Numerical simulation of enhanced external counterpulsation.

Enhanced external counterpulsation (EECP) is a noninvasive, counterpulsative method to provide temporary aid to the failing heart by sequentially inflating cuffs on the lower extremity out-of-phase with the left ventricle. Optimization of the method necessitates consideration of the hemodynamics created by EECP and the mode of action providing patient benefit. A computational model based on the governing one-dimensional equations is developed that simulates cardiovascular hemodynamics during EECP. The model includes a 30-element arterial system including the left ventricle, bifurcations, and peripheral arterial vessels. Effects of vessel collapse as external pressure is applied, arterial refilling on pressure release, changes in aortic pressure, and shear stress generated in the arteries are each investigated. Device parameters are systematically varied to determine their effect on system performance. Results show the potential for significant collapse and shear augmentation throughout the arteries of the lower extremity. Performance is strongly influenced by the mean level of external pressurization and the timing of cuff inflation, but less so by the relative timing and pressure differences between cuff segments.

Desmuslin, an intermediate filament protein that interacts with alpha -dystrobrevin and desmin.

Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, alpha1-syntrophin, and the sarcoglycan complex. The precise role of alpha-dystrobrevin in skeletal muscle has not yet been determined. To study alpha-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and alpha-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.

Analysis of the spatial, temporal and tissue-specific transcription of gamma-sarcoglycan gene using a transgenic mouse.

To evaluate the promoter function of the 5'-flanking sequence of mouse gamma-sarcoglycan (gamma-SG) gene in vivo, we generated transgenic mice harboring this sequence fused with enhanced green fluorescent protein reporter gene. The reporter expression was restricted in striated muscles and particularly strong in all myofibers in skeletal muscles. Using these mice, we examine the spatial and temporal transcriptional patterns of the gamma-SG gene during mouse skeletal muscle development. The expression of basic helix loop helix transcriptional factors preceded that of the reporter. Differences between the expression of reporter and endogenous gamma-SG genes in non-muscle tissues suggested the existence of additional promoter elements in the endogenous gene, and the analysis of endogenous mRNAs demonstrated the existence of a novel upstream exon and promoter active in non-muscle tissues.

Sarcolemmopathy: muscular dystrophies with cell membrane defects.

In this article, we review the molecular pathology of muscular dystrophies caused by defects of proteins located within or near cell membranes. These disorders include Bethlem myopathy, merosinopathy, dystrophinopathy, sarcoglycanopathies, integrinopathy, dysferlinopathy and caveolinopathy. We refer to these diseases collectively as sarcolemmopathy. Here, we describe the biological functions of these proteins in the context of muscular contractions and their roles in the infrastructure of muscle; defects of muscle infrastructures cause those diseases. As an example, in dystrophinopathy, cell membranes have mechanical defects due to the absence of dystrophin. Cracks of the cell membrane induced by muscle contraction may allow the influx and efflux of substances that trigger muscle cell degeneration. However, such cracks may be resealed on relaxation. In addition, dystrophinopathy causes secondary defects of various dystrophin-associated proteins suggesting that defects in cell signaling participate in the pathologic process. With regard to other sarcolemmopathies, we discuss pathological mechanisms based on available data.

Identification of myogenesis-dependent transcriptional enhancers in promoter region of mouse gamma-sarcoglycan gene.

Four sarcoglycan subunit proteins, alpha-, beta-, gamma- and delta-sarcoglycans, form a complex on the skeletal muscle cell surface membrane and a gene defect in any one of them causes the loss or marked decrease of whole sarcoglycan complex, resulting in an autosomal recessive muscular dystrophy, sarcoglycanopathy. To characterize the regulation of sarcoglycan transcription during myocyte differentiation, we isolated the promoter regions for all sarcoglycan transcripts and measured the level of transcriptional activity of these promoter regions in the C2C12 skeletal muscle cell line. The promoters of gamma-sarcoglycan and one of two promoters of alpha-sarcoglycan exhibited marked transcriptional activation following differentiation to myotubes. Then, we characterized the 1.5-kb region of the gamma-sarcoglycan promoter by generating reporter-constructs having various deletions and measuring their transcriptional activities. In this promoter, we identified a basal promoter region and two enhancer regions dependent on differentiation. We also showed that A/T-rich and E box elements in the upstream enhancer region are essential for the activation of gamma-sarcoglycan transcription following myotube formation. Furthermore, from the identification of binding proteins to these elements together with the cotransfection experiments with the gamma-sarcoglycan promoter reporter construct and cDNAs encoding these binding factors to 10T1/2 fibroblast cell line, it was suggested that MyoD directs the transcription of gamma-sarcoglycan gene as one of the trans activators.

Caveolin-3 deficiency causes muscle degeneration in mice.

Caveolin-3 is a muscle-specific protein integrated in the caveolae, which are small invaginations of the plasma membrane. Mutations of the caveolin-3 gene, localized at 3p25, have been reported to be involved in the pathogenesis of limb-girdle muscular dystrophy (LGMD1C or caveolinopathy) with mild clinical symptoms, inherited through an autosomal dominant form of genetic transmission. To elucidate the pathogenetic mechanism, we developed caveolin-3-deficient mice for use as animal models of caveolinopathy. Caveolin-3 mRNA and its protein were absent in homozygous mutant mice. In heterozygous mutant mice, both the mRNA and its protein were normal in size, but their amounts were reduced by about half. The density of caveolae in skeletal muscle plasma membrane was roughly proportional to the amount of caveolin-3. In homozygous mutant mice, muscle degeneration was recognized in soleus muscle at 8 weeks of age and in the diaphragm from 8 to 30 weeks, although there was no difference in growth and movement between wild-type and mutant mice. No apparent muscle degeneration was observed in heterozygous mutant mice, indicating that pathological changes caused by caveolin-3 gene disruption were inherited through the recessive form of genetic transmission.

A sarcoglycan-dystroglycan complex anchors Dp116 and utrophin in the peripheral nervous system.

The dystrophin-associated membrane-integrated protein complex anchors dystrophin in the sarcolemma of striated muscles and is composed of two glycoprotein subcomplexes, the dystroglycan and the sarcoglycan (SG) complexes, and a small membrane protein termed sarcospan (SPN). The SG complex consists of four transmembrane glycoproteins, alpha-SG, beta-SG, gamma-SG and delta-SG. We found that beta-SG and delta-SG were co-expressed with epsilon-SG, a alpha-SG homolog, in the peripheral nerve, but not with alpha-SG or gamma-SG. SPN, which tightly links to the SG complex in the muscle cell membrane, was absent in the peripheral nerve. These peripheral nerve SGs were colocalized at the outermost layer of the myelin sheath of nerve fibers together with the dystroglycan complex, utrophin, and a short dystrophin isoform (Dp116). Immunocytochemical analysis using SG-deficient animals showed that a defect in beta- or delta-SG led to a great reduction of all residual SGs, but not of the other proteins, i.e., dystroglycans, Dp116 and utrophin, in the peripheral nerve. This observation suggests that the epsilon-, beta- and delta-SG molecules form a complex behaving as a single unit similar to the SG complex in muscle cells. An immunoprecipitation study indicated that the SG complex is associated with the dystroglycan complex and Dp116 or utrophin. These results demonstrated that Dp116 and utrophin are anchored to a novel membrane protein architecture, which consists of the SG and dystroglycan complexes, but not SPN, in the Schwann cell membrane.

Unilateral spatial neglect in AD: significance of line bisection performance.

BACKGROUND: Unilateral spatial neglect has been rarely reported in patients with AD, although they often have right and left asymmetry of temporoparietal dysfunction. OBJECTIVE: To investigate if patients with AD would show unilateral spatial neglect in the line bisection test, and to reveal the relationship between their neglect and the area of cerebral dysfunction. METHOD: Thirty-two patients with mild to moderate AD and 32 age-matched healthy control subjects underwent an extensive line bisection test. SPECT was also obtained for the patients. RESULTS: Rightward bisection errors exceeded the normal range in 25% of patients with AD. They exhibited greater rightward errors for the longer lines in the left hemispace than in the right hemispace, and with the right hand than with the left hand; this corresponds to the characteristics of neglect seen after right hemisphere lesions. All patients who bisected 200 mm lines with errors over 10 mm showed disproportionate lowering of performance IQ and asymmetric right hemisphere hypoperfusion, especially in the temporoparietal region. Seventy-five percent of the patients performed normally in the center presentation but erred slightly toward the body midline in the right and left hemispaces. CONCLUSION: Left unilateral spatial neglect in mild to moderate AD may be rather common if tested with the line bisection test. Rightward errors over 10 mm suggest right temporoparietal dysfunction. In AD, three or more bisections of 200 mm lines in the center presentation are recommended for detection of neglect. Patients with AD but without neglect may have difficulty in shifting attention into the peripheral sector of the egocentric space.

Constant involvement of the Betz cells and pyramidal tract in multiple system atrophy: a clinicopathological study of seven autopsy cases.

We investigated clinicopathologically the pyramidal signs, including spasticity, hyperreflexia, and Babinski's sign, and the involvement of the pyramidal tract and primary motor cortex, in seven Japanese autopsy cases of multiple system atrophy (MSA). Pyramidal signs were observed in six (86%) of the seven autopsy cases. Hyperreflexia and Babinski's sign were each evident in five patients, but spasticity was observed in only one patient. Loss of Betz cells and presence of glial cytoplasmic inclusions in the primary motor cortex were noticed in all seven cases. Astrocytosis in the fifth layer of the primary motor cortex was noticed in five cases, but its presence was not related to the duration of the disease. Involvement of the pyramidal tract in the spinal cord, particularly of the small myelinated fibers, was observed in all seven cases, but no involvement of the pyramidal tract in the midbrain was evident in any of the six cases in which this structure was examined. In MSA, pyramidal signs were shown to be present more frequently than believed before, and the clinicopathological correlation between pyramidal signs and involvement of the pyramidal tract was obvious. Constant involvement of Betz cells in MSA has not been reported. Our clinicopathological findings may also make a contribution to the understanding of the clinicopathological hallmarks of MSA.