Potential acetylcholine receptor (AChR) mutants of the nematode are selectable by resistance to the neurotoxic drug levamisole, a probable cholinergic agonist. To determine which mutants may have achieved resistance through loss of levamisole receptor function, we have assayed mutant extracts for specific 3H-meta-aminolevamisole binding activity in the presence and absence of mecamylamine. We find that mutants in 3 of the 7 genes associated with extreme levamisole resistance are obviously deficient in saturable specific 3H-meta-aminolevamisole binding activity. Mutants of the 4 other genes have abnormal binding activities that fail to undergo the apparent allosteric activation of saturable specific 3H-meta-aminolevamisole binding activity caused by mecamylamine. Thus, all 7 genes appear to be required to produce a fully functional levamisole receptor. Mutants of several other genes associated only with partial resistance to levamisole have at least grossly normal receptor binding activities.

Gene therapy holds great promise for curing Duchenne muscular dystrophy (DMD), the most common fatal inherited childhood muscle disease. Success of DMD gene therapy depends upon functional improvement in both skeletal and cardiac muscle. Numerous gene transfer studies have been performed to correct skeletal muscle pathology, yet little is known about cardiomyopathy gene therapy. Since complete transduction of the entire heart is an impractical goal, it becomes critical to determine the minimal level of correction needed for successful DMD cardiomyopathy gene therapy. To address this question, we generated heterozygous mice that persistently expressed the full-length dystrophin gene in 50% of the cardiomyocytes of mdx mice, a model for DMD. We questioned whether dystrophin expression in half of the heart cells was sufficient to prevent stress-induced cardiomyopathy. Heart function of mdx mouse is normal in the absence of external stress. To determine the therapeutic effect, we challenged 3-month-old mice with beta-isoproterenol. Cardiomyocyte sarcolemma integrity was significantly impaired in mdx but not in heterozygous and C57Bl/10 mice. Importantly, in vivo closed-chest hemodynamic assays revealed normal left ventricular function in beta-isoproterenol-stimulated heterozygous mice. Since the expression profile in the heterozygous mice mimicked viral transduction, we conclude that gene therapy correction in 50% of the heart cells may be sufficient to treat cardiomyopathy in mdx mice. This finding may also apply to the gene therapy of other inherited cardiomyopathies.

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