Contractile properties of diaphragm muscle segments from old mdx and old transgenic mdx mice.

Diaphragm muscles of young (4- to 6-mo-old) mdx mice show severe fiber necrosis and have normalized forces and powers 60 and 46% of the values for control C57BL/10 mice. In contrast, microinjection of mdx mouse embryos with a truncated dystrophin minigene has produced young transgenic mdx (tg-mdx) mice with a level of dystrophin expression and structural and functional properties of diaphragm muscle strips measured in vitro not different from those of control mice. Whether dystrophin expression and functional corrections persist for the life span of these animals is not know. We tested the null hypothesis that, in old (24 mo) tg-mdx mice, dystrophin expression is adequate and diaphragm muscle strips have forces and powers not different from values for diaphragm muscle strips from young tg-mdx mice or control mice. Compared with control values, diaphragm muscle strips from old mdx mice had normalized forces and powers of 48 and 31%, respectively. Expression of dystrophin persisted in diaphragm muscles of old tg-mdx mice, and functional properties were not different from diaphragm muscles of young tg-mdx or young or old control mice. These results suggest that, with a transgenic animal approach, dystrophin expression and functional corrections persist for the life span of the animals.

Transgenic mdx mice expressing dystrophin with a deletion in the actin-binding domain display a "mild Becker" phenotype.

The functional significance of the actin-binding domain of dystrophin, the protein lacking in patients with Duchenne muscular dystrophy, has remained elusive. Patients with deletions of this domain (domain I) typically express low levels of the truncated protein. Whether the moderate to severe phenotypes associated with such deletions result from loss of an essential function, or from reduced levels of a functional protein, is unclear. To address this question, we have generated transgenic mice that express wild-type levels of a dystrophin deleted for the majority of the actin-binding domain. The transgene derived protein lacks amino acids 45-273, removing 2 of 3 in vitro identified actin interacting sites and part of hinge 1. Examination of the effect of this deletion in mice lacking wild-type dystrophin (mdx) suggests that a functional domain I is not essential for prevention of a dystrophic phenotype. However, in contrast to deletions in the central rod domain and to full-length dystrophin, both of which are functional at only 20% of wild-type levels, proteins with a deletion in domain I must be expressed at high levels to prevent a severe dystrophy. These results are also in contrast to the severe dystrophy resulting from truncation of the COOH-terminal domain that links dystrophin to the extracellular matrix. The mild phenotype observed in mice with domain I-deletions indicates that an intact actin-binding domain is not essential, although it does contribute to an important function of dystrophin. These studies also suggest the link between dystrophin and the subsarcolemmal cytoskeleton involves more than a simple attachment of domain I to actin filaments.

Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice.

Duchenne and Becker muscular dystrophy are caused by defects in the dystrophin gene, and are candidates for treatment by gene therapy. We have shown previously that overexpression of a full-length dystrophin cDNA prevents the development of dystrophic symptoms in mdx mice. We show here that this functional correction can be achieved by expressing the full-length muscle isoform at a lower level than is present in control animals. Gene therapy for DMD may necessitate the use of truncated dystrophin mini-genes to accommodate the limited cloning capacity of current-generation viral delivery vectors. We have constructed both murine and human mini-genes deleted for exons 17-48, and have demonstrated that expression of either mini-gene can almost completely prevent the development of dystrophic symptoms in transgenic mdx mice. These results suggest that viral-mediated expression of moderate levels of a truncated dystrophin could be an effective treatment for DMD.

Prevention of dystrophic pathology in mdx mice by a truncated dystrophin isoform.

The C-terminal domain of dystrophin is alternatively spliced to produce a variety of tissue and developmental stage-specific isoforms. Recent studies suggest that the C-terminal domain binds to the dystrophin-associated glycoprotein complex (DGC) in muscle, but little is known about the functional significance of the alternative splicing or what role individual isoforms may play in specific tissues. The major dystrophin transcript in brain lacks exons 71-74, and encodes an isoform not observed in skeletal muscle. To explore the capacity of this truncated isoform to function in muscle, we have generated transgenic mice expressing a murine dystrophin mini-gene missing exons 71-74. Uniform expression of this construct on a mutant mdx mouse background results in normal muscle morphology and physiology, and prevents the development of muscular dystrophy. These mice also display normal expression and localization of the DGC, suggesting that the alternatively spliced exons are not required for dystrophin function in skeletal muscle. An additional line of mice was analyzed that had a mosaic pattern of expression. These mice display a markedly milder phenotype than mdx mice, despite the expression of dystrophin in only half the muscle fibers. These results indicate that viral delivery of dystrophin to a simple majority of fibers in a muscle group would greatly reduce the dystrophic pathology associated with Duchenne muscular dystrophy.

Muscle sound frequencies of the frog are modulated by skeletal muscle tension.

The purpose of this study was to determine the relationships among muscle sound frequencies, muscle tension, and stiffness. Time-frequency transformations of nonstationary acoustic signals provided measures of resonant frequency during isometric contractions of frog (Rana pipiens) semitendinosus and gastrocnemius muscles. A mathematical expression for muscle transverse resonant frequency, elastic modulus and tension, based on elastic beam theory, was formulated by the Rayleigh method adapted for muscles. For thin muscles, the elastic modulus was found to have negligible influence on transverse muscle resonant frequency. Changes in muscle tension were the major determinants of changes in transverse resonant frequency. Consequently, for thin muscles, the time course of muscle tension, but not elastic modulus, can be monitored acoustically during the early phase of contraction when muscles give rise to sounds. Muscles were found to be anisotropic with a modulus of elasticity, EL, measured via length perturbations near 0.1% muscle length peak-to-peak, that was much larger than the modulus of elasticity, Eb, that resists the lateral bending that causes sound production. The elastic and resonant behavior of a thin muscle is similar to a tensioned fibrous cable with distributed mass.

Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity.

Duchenne and Becker muscular dystrophy (DMD and BMD) are X-linked recessive diseases caused by defective expression of dystrophin. The mdx mouse, an animal model for DMD, has a mutation that eliminates expression of the 427K muscle and brain isoforms of dystrophin. Although these animals do not display overt muscle weakness or impaired movement, the diaphragm muscle of the mdx mouse is severely affected and shows progressive myofibre degeneration and fibrosis which closely resembles the human disease. Here we explore the feasibility of gene therapy for DMD by examining the potential of a full-length dystrophin transgene to correct dystrophic symptoms in mdx mice. We find that expression of dystrophin in muscles of transgenic mdx mice eliminates the morphological and immunohistological symptoms of muscular dystrophy. In addition, overexpression of dystrophin prevents the development of the abnormal mechanical properties associated with dystrophic muscle without causing deleterious side effects. Our results provide functional evidence for the feasibility of gene therapy for DMD.

Muscle sounds are emitted at the resonant frequencies of skeletal muscle.

The changes in mechanical resonant frequency of whole muscles during twitch and tetanic contractions were compared to changes in frequency components of the pressure wave produced by muscles during contraction. Resonant frequencies were determined by imposing sinusoidal length changes on a muscle and observing transverse standing waves when the frequency of length change matched the muscle's resonant frequency or a harmonic of the resonant frequency. Acoustic signal instantaneous frequency spectrums were calculated using time-frequency transformations including the Wigner transform and the exponential distribution. During a tetanic muscle contraction, the peak instantaneous frequency initially increased and then became constant as the force plateau was reached. The resonant frequencies determined during the force plateau and during relaxation spanned the same range as the peak instantaneous frequency of the acoustic signal. These results suggest that the acoustic signal may be useful as a non-invasive monitor of muscle resonant frequency during contraction.

Informed consent: considerations in aesthetic and reconstructive surgery of the breast.

A definite standard of care exists for establishing informed consent. The plastic surgeon has an obligation to meet these requirements with each patient undergoing surgery. The difficulties of adequately informing have been described and suggestions made for supplementing the consultation with a variety of adjuncts. The goal is to inform and use whatever methods are effective in achieving this goal. Establishing valid informed consent will not prevent all medical liability claims. Despite the most conscientious efforts to inform, some claims will still occur alleging "lack of informed consent." The surgeon must then prove that sufficient information was provided. However, many patients who feel that the surgeon made every effort to inform and felt that the surgeon was honest, conscientious, concerned, and thorough will be less inclined to generate claims even in the face of unsatisfactory results or untoward complications. Informed consent is the patient's right and the surgeon's obligation. The medicolegal benefits from fully informing are tangible and one of the most effective methods to reduce claims and modify risk.

Fluid mechanics of muscle vibrations.

The pressure field produced by an isometrically contracting frog gastrocnemius muscle is described by the fluid mechanics equations for a vibrating sphere. The equations predict a pressure amplitude that is proportional to the lateral acceleration of the muscle, inversely proportional to the square of the distance from the muscle, and cosinusoidally related to the major axis of lateral movement. The predictions are confirmed by experiments that measure the pressure amplitude distribution and by photographs of muscle movement during contraction. The lateral movement of muscle has the appearance of an oscillating system response to a step function input--the oscillation may be at the resonant frequency of the muscle and therefore may provide a means to measure muscle stiffness without actually touching the muscle.

The effectiveness of benzodiazepines and narcotics in outpatient surgery.

In a prospective study of 211 outpatients undergoing cosmetic surgical procedures under local anesthesia supplemented with intravenous diazepam and morphine, patient pain response to the local anesthetic infiltration was graded and the recall of the operative experience was assessed. One hundred and fifty-five patients (73%) had either no pain response or a mild response, 41 (19%) had a moderate response, and 15 (7%) had a severe response. One hundred and eighty-seven patients (88%) had total amnesia for the local anesthetic infiltration and the operative procedure, and 183 (87%) could not recall pain, but had recall of events during surgery. There were no untoward drug reactions nor episodes of cardiorespiratory depression. We conclude that intravenous diazepam with or without intravenous morphine following triazolam, morphine, and premethazine premedication is a safe and effective regimen for outpatient surgery.