␣1-Syntrophin is a member of the family of dystrophin-associated proteins and is strongly expressed in the sarcolemma and the neuromuscular junctions. All three syntrophin isoforms have a PDZ domain that appears to participate in protein-protein interactions at the plasma membrane. ␣1-Syntrophin has additionally been shown to associate with neuronal nitric-oxide synthase (nNOS) through PDZ domains in vitro. These observations suggest that ␣1-syntrophin may work as a modular adaptor protein that can link nNOS or other signaling enzyme to the sarcolemmal dystrophin complex. In the sarcolemma, nNOS regulates the homeostasis of reactive free radical species and may contribute to the oxidative damage to muscle protein in muscle disease such as Duchenne muscular dystrophy. In this study, we generated ␣1-syntrophin knock-out mice to clarify the interaction between ␣1-syntrophin and nNOS in the skeletal muscle. We observed that nNOS, normally expressed in the sarcolemma, was largely absent from the sarcolemma, but considerably remained in the cytosol of the knock-out mice. Even though the distribution of nNOS was altered, the knock-out mice displayed no gross histological changes in the skeletal muscle. We also discovered that muscle contractile properties have not been influenced in the knock-out mice.

While subtypes 1 and 2 of the ryanodine receptor (RyR) function as intracellular Ca2+ release channels, little is known about the function of the third subtype (RyR-3), first identified in brain. Myocytes from mice homozygous for a targeted mutation in the RyR-1 gene (dyspedic mice) can now be used for a study on the function of RyR-3, which is predominantly expressed in these cells according to our reverse transcriptionpolymerase chain reaction analysis. We here demonstrate in these myocytes caffeine-, ryanodine-and adenine nucleotide-sensitive Ca>+-induced Ca2> release with -10 times lower sensitivity to Ca2+ than that of RyR-1. Although RyR-3 does not mediate excitationcontraction coupling of the skeletal muscle type, we propose that RyR-3 may induce intracellular Ca2+ release in response to a Ca2+ rise with a high threshold.

The ryanodine receptor type 3 (RyR-3) functions as a Ca 2؉-induced Ca 2؉ release (CICR) channel and is distributed in a wide variety of cell types including skeletal muscle and smooth muscle cells, neurons, and certain non-excitable cells. However, the physiological roles of RyR-3 are totally unclear. To gain an insight into the function of RyR-3 in vivo, we have generated mice lacking RyR-3 by means of the gene targeting technique. The mutant mice thus obtained showed apparently normal growth and reproduction. Although Ca 2؉ -induced Ca 2؉ release from intracellular Ca 2؉ stores of the mutant skeletal muscle differed in Ca 2؉ sensitivity from that of wild-type muscle, excitation-contraction coupling of the mutant muscle seemed to be normal. Moreover, we could not find any significant disturbance in the smooth muscle and lymphocytes from the mutant mice. On the other hand, the mutant mice showed increased locomotor activity, which was about 2-fold greater than that of the control mice. These results indicate that the loss of RyR-3 causes no gross abnormalities and suggest that the lack of RyR-3-mediated Ca 2؉ signaling results in abnormalities of certain neurons in the central nervous system.

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α1-Syntrophin is a member of the family of dystrophin-associated proteins; it has been shown to recruit neuronal nitric oxide synthase and the water channel aquaporin-4 to the sarcolemma by its PSD-95/SAP-90, Discs-large, ZO-1 homologous domain. To examine the role of α1-syntrophin in muscle regeneration, we injected cardiotoxin into the tibialis anterior muscles of α1-syntrophin–null (α1syn−/−) mice. After the treatment, α1syn−/− muscles displayed remarkable hypertrophy and extensive fiber splitting compared with wild-type regenerating muscles, although the untreated muscles of the mutant mice showed no gross histological change. In the hypertrophied muscles of the mutant mice, the level of insulin-like growth factor-1 transcripts was highly elevated. Interestingly, in an early stage of the regeneration process, α1syn−/− mice showed remarkably deranged neuromuscular junctions (NMJs), accompanied by impaired ability to exercise. The contractile forces were reduced in α1syn−/− regenerating muscles. Our results suggest that the lack of α1-syntrophin might be responsible in part for the muscle hypertrophy, abnormal synapse formation at NMJs, and reduced force generation during regeneration of dystrophin-deficient muscle, all of which are typically observed in the early stages of Duchenne muscular dystrophy patients.