transmembrane protein isolated from the triad junction in skeletal muscle, were examined by immunohistochemistry during the development of rabbit skeletal muscle. MG29 appeared in the sarcoplasmic reticulum (SR) in muscle cells at fetal day 15 before the onset of transverse tubule (T tubule) formation. In muscle cells at fetal day 27, in which T tubule and triad formation is ongoing, both SR and triad were labeled for MG29. In muscle cells at newborn 1 day, the labeling of the SR had become weak and the triads were well developed and clearly labeled for MG29. Specific and clear labeling for MG29 was restricted to the triads in adult skeletal muscle cells. When MG29 was expressed in amphibian embryonic cells by injection of the cRNA, a large quantity of tubular smooth-surfaced endoplasmic reticulum (sER) was formed in the cytoplasm. The tubular sER was 20-40 nm in diameter and appeared straight or reticular in shape. The tubular sER was formed by the fusion of coated vesicles [budded off from the rough-surfaced endoplasmic reticulum (rER)] and vacuoles of rER origin. The present results suggest that MG29 may play important roles both in the formation of the SR and the construction of the triads during the early development of skeletal muscle cells.