Increased contractility in vascular smooth muscle of dystrophic hamsters

To investigate the "vascular" hypothesis of muscular dystrophy, the sensitivity and contractility of aortic spiral strips of dystrophic (BIO 14.6) and normal (FIB) hamsters have been determined to various smooth muscle agonists. The results obtained with cumulative dose-response curves show that there is no increase in the sensitivity of the dystrophic compared with the normal aorta to noradrenaline, phenylephrine, isoproterenol, histamine, or 5-hydroxytryptamine. However, there was a significant increase in the force generated by aortic strips of the dystrophic animals to all agonists. Determination of noncollagen and collagen protein showed that there was no difference in the relative proportions of these proteins in the aortas from the two strains. The results show that in this animal model of dystrophy an increased response to vasopressor amines occurs and is in accordance with that expected of the vascular hypothesis.     

Electrophysiological observations on diaphragm muscle from normal and dystrophic hamsters

The cellular electrical activity of diaphragm from F1B normal and BIO 14.6 dystrophic hamsters has been investigated using microelectrodes. Resting membrane potentials and action potentials were recorded from control muscles and from muscles exposed to 2,4-dinitrophenol. The action potentials of normal and dystrophic diaphragms were similar in amplitude and configuration. Treatment with 2,4-dinitrophenol caused the action potential amplitude of both diaphragms to decline by similar amounts. The control resting membrane potential of diaphragm from dystrophic hamsters is not significantly different from that of normal hamsters. Treatment with 2,4-dinitrophenol caused a linear decrease in the resting membrane potentials of both groups of muscles. Dystrophic muscle, however, showed a more rapid decline in excitability when exposed to 2,4-dinitrophenol. This suggests that adenosine triphosphate production in dystrophic muscle is partially inhibited as has been suggested by other workers.     

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.     

Acid phosphatase activity in soleus and plantaris muscle fibres of normal and dystrophic hamsters

Summary The activity of acid phosphatase in skeletal muscle fibres of the plantaris and soleus of normal and dystrophic male hamsters was quantified using a histochemical post-coupling semipermeable membrane technique. Althoug the absolute levels of activity were found to vary widely from one animal to another, the ratio of the mean activities in the two muscles in each animal was virtually constant. In normal muscles, the ratio was about 0.73 and in dystrophic muscles, about 0.77. The activity in plantaris muscle fibres was always significantly lower than that in the corresponding soleus fibres, and in normal fibres compared to dystrophic ones. Another difference was that in normal fibres the mean activity declined to a constant level in mature animals older than about 3 months. In contrast, the activity in dystrophic muscles appeared to fall exponentially throughout life. The functional significance of these findings is discussed.In honour of Prof. P. van Duijn     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

An evaluation of ventilation in dystrophic Syrian hamsters

Ventilatory responses of 10 control and 10 dystrophic male hamsters to air, hypercapnia, and hypoxia were evaluated at four ages (40, 70, 100, and 140 days). Tidal volume (VT), frequency (f), minute ventilation (VE) as well as inspiratory and expiratory time of awake animals were measured with a plethysmograph. There was a small increase of VT in both groups with age. Although there was no change of f in the control group with age, there was a progressive decrease in f (means +/- SE: 92 +/- 8, 97 +/- 9, 74.5 +/- 10, and 68 +/- 8 breaths/min) in the dystrophic group. Consequently VE on air decreased in the dystrophic group. Both groups showed similar responses to hypoxia (13 and 10% O2) and hypercapnia (3, 5, and 8% CO2) at 40 days. By 70 days the hypercapnic, but not hypoxic, response of the dystrophic animals was significantly decreased compared with that of the control group (at 8% CO2, VE = 47.4 +/- 4.1 vs. 75.7 +/- 7.6 ml/min, P less than 0.01). At both 100 and 140 days the response of the dystrophic group to CO2 was flat; i.e., the slope VE vs. fractional concentration of inspired CO2 was close to zero, and the hypoxic responses were greatly diminished. Because hamsters increase VE in response to CO2 primarily by increasing VT, the data suggest that dystrophic hamsters are unable to increase VT at a very early age, presumably due to muscle weakness. The normal response of hamsters to hypoxia, which is primarily to increase f, appears to be maintained for a longer time.     

Beta-adrenergic ([3H] CGP-12177) receptors are elevated in slices of soleus muscle from CHE 147 dystrophic hamsters

We have utilized a muscle slice technique to compare the ontogeny of cell surface beta-adrenergic receptor binding in soleus and extensor digitorum longus (EDL) muscles of male Golden Syrian (GS) and Canadian Hybrid Farms 147 (CHF 147) dystrophic hamsters. Binding of the beta-adrenergic antagonist, [3H] CGP-12177 (CGP), to GS muscle slices was reversible, saturable, stereospecific and of high affinity. Bmax was higher in the soleus (2.57 +/- .12 fmol/mg wet wt) than in the EDL (1.6 +/- .17 fmol/mg wet wt) of adult animals while affinities were similar (0.35 +/- .06 and 0.24 +/- .04 nM respectively). No differences in binding characteristics were seen in EDL of GS compared to CHF 147 animals. In soleus slices frm GS hamsters, Bmax was highest at 16 days of age (5.72 +/- 0.26 fmol/mg), decreased between 16 and 29 days and remained constant until 300 days (2.51 +/- 0.52 fmol/mg). In dystrophic soleus slices, Bmax was also higher at 16 days than at any other age but receptor number decreased gradually, remaining higher than in GS until 90 days of age (p less than 0.05). The failure of beta-adrenergic receptor number to decrease at a normal rate may be implicated in the pathogenesis of hamster polymyopathy.     

Analysis of erythrocyte membrane proteins from dystrophic hamsters

To search for potentially mutant proteins, we have investigated erythrocyte ghost proteins from normal and dystrophic hamster by two-dimensional gel electrophoresis. No significant differences are observed between dystrophic and normal erythrocytes in their peptide patterns on SDS-polyacrylamide gel electrophoresis while on two-dimensional gels a protein spot of approximate Mr 20 000 with an approximate isoelectric point of 4.5 is found in erythrocytes from dystrophic animals and is consistently absent in normal erythrocytes. A large population of erythrocyte (60%) from dystrophic hamsters shows distorted shape as visualized by scanning electron microscopy. The nature of this protein and its relevance in hamster muscular dystrophy are at present not known.     

Programmed cell death in dystrophic (mdx) muscle is inhibited by IGF-II

The pathology of Duchenne Muscular Dystrophy (DMD) is characterised by unstable muscle fibres and by increased cell turnover due to the absence of functional dystrophin protein. We have used skeletal muscle, primary muscle stem cell cultures (Smith and Schofield, 1994; Smith et al., paper submitted) and clonal cell lines of the mouse DMD model (mdx) and its congenic control (C57BI) to demonstrate that programmed cell death (PCD) and apoptotic morphology is increased in dystrophic (mdx) muscle and in cultured muscle cells. We also show that the peptide growth factor (IGF-II), which is thought to play a role in mammalian myogenesis, reduces PCD in mammalian skeletal muscle myoblasts both in vivo and in vitro. This is the first time that apoptosis or PCD have been demonstrated in normal mammalian skeletal muscle. We discuss the potential of this system in determining the role of PCD in mammalian myogenesis and skeletal muscle maturation, its significance in dystrophic muscle, and suggest a novel therapeutic route whereby the pathology of DMD may be alleviated using the survival properties of IGF-II.     

Fiber regeneration is not persistent in dystrophic (MDX) mouse skeletal muscle

Fiber replacement has been measured in adult mdx mouse limb skeletal muscles. During the first 10 days after birth all fibers appear normal; between Week 3 and 4 there is massive fiber degeneration followed by regeneration in which close to 100% of the fibers are repaired or replaced. New fibers arising in adult mice are characterized by expression of fetal myosin mRNAs in whole muscle extracts, and by staining of individual fibers with an embryonic myosin heavy chain-specific antibody. By 10 weeks of age new fiber replacement rate, indicated by frequency of fibers reacting with antibody, is reduced to about 10%, and by 1 year of age less than 1% of the fibers are being replaced at rates above control. Total fiber number also remains fairly constant. We conclude that the fibers regenerating up to 10 weeks of age become stabilized and do not undergo further rounds of degeneration and regeneration. This is consistent with the observed benign phenotype of adult mdx animals and with the idea that once-regenerated fibers escape the catastrophic dystrophic phenotype by acquiring a function that compensates for their mdx mutation. The mechanism by which regenerated mdx fibers restore adequate function in the absence of dystrophin may, when understood, provide clues to effective nongenetic interventions for muscular dystrophy in humans where regenerated fibers continue to degenerate and where the disease is often fatal.     

Halothane anaesthesia of normal and dystrophic hamsters.

Induction, carried out in a small clear-plastic box with 3-5% (v/v) halothane in 30:70 (v/v) oxygen: nitrous oxide, was quiet and rapid. Recovery was almost instantaneous. 2% halothane in the oxygen-nitrous oxide mixture was sufficient for maintenance anaesthesia. The anaesthetic mixture was given by face mask in an open circuit specially designed to function at low gas-flow rates. The halothane content of the muscle and blood after 25 min anaesthesia was estimated by gas chromatography of n-heptane extracts. The mean level (+/- s.e.m.) in blood was 22-8 +/- 2-7 mg/100 ml (n=4), and in dystrophic muscle 226 +/- 36-8 mg/100 g wet weight of tissue (n=4): there was a positive correlation (r=0-94) between them (p less than 0-02).