Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products.

Using murine models, we have previously demonstrated that recombinant adeno-associated virus (rAAV)-mediated microdystrophin gene transfer is a promising approach to treatment of Duchenne muscular dystrophy (DMD). To examine further therapeutic effects and the safety issue of rAAV-mediated microdystrophin gene transfer using larger animal models, such as dystrophic dog models, we first investigated transduction efficiency of rAAV in wild-type canine muscle cells, and found that rAAV2 encoding beta-galactosidase effectively transduces canine primary myotubes in vitro. Subsequent rAAV2 transfer into skeletal muscles of normal dogs, however, resulted in low and transient expression of beta-galactosidase together with intense cellular infiltrations in vivo, where cellular and humoral immune responses were remarkably activated. In contrast, rAAV2 expressing no transgene elicited no cellular infiltrations. Co-administration of immunosuppressants, cyclosporine and mycophenolate mofetil could partially improve rAAV2 transduction. Collectively, these results suggest that immune responses against the transgene product caused cellular infiltration and eliminated transduced myofibers in dogs. Furthermore, in vitro interferon-gamma release assay showed that canine splenocytes respond to immunogens or mitogens more susceptibly than murine ones. Our results emphasize the importance to scrutinize the immune responses to AAV vectors in larger animal models before applying rAAV-mediated gene therapy to DMD patients.

Localization of plectin and other related proteins along the sarcolemma in smooth muscle cells of rat colon.

Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments. To better understand the functional roles of plectin in smooth muscle cells, we examined the distribution of plectin and other related proteins in rat colon smooth muscles by confocal laser and electron microscopy. The sarcolemma of smooth muscle cells exhibits two ultrastructurally distinct domains, domains associated with dense plaques and caveola-rich domains. Staining with anti-plectin and anti-desmin antibodies showed that plectin was localized along the sarcolemma in an intermittent manner and desmin was distributed in the sarcoplasm and intermittently at the cell periphery where it was codistributed with desmin. Plectin exhibited complementary and non-overlapping distribution to caveolin-1 and dystrophin, components of caveola domains, whereas plectin was codistributed with vinculin, talin and integrin beta1, components of dense plaques. Plectin was also codistributed with beta2-chain laminin but not with beta1-chain laminin. Electron microscopic observations on the sarcolemma revealed close association of intermediate filaments with dense plaques. Correlated confocal and electron microscopy clearly demonstrated that anti-plectin fluorescence corresponded to dense plaques but not to caveola domains in electron microscopic images. These findings indicate that plectin is confined to dense plaques to which desmin intermediate filaments may be anchored in rat colon smooth muscle cells.

Initiation and maturation of I-Z-I bodies in the growth tips of transfected myotubes.

While over a dozen I-Z-I proteins are expressed in postmitotic myoblasts and myotubes it is unclear how, when, or where these first assemble into transitory I-Z-I bodies (thin filament/Z-band precursors) and, a short time later, into definitive I-Z-I bands. By double-staining the growth tips of transfected myotubes expressing (a) MYC-tagged s-alpha-actinins (MYC/s-alpha-actinins) or (b) green fluorescent protein-tagged titin cap (GFP/T-cap) with antibodies against MYC and I-Z-I band proteins, we found that the de novo assembly of I-Z-I bodies and their maturation into I-Z-I bands involved relatively concurrent, cooperative binding and reconfiguration of, at a minimum, 5 integral Z-band molecules. These included s-alpha-actinin, nebulin, titin, T-cap and alpha-actin. Resolution of the approximately 1.0 microm polarized alpha-actin/nebulin/tropomyosin/troponin thin filament complexes occurred subsequent to the maturation of Z-bands into a dense tetragonal configuration. Of particular interest is finding that mutant MYC/s-alpha-actinin peptides (a) lacking spectrin-like repeats 1-4, or consisting of spectrin-like repeats 1-4 only, as well as (b) mutants/fragments lacking titin or alpha-actin binding sites, were promptly and exclusively incorporated into de novo assembling I-Z-I bodies and definitive I-Z-I bands as was exogenous full length MYC/s-alpha-actinin or GFP/T-cap.

Plectin is a linker of intermediate filaments to Z-discs in skeletal muscle fibers.

Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments, and its deficiency causes the disruption of myofibrils, or muscular dystrophy. To better understand the functional role of plectin in skeletal muscle fibers, we have examined the topological and structural relationships of plectin to intermediate filaments and Z-discs in rat diaphragm muscles by confocal and immunoelectron microscopy. Immunofluorescence analysis revealed that plectin was colocalized with desmin at the periphery of Z-discs. This plectin localization around Z-discs was constantly maintained irrespective of the contracted or extended state of the muscle fibers, suggesting either direct or indirect association of plectin with Z-discs. Immunogold labeling in skinned muscle fibers clearly demonstrated that plectin-labeled fine threads linked desmin intermediate filaments to Z-discs and connected intermediate filaments to each other. These results indicate that through plectin threads desmin intermediate filaments form lateral linkages among adjacent Z-discs, preventing individual myofibrils from disruptive contraction and ensuring effective force generation.

Dispensability of the actin-binding site and spectrin repeats for targeting sarcomeric alpha-actinin into maturing Z bands in vivo: implications for in vitro binding studies.

To explore the roles of specific domains of sarcomeric alpha-actinin (s-alpha-actinin) in the assembly and maintenance of striated myofibrils, myogenic cultures were transfected with four MYC-tagged s-alpha-actinin peptides. They were: (1) full-length sarcomeric alpha-actinin, (2) an N-terminal deletion that removed the actin-binding site only (MYC/A-), (3) a peptide that consisted of the actin-binding site only (MYC/A+), and (4) an N-terminal deletion that removed the EF-hands and titin-binding domains (MYC/EFT-). While cytotoxic in replicating myogenic cells, as they were in PtK2 cells, the four MYC peptides were not cytotoxic in postmitotic myotubes. In myotubes each of the four different MYC peptides were promptly and selectively incorporated into normal Z bands. The incorporation of MYC/A-, MYC/A+, and MYC/EFT- into Z bands suggests that (a) the actin-binding site, (b) the spectrin-repeats believed to be responsible for anti-parallel dimerization, and (c) the C-terminal EF-hands and titin-binding domains are each dispensable for targeting s-alpha-actinin/MYC peptides into Z bands. These findings could not have been predicted from the behavior of alpha-actinin (a) in binding assays in cell-free systems or (b) when expressed in transfected nonmuscle cells.

The direct visualization of structural array from laminin to dystrophin in sarcolemmal vesicles prepared from rat skeletal muscles.

It has been biochemically shown that dystrophin and alpha- and beta-dystroglycan form an oligomeric complex which links laminin, a component of the basement membrane, to components of the subsarcolemmal cytoskeleton in skeletal muscle fibers. In the present study the dystrophin-glycoprotein complex and its structural relationships to laminin and subsarcolemmal cytoskeleton were ultrastructurally examined in crude surface membranes prepared from rat skeletal muscles. Sarcolemmal vesicles within crude surface membranes were identified and characterized by fine protrusions on their outer surface and electron-dense materials or patches associated with the inner surface. These two components were seen to be in register with each other across the sarcolemma. The fine protrusions were immunolabeled by anti-alpha-dystroglycan and reassociated with exogenous laminin. Immunolabeling in combination with laminin reassociation demonstrated that the electron-dense materials contained dystrophin at laminin-binding domains of the membrane. In addition, they were often associated with very fine filaments. These results provide morphological evidence for the biochemically proposed model of molecular array of dystrophin complex from the basement membrane to the subsarcolemmal cytoskeleton.

Unanticipated temporal and spatial effects of sarcomeric alpha-actinin peptides expressed in PtK2 cells.

To understand the multiple roles of alpha-actinin in the assembly of (1) Z bands in muscle, and (2) a variety of cytoskeletal structures in non-muscle cells, 4 sarcomeric alpha-actinin derived cDNAs tagged with a MYC epitope were constructed. The constructs were: (1) full-length (FL/MYC); (2) minus EF-hands (-EF/MYC); (3) actin-binding site (+A/MYC); and (4) minus actin-binding site (-A/MYC). These four cDNAs were individually transfected into PtK2 cells. The exogenous sarcomeric alpha-actinin (s-alpha-actinin/MYC) was followed with labeled anti-MYC, the endogenous non-sarcomeric alpha-actinin (non-s-alpha-actinin) with labeled anti-non-s-alpha-actinin. The salient findings were: (1) the selective intracellular localizations of each expressed MYC-tagged peptide differed one from the other; (2) their respective localizations in the 10-24-h post-transfection (p.t.) period differed from their localizations in the 48-72-h p.t. period; (3) each MYC-positive peptide was cytotoxic, but each in a distinctive way; and (4) while the selective targeting of FL/MYC to dense bodies, adhesion plaques, adherens junctions, and ruffled membranes was consistent with binding studies in cell-free systems, the incorporation of the mutated peptides, particularly +A/MYC and -A/MYC was not. Changes in localization over time and the distinctive cytopathologies probably reflect domain-specific targeting. They also suggest unexpected cooperative involvement of multiple domains of alpha-actinin with specific receptors in distal cytoskeletal structures. To date, such qualitative in vivo interactions have not been described either in in vitro binding studies, or in short-term experiments involving localization and/or fate of microinjected labeled molecules into living cells.

Functional morphology of serially linked skeletal muscle fibers.

In the skeletal muscle fiber organization of many vertebrate muscles, serial arrangements or linkages of muscle fibers along the muscle or fascicle are commonly found. These serially linked muscle fibers employ distinct junctional morphologies from muscle to muscle. Notable are the end-to-end linkages of muscle fibers through tendinous intersections (TIs), where many fibers end onto a continuous connective tissue plate with folded terminations similar to myotendinous junctions. Besides this end-to-end linkage, overlapping linkages or arrangements occur among nonspanning fibers terminating intrafascicularly. These nonspanning fibers bear tapering terminations with direct cell-cell (myomuscular) junctions or without any specialized junctions. Despite their overlapping linkages or tapering profiles, nonspanning fibers maintain a uniform sarcomere length along the linked fibers, suggesting that the overlapping-linked nonspanning fibers are equivalent to the end-to-end linked fibers in their mechanical capacity. However, the junctional compliance could differ in their extracellular elastic components and their organization at junctional sites, e.g., direct mechanical (myomuscular) junctions vs. indirect linkages through connective tissue. Increasing evidence suggests that the elastic components, including muscle fibers as well as connective tissues, are more critical than previously thought for the mode and/or the efficiency of tension transmission among serially arranged fibers and thus for the mechanical properties of the muscle.

Truncated desmin in PtK2 cells induces desmin-vimentin-cytokeratin coprecipitation, involution of intermediate filament networks, and nuclear fragmentation: a model for many degenerative diseases.

The earliest expression of truncated desmin in transfected PtK2 cells results in the formation of dispersed microprecipitates containing not only the truncated desmin, but also endogenous vimentin and cytokeratin proteins. Desmin microprecipitates without vimentin or vimentin microprecipitates without desmin are not observed. The microprecipitates involving cytokeratin invariably are also positive for desmin and vimentin. Over time, the precipitates enlarge into 1- to 2-microns spheroids and then fuse into amorphous chimeric juxtanuclear masses that can occupy > 30% of the cell volume. Concurrently, first the vimentin and then the cytokeratin networks are resorbed. The chimeric precipitates are not recognized or marked for degradation by the lysosomal system. Ultimately the cell nucleus fragments and the cell dies. Similar protein complexes appear in many human and animal pathologies, suggesting that a similar protein-precipitation sequence initiated by the introduction of a mutationally or environmentally altered protein molecule is at work.

Functional combination of tapering profiles and overlapping arrangements in nonspanning skeletal muscle fibers terminating intrafascicularly.

Using digital image analysis and several anatomical methods, morphometric analysis of nonspanning fibers which had tapering profiles at their intrafascicular termination sites and represented overlapping arrangements within the fiber fascicles was performed in the rat rectus abdominis. Special emphasis was focused on dimensional relationships occurring between overlapping portions and tapering segments and sarcomere lengths in non- and overlapping portions. Nonspanning fibers were found to overlap each other for more than 40% of their length. In length, their overlapping portions generally corresponded to their tapering segments, which were also greater than 40% of the fiber length. In addition, despite the presence of overlapping linkages, nonspanning fibers maintained a fairly uniform length irrespective of their overlapping and nonoverlapping portions. Overlapping linkages in fibers without tapering profiles have a larger cross-sectional area in the overlapping portion than in the non-overlapping one, resulting in a phenomenon which will cause different sarcomere lengths between the two portions during fiber stretching. The present results suggest that tapering profiles in the overlapping portion ensure uniform sarcomere lengths within nonspanning fibers, thereby providing mechanical stability in each fiber.

Architectural design, fiber-type composition, and innervation of the rat rectus abdominis muscle.

The rectus abdominis muscle is architecturally compartmentalized by tendinous intersections and is supplied by multiple thoracic nerves. In this study, the rectus abdominis of the rat has been qualitatively and quantitatively examined with regard to muscle dimensions, fiber organization, fiber-type composition, and innervation. The muscle exhibits architectural heterogeneity and different patterns of innervation among its thoracic, epigastric, and hypogastric parts. The epigastric part, adherent to the rectus sheath via tendinous intersections, represents relatively simple design. It is formed by serially arranged compartments with shorter fibers, compared with the other parts. These compartments are segmentally supplied by thoracic nerves. The hypogastric part is more complex, forms an interdigitation of muscular slips, and has segmental distribution of thoracic nerves in mediolateral direction. The thoracic part much differs from the other parts. It has smaller cross-sectional areas, compartments composed of abundant nonspanning fibers with intrafascicular termination, and non-segmental distribution of thoracic nerves. In addition to these craniocaudal specializations among the three parts, the muscle exhibits mediolateral differences in fiber-type composition. Slow-twitch oxidative fibers are more densely distributed in the medial half region than the lateral, whereas fast-twitch glycolytic fibers follow an inverse pattern. The mediolateral differences in fiber-type composition as well as the craniocaudal specializations in architectural design and innervation imply regionally differentiated recruitments of the muscle in various behaviors.

Structural heterogeneity of the basement membrane in the rat proximal tubule.

The ultrastructure of the basement membrane of the rat proximal tubule was observed by transmission electron microscopy after the use of a cold dehydration technique. The basement membrane of the P1 segment is thick and possesses several structural specializations that are rare in other basement membranes; these include intraepithelial ridges, dense bars, and basement membrane vesicles. The intraepithelial ridges are found in the intercellular spaces between interdigitating processes of the proximal tubule cells. The ridges and the interdigitating processes run circumferentially around the tubule. The dense bars are frequently found in the intraepithelial ridges. They are especially prominent on the concave side of the tubular bends and to a lesser extent near sites where intracellular actin filaments anchor onto the basal cell membranes. The basement membrane vesicles are bounded by unit membranes; they are variable in both their electron density and their size. They are usually found in association with dense bars, and the grade of their accumulation is positively correlated with the development of the dense bars. These three specializations have no topographical relationship with the interstitial structures, such as fibroblasts and collagen fibrils. The specializations are best developed on the concave side of tubular bends where the circumferential stresses caused by the intraluminal hydraulic pressure are presumably the largest; we therefore propose that they are an adaptation to, or a manifestation of, the increased wall stress in the proximal tubule.

In vivo evaluation of fixation of metal implants coated by melt-sprayed alumina--experiments in rabbit.

Skeletal fixation characteristics of the stainless steel implants coated by 60 microns thick porous alumina layer using a flame spray method were evaluated in vivo for the duration of up to 12 weeks. Surface roughness of the implants appeared to be the determining factor for the fixation characteristics, irrespective of the implant materials.

Lower axial skeleton of the musk shrew (Suncus murinus).

Macroscopic structure as well as pre- and postnatal development of the lumbar, sacral, and caudal vertebrae of the musk shrew (Suncus murinus, Insectivora) were observed. The lumbar vertebrae possess two pairs of unusual processes, hyperapophyses and hypapophyses. The hyperapophyses are located on the dorsal surface of the caudal articular processes of all the lumbar vertebrae, whereas the hypapophyses are found on the caudal part of the ventral surface of the bodies in the first few lumbar vertebrae. The former gives attachment to the Mm. rotatores lumborum and the latter to the Mm. psoas major and minor. The articular processes of the lumbar vertebrae are oriented more horizontally compared with those in other mammals. The sacrum is very narrow transversely due to poor development of the ventrolateral wing. The auricular surface includes cranial parts of the wing and of the fused vertebral arches as well as the cranial articular process of the first sacral vertebra. In the caudal vertebrae, chevron bones are H-shaped when viewed ventrally, and give attachment to tendons of the caudal muscles. This report describes the relationships between the structural peculiarities of the lower axial skeleton and the locomotive habits of the musk shrew.

Serum constituents and liver photomicrographs of wild Suncus murinus in the south of Taiwan.

Serum constituents and liver of the wild male suncus as well as of those bred and fed in our laboratory were examined serologically and histologically. The following results were obtained: The serum glutamic-oxaloacetic transaminase, gamma-glutamyl transaminase, inorganic phosphate, blood urea nitrogen, creatinine, uric acid, and zinc turbidity test levels of wild suncus were higher than those of 4 and 8 week old fed suncus. The total cholesterol level of wild suncus was lower than those of 4 and 8 week old fed suncus. In wild suncus, the total bilirubin and direct bilirubin levels were higher, and the Ca and albumin levels were lower than those of 4 week old fed suncus. However, no significant differences were observed between the wild and the 8 week old fed suncus. Although the alkaline phosphatese and thymol turbidity test levels of wild suncus were larger than those of 8 week old fed suncus, no significant differences were observed between the wild and the 4 week old fed suncus. The histological study revealed the presence of fatty droplets in only 2 of the 67 wild suncus examined while fatty droplets (grade +) were observed in almost all the fed suncus. In one case of the wild suncus, moderate round cell infiltration in interlobular connective tissue was found.