Analysis Tools
cellular
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• both cardiomyocytes and skeletal muscle fibers show a severe reduction in the number of caveolin3-coated deep membrane invaginations or caveolae at the plasma membrane
• chemically fixed primary cardiomyocytes from newborn pups show only 15% of the deeply invaginated membrane profiles with caveolar morphology found in wild-type controls
• analysis of freeze-fractured and immunolabeled heart tissue shows that caveolar invagination is even more impaired than in dissociated cells, with only 11.7% of the caveolae found in wild-type controls
• even the density of shallow caveolin3-positive indentations is significantly decreased in heart tissue; in line with the decreased densities of caveolae and caveolin3-positive indentations, caveolin3 labeling at deep and shallowly invaginated caveolar structures is strongly decreased
• skeletal muscle fibers also show a severe loss of deeply invaginated caveolae while shallow caveolin3-positive indentations are reduced to a lesser extent; the amount of caveolin3 at deep and shallow caveolin3-marked caveolar invaginations is strongly reduced, but caveolin3 clusters persist at the plasma membrane with a moderate (30%) reduction in cluster density and a minor (7%) decrease in caveolin3 abundance within clusters
• biochemical analysis of membrane preparations of heart and skeletal muscle tissue shows alterations in the biophysical properties of caveolin3-containing detergent-resistant membranes (DRMs), suggesting that syndapin III organizes caveolin3-coated caveolae and caveolin3-associated lipid domains
• primary cardiomyocytes show a 75% reduction of deeply invaginated caveolin3-positive caveolar structures in response to increased membrane tension induced by hypoosmotic conditions
• however, overall anti-caveolin3 labeling density at membranes of freeze-fractured hearts and skeletal muscle is unaffected and caveolin3 levels remain normal in DRM fractions
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cardiovascular system
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• hearts of 20-week-old mice show no cellular damage or alterations in the cross-sectional area of cardiac myocytes or left ventricular wall thickness; heart weights and heart/body weight ratios are normal at 4 months of age
• immunoblot analyses of ERK1/2 and phosphoERK1/2 in heart homogenates show no alteration in MAPK signaling
• no signs of cardiomyocyte hypertrophy or necrosis are observed in untrained mice even at >64 weeks of age; moreover, no signs of exercise-induced cellular damage or necrosis are detected in trained mice
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• plasma membranes of chemically fixed primary cardiomyocytes isolated from newborn pups show an 85% reduction of deeply invaginated membrane profiles with caveolar appearance relative to wild-type membranes
• immunogold labeling of freeze-fractured plasma membranes shows a significant decrease in the density of caveolar coat protein caveolin3 in deeply invaginated caveolae concomitant with a severe reduction in the density of caveolin3-marked deep invaginations (classical caveolae) in cardiomyocytes
• caveolin1-positive caveolae found in primary cardiomyocytes are invaginated normally, whereas caveolin3-positive membranes of cardiomyocytes appear flattened
• interestingly, the density of cavin-1 (caveolae associated 1) is increased by ~30% at the plasma membrane of cardiomyocytes, concentrating in large clusters which are often negative for caveolin3
• however, the overall anti-caveolin3 immunolabeling density at the plasma membrane is similar to that of wild-type cardiomyocytes
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• Trypan Blue assays show an increased % of Trypan Blue-positive ruptured primary cardiomyocytes in response to mild and stronger hypoosmotic stress
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muscle
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• no localization defects of dystrophin and beta-dystroglycan are detected in sections of skeletal muscles; protein levels of dystrophin and beta-dystroglycan are unchanged in heart and skeletal muscle homogenates
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• upon physical exercise, skeletal muscles from aged mice display necrotic fibers invaded by immune cells as well as inflammation secondary to degeneration
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• both untrained and trained skeletal muscles from aged (>64 weeks old) mice show altered caliber spectra
• upon physical exercise, skeletal muscles show a widened caliber spectrum, necrotic fibers invaded by immune cells, inflammation secondary to degeneration, and a ~3-fold increase in the frequency of detached nuclei
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• untrained skeletal muscles from aged mice show an increased abundance of fibers with only small cross-sections
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• trained skeletal muscles from aged mice show exercise-induced skeletal muscle fiber hypertrophy with a significant increase in mean cross sectional area
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• upon physical exercise, skeletal muscle fibers from aged mice show signs of centronuclear myopathy, with a ~3-fold increase in the frequency of detached nuclei
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• upon physical exercise, skeletal muscle fibers from aged mice show degenerative changes similar to those seen in human skeletal muscle diseases linked to CAV3 mutation
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• upon physical exercise, skeletal muscle fibers from aged mice show clear necrotic events by acidic phosphatase staining
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immune system
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• upon physical exercise, skeletal muscles from aged mice display necrotic fibers invaded by immune cells as well as inflammation secondary to degeneration
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