Original Paper Cellular Physiology Cell Physiol Biochem 2012;29:905-918 Accepted: March 29, 2012 and Biochemistry Cholesterol Depletion Uncouples β-dystroglycans from Discrete Sarcolemmal Domains, Reducing the Mechanical Activity of Skeletal Muscle Jesús Vega-Moreno1, Aldo Tirado-Cortes1, Rocío Álvarez1, Claudine Irles2, Jaime Mas-Oliva3 and Alicia Ortega1,2 1Departamento de Bioquímica, Facultad de Medicina, Universidad NacionalAutónoma de México, México City, 2Departamento de Bioquímica, Instituto Nacional de Perinatología, México City, 3Departamento de Bioquímica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México City Key Words tigue is unaffected, but fatigue recovery is depend- Cholesterol • Skeletal muscle • β-dystroglycan • ent upon cholesterol restoration. MβCD modifies the Sarcolemma • Lipid raft structures of lipid rafts obtained from MβCD-treated muscles by, displacing the membrane proteins β-DG and caveolin-3 f from the lipid raft, thus reducing the Abstract interaction of β-DG with dystrophin. Conclusion: Cho- Background/Aims: β-Dystroglycan (β-DG) is a trans- lesterol depletion weakens the muscle contractile membrane glycoprotein that links the intracellular force by disturbing the sarcolemmal distribution of β- cytoskeleton to the extracellular matrix and is crucial dystroglycan and its interaction with dystrophin, two for the molecular pathway of lateral force transmis- key proteins in the alignment of lateral force trans- sion in muscle. We aimed to investigate the effect of mission pathway. decreasing sarcolemmal cholesterol on the distribu- Copyright © 2012 S. Karger AG, Basel tion of β-DG, its interaction with dystrophin and the impact on the contraction efficiency of muscle. Meth- ods: Isolated rat extensor digitorum longus muscles were incubated with methyl β-cyclodextrin (MβCD) to deplete cholesterol and with MβCD-cholesterol to restore cholesterol. Electric stimulation protocols were used to determine muscle force and fatigue. Deter- gent-resistant membranes (lipid rafts) were separated Introduction from isolated skeletal muscle sarcolemma. The dis- tribution and interactions of β-DG, caveolin-3 and Dystroglycan (DG) is a 2-subunit protein derived dystrophin were determined by an immunoreactivity from a single gene, DAG1, which encodes an 895- analysis. Results: Cholesterol depletion in muscle aminoacid precursor and is highly conserved in verte- results in a weakened force of contraction, which re- brates. The precursor of DG is post-translationally covers after cholesterol restoration. The rate of fa- cleaved between residues 653 and 654 to generate the © 2012 S. Karger AG, Basel Alicia Ortega 905 1015-8987/12/0296-0905$38.00/0 Departamento de Bioquímica y Biología Molecular, Fax +41 61 306 12 34 Facultad de Medicina,Universidad NacionalAutónoma de México, E-Mail [email protected] Accessible online at: México City AP 70-159, CP 04510, (México) www.karger.com www.karger.com/cpb Tel. +5255 56232511, Fax +5255) 56162419, E-Mail [email protected] α-DG and β-DG subunits [1]. α-DG is an extracellular ere [21, 22]. Caveolin-3, the muscle-specific form of peripheral membrane protein that associates with the caveolin, is also a major structural and regulatory integral muscle cell plasma membrane (sarcolemma) through an membrane protein at the sarcolemma and in the interaction with β-DG. Residues 550 and 565 of α-DG TT-membrane. Caveolin-3-null mice exhibit abnormali- are non-covalently anchored to residues 691 and 719 of ties in the formation [23] and organisation of the T-tubule β-DG [2]. Importantly, the Cys669 and Cys713 residues of system [24] and have dilated and longitudinally β-DG are redox regulated to control the interaction with oriented TT-membranes [25], thereby affecting sarcom- the α-DG subunit [3]. DG is the central component of eric fibre structure and fibre development [26]. Caveolin- the dystrophin-glycoprotein complex (DGC) in muscle, 3 is associated with different membrane which is linked to the transmission of lateral force during regions of the cardiac sarcolemma and the TT-membrane muscle activity [4, 5]. α-DG functions as a receptor for that are related to hormone and drug signalling [27]. extracellular matrix proteins, such as laminin, thus ensur- Signal transduction through cellular membranes can ing contact between the basal membrane and the plasma be regulated by the interaction of the cytoskeleton with membrane [6]. β-DG is a sarcolemmal transmembrane caveolin-enriched membrane domains in striated glycoprotein and contains a single 24-amino acid mem- muscle [28]. brane-crossing helix. β-DG links the cytoskeleton to the Caveolin-3 and β-DG are influenced by the lipid extracellular matrix [1, 6, 7]. β-DG is aligned mainly with composition of the membrane. High cholesterol and sphin- the Z-disk and exhibits a sarcomeric distribution along golipid concentrations in the plasma membrane are asso- the myofibre [8]. The intracellular domain of β-DG inter- ciated with discrete membrane domains [29] that are acts with the costameric proteins dystrophin and utrophin enriched with caveolins. The extraction of cholesterol from [9, 10]. Laterally, β-DG is also linked to the sarcoglycan skeletal muscle fibre diminishes the interaction of caveolin- protein complex (SGC) [11, 12]. The absence or muta- 3 with dystrophin [30] and affects the contraction force tion of any of the costameric proteins associated with the of the muscle [31]. Because TT-membranes are enriched DG complex, such as dystrophin [13], dystroglycans [14], in cholesterol, cholesterol depletion may alter muscle SGC components [10] and laminin [15], strongly perturbs mechanical activity by affecting the activity of the the morphology of muscle fibres, resulting in weak and dihydropyridine receptor [31]. dystrophic muscle. The absence of dystrophin in skeletal In the present study, we investigated the effects of muscle leads to a dramatic perturbation of the sarcolem- decreasing the cholesterol concentration with methyl- β- mal location of both α- and β-DG [16]. Ciclodextryn (MβCD) on the mechanical properties of Sarcoglycanopathies are a group of four autosomal fast skeletal muscle. MβCD is a cyclic oligomer of recessive limb girdle muscular dystrophies (LGMDs) glucopyranoside that does not incorporate into mem- caused by mutations of the α, β, γ and δ sarcoglycan branes, is not membrane permeable and selectively ex- genes, resulting in the disruption of the sarcomeric ar- tracts membrane cholesterol by sequestering it in a cen- rangement of the SGC in the sarcolemma [17] and sar- tral non-polar cavity [32]. From the isolated sarcolemma, coplasmic reticulum (SR) [18]. Some naturally occurring we separated a ganglioside-enriched membrane with a mutations in the DG gene have been associated with spe- detergent-resistant membrane domain (lipid raft) and cific types of muscular dystrophy [19], but the absence analysed the distribution of β-DG. The depletion of sar- of β-DG in skeletal muscle is embryonically lethal [20]. colemmal cholesterol with MβCD modified the distribu- β-DG is the central component of the dystrophin- tion of β-DG in the membrane and the interaction of β- dystroglycan- laminin axis in striated muscle. During iso- DG with dystrophin and caveolin-3. These effects were tonic contraction, these proteins interact to transmit lat- associated with a decrease in force development, which eral force. reversibly affected the mechanical properties of skeletal In adult skeletal muscle, the sarcolemma invaginates muscle. We propose that the disruption of discrete cho- periodically, giving rise to the transverse tubule membrane lesterol-enriched domains in the sarcolemma affects the network (TT-membrane). The TT-membrane openings distribution of β-DG and, consequently, its interaction with to the sarcolemma are in close proximity to the Z-disk, dystrophin, resulting in a decreased force of contraction, and TT-membrane genesis is related to the formation of which is immediately reversible upon the restoration of multiple caveolae, primarily in the I-band of the sarcom- cholesterol content. 906 Cell Physiol Biochem 2012;29:905-918 Vega-Moreno/Tirado-Cortes/Álvarez/Irles/Mas-Oliva/Ortega Materials and Methods tion was followed by 5 minutes of rest before the tension re- covery experiment; the same stimulation protocol described Animals above was used. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Insti- Cholesterol concentration in muscle fibre modified by tute of Laboratory Animal Resources of the United States as MβCD approved in Mexico by the Ethics Committee of the School of To study the effect of cholesterol removal on mechanical Medicine of the National Autonomous University of Mexico properties, the EDL muscle was incubated for 30 min with 15 (UNAM) (NOM-062-ZOO1999). mM MβCD and washed before the initiation of the fatigue protocol. To study the reversibility of the effect of cholesterol Muscle preparation for mechanical studies depletion on muscle activity, the EDL muscle was incubated Male Wistar rats weighing 240 to 280 g were euthanised with 15 mM MβCD and subsequently incubated with either 10 by cervical dislocation; the extensor digitorum longus (EDL) or 15 mM MβCD-cholesterol and washed before the initiation was then isolated at room temperature. The isolated muscle of the fatigue protocol. The normalised force was calculated was placed in an acrylic chamber equipped with platinum elec- from the maximal force produced in