Specific Tetanic Force Research Articles

Progressive resistance voluntary wheel running in the mdx mouse

Exercise training has been minimally explored as a therapy to mitigate the loss of muscle strength for individuals with Duchenne muscular dystrophy (DMD). Voluntary wheel running is known to elicit beneficial adaptations in the mdx mouse model for DMD. The aim of this study was to examine progressive resistance wheel running in mdx mice by comprehensively testing muscle function before, during, and after a 12-week training period. Male mdx mice at ~4 weeks age were randomized into three groups: Sedentary, Free Wheel, and Resist Wheel. Muscle strength was assessed via in vivo dorsiflexion torque, grip strength, and whole body tension intermittently throughout the training period. Contractility of isolated soleus muscles was analyzed at the study's conclusion. Both Free and Resist Wheel mice had greater grip strength (~22%) and soleus muscle specific tetanic force (26%) compared with Sedentary mice. This study demonstrates that two modalities of voluntary exercise are beneficial to dystrophic muscle and may help establish parameters for an exercise prescription for DMD.

381. Self-Cleaving Multimeric Hammerhead Ribozymes Abolish Mutant Collagen Transcripts In Cellulo and Exhibit Activity In Vivo

Current progress in adenoviral (Ad) gene therapy for Duchenne muscular dystrophy (DMD), a degenerative, inherited neuromuscular disease characterized by a lack of a functional dystrophin protein, has revealed many physical and biochemical obstacles affecting the transduction of muscle. To avoid barriers such as the basal lamina surrounding each muscle fiber and a mature immune system, fetal gene therapy could be an attractive method to deliver therapeutic genes to afflicted individuals. The ability to use a small vector load to transduce target organs in utero, combined with the possibility of transducing progenitor or stem cells, provides a key advantage over post-natal treatment. High-capacity Ad (HC-Ad) vectors devoid of all viral genes have shown more persistent expression after transduction in utero, as compared to first-generation Ad vectors, and have the added ability to carry the full-length 14 kb murine or human dystrophin cDNA. In a previous transduction study in C57BL/6 mice in utero, transgene expression was stable for at least 5 months by intramuscular injection. Furthermore, a lack of neutralizing antibody response to the Ad vector in injected animals was observed, which may allow for repeat administration of vector later in life. For this study we wanted to determine if a single intramuscular in utero injection of an HC-Ad vector carrying the full-length murine dystrophin cDNA could ameliorate the dystrophic phenotype, restore the dystrophin-glycoprotein complex, and provide functional correction of transduced muscles in the mdx mouse, an animal model for DMD. Using a surgical approach on pregnant mdx mice carrying embryonic gestation day 16 fetuses (E-16), one hind limb of fetal mice was injected with an HC-Ad vector carrying dystrophin. The injected pups were delivered naturally and analyzed at 9 weeks of age. In vitro physiological studies showed modest functional correction as assessed by specific tetanic force and muscle weight in an in utero transduced tibialis anterior muscle demonstrating recombinant dystrophin expression. Furthermore, the dystrophin-glycoprotein complex was restored in those fibers expressing dystrophin. These studies provide the first evidence that dystrophin gene delivery in utero has the potential for therapeutic effect in DMD.

378. An AAV Vector-Mediated Micro-Dystrophin Expression in Relatively Small Percentage of Dystrophin-Deficient mdx Myofibers Still Improved the mdx Phenotype through Compensatory Hypertrophy

Top of pageAbstract [Background] Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene. An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, though it has a limitation in insertion size up to 4.9 kb. Therefore, a full-length dystrophin cDNA (14 kb) cannot be incorporated into an AAV vector. We previously generated micro-dystrophin transgenic mdx mice. Micro-dystrophin CS1 transgenic mdx mice showed almost complete amelioration of dystrophic phenotypes (BBRC. 293: 1265, 2002). [Objective] We constructed an AAV vector expressing micro-dystrophin ΔCS1, and introduced it into skeletal muscles of mdx mice and examined whether the dystrophic process had been ameliorated or not. [Method] To incorporate micro-dystrophin CS1 cDNA (4.9 kb) into an AAV vector, we deleted 5′- and 3′-UTRs and exons 71-78 (alternative splicing regions), resulting 3.8 kb ΔCS1 cDNA. We produced type 2 AAV vector expressing ΔCS1 under the control of muscle specific MCK promoter to avoid immune response against transgene product (Gene Ther. 9: 1576, 2002), designated AAV2-MCKΔCS1. The vector was injected into anterior tibial (TA) muscles of 10-day-old and 5-week-old mdx mice. Mdx muscles show no obvious changes of degeneration at 10-day, whereas 5-week-old mdx muscles exhibit active cycles of muscle degeneration/regeneration. [Result] When the AAV2-MCKΔCS1 was injected at 5 weeks of age, dystrophin-positive fibers were 51.5 ± 17.3% at 24 weeks after the injection. The ratio of centrally nucleated fibers in ΔCS1-positive fibers was significantly reduced compared with that of ΔCS1-negative fibers, indicating protective function of ΔCS1 against muscle degeneration. Furthermore, AAV-injected muscles revealed complete recovery of the specific tetanic force. When injected at 10-day-old, ΔCS1-positive fibers was 16.5 ± 7.0% at 24 weeks. Most of ΔCS1-positive fibers were peripherally nucleated. Surprisingly, there was no statistical difference in specific tetanic force between AAV-injected mdx muscles and B10 muscles. To clarify the mechanism of physiological recovery due to small numbers of ΔCS1-positive fibers, we examined the relationship between myofiber hypertrophy and force generation. We found positive correlation between the muscle weight and the force generation when injected at 10-day-old. To confirm whether increased muscle weight reflected myofiber hypertrophy, we measured cross section areas (CSAs) of individual fibers. Fiber CSAs were remarkably larger in ΔCS1-positive mdx fibers compared with ΔCS1-nagative, non-treated mdx fibers, and even B10 fibers. Thus, selective hypertrophy of ΔCS1-positive fibers seemed to greatly assist contractile force generation. [Conclusion] The AAV2-MCKΔCS1 could be a powerful tool for the gene therapy of DMD. For clinical application of this strategy to DMD patients, experiments using a bigger animal model, e.g. canine X-linked muscular dystrophy will be very important.

Insulin-like growth factor-1 prevents age-related decrease in specific force and intracellular Ca2+ in single intact muscle fibres from transgenic mice.

In the present work we test the hypothesis that sustained transgenic overexpression of insulin-like growth factor-1 (IGF-1) in skeletal muscle prevents age-related decreases in myoplasmic Ca2+ concentration and consequently in specific force in single intact fibres from the flexor digitorum brevis (FDB) muscle from the mouse. Measurements of IGF-1 concentration in FDB muscle showed higher levels in transgenic than in wild-type mice at all ages. The specific tetanic force decreased significantly in single muscle fibres from old (286 +/- 22 kPa) compared to young wild-type (455 +/- 28 kPa), young transgenic (423 +/- 43 kPa), and old transgenic mice (386 +/- 15 kPa) (P < 0.05). These results are consistent with measurements in whole FDB muscles. The peak Ca2+ concentration values in response to prolonged stimulation were: 1.47 +/- 0.15, 1.70 +/- 0.29, 0.97 +/- 0.13 and 1.7 +/- 0.22 microM, in fibres from young wild-type, young transgenic, old wild-type and old transgenic mice, respectively. The effects of caffeine on FDB fibres support the conclusion that the age-related decline in peak myoplasmic Ca2+ and specific force is not explained by sarcoplasmic reticulum Ca2+ depletion. Immunohistochemistry in muscle cross-sections was performed to determine whether age and/or IGF-1 overexpression induce changes in fibre type composition. The relative percentages of type IIa, IIx and I myosin heavy chain (MHC) isoforms did not change significantly with age or genotype. Therefore, IGF-1 prevents age-related decline in peak intracellular Ca2+ and specific force in a muscle that does not exhibit changes in fibre type composition with senescence.

Beneficial effects of GH/IGF-1 on skeletal muscle atrophy and function in experimental heart failure.

Muscle atrophy is a determinant of exercise capacity in heart failure (CHF). Myocyte apoptosis, triggered by tumor necrosis factor-alpha (TNF-alpha) or its second messenger sphingosine (SPH), is one of the causes of atrophy. Growth hormone (GH) improves hemodynamic and cardiac trophism in several experimental models of CHF, but its effect on skeletal muscle in CHF is not yet clear. We tested the hypothesis that GH can prevent skeletal muscle apoptosis in rats with CHF. CHF was induced by injecting monocrotaline. After 2 wk, 2 groups of rats were treated with GH (0.2 mg.kg(-1).day(-1) and 1.0 mg.kg(-1).day(-1)) subcutaneously. A third group of controls had saline. After 2 additional weeks, rats were killed. Tibialis anterior cross-sectional area, myosin heavy chain (MHC) composition, and a study on myocyte apoptosis and serum levels of TNF-alpha and SPH were carried out. The number of apoptotic nuclei, muscle atrophy, and serum levels of TNF-alpha and SPH were decreased with GH at high but not at low doses compared with CHF rats. Bcl-2 was increased, whereas activated caspases and bax were decreased. The MHC pattern in GH-treated animals was similar to that of controls. Monocrotaline slowed down both contraction and relaxation but did not affect specific tetanic force, whereas absolute force was decreased. GH treatment restored contraction and relaxation to control values and brought muscle mass and absolute twitch and tetanic tension to normal levels. These findings may provide an insight into the therapeutic strategy of GH given to patients with CHF to improve exercise capacity.

The capacity of mdx mouse diaphragm muscle to do oscillatory work

1. Mdx mice were used as a model for Duchenne muscular dystrophy; both lack dystrophin. It was hypothesized that the mdx condition would have a marked effect on the ability of diaphragm muscle from mdx mice to do active net work and generate power. This hypothesis was tested using the work-loop technique. 2. Specific twitch force, specific tetanic force and maximum power were all significantly less in diaphragm strips from mdx mice than those from control mice. 3. In all preparations muscle length at which maximum power was achieved (Lw) was about 8% less than that at which maximum tetanic force was achieved (L0), both in mdx and control muscle. 4. The isometric force-length curve for mdx muscle was steeper on both sides of the plateau. Similarly, the curve relating net work per cycle to muscle length was steeper for mdx muscle on both sides of the plateau. 5. Maximum power of mdx muscle was achieved at a lower strain than for control muscle; maximum power occurred at a strain of 10.2% for mdx and 14.7% for control. Further increases in strain caused a marked decrease of power production in mdx muscle, whereas they caused a smaller decrease in control muscle. 6. In summary, at muscle lengths longer than Lw and at high strains, performance of mdx muscle was compromised relative to that of control muscle. Work and power were compromised more than isometric force.

Full functional rescue of a complete muscle (TA) in dystrophic hamsters by adeno-associated virus vector-directed gene therapy.

Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the delta-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74-82, 1999). In this report, we show efficient and long-term delta-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose AAV vector injection into the tibialis anterior muscle of the dystrophic hamsters. AAV vector treatment led to more than 97% recovery in muscle strength for both the specific twitch force and the specific tetanic force, when compared to the age-matched control. Vector treatment also prevented pathological muscle hypertrophy and resulted in normal muscle weight and size. Finally, vector-treated muscle showed substantial improvement of the histopathology. This is the first report of successful functional rescue of an entire muscle after AAV-mediated gene delivery. This report also demonstrates the feasibility of in vivo gene therapy for LGMD patients by using AAV vectors.

Effect of undernutrition on contractile and fatigue properties of rat diaphragm during development

The present study was designed to assess the effects of combined pre- and postnatal undernutrition on the in vitro contractile and fatigue properties of the rat diaphragm during development. In vitro direct stimulation of costal diaphragm from control (CTL) and undernourished (UN) rats was done on postnatal days 1, 4, 14, 21, 30, 40, 50, and 60. Combined pre- and postnatal undernutrition resulted in stunted animal growth but did not alter the diaphragm-to-total body weight ratio. Twitch contraction time, half-relaxation time, and force-frequency relationships were not consistently affected by undernutrition. Specific twitch force and specific tetanic force were also unchanged in the UN group. Fatigue resistance was high and comparable in UN and CTL groups at days 1 and 4. At day 14 and thereafter, fatigue resistance declined but was consistently higher in the UN than in the CTL group. We conclude that combined pre- and postnatal undernutrition results in a significant increase in fatigue resistance of the diaphragm compared with CTL, whereas diaphragm muscle contractile properties are not appreciably affected by prolonged undernutrition.