Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene

Fast skeletal muscles of mdx (X chromosome-linked muscular dystrophy) mice were injected after birth with a recombinant adenovirus containing a minidys- trophin gene, a 6.3-kbp cDNA coding for the N- and C-terminal ends of dystrophin. Adult muscles were challenged by forced lengthening during tetanic contractions. Stretch-induced mechanical and histological damages were much reduced in injected muscles, in direct proportion of the Miniber of fibers expressing minidystrophin. Damaged fibers were preferentially found among minidystrophin-negative regions. Minidystrostrophin confers an important functional and structural protection of limb muscles against high mechanical stress, even after a partial somatic gene transfer.     

Adenovirus-mediated dystrophin minigene transfer improves muscle strength in adult dystrophic (MDX) mice

Tablets have been prepared at known compaction force from three types of pellets in different proportions: (1) pellets containing 80% theophylline as a model drug coated with different thicknesses of a polymer film coat, (2) pellets containing glyceryl monostearate as a deformable material, and (3) pellets containing a disintegrant. The breaking load, friability, disintegration and drug release properties were evaluated, the latter as a mean dissolution time (MDT) and its variance (VDT). The mechanism of dissolution was assessed from the value of the relative dispersion (RD) of the mean dissolution time. The possible relationship between the properties of the pellets and those of the tablets was evaluated by canonical analysis followed by multiple regression analysis. It was found that only about 51% of the tablet properties could be predicted from the properties of the pellets. The quantitative relationship between pellet properties and tablet properties was found to vary in type and level of quality. Reduction of breaking load, friability and disintegration was less predictable than that of dissolution represented as the MDT. The values of RD for the different preparations clearly identified those preparations where the film coat still retained control of the dissolution process. Such formulations contained at least 40% of soft pellets and coated pellets with at least a weight gain of 8%.     

Developmental expression of dystrophin, dystrophin-associated glycoproteins and other membrane cytoskeletal proteins in human skeletal and heart muscle

Dystrophin, utrophin and the dystrophin-associated glycoproteins, beta-dystroglycan and adhalin, were analyzed, together with the membrane cytoskeletal proteins beta-spectrin, vinculin and talin, and adult and fetal myosin heavy chains, in 25 normal human fetuses from 8 to 24 weeks of gestation. Dystrophin was present in heart and skeletal muscle from 8 weeks although in the latter was mainly in the cytoplasm at this stage. Utrophin expression increased until around gestational weeks 19/21, but by 24 weeks immunostaining and immunoblot band intensities had reduced. Beta-dystroglycan was scarce in skeletal muscle at 8 weeks, increased with maturation and was more abundant in heart of the same age. Adhalin appeared later than beta-dystroglycan on skeletal muscle fiber surfaces, positivity became more intense as the fibers matured. In heart adhalin was detectable only in groups of cells at 12-16 weeks. From 8 weeks all fetal myotubes expressed beta-spectrin on their surfaces, while vinculin and talin positivity was mainly at the periphery of the fascicles, increasing with age. Adult slow myosin was seen in most myotubes at 10 weeks. Secondary myotubes then formed which increasingly expressed adult fast myosin, while still retaining fetal myosin. By 24 weeks most fibers expressing adult slow myosin had lost fetal myosin and were more mature in the expression of most membrane proteins. Muscle membrane organization during human fetal development is a complex process and takes place earlier in heart than skeletal muscle.     

Decreased proportion of type I myofibers in skeletal muscle of dogs with chronic heart failure

BACKGROUND: Whether biochemical and histological abnormalities of skeletal muscle (SM) develop in patients with chronic heart failure (HF) remains controversial. In the present study, dogs with chronic HF were used to examine potential alterations of SM fiber type, fiber size, number of capillaries per fiber (C/F), beta-adrenergic receptor density (Bmax), and fiber ultrastructural integrity. METHODS AND RESULTS: HF was produced in 17 dogs by sequential intracoronary microembolizations. Biopsies of the lateral head of the triceps muscle were used in all studies. Type I and type II fibers were differentiated by myofibrillar ATPase (pH 9.4 or 4.2). Bmax was assessed by radioligand binding and SM ultrastructure by transmission electron microscopy. Comparisons were made with biopsies obtained from nine control dogs. The percentage of SM type I fibers was reduced in HF dogs compared with control dogs (19 +/- 2% versus 32 +/- 5%) (p < 0.001), whereas the percentage of SM type II fibers was increased (81 +/- 2% versus 68 +/- 5%) (p < 0.001). The change in fiber type composition was not associated with a preferential atrophy or hypertrophy of either fiber type. There was no difference in SM Bmax (198.9 +/- 14.3 versus 186.8 +/- 17.3 fmol/mg protein) or in C/F (5.37 +/- 0.26 versus 5.84 +/- 0.21) between HF dogs and control dogs. No ultrastructural abnormalities were present in SM fibers of HF dogs. CONCLUSIONS: In dogs with HF, there is a decrease in the relative composition of the slow-twitch type I SM fibers and an increase in fast-twitch type II fibers. The shift in fiber type composition is not associated with preferential atrophy of either fiber type or with a reduction in C/F, beta-adrenergic receptor density, or structural abnormalities of the myofibers.     

Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts: method and efficacy of adenovirus-mediated transduction

OBJECTIVE: Allograft-targeted immunosuppressive gene therapy may inhibit recipient immune activation and provide an alternative to systemic immunosuppression. We studied the optimal technique and efficacy of intracoronary gene transfer of viral interleukin-10 and human transforming growth factor-beta 1 in a rabbit model of heterotopic heart transplantation. METHODS: Replication-defective adenoviral vectors were constructed, expressing viral interleukin-10 (AdSvIL10) or transforming growth factor-beta 1 (AdCMVTGF-beta 1). Intracoronary delivery of vectors was accomplished ex vivo by either bolus injection or slow infusion. The allografts were implanted heterotopically in recipient rabbits and collected 4 days after the operation. Vector dose was 4 x 10(9) to 6 x 10(10) pfu/gm of donor heart. Transfer was confirmed by DNA amplification for both genes. Gene product expression in tissue was quantified by immunoassay and visualized by immunohistochemical staining. RESULTS: Allograft viral uptake was only 9.9% +/- 2.4% with bolus injection, but increased to 80.5% +/- 6.8% at 1 ml/min infusion rate (p = 5 x 10(-14)). Uptake ratio was not affected by vector quantity or slower infusion rates. Transforming growth factor-beta 1 was consistently detected in allografts infected with AdCMVTGF-beta 1, but not with control adenovirus or AdSvIL10. Expression was proportional to infused vector quantity and reached 10 ng/gm of allograft at infused 10(10) pfu/gm. Transforming growth factor-beta 1 was also detected in recipient's serum at less than 1 ng/ml. Viral interleukin-10 was detected in minor amounts only (< 1 ng/gm) in allografts infected with AdvIL10 up to 5 x 10(10) pfu/gm. Nevertheless, it was detected in recipient serum at concentrations up to 0.4 ng/ml. CONCLUSIONS: Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts during cold preservation is feasible. Slow infusion is superior to bolus injection. In vivo effects on allograft rejection remain to be determined.     

Position-dependent variegation of a CD4 minigene with targeted expression to mature CD4+ T cells

The CD4 gene follows a complex and highly regulated pattern of expression throughout T cell development. This expression is governed by different regulatory elements that have been partly identified, including a promoter, a proximal enhancer, and a silencer. Here we show that a CD4 minigene comprising a combination of these elements is specifically expressed in mature CD4+ T cells of transgenic mice, but not in CD4+CD8+ double positive thymocytes. The proportion of transgene-expressing CD4+ T cells was constant within a given transgenic line, but varied greatly from one line to another. We demonstrate that this pattern of expression is due to integration of the transgene within or in the vicinity of centromeric heterochromatin. This position-effect variegation demonstrated with a short CD4 transgene has not been observed with larger ones containing additional regulatory sequences, suggesting that the CD4 gene contains a locus control region. Such position-dependent effects must be taken into consideration when developing transgenic models or gene transfer vectors because they can result in the absence of transgene expression in a subpopulation of target cells. Finally, the combination of the CD4 gene silencer, proximal enhancer, and promoter provides an interesting tool to selectively express genes of interest in mature CD4+ T cells of transgenic mice and for the development of gene therapy vectors.     

Suppression of proliferative cholangitis in a rat model with direct adenovirus-mediated retinoblastoma gene transfer to the biliary tract

Two genes for Ca2+-dependent protein kinases, PCaPK-alpha and PCaPK-beta, were isolated from a Paramecium genomic DNA library. The coding region of PCaPK-alpha encoded 481 amino acids and that of PCaPK-beta encoded 493 amino acids, predicting molecular masses of 55603 Da and 57131 Da for each putative protein. The sequences of the protein kinase catalytic domains of PCaPK-alpha and PCaPK-beta were closely related to those of the Ca2+-dependent protein kinases (CDPKs) from Plasmodium, Eimeria, and several plants, and the catalytic region of the Ca2+/calmodulin-dependent protein kinase family (35-48% identity). In the junction region between the catalytic and regulatory regions, only 9 of 31 amino acid residues are the same in the two Paramecium genes, and the sequences encoded in the Paramecium genes differ from those in the plant CDPK genes in about 20 of 31 residues in the junction region. The C-terminal region of the Paramecium kinases shared sequence similarity with Paramecium calmodulin (30-34% identity). Two Ca2+-dependent protein kinases previously characterized from Paramecium (52 kDa CaPK-1, and 50 kDa CaPK-2) are activated by Ca2+ in the micromolar concentration range and they directly bind Ca2+ in a 45Ca2+ overlay blot assay. The size predicted from the genes, the presence of four putative Ca2+-binding motifs encoded in PCaPK-alpha and PCaPK-beta, and the immunological cross-reaction of expressed cloned fragments of these genes with CaPK-2, suggest that they encode proteins of the same family.     

Apoptosis by retrovirus- and adenovirus-mediated gene transfer of Fas ligand to glioma cells: implications for gene therapy

Astrocytic tumors frequently express Fas/APO-1 (Fas), in sharp contrast to surrounding normal brain cells, providing a potential window through which selective killing of tumor cells could be pursued. To assess this possibility, we transduced Fas into U251, a glioma cell line resistant to anti-Fas antibody-mediated apoptosis, and obtained transfectants with high levels of Fas expression. Anti-Fas antibody showed significantly enhanced cytotoxicity for the transfectants, suggesting that U251 cells maintained an intercellular cascade of Fas-mediated apoptosis. When U251 transfectants with high-level Fas expression were transduced with Fas ligand-encoding gene via retrovirus, they were unaffected by exposure to anti-Fas antibody or Fas ligand adenovirus (Adeno-FL). Thus, retroviral induction of Fas ligand into the glioma cells with high levels of Fas led to the selection of cells that were resistant to Fas-dependent apoptosis. These resistant U251 transfectants were susceptible to FADD adenovirus (Adeno-FADD)-induced apoptosis, indicating that a cascade of death signals was blocked at the steps between Fas ligand and FADD. As for adenoviral transduction of Fas ligand into gliomas, gliomas with a relatively high level of expression of Fas were remarkably sensitive to Adeno-FL-induced apoptosis. Besides, Adeno-FADD induced pronounced apoptosis in all glioma cells. Our data suggest the possibility of using adenovirus-mediated transduction of Fas ligand and FADD genes as a therapeutic approach to target gliomas.     

Transient changes in growth and in calpain and calpastatin expression in ovine skeletal muscle after short-term dietary inclusion of cimaterol

Prolonged dietary inclusion of beta-adrenergic agonists can induce skeletal muscle hypertrophy in meat animals, by a mechanism probably related to the calcium-dependent proteolytic enzymes, or calpains, and in particular to their specific inhibitor calpastatin. Calpain and calpastatin activities are also believed to be important factors during post-mortem tenderisation of meat. beta-Agonist treatment is generally associated with increased calpastatin activity, which may lead to meat toughness. The aim of the present study was to examine the effect of a short period of cimaterol (feeding for 8 days, followed by reversion to a normal diet for a further 24 days) on muscle growth and on calpain isoform and calpastatin activities and specific mRNA abundance in the longissimus dorsi (LD) muscle. Significant changes were detected in LD wet weight and in calpastatin activity and mRNA after only 8 days treatment with cimaterol. After 24 further days on a control diet, both LD wet weight and calpastatin activity were not significantly different (P > 0.05) from untreated controls of the same age, although calpastatin mRNA stayed surprisingly high. In contrast to several earlier studies, changes in calpain I (or mu-calpain) and calpain II (or m-calpain) activity and calpain I mRNA were not significantly different (P > 0.05) from controls in any groups. These data suggest that calpastatin activity rather than the activity of either calpain isoform is closely linked to beta-agonist-induced muscle hypertrophy. Changes in calpastatin mRNA are not directly proportional to inhibitory activity, suggesting that variable mRNA species may be transcribed, spliced or stabilised, but not necessarily translated as part of the beta-agonist response.     

Adenovirus-mediated transfer of human acid maltase gene reduces glycogen accumulation in skeletal muscle of Japanese quail with acid maltase deficiency

Acid maltase deficiency (AMD) causes a lysosomal glycogenosis inherited as an autosomal recessive trait. The infantile type of AMD (Pompe disease) leads to early death due to severe dysfunction of cardiac and respiratory muscles and no effective therapy is available. Replication-defective adenovirus vectors offer a promising tool for in vivo gene delivery and gene therapy. We constructed a recombinant adenovirus containing the human acid maltase (AM) cDNA downstream of the CAG promoter, composed of modified chicken beta-actin promoter and CMV IE enhancer (AxCANAM). Japanese quail with AMD was used for this study as an animal model for human AMD. When cultured fibroblasts from AMD quail were infected with AxCANAM, AM activity in the cells increased in proportion to the multiplicity of infection (MOI). When AxCANAM (4.5 x 10(8) PFU) was injected into unilateral superficial pectoral muscle of AMD quail, PAS staining showed that glycogenosomes disappeared and stainability of acid phosphatase was reduced in the injected area as compared with the contralateral muscle of the same birds. Biochemically, AM activity increased and glycogen content decreased in the injected muscle. Western blot analysis showed that AMD quail muscle injected with AxCANAM expressed human AM protein processed to active forms. These results suggest that the human AM cDNA transferred by an adenovirus vector was sufficiently expressed, leading to a marked reduction of the glycogen accumulation in the skeletal muscle of AMD quail.     

Improved exercise tolerance after losartan and enalapril in heart failure: correlation with changes in skeletal muscle myosin heavy chain composition

BACKGROUND: In congestive heart failure, fatigue-resistant, oxidative, slow type I fibers are decreased in leg skeletal muscle, contributing to exercise capacity (EC) limitation. The mechanisms by which ACE inhibitors and AII antagonists improve EC is still unclear. We tested the hypothesis that improvement in EC is related to changes in skeletal muscle composition toward type I fibers. METHODS AND RESULTS: Eight patients with congestive heart failure, NYHA classes I through IV, were treated for 6 months with enalapril (E) 20 mg/d, and another 8 with losartan (L) 50 mg/d. EC was assessed with maximal cardiopulmonary exercise testing at baseline and after treatment. Myosin heavy chain (MHC) composition of the gastrocnemius was studied after electrophoretic separation of slow MHC1, fast oxidative MHC2a, and fast glycolytic MHC2b isoforms from needle microbiopsies obtained at baseline and after 6 months. EC improved in both groups. Peak V(O2) increased from 21.0+/-4.7 to 27.6+/-4.3 mL . kg-1 . min -1 (P=0.011) in the L group and from 17.5+/-5.0 to 25.0+/-5.5 mL . kg-1 . min -1 (P=0.014) in the E group. Similarly, ventilatory threshold changed from 15.0+/-4.0 to 19.9+/-4.9 mL (P=0. 049) with L and from 12.0+/-1.9 to 15.4+/-3.5 mL (P=0.039) with E. MCH1 increased from 61.2+/-11.2% to 75.4+/-7.6% with L (P=0.012) and from 60.6+/-13.1% to 80.1+/-10.9% (P=0.006) with E. Similarly, MHC2a decreased from 21.20+/-9.5% to 12.9+/-4.4% (P=0.05) with L and from 19.9+/-7.8% to 11.8+/-7.9% (P=0.06) with E. MHC2b changed from 17. 5+/-6.5% to 11.7+/-5.2% (P=0.07) with L and from 19.5+/-6.4% to 8. 1+/-4.6% (P=0.0015) with E. There was a significant correlation between net changes in MHC1 and absolute changes in peak V(O2) (r2=0.29, P=0.029) and a trend to significance for MHC2a and 2b. CONCLUSIONS: Six months' treatment with L and with E produces an improvement in EC of similar magnitude. These changes are accompanied by a reshift of MHCs of leg skeletal muscle toward the slow, more fatigue-resistant isoforms. Magnitude of MHC1 changes correlates with the net peak V(O2) gain, which suggests that improved EC may be caused by favorable biochemical changes occurring in the skeletal muscle.     

The effects of pretreatment with soman simulator in the skeletal muscle: direct interactions with acetylcholinesterase

Soman simulator PDP is a compound that has a chemical structure identical to soman, except that the fluorine atom is replaced by a methyl group which makes PDP unable to bind covalently to the AChE active center. In rats, late mortality observed after treatment with high doses of soman could be prevented by PDP pretreatment. Such pretreatment has been much less efficient in primates. The effect of PDP in rats has been explained by blocking the deposition of soman in so-called soman depots in which soman is stored intact and subsequently released. In this paper we demonstrate that in the presence of PDP, inhibition of rat muscle AChE by soman is reduced in rat but not in human muscle homogenates. This result suggests that at least part of the beneficial effects of PDP pretreatment in rat might be due to the direct interaction of PDP with AChE resulting in reduced AChE phosphorylation by soman.     

Brain nucleic acid composition and fractional rates of protein synthesis in response to chronic ethanol feeding: comparison with skeletal muscle

Soybeans were treated with proteases to reduce allergenicity. By immunoblotting with a monoclonal antibody against a major soybean allergen (Gly m Bd 30K), two of eight proteases so far tested were selected to achieve a reduction in allergenicity. Both antigenicity to the monoclonal antibody and allergenicity to the sera from soybean-sensitive patients proved to be markedly reduced by processing with either protease. Thus, soybeans treated with an appropriate protease may possibly be supplied as a hypoallergenic foodstuff for patients.     

Increased gene expression after liposome-mediated arterial gene transfer associated with intimal smooth muscle cell proliferation. In vitro and in vivo findings in a rabbit model of vascular injury

Arterial gene transfer represents a novel strategy that is potentially applicable to a variety of cardiovascular disorders. Attempts to perform arterial gene transfer using nonviral vectors have been compromised by a low transfection efficiency. We investigated the hypothesis that cellular proliferation induced by arterial injury could augment gene expression after liposome-mediated gene transfer. Nondenuded and denuded rabbit arterial strips were maintained in culture for up to 21 d, after which transfection was performed with a mixture of the plasmid encoding firefly luciferase and cationic liposomes. In non-denuded arteries, the culture interval before transfection did not affect the gene expression. In contrast, denuded arteries cultured for 3-14 d before transfection yielded 7-13-fold higher expression (vs. day 0; P < 0.005). Transfection was then performed percutaneously to the iliac arteries of live rabbits with or without antecedent angioplasty. Gene expression increased when transfection was performed 3-7 d postangioplasty (P < 0.05). Proliferative activity of neointimal cells assessed in vitro by [3H]thymidine incorporation, and in vivo by immunostaining for proliferating cell nuclear antigen, increased and declined in parallel with gene expression. These findings thus indicate that the expression of liposome-mediated arterial gene transfer may be augmented in presence of ongoing cellular proliferation.     

Projections of sympathetic non-noradrenergic neurons to skeletal muscle arteries in guinea-pig limbs vary with the metabolic character of muscles

This study set out to examine in detail the distribution of axons of sympathetic non-noradrenergic neurons innervating the arterial bed in skeletal muscles of the forelimb and hindlimb of guinea-pigs. The distribution of non-noradrenergic axons with immunoreactivity to vasoactive intestinal peptide (VIP) was examined in limb muscles of different histochemical character. The immunohistochemical demonstration of myosin heavy chain from fast-twitch muscle, and the histochemical demonstration of adenosine triphosphatase and succinic dehydrogenase, were used to determine the muscle fibre profile of 6 different limb muscles. Muscles included the oxidative type I muscle fibre-rich accessory semimembranosus muscle, the predominantly glycolytic type II muscle fibre-rich cranial gracilis and biceps brachii muscles and the plantaris, gastrocnemius medial head and triceps brachii long head of mixed muscle fibre composition. The frequency with which the VIP-immunoreactive (VIP-IR) axons innervated intramuscular arterial vessels was compared between categories of muscles defined by their muscle fibre profile. This study demonstrated that the projection of non-noradrenergic sympathetic neurons to skeletal muscle vasculature was widespread in guinea-pig limb muscles, but that it was not uniform. VIP-IR axons were more likely to innervate the arterial vasculature of muscles with a high proportion of type I and/or oxidative muscle fibres than of muscles with a large proportion of type IIb muscle fibres. This relationship between the distribution of sympathetic non-noradrenergic axons and the metabolic characteristics of muscle suggests that these presumed vasodilator neurons have an important role in matching blood flow to the particular metabolic demands of different limb muscles.     

Myotonia dystrophica with heart involvement: an electron microscopic study of skeletal, cardiac, and smooth muscle

The electron microscopic features of the striated skeletal muscle, the striated cardiac muscle, and the smooth muscle from a woman who had been suffering for many years from myotonia dystrophica with cardiac involvement are described. The skeletal muscle was studied at two different stages of the disease. In the first material the main changes consisted of centrally situated nuclei, disorganisation of the sarcomeres, and focal disruption of the Z-line. The satellite cells were well represented. Three years later atrophy and degenerative, necrotic changes of the skeletal muscle were evident. The satellite cells were absent. Few changes were seen in the striated cardiac muscle. These consisted of slight interstitial fibrosis and large accumulations of mitochondria with intramitochondrial dense granules. The smooth muscle cells of the oesophagus showed disorientated filaments and mild degenerative changes. It is concluded that the skeletal muscle was more severely affected than the other types of muscle.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.