Goro Kuwajima

Alterations of Sarcoplasmic Reticulum Proteins in Failing Human Dilated Cardiomyopathy

BACKGROUND Previous studies provide considerable evidence that excitation-contraction coupling may be disturbed at the level of the sarcoplasmic reticulum (SR) in the failing human heart. Disturbed SR function may result from altered expression of calcium-handling proteins. METHODS AND RESULTS Levels of SR proteins involved in calcium release (ryanodine receptor), calcium binding (calsequestrin, calreticulin), and calcium uptake (calcium ATPase, phospholamban) were measured by Western blot analysis in nonfailing human myocardium (n = 7) and in end-stage failing myocardium due to dilated cardiomyopathy (n = 14). The levels of the ryanodine receptor, calsequestrin, and calreticulin were not significantly different in nonfailing and failing human myocardium. Phospholamban protein levels (pentameric form) normalized per total protein were decreased by 18% in the failing myocardium (P < .05). However, phospholamban protein levels were not significantly different in failing and nonfailing myocardium when normalization was performed per calsequestrin. Protein levels of SR calcium ATPase, normalized per total protein or per calsequestrin, were decreased by 41% (P < .001) or 33% (P < .05), respectively, in the failing myocardium. Furthermore, SR calcium ATPase was decreased relative to ryanodine receptor by 37% (P < .05) and relative to phospholamban by 28% (P < .05). CONCLUSIONS Levels of SR proteins involved in calcium binding and release are unchanged in failing dilated cardiomyopathy. In contrast, protein levels of calcium ATPase involved in SR calcium uptake are reduced in the failing myocardium. Moreover, SR calcium ATPase is decreased relative to its inhibitory protein, phospholamban.(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitation of NMDAR-Independent LTP and Spatial Learning in Mutant Mice Lacking Ryanodine Receptor Type 3

To evaluate the role in synaptic plasticity of ryanodine receptor type 3 (RyR3), which is normally enriched in hippocampal area CA1, we generated RyR3-deficient mice. Mutant mice exhibited facilitated CA1 long-term potentiation (LTP) induced by short tetanus (100 Hz, 100 ms) stimulation. Unlike LTP in wild-type mice, this LTP was not blocked bythe NMDA receptor antagonist D-AP5 but was partially dependent on L-type voltage-dependent Ca2+ channels (VDCCs) and metabotropic glutamate receptors (mGluRs). Long-term depression (LTD) was not induced in RyR3-deficient mice. RyR3-deficient mice also exhibited improved spatial learning on a Morris water maze task. These results suggest that in wild-type mice, in contrast to the excitatory role of Ca2+ influx, RyR3-mediated intracellular Ca2+ ([Ca2+]i) release from endoplasmic reticulum (ER) may inhibit hippocampal LTP and spatial learning.

Two types of ryanodine receptors in mouse brain: Skeletal muscle type exclusively in Purkinje cells and cardiac muscle type in various neurons

Two types of ryanodine receptors, channels for Ca2+ release from intracellular stores, are known. We detected the skeletal muscle type only in cerebellum by immunoblot analysis of microsomes and partially purified proteins. The cardiac muscle type was found in all parts of the mouse brain. Immunohistochemical study showed that the cardiac muscle type was localized mainly at the somata of most neurons. Analysis of mutant cerebella suggested that the skeletal muscle type was present exclusively in Purkinje cells. These results suggest that Ca(2+)-induced Ca2+ release, probably mediated by the cardiac muscle receptor, functions generally in various neurons, whereas depolarization-induced Ca2+ release, probably mediated by the skeletal muscle receptor, functions specifically in Purkinje cells.

Thymomas express epitopes shared by the ryanodine receptor

Myasthenia gravis (MG) patients with thymoma have antibodies against ryanodine receptor (RyR) of skeletal and heart muscle. In this study, thymomas were examined for reactivity with a panel of polyclonal rabbit antibodies against various short peptides of RyR. An antibody against peptide C2 in the transmembrane region of RyR stained thymoma epithelial cells in cryosections of 17/23 thymomas, and detected a 40-kDa peptide in Western blotting of a thymoma membrane fraction. The other RyR antibodies did not react with thymoma tissue. The anti-C2 RyR antibody did not react with normal thymus, tonsil or carcinoma of colon. The results strongly indicate that epithelial thymoma cells express an epitope shared by the transmembrane region of skeletal and cardiac muscle RyR.

Unaltered ryanodine receptor protein levels in ischemic cardiomyopathy.

Previous studies on sarcoplasmic reticulum calcium release channel (ryanodine receptor) demonstrated that protein levels are unchanged in myocardium from hearts with end-stage failing dilated cardiomyopathy. In ischemic cardiomyopathy, ryanodine receptor mRNA levels were shown to be decreased but no data on protein levels are available. Accordingly, protein levels of ryanodine receptor, calsequestrin, and sarcoplasmic reticulum calcium-ATPase (SR-Ca2+-ATPase) were measured by Western blot analysis in nonfailing human myocardium (n = 7) and in end-stage failing myocardium due to ischemic cardiomyopathy (n = 14). Protein levels of calsequestrin which is the major sarcoplasmic reticulum calcium storage protein were similar in nonfailing myocardium and in myocardium from end-stage failing hearts with ischemic cardiomyopathy. Ryanodine receptor protein levels, normalized to total protein or calsequestrin were also unchanged in ischemic cardiomyopathy. In contrast, protein levels of SR-Ca2+-ATPase normalized to total protein or calsequestrin were decreased by 31 and 30%, respectively (p < 0.05). The data indicate that (I) sarcoplasmic reticulum calcium uptake sites are decreased relative to the release sites in ischemic cardiomyopathy, and (2) alterations of sarcoplasmic proteins are similar in ischemic and dilated cardiomyopathy.

Immunohistochemical localization of ryanodine receptors in mouse central nervous system

The distribution of ryanodine receptor-like immunoreactivity in the mouse central nervous system was studied using two antibodies raised against synthetic peptides. These peptides represented a region conserved between the cardiac and skeletal muscle forms and a region specific to the cardiac form. Western blotting analysis and [3H]ryanodine binding analysis showed ryanodine receptors are expressed in all the brain regions. The activity was prominent in hippocampus and cerebral cortex. Immunohistochemical study demonstrated that the ryanodine receptors were localized unevenly in somata. Some apical and proximal dendrites in some cells were also labeled. In hippocampus pyramidal neurons in CA2-3 region were more labeled than CA1 region. Immunohistochemical distribution revealed by two antibodies was essentially the same but the fibers were more immunoreactive with the antibody raised against the cardiac muscle ryanodine form. The localization of ryanodine receptors was quite different from that of inositol 1,4,5-trisphosphate receptors.

Thymomas Express Ryanodine Receptor Epitopes

Myasthenia gravis (MG) patients with thymoma have antibodies against the Ca2+release channel of striated muscle, the ryanodine receptor (RyR). Thymomas were examined for immunoreactivity with a panel of polyclonal antibodies against RyR peptides. An antibody raised against a peptide in the transmembrane segment of cardiac and skeletal muscle RyR immunostained thymoma epitehlial cells in sections of 17/23 thymomas, and detected a 40 kDa peptide in membrane fractions of thymoma. The RyR peptide was not detected in normal thymus, tonsil or carcinoma of colon. The results indicate that neoplastic thymoma cells express epitopes shared by skeletal and cardiac muscle RyR.