Decrease in the density of t-tubular L-type Ca2+ channel currents in failing ventricular myocytes.

In some forms of cardiac hypertrophy and failure, the gain of Ca(2+)-induced Ca(2+) release [CICR; i.e., the amount of Ca(2+) released from the sarcoplasmic reticulum normalized to Ca(2+) influx through L-type Ca(2+) channels (LTCCs)] decreases despite the normal whole cell LTCC current density, ryanodine receptor number, and sarcoplasmic reticulum Ca(2+) content. This decrease in CICR gain has been proposed to arise from a change in dyad architecture or derangement of the t-tubular (TT) structure. However, the activity of surface sarcolemmal LTCCs has been reported to increase despite the unaltered whole cell LTCC current density in failing human ventricular myocytes, indicating that the "decreased CICR gain" may reflect a decrease in the TT LTCC current density in heart failure. Thus, we analyzed LTCC currents of failing ventricular myocytes of mice chronically treated with isoproterenol (Iso). Although Iso-treated mice exhibited intact t-tubules and normal LTCC subunit expression, acute occlusion of t-tubules of isolated ventricular myocytes with osmotic shock (detubulation) revealed that the TT LTCC current density was halved in Iso-treated versus control myocytes. Pharmacological analysis indicated that kinases other than PKA or Ca(2+)/calmodulin-dependent protein kinase II insufficiently activated, whereas protein phosphatase 1/2A excessively suppressed, TT LTCCs in Iso-treated versus control myocytes. These results indicate that excessive ß-adrenergic stimulation causes the decrease in TT LTCC current density by altering the regulation of TT LTCCs by protein kinases and phosphatases in heart failure. This phenomenon might underlie the decreased CICR gain in heart failure.

Follow-up of three patients with a large in-frame deletion of exons 45-55 in the Duchenne muscular dystrophy (DMD) gene.

We review the clinical status of skeletal involvement and cardiac function in three unrelated patients harboring an in-frame deletion of exons 45 to 55 in the DMD gene followed up for 2 to 7 years. Two younger patients diagnosed as having X-linked dilated cardiomyopathy (XLDCM) developed congestive heart failure without overt skeletal myopathy. Heart failure recurred after viral infection but responded well to diuretics and angiotensin-converting enzyme inhibitors. One older patient diagnosed with Becker muscular dystrophy showed limb-girdle muscular atrophy and weakness at the age of 50, but did not have any cardiac symptoms. Skeletal muscle involvement in each patient remained unchanged, and cardiac function did not worsen in any of the patients during the study. In a younger XLDCM patient, the amount and molecular weight of mutant dystrophin were equally slightly decreased in both skeletal and cardiac muscles. Immunostaining for dystrophin and dystrophin-associated proteins was slightly reduced in both skeletal and cardiac muscle, with no discernible difference between the two. The phenotype of this dystrophinopathy can manifest as XLDCM in younger patients; however, careful attention to cardiac management may result in a favorable prognosis.

Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ).

BACKGROUND: Matrix metalloproteinases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of all extracellular matrix (ECM) components in both physiological and pathological processes in various tissues. The aim of this study was to examine the involvement of gelatinase MMP family members, MMP-2 and MMP-9, in dystrophin-deficient skeletal muscle. Towards this aim, we made use of the canine X-linked muscular dystrophy in Japan (CXMDJ) model, a suitable animal model for Duchenne muscular dystrophy. METHODS: We used surgically biopsied tibialis cranialis muscles of normal male dogs (n = 3) and CXMDJ dogs (n = 3) at 4, 5 and 6 months of age. Muscle sections were analyzed by conventional morphological methods and in situ zymography to identify the localization of MMP-2 and MMP-9. MMP-2 and MMP-9 activity was examined by gelatin zymography and the levels of the respective mRNAs in addition to those of regulatory molecules, including MT1-MMP, TIMP-1, TIMP-2, and RECK, were analyzed by semi-quantitative RT-PCR. RESULTS: In CXMDJ skeletal muscle, multiple foci of both degenerating and regenerating muscle fibers were associated with gelatinolytic MMP activity derived from MMP-2 and/or MMP-9. In CXMDJ muscle, MMP-9 immunoreactivity localized to degenerated fibers with inflammatory cells. Weak and disconnected immunoreactivity of basal lamina components was seen in MMP-9-immunoreactive necrotic fibers of CXMDJ muscle. Gelatinolytic MMP activity observed in the endomysium of groups of regenerating fibers in CXMDJ did not co-localize with MMP-9 immunoreactivity, suggesting that it was due to the presence of MMP-2. We observed increased activities of pro MMP-2, MMP-2 and pro MMP-9, and levels of the mRNAs encoding MMP-2, MMP-9 and the regulatory molecules, MT1-MMP, TIMP-1, TIMP-2, and RECK in the skeletal muscle of CXMDJ dogs compared to the levels observed in normal controls. CONCLUSION: MMP-2 and MMP-9 are likely involved in the pathology of dystrophin-deficient skeletal muscle. MMP-9 may be involved predominantly in the inflammatory process during muscle degeneration. In contrast, MMP-2, which was activated in the endomysium of groups of regenerating fibers, may be associated with ECM remodeling during muscle regeneration and fiber growth.

Up-regulation of mitogen activated protein kinases in mdx skeletal muscle following chronic treadmill exercise.

Dystrophin, a product of the Duchenne muscular dystrophy gene, is a cytoskeletal protein of skeletal and cardiac muscle fibers. Dystrophin-deficient muscle fibers are abnormally vulnerable to mechanical stress including physical exercise, which is a powerful stimulator of mitogen-activated protein kinases (MAPKs). To examine how treadmill exercise affects MAPK family members in dystrophin-deficient skeletal muscle, we subjected both mdx mice, an animal model for Duchenne muscular dystrophy, and C57BL/10 mice to treadmill exercise and examined the phosphorylated protein levels of extracellular-signal regulated kinase (ERK1/2), p38 MAPK and c-Jun N terminal kinase 1 and 2 (JNK1 and JNK2) in the gastrocnemius muscle. Phosphorylation of ERK1/2, p38 MAPK and JNK2, but not JNK1, increased more in the muscles of exercise trained mdx mice than in muscles of trained C57BL/10 or untrained mdx mice. These results show that physical exercise aberrantly up-regulates the phosphorylated form of ERK1/2, p38 MAPK and JNK2 in dystrophin-deficient skeletal muscle and that their up-regulation might play a role in the degeneration and regeneration process of dystrophic features.

Neuregulin receptor ErbB2 localization at T-tubule in cardiac and skeletal muscle.

Previous studies have indicated that ErbB receptors for neuregulins play an important role in cardiac development and muscle spindle formation during embryogenesis; however, little is known about their functions in adulthood. Recent reports indicate that breast cancer therapy with humanized monoclonal ErbB2 antibody induces cardiomyopathy, suggesting that ErbB2 serves as a crucial signaling receptor, even in the adult heart. Here, we examine ErbB2 expression and localization in both cardiac and skeletal muscle of adult mice via immunoblot and immunohistochemistry. ErbB2 was detected as a band approximately 185 kD molecular mass in each cardiac and skeletal muscle extraction. Confocal images of double labeling showed that ErbB2 was colocalized with caveolin-3 in cardiac muscle and with dihydropyridine receptor in skeletal muscle, suggesting that ErbB2 was localized at the T-tubule. In addition, immunoelectron micrographs clearly demonstrated that ErbB2 was located at the T-tubule in both types of muscle. Taken together, the results of the present study suggest that neuregulin-ErbB2 signaling plays a role in the physiological function of cardiac and skeletal muscle, even in adulthood.

Oligodendrocyte precursor hypercellularity and abnormal retina development in mice overexpressing PDGF-B in myelinating tracts.

Platelet-derived growth factor (PDGF) influences the generation of neurons and glia during embryogenesis and in early postnatal life. In an attempt to determine the consequences of an overexpression of PDGF-B during the first weeks of life, we targeted transgenic expression of a human PDGF-B cDNA to myelinating tracts using the promoter region of the myelin basic protein (MBP) gene. Transgenic mRNA and protein were expressed in the brain and the expression profile of the human PDGF-B during early postnatal development closely paralleled that of the endogenous mouse MBP gene. The gross morphological appearance of transgenic brains was normal but at the cellular level several phenotypic alterations could be identified. In white matter tracts such as the corpus callosum and cerebellar medulla, there was a marked hypercellularity. The number of oligodendrocyte precursors was increased and astrocytes were more abundant. In adult mice carrying the MBP-PDGF-B transgene, however, myelination appeared normal and the amount of oligodendrocytes was similar to that of control littermates. In addition to the phenotypic alterations in the brain, investigation of eye structure revealed a striking disorganization of retinal architecture. The retina was folded with cells collected in papillar or follicular-like structures. Retinal whole mount preparations after India ink perfusion revealed capillary disorganization with large-caliber vessels supporting only a few fine branches. Our observations strengthen the notion that PDGF is an important effector molecule in postnatal CNS development.

Actin-rich spherical extrusion induced in okadaic acid-treated K562 cells by crosslinking of membrane microdomains.

Interconnection between surface microdomains and the actin cytoskeleton is vital to various cellular activities. We studied the responses of okadaic acid (OKA)-treated K562 leukemia cells to crosslinking of membrane microdomains. Although OKA alone induced clustering of surface-bound F-actin, addition of a biotinylated poly(ethylene glycol) derivative of cholesterol (bPEG-Chol) and subsequent binding of streptavidin (SA) further induced accumulation of the clusters, resulting in the formation of a spherical cell extrusion. This extrusion was also induced by direct crosslinking of a raft marker, CD59, and ganglioside GM1. In addition, we found that knockout of the gene encoding Fyn kinase inhibited formation of the spherical extrusion in murine T-cells. In bPEG-Chol/SA-treated cells, CD59, ganglioside GM1, and clathrin/AP-2 were all accumulated on the surface of the actin-rich extrusion, whereas dynamin and transferrin receptors were unaffected. Intermediate filaments, mitochondria, and other vesicles also accumulated. These results suggest that crosslinking of membrane domains exaggerates the linkage between actin and a defined set of membrane proteins in OKA-treated cells.