Myozenin: An α-actinin- and γ-filamin-binding protein of skeletal muscle Z lines

To better understand the structure and function of Z lines, we used sarcomeric isoforms of α-actinin and γ-filamin to screen a human skeletal muscle cDNA library for interacting proteins by using the yeast two-hybrid system. Here we describe myozenin (MYOZ), an α-actinin- and γ-filamin-binding Z line protein expressed predominantly in skeletal muscle. Myozenin is predicted to be a 32-kDa, globular protein with a central glycine-rich domain flanked by α-helical regions with no strong homologies to any known genes. The MYOZ gene has six exons and maps to human chromosome 10q22.1-q22.2. Northern blot analysis demonstrated that this transcript is expressed primarily in skeletal muscle with significantly lower levels of expression in several other tissues. Antimyozenin antisera stain skeletal muscle in a sarcomeric pattern indistinguishable from that seen by using antibodies for α-actinin, and immunogold electron microscopy confirms localization specifically to Z lines. Thus, myozenin is a skeletal muscle Z line protein that may be a good candidate gene for limb-girdle muscular dystrophy or other neuromuscular disorders.

Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP- dependent protein kinase

In skeletal muscle, transcription of the gene encoding the mouse type Iα (RIα) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RIα protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIα or RIIα fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIα subunits and requires the amino-terminal residues 1–81. Mutagenesis of Phe-54 to Ala in the full-length RIα–green fluorescent protein template abolishes localization, indicating that dimerization of RIα is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIα at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Iα homologue RCE with AKAPCE and for in vitro binding of RIα to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RIα tethering at this site.

Neuromuscular synapses mediate motor axon branching and motoneuron survival during the embryonic period of programmed cell death

The embryonic period of motoneuron programmed cell death (PCD) is marked by transient motor axon branching, but the role of neuromuscular synapses in regulating motoneuron number and axonal branching is not known. Here, we test whether neuromuscular synapses are required for the quantitative association between reduced skeletal muscle contraction, increased motor neurite branching, and increased motoneuron survival. We achieved this by comparing agrin and rapsyn mutant mice that lack acetylcholine receptor (AChR) clusters. There were significant reductions in nerve-evoked skeletal muscle contraction, increases in intramuscular axonal branching, and increases in spinal motoneuron survival in agrin and rapsyn mutant mice compared with their wild-type littermates at embryonic day 18.5 (E18.5). The maximum nerve-evoked skeletal muscle contraction was reduced a further 17% in agrin mutants than in rapsyn mutants. This correlated to an increase in motor axon branch extension and number that was 38% more in agrin mutants than in rapsyn mutants. This suggests that specializations of the neuromuscular synapse that ensure efficient synaptic transmission and muscle contraction are also vital mediators of motor axon branching. However, these increases in motor axon branching did not correlate with increases in motoneuron survival when comparing agrin and rapsyn mutants. Thus, agrin-induced synaptic specializations are required for skeletal muscle to effectively control motoneuron numbers during embryonic development. (C) 2003 Elsevier Science (USA). All rights reserved.

Constraints on muscular performance: trade-offs between power output and fatigue resistance

An important functional and evolutionary constraint on the physical performance of vertebrates is believed to be the trade-off between speed and endurance capacity. However, despite the pervasiveness of physiological arguments, most studies have found no evidence of the trade-off when tested at the whole-animal level. We investigated the existence of this trade-off at the whole-muscle level, the presumed site of this physiological conflict, by examining inter-individual variation in both maximum power output and fatigue resistance for mouse extensor digitorum longus (EDL) muscle using the work-loop technique. We found negative correlations between several measures of in vitro maximum power output and force production with fatigue resistance for individual mouse EDL muscles, indicating functional trade-offs between these performance parameters. We suggest that this trade-off detected at the whole-muscle level has imposed an important constraint on the evolution of vertebrate physical performance.

Clathrin isoform CHC22, a component of neuromuscular and myotendinous junctions, binds sorting nexin 5 and has increased expression during myogenesis and muscle regeneration

The muscle isoform. of clathrin heavy chain, CHC22, has 85% sequence identity to the ubiquitously expressed CHC17, yet its expression pattern and function appear to be distinct from those of well-characterized clathrin-coated vesicles. In mature muscle CHC22 is preferentially concentrated at neuromuscular and myotendinous junctions, suggesting a role at sarcolemmal contacts with extracellular matrix. During myoblast differentiation, CHC22 expression is increased, initially localized with desmin and nestin and then preferentially segregated to the poles of fused myoblasts. CHC22 expression is also increased in regenerating muscle fibers with the same time course as embryonic myosin, indicating a role in muscle repair. CHC22 binds to sorting nexin 5 through a coiled-coil domain present in both partners, which is absent in CHC17 and coincides with the region on CHC17 that binds the regulatory light-chain subunit. These differential binding data suggest a mechanism for the distinct functions of CHC22 relative to CHC17 in membrane traffic during muscle development, repair, and at neuromuscular and myotendinous junctions.

Effect of prolonged inactivity on skeletal motor nerve terminals during aestivation in the burrowing frog, Cyclorana alboguttata

This study examined the effect of prolonged inactivity, associated with aestivation, on neuromuscular transmission in the green-striped burrowing frog, Cyclorana alboguttata. We compared the structure and function of the neuromuscular junctions on the iliofibularis muscle from active C. alboguttata and from C. alboguttata that had been aestivating for 6 months. Despite the prolonged period of immobility, there was no significant difference in the shape of the terminals (primary, secondary or tertiary branches) or the length of primary terminal branches between aestivators and non-aestivators. Furthermore, there was no significant difference in the membrane potentials of muscle fibres or in miniature end plate potential (EPP) frequency and amplitude. However, there was a significant decrease in evoked transmitter release characterised by a 56% decrease in mean EPP amplitude, and a 29% increase in the failure rate of nerve terminal action potentials to evoke transmitter release. The impact of this suite of neuromuscular characteristics on the locomotor performance of emergent frogs is discussed.

Glycinergic and GABAergic synaptic activity differentially regulate motoneuron survival and skeletal muscle innervation

GABAergic and glycinergic synaptic transmission is proposed to promote the maturation and refinement of the developing CNS. Here we provide morphological and functional evidence that glycinergic and GABAergic synapses control motoneuron development in a region-specific manner during programmed cell death. In gephyrin-deficient mice that lack all postsynaptic glycine receptor and some GABA(A) receptor clusters, there was increased spontaneous respiratory motor activity, reduced respiratory motoneuron survival, and decreased innervation of the diaphragm. In contrast, limb-innervating motoneurons showed decreased spontaneous activity, increased survival, and increased innervation of their target muscles. Both GABA and glycine increased limb-innervating motoneuron activity and decreased respiratory motoneuron activity in wild-type mice, but only glycine responses were abolished in gephyrin-deficient mice. Our results provide genetic evidence that the development of glycinergic and GABAergic synaptic inputs onto motoneurons plays an important role in the survival, axonal branching, and spontaneous activity of motoneurons in developing mammalian embryos.

Muscle fiber and motor unit behavior in the longest human skeletal muscle

The sartorius muscle is the longest muscle in the human body. It is strap-like, up to 600 mm in length, and contains five to seven neurovascular compartments, each with a neuromuscular endplate zone. Some of its fibers terminate intrafascicularly, whereas others may run the full length of the muscle. To assess the location and timing of activation within motor units of this long muscle, we recorded electromyographic potentials from multiple intramuscular electrodes along sartorius muscle during steady voluntary contraction and analyzed their activity with spike-triggered averaging from a needle electrode inserted near the proximal end of the muscle. Approximately 30% of sartorius motor units included muscle fibers that ran the full length of the muscle, conducting action potentials at 3.9 +/- 0.1 m/s. Most motor units were innervated within a single muscle endplate zone that was not necessarily near the midpoint of the fiber. As a consequence, action potentials reached the distal end of a unit as late as 100 ms after initiation at an endplate zone. Thus, contractile activity is not synchronized along the length of single sartorius fibers. We postulate that lateral transmission of force from fiber to endomysium and a wide distribution of motor unit endplates along the muscle are critical for the efficient transmission of force from sarcomere to tendon and for the prevention of muscle injury caused by overextension of inactive regions of muscle fibers.

Proprioceptive neuromuscular facilitation stretching - Mechanisms and clinical implications

Proprioceptive neuromuscular facilitation (PNF) stretching techniques are commonly used in the athletic and clinical environments to enhance both active and passive range of motion (ROM) with a view to optimising motor performance and rehabilitation. PNF stretching is positioned in the literature as the most effective stretching technique when the aim is to increase ROM, particularly in respect to short-term changes in ROM. With due consideration of the heterogeneity across the applied PNF stretching research, a summary of the findings suggests that an 'active' PNF stretching technique achieves the greatest gains in ROM, e.g. utilising a shortening contraction of the opposing muscle to place the target muscle on stretch, followed by a static contraction of the target muscle. The inclusion of a shortening contraction of the opposing muscle appears to have the greatest impact on enhancing ROM. When including a static contraction of the target muscle, this needs to be held for approximately 3 seconds at no more than 20% of a maximum voluntary contraction. The greatest changes in ROM generally occur after the first repetition and in order to achieve more lasting changes in ROM, PNF stretching needs to be performed once or twice per week. The superior changes in ROM that PNF stretching often produces compared with other stretching techniques has traditionally been attributed to autogenic and/or reciprocal inhibition, although the literature does not support this hypothesis. Instead, and in the absence of a biomechanical explanation, the contemporary view proposes that PNF stretching influences the point at which stretch is perceived or tolerated. The mechanism(s) underpinning the change in stretch perception or tolerance are not known, although pain modulation has been suggested.