Mechanical strain increases gene transfer to skeletal muscle cells

Gene transfer techniques possess tremendous potential for treating diseases and for facilitating the study of basic physiological processes. However, further development of efficient and safe methods for gene transfer is needed. The purpose of this study was to test the hypothesis that mechanical strain increases the transfer of DNA to differentiated skeletal muscle cells. We tested this hypothesis by applying cyclic strain to cultured skeletal myotubes either prior to or immediately after the introduction of exogenous DNA complexed with lipids, with strains of varying magnitude (10%, 20% and 30%), number (1800, 3600 and 7200 strain cycles) and frequency (0.5, 1.0 and 1.5 Hz). Results demonstrated that DNA transfection was increased by exposing muscle cells to cyclic strain, and that strain magnitude, number and frequency each influenced DNA transfection. Optimal strain conditions (20% strain magnitude, 3600 cycles applied at 1 Hz) were utilized to examine the role of membrane transport systems in strain-induced increases in DNA transfection. Filipin III was used to inhibit caveolar transport and was found to inhibit strain-mediated increases in DNA transfection, whereas chlorpromazine, used to inhibit clathrin-coated vesicle transport, had no effect. These results indicate that mechanical strain may be an effective method for increasing DNA transfection in skeletal muscle through enhanced caveolar transport.     

Systemic administration of naked DNA: gene transfer to skeletal muscle

Skeletal muscle is a promising target tissue for the gene therapy of both muscle and non-muscle disorders. Gene transfer into muscle tissue can produce a variety of physiologically active proteins and may ultimately be applied to the treatment of many diseases. A variety of methods have been studied to transfer genes into skeletal muscle, including viral and non-viral vectors. In this review, we discuss recent developments in the non-viral delivery of genes to muscles.     

Isoproterenol ameliorates workstress-induced rat skeletal muscle degeneration

Beta-Agonists though have been widely studied for their protein anabolic effects in skeletal muscles, but the lipid status under work stress and agonist treatment have not been understood well in the skeletal muscles and heart of rat. In the present study, adult male Wistar rats were subjected to work overload stress and beta agonist isoproterenol treatment (2 mg kg(-1) day(-1) intraperitoneally) to examine, whether it attenuates work stress-induced changes or not. Simultaneously, beta2 antagonist butoxamine (2 mg kg(-1) day(-1) intraperitoneally) was administered to another isoproterenol-treated group. Work stress led to myofibrillar degeneration as well as rapid utilization of lipid to meet increased energy demands and for muscle repair, which was reflected through histochemical localization of lipids and biochemical estimation of cholesterol and triglycerides. Significantly decreased cholesterol levels in skeletal muscles and heart muscles were noticed. As expected, isoproterenol reversed the conditions by raising cholesterol and triglyceride levels significantly in the skeletal muscles and also by ameliorating the degenerative changes in muscle fibres as induced by work overload. However, severe accumulation of lipids in heart infers towards deleterious effects of isoproterenol on heart and thus remains a limiting factor for its immediate clinical application. Further research is needed to separate desirable effects of beta agonists on skeletal muscles from any undesirable effects on the heart, so as to optimize their therapeutic potential.     

Guava leaves extract ameliorates STZ induced diabetes mellitus via activation of PI3K/AKT signaling in skeletal muscle of rats

Molecular Biology Reports - Blood glucose homeostasis and insulin signaling pathway regulation take a vital role in the management of diabetes mellitus. Our present was designed to explore the...     

Nonviral gene transfer to skeletal, smooth, and cardiac muscle in living animals

The study of muscle physiology has undergone many changes over the past 25 years and has moved from purely physiological studies to those intimately intertwined with molecular and cell biological questions. To ask these questions, it is necessary to be able to transfer genetic reagents to cells both in culture and, ultimately, in living animals. Over the past 10 years, a number of different chemical and physical approaches have been developed to transfect living skeletal, smooth, and cardiac muscle systems with varying success and efficiency. This review provides a survey of these methods and describes some more recent developments in the field of in vivo gene transfer to these various muscle types. Both gene delivery for overexpression of desired gene products and delivery of nucleic acids for downregulation of specific genes and their products are discussed to aid the physiologist, cell biologist, and molecular biologist in their studies on whole animal biology. electroporation; liposomes; plasmids; transfection; gene expression     

Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass

BACKGROUND: Myostatin negatively regulates skeletal muscle growth. Myostatin knockout mice exhibit muscle hypertrophy and decreased interstitial fibrosis. We investigated whether a plasmid expressing a short hairpin interfering RNA (shRNA) against myostatin and transduced using electroporation would increase local skeletal muscle mass. METHODS: Short interfering RNAs (siRNAs) targeting myostatin were co-transfected with a myostatin-expressing plasmid into HEK293 cells and identified for myostatin silencing by Western blot. Corresponding shRNAs were cloned into plasmid shRNA expression vectors. Myostatin or a randomer negative control shRNA plasmid was injected and electroporated into the tibialis anterior or its contralateral muscle, respectively, of nine rats that were sacrificed after 2 weeks. Six other rats received a beta-galactosidase reporter plasmid and were sacrificed at 1, 2, and 4 weeks. Uptake of plasmid was examined by beta-galactosidase expression, whereas myostatin expression was determined by real-time polymerase chain reaction (PCR) and Western blotting. Muscle fiber size was determined by histochemistry. Satellite cell proliferation was determined by PAX7 immunohistochemistry. Myosin heavy chain type II (MHCII) expression was determined by Western blot. RESULTS: beta-Galactosidase reporter plasmid was expressed at 1 and 2 weeks but diminished by 4 weeks in tibialis anterior skeletal muscle. Myostatin shRNA reduced myostatin mRNA and protein expression by 27 and 48%, respectively. Tibialis anterior weight, fiber size, and MHCII increased by 10, 34, and 38%, respectively. Satellite cell number was increased by over 2-fold. CONCLUSIONS: This is the first demonstration that myostatin shRNA gene transfer is a potential strategy to increase muscle mass.     

Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle

Duchenne muscular dystrophy (DMD) is an X-linked, lethal, degenerative disease that results from mutations in the dystrophin gene, causing necrosis and inflammation in skeletal muscle tissue. Treatments that reduce muscle fiber destruction and immune cell infiltration can ameliorate DMD pathology. We treated the mdx mouse, a model for DMD, with the immunosuppressant drug rapamycin (RAPA) both locally and systemically to examine its effects on dystrophic mdx muscles. We observed a significant reduction of muscle fiber necrosis in treated mdx mouse tibialis anterior (TA) and diaphragm (Dia) muscles 6 wks post-treatment. This effect was associated with a significant reduction in infiltration of effector CD4(+) and CD8(+) T cells in skeletal muscle tissue, while Foxp3(+) regulatory T cells were preserved. Because RAPA exerts its effects through the mammalian target of RAPA (mTOR), we studied the activation of mTOR in mdx TA and Dia with and without RAPA treatment. Surprisingly, mTOR activation levels in mdx TA were not different from control C57BL/10 (B10). However, mTOR activation was different in Dia between mdx and B10; mTOR activation levels did not rise between 6 and 12 wks of age in mdx Dia muscle, whereas a rise in mTOR activation level was observed in B10 Dia muscle. Furthermore, mdx Dia, but not TA, muscle mTOR activation was responsive to RAPA treatment.     

Functional in vivo gene transfer into the myofibers of adult skeletal muscle

The postmitotic nature and longevity of skeletal muscle fibers permit stable expression of any transfected gene. Direct in vivo injection of plasmid DNA, in both adult and regenerating muscles, is a safe, inexpensive, and easy approach. Here we present an optimized electroporation protocol based on the use of spatula electrodes to transfer cDNA in vivo into the adult myofibers of an anatomically defined muscle, which could be functionally characterized. In our hands, about 80% of adult myofibers were transfected in vivo by different plasmids for GFP fusion proteins or for beta-galactosidase. The luciferase activity increased several orders of magnitude when compared to standard DNA delivery. In an anatomical defined muscle, the wide gene transfer was comparable to or better than that of retrovirus delivery, that recently has been shown to be prone to severe side-effects in human clinical studies. Furthermore, with our method the tissue damage was greatly decreased. Thus, the present work describes in vivo functional electrotransfer of genes in adult skeletal muscle fibers by a protocol that is of great potential for gene therapy, as well as for basic research.     

Optimization of ectopic gene expression in skeletal muscle through DNA transfer by electroporation

Background  

Electroporation (EP) is a widely used non-viral gene transfer method. We have attempted to develop an exact protocol to maximize DNA expression while minimizing tissue damage following EP of skeletal muscle in vivo. Specifically, we investigated the effects of varying injection techniques, electrode surface geometry, and plasmid mediums.     

Long-term,high level in vivo gene expression after electric pulse-mediated gene transfer into skeletal muscle

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the local or systemic secretion of therapeutic proteins. However, current DNA delivery technologies have to be improved. We report very efficient luciferase gene transfer into muscle fibres obtained through the delivery of squarewave electric pulses of moderate field strength (100–200 V/cm) and of long duration (20 ms) to muscle previously injected with plasmid DNA. This intramuscular ‘electrotransfer’ method increases reporter gene expression by more than 100 times. It is noteworthy that this expression remains high and stable for at least 9 months. Moreover, intramuscular electrotransfer strongly decreases the interindividual variability usually observed after plasmid DNA injection into muscle fibres. Therefore, DNA electrotransfer in muscle possesses broad potential applications in gene therapy and for physiological, pharmacological and developmental studies.     

Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation

Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ～80% from pre-EP levels. Within 3 h, torque recovered to ～50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ～25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ～20% and ～70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.     

Towards gene therapy of diabetes mellitus

A definitive treatment for diabetes mellitus will be one that maintains a normal blood glucose concentration in the face of fluctuating dietary intake. To accomplish this, there must be mechanisms to sense the amount of blood glucose coupled to rapid release of the right amount of insulin. While mechanical devices to accomplish this are being developed, ultimately the best approach is likely to be based on genetic modification of cells. These could be pancreatic beta-cells, which are the only cells that produce insulin, or other cells that are involved in the pathogenesis of diabetes. Although definitive treatment of diabetes using genetically modified cells is a long-term goal, much progress is being made. This review discusses various approaches to modifying cells genetically, both in vitro and in vivo, for the treatment of diabetes.     

Muscle-specific overexpression of the adenovirus primary receptor CAR overcomes low efficiency of gene transfer to mature skeletal muscle

Significant levels of adenovirus (Ad)-mediated gene transfer occur only in immature muscle or in regenerating muscle, indicating that a developmentally regulated event plays a major role in limiting transgene expression in mature skeletal muscle. We have previously shown that in developing mouse muscle, expression of the primary Ad receptor CAR is severely downregulated during muscle maturation. To evaluate how global expression of CAR throughout muscle affects Ad vector (AdV)-mediated gene transfer into mature skeletal muscle, we produced transgenic mice that express the CAR cDNA under the control of the muscle-specific creatine kinase promoter. Five-month-old transgenic mice were compared to their nontransgenic littermates for their susceptibility to AdV transduction. In CAR transgenics that had been injected in the tibialis anterior muscle with AdVCMVlacZ, increased gene transfer was demonstrated by the increase in the number of transduced muscle fibers (433 +/- 121 in transgenic mice versus 8 +/- 4 in nontransgenic littermates) as well as the 25-fold increase in overall beta-galactosidase activity. Even when the reporter gene was driven by a more efficient promoter (the cytomegalovirus enhancer-chicken beta-actin gene promoter), differential transducibility was still evident (893 +/- 149 versus 153 +/- 30 fibers; P < 0.001). Furthermore, a fivefold decrease in the titer of injected AdV still resulted in significant transduction of muscle (253 +/- 130 versus 14 +/- 4 fibers). The dramatic enhancement in AdV-mediated gene transfer to mature skeletal muscle that is observed in the CAR transgenics indicates that prior modulation of the level of CAR expression can overcome the poor AdV transducibility of mature skeletal muscle and significant transduction can be obtained at low titers of AdV.     

Gene transfer to skeletal muscle by site-specific delivery of electroporation and ultrasound

Lipid metabolism drastically changes in response to the environmental factors in metazoans. Lipid is accumulated at the food rich condition, while mobilized in adipocyte tissue in starvation. Such lipid mobilization is also evident during the pupation of the insects. Pupation is induced by metamorphosis hormone, ecdysone via ecdysone receptor (EcR) with lipid mobilization, however, the molecular link of the EcR-mediated signal to the lipid mobilization remains elusive. To address this issue, EcR was genetically knocked-down selectively in 3rd instar larva fat body of Drosophila, corresponding to the adipocyte tissues in mammalians, that contains adipocyte-like cells. In this mutant, lipid accumulation was increased in the fat body. Lipid accumulation was also increased when knocked-down of taiman, which served as the EcR co-activator. Two lipid metabolism regulatory factor, E75B and adipose (adp) as well as cell growth factor, dMyc, were found as EcR target genes in the adipocyte-like cells, and consistently knock-down of these EcR target genes brought phenotypes in lipid accumulation supporting EcR function. These findings suggest that EcR-mediated ecdysone signal is significant in lipid metabolism in insects.