Biomedical subjects

Kay Ohlendieck

Publications and source records attributed to Kay Ohlendieck.

10 recordsLinked to original sources

PubMed2003

Increased sensitivity of the ryanodine receptor to halothane-induced oligomerization in malignant hyperthermia-susceptible human skeletal muscle.

Mutations in the skeletal muscle RyR1 isoform of the ryanodine receptor (RyR) Ca2+-release channel confer susceptibility to malignant hyperthermia, which may be triggered by inhalational anesthetics such as halothane. Using immunoblotting, we show here that the ryanodine receptor, calmodulin, junctin, calsequestrin, sarcalumenin, calreticulin, annexin-VI, sarco(endo)plasmic reticulum Ca2+-ATPase, and the dihydropyridine receptor exhibit no major changes in their expression level between normal human skeletal muscle and biopsies from individuals susceptible to malignant hyperthermia. In contrast, protein gel-shift studies with halothane-treated sarcoplasmic reticulum vesicles from normal and susceptible specimens showed a clear difference. Although the alpha2-dihydropyridine receptor and calsequestrin were not affected, clustering of the Ca2+-ATPase was induced at comparable halothane concentrations. In the concentration range of 0.014-0.35 mM halothane, anesthetic-induced oligomerization of the RyR1 complex was observed at a lower threshold concentration in the sarcoplasmic reticulum from patients with malignant hyperthermia. Thus the previously described decreased Ca2+-loading ability of the sarcoplasmic reticulum from susceptible muscle fibers is probably not due to a modified expression of Ca2+-handling elements, but more likely a feature of altered quaternary receptor structure or modified functional dynamics within the Ca2+-regulatory apparatus. Possibly increased RyR1 complex formation, in conjunction with decreased Ca2+ uptake, is of central importance to the development of a metabolic crisis in malignant hyperthermia.

Anesthetics, Inhalation↗

PubMed2003

Use of continuous-elution gel electrophoresis as a preparative tool for blot overlay analysis.

Blot overlay techniques have long been used to directly visualize protein-protein interactions within membrane complexes. However, this approach is often hampered by the limited quantities of purified membrane proteins available for conjugation with marker molecules. Here we applied continuous-elution gel electrophoresis as a preparative alternative to isolate sufficient amounts of a homogeneous protein sample to be used as a peroxidase-labeled probe in blot overlays. Microsomal muscle proteins ranging from approximately 20 to 600 kDa were electrophoretically separated and various marker proteins present in eluted fractions were identified by immunoblotting. Since the supramolecular structure of calsequestrin has recently been determined, this terminal cisternae protein was isolated as a model protein for studying protein-protein interactions. In blot overlay assays, peroxidase-conjugated calsequestrin specifically bound to the ryanodine receptor, triadin, calsequestrin itself, and junctin, illustrating that the biological binding affinities are retained in electrophoretically prepared muscle proteins. Potential applications for differential blot overlay approaches and for analyzing pathophysiological preparations from dystrophic muscle were evaluated. Since continuous-elution gel electrophoresis can separate a wide range of differently sized proteins from subcellular fractions, our report indicates that this technique can be utilized for the rapid identification of protein-protein interactions in future high-throughput analyses of subproteomes.

Animals↗

PubMed2003

Increased expression of the nicotinic acetylcholine receptor in stimulated muscle.

Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts.

Animals↗

PubMed2003

The importance of alcohol-induced muscle disease.

Alcohol-induced muscle disease (AIMD) is a composite term to describe any muscle pathology (molecular, biochemical, structural or physiological) resulting from either acute or chronic alcohol ingestion or a combination thereof. The chronic form of AIMD is arguably the most prevalent skeletal muscle disorder in the Western Hemisphere affecting more than 2000 subjects per 100,000 population and is thus much more common than hereditary disorders such as Becker or Duchenne muscular dystrophy. Paradoxically, most texts on skeletal myopathies or scientific meetings covering muscle disease have generally ignored chronic alcoholic myopathy. The chronic form of AIMDs affects 40-60% of alcoholics and is more common than other alcohol-induced diseases, for example, cirrhosis (15-20% of chronic alcoholics), peripheral neuropathy (15-20%), intestinal disease (30-50%) or cardiomyopathy (15-35%). In this article, we summarise the pathological features of alcoholic muscle disease, particularly biochemical changes related to protein metabolism and some of the putative underlying mechanisms. However, the intervening steps between the exposure of muscle to ethanol and the initiation of the cascade of responses leading to muscle weakness and loss of muscle bulk remain essentially unknown. We argue that alcoholic myopathy represents: (a) a model system in which both the causal agent and the target organ is known; (b) a myopathy involving free-radical mediated pathology to the whole body which may also target skeletal muscle and (c) a reversible myopathy, unlike many hereditary muscle diseases. A clearer understanding of the mechanisms responsible for alcoholic myopathy is important since some of the underlying pathways may be common to other myopathies.

Alcohol Drinking↗

PubMed2003

Comparative analysis of Dp427-deficient mdx tissues shows that the milder dystrophic phenotype of extraocular and toe muscle fibres is associated with a persistent expression of beta-dystroglycan.

The cell biological hypothesis of Duchenne muscular dystrophy assumes that deficiency in the membrane cytoskeletal element dystrophin triggers a loss in surface glycoproteins, such as beta-dystroglycan, thereby rendering the sarcolemmal membrane more susceptible to micro-rupturing. Secondary changes in ion homeostasis, such as increased cytosolic Ca2+ levels and impaired luminal Ca2+ buffering, eventually lead to Ca2+-induced myonecrosis. However, individual muscle groups exhibit a graded pathological response during the natural time course of x-linked muscular dystrophy. The absence of the dystrophin isofom Dp427 does not necessarily result in a severe dystrophic phenotype in all muscle groups. In the dystrophic mdx animal model, extraocular and toe muscles are not as severely affected as limb muscles. Here, we show that the relative expression and sarcolemmal localization of the central trans-sarcolemmal linker of the dystrophin-glycoprotein complex, beta-dystroglycan, is preserved in mdx extraocular and toe fibres by means of two-dimensional immunoblotting and immunofluorescence microscopy. Thus, with respect to improving myology diagnostics, the relative expression levels of beta-dystroglycan appear to represent reliable markers for the severity of secondary changes in dystrophin-deficient fibres. Immunoblotting and enzyme assays revealed that mdx toe muscle fibres exhibit an increased expression and activity of the sarcoplasmic reticulum Ca2+-ATPase. Chemical crosslinking studies demonstrated impaired calsequestrin oligomerization in mdx gastrocnemius muscle indicating that abnormal calsequestrin clustering is involved in reduced Ca2+ buffering of the dystrophic sarcoplasmic reticulum. Previous studies have mostly attributed the sparing of certain mdx fibres to the special protective properties of small-diameter fibres. Our study suggests that the rescue of dystrophin-associated glycoproteins, and possibly the increased removal of cytosolic Ca2+ ions, might also play an important role in protecting muscle cells from necrotic changes.

Animals↗

PubMed2003

Persistent expression of the alpha1S-dihydropyridine receptor in aged human skeletal muscle: implications for the excitation-contraction uncoupling hypothesis of sarcopenia.

Previous studies on aged animal muscle suggest that excitation-contraction uncoupling and fibre transitions play a central role in sarcopenia, the progressive loss and functional decline of aging skeletal muscle fibres. A drastic reduction in the voltage-sensing alpha1S-subunit of the transverse-tubular dihydropyridine receptor is believed to be the underlying cause for a decreased transmission of the surface depolarization signal into Ca2+-mediated muscle contraction. Extending these studies to human muscle, we asked whether potential changes in the relative expression of the voltage sensor occur in senescent human fibres. For internal standardization and as markers of potential fast-to-slow transitions, the fast isoforms of the Ca2+-binding element calsequestrin and the myosin heavy chain were employed. Besides small inter-individual variations in expression levels, the microsomal immunoblot analysis of vastus lateralis autopsy specimens from male humans aged 18 to 82 years of age showed no major changes in the relative abundance of the alpha1S- and alpha2-dihydropyridine receptor, fast calsequestrin and the slow/fast myosin heavy chains. The oligomeric status of the alpha1S-dihydropyridine receptor was unaltered in aged fibres. Biochemical assays revealed no significant modifications in Ca2+-ATPase activity and a reduced Ca2+-binding capacity in aged human muscle preparations. Although impairments of other Ca2+-regulatory proteins and/or disturbed protein-protein interactions might be involved in the pathophysiological changes of sarcopenia, dihydropyridine receptor and calsequestrin expression seem to be preserved during the aging process of human skeletal muscle fibres. Hence, the supposition that excitation-contraction uncoupling is responsible for sarcopenia can not be transferred from animal models to senescent human muscle without modifications.

Adult↗

PubMed2002

Distal mdx muscle groups exhibiting up-regulation of utrophin and rescue of dystrophin-associated glycoproteins exemplify a protected phenotype in muscular dystrophy.

Unique unaffected skeletal muscle fibres. unlike necrotic torso and limb muscles, may pave the way for a more detailed understanding of the molecular pathogenesis of inherited neuromuscular disorders and help to develop new treatment strategies for muscular dystrophies. The sparing of extraocular muscle in Duchenne muscular dystrophy is mostly attributed to the special protective properties of extremely fast-twitching small-diameter fibres, but here we show that distal muscles also represent a particular phenotype that is more resistant to necrosis. Immunoblot analysis of membranes isolated from the well established dystrophic animal model mdx shows that, in contrast to dystrophic limb muscles, the toe musculature exhibits an up-regulation of the autosomal dystrophin homologue utrophin and a concomitant rescue of dystrophin-associated glycoproteins. Thus distal mdx muscle groups provide a cellular system that naturally avoids myofibre degeneration which might be useful in the search for naturally occurring compensatory mechanisms in inherited skeletal muscle diseases.

Animals↗

PubMed2002

Supramolecular calsequestrin complex.

As recently demonstrated by overlay assays using calsequestrin-peroxidase conjugates, the major 63 kDa Ca(2+)-binding protein of the sarcoplasmic reticulum forms complexes with itself, and with junctin (26 kDa), triadin (94 kDa) and the ryanodine receptor (560 kDa) [Glover, L., Culligan, K., Cala, S., Mulvey, C. & Ohlendieck, K. (2001) Biochim. Biophys. Acta1515, 120-132]. Here, we show that variations in the relative abundance of these four central elements of excitation-contraction coupling in different fiber types, and during chronic electrostimulation-induced fiber type transitions, are reflected by distinct alterations in the calsequestrin overlay binding patterns. Comparative immunoblotting with antibodies to markers of the junctional sarcoplasmic reticulum, in combination with the calsequestrin overlay binding patterns, confirmed a lower ryanodine receptor expression in slow soleus muscle compared to fast fibers, and revealed a drastic reduction of the RyR1 isoform in chronic low-frequency stimulated tibialis anterior muscle. The fast-to-slow transition process included a distinct reduction in fast calsequestrin and triadin and a concomitant reduction in calsequestrin binding to these sarcoplasmic reticulum elements. The calsequestrin-binding protein junctin was not affected by the muscle transformation process. The increase in calsequestrin and decrease in junctin expression during postnatal development resulted in similar changes in the intensity of binding of the calsequestrin conjugate to these sarcoplasmic reticulum components. Aged skeletal muscle fibers tended towards reduced protein interactions within the calsequestrin complex. This agrees with the physiological concept that the key regulators of Ca(2+) homeostasis exist in a supramolecular membrane assembly and that protein-protein interactions are affected by isoform shifting underlying the finely tuned adaptation of muscle fibers to changed functional demands.

Aging↗

PubMed2002

Drastic reduction of calsequestrin-like proteins and impaired calcium binding in dystrophic mdx muscle.

Although the reduction in dystrophin-associated glycoproteins is the primary pathophysiological consequence of the deficiency in dystrophin, little is known about the secondary abnormalities leading to x-linked muscular dystrophy. As abnormal Ca(2+) handling may be involved in myonecrosis, we investigated the fate of key Ca(2+) regulatory membrane proteins in dystrophic mdx skeletal muscle membranes. Whereas the expression of the ryanodine receptor, the dihydropyridine receptor, the Ca(2+)-ATPase, and calsequestrin was not affected, a drastic decline in calsequestrin-like proteins of 150-220 kDa was observed in dystrophic microsomes using one-dimensional immunoblotting, two-dimensional immunoblotting with isoelectric focusing, diagonal two-dimensional blotting technique, and immunoprecipitation. In analogy, overall Ca(2+) binding was reduced in the sarcoplasmic reticulum of dystrophic muscle. The reduction in Ca(2+) binding proteins might be directly involved in triggering impaired Ca(2+) sequestration within the lumen of the sarcoplasmic reticulum. Thus disturbed sarcolemmal Ca(2+) fluxes seem to influence overall Ca(2+) homeostasis, resulting in distinct changes in the expression profile of a subset of Ca(2+) handling proteins, which might be an important factor in the progressive functional decline of dystrophic muscle fibers.

Animals↗

PubMed2002

Diversity of the Brain Dystrophin-Glycoprotein Complex.

Duchenne muscular dystrophy (DMD), the most common inherited neuromuscular disorder, is characterized by progressive muscle wasting and weakness. One third of Duchenne patients suffer a moderate to severe, nonprogressive form of mental retardation. Mutations in the DMD gene are thought to be responsible, with the shorter isoforms of dystrophin implicated in its molecular brain pathogenesis. It is becoming clear that region-specific variations in dystrophin isoforms delegate the composition of the dystrophin-glycoprotein complex in brain, and hence, the function of the specific membrane assembly. Here we summarize the recent advances in the understanding of brain dystrophin, dystrophin-related proteins and dystrophin-associated proteins.

Journal Article↗