MRI and proteomic studies in normal and
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1 MRI and proteomic studies in normal and pathological muscles. Isabel ILLA. Catedrática Medicina de la Universitat Autònoma de Barcelona. Chief Neuromuscular Diseases Unit. Hospital Santa Creu i Sant Pau. Barcelona 2nd MuscleTech Network Workshop September 27 29, 2010 Barcelona
2 HOSPITAL SANTA CREU I SANT PAU INSTITUT RECERCA (IR-HSCSP) Clinical and basic researchers working In Neuromuscular Diseases in the Hospital campus.
3 Clinical and Lab facilities Clinical Unit: Out-patients In- patients Day hospital ( infusions IvIg, Rituximab, ) EMG Muscle biopsy 3T-MRI Laboratory: Sample collection ( muscle biopsies> 1000, sera > 5000, Immunohistochem, ELISA, WB, 2D gel electrophoresis, IP, Blue-N electrophoresis, tissue culture, rtpcr, flow cytometry, confocal microscopy FLIM, itraq analysis QMT, Mouse models
4 Research interests Immune diseases: Biomarkers (CIDP and MG), immunopathology, new therapies and their mechanisms. Muscular Dystrophies Dysferlin: Protein function, interacting partners, cell therapy. MRI IP of a national project funded d by CIBERNED ( Ministery of Health) to create a national database of fneuromuscular Diseases
5 Muscular dystrophies They are muscle diseases. Due to mutations in different genes. Patients have a progressive muscle weakness.
6 normal muscle muscular dystrophy
7 Diagnostic Strategy Pattern of muscle weakness Pattern of muscle weakness. MRI. Muscle biopsy. Immunohistochemistry, Western Blot,.. Genetic analysis.
8 Proximal weakness Distal weakness
9 Diagnostic strategy Pattern of muscle weakness. RNM MRI. Muscle biopsy. Immunohistochemistry, Western Blot,.. Genetic analysis. Mercuri et alnmd Fischer D et al J Neurol
10 Diagnostic strategy Pattern of muscle weakness. MRI. Muscle biopsy. Immunohistochemistry, Western Blot,.. Genetic analysis.
11 Diagnostic strategy Pattern of muscle weakness. MRI. Muscle biopsy. Immunohistochemistry, Western Blot,.. Genetic analysis.
12 MRI studies Same gene mutated = same pattern of muscle involvement?. Different diseases = pattern? Are all muscles equally affected? Are some muscles spared regardless of the type of muscular dystrophy?
13 1 2 Mutations in TTN, encoding the protein titin (TTN)
14 Oculopharyngeal muscular dystrophy Polyadenylate-binding l di protein, Nuclear, 1 (PABN1; PABP2) Genetics: Expansion of GCG repeat Ptosis + dysphagia
15 MRI, moderate phenotype LGMD 2I: LGMD 2A(calpain): Adductor, biceps femoris gastrocnemius median head soleus LGMD2B (dysferlin): thigh : anterior and posterior both gastrocnemius Mercuri et alnmd Fischer D et al J Neurol
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18 LIMB GIRDLE WEAKNESS Sarcolemmal proteins Ervasti JM BBA 2007
19 DISTAL WEAKNESS Lange S., Ehler E., Gautel M. TRENDS in Cell Biology. January 2006
20 MRI studies Same gene mutated = same pattern of muscle involvement? yes Are all muscles equally affected? No. Why? Are some muscles spared regardless of the type Are some muscles spared regardless of the type of muscular dystrophy? Yes. Why?
21 Proteomic study Same muscle (triceps) in controls and different muscular dystrophies: calpainopathy, FSH and dysferlin myopathy. Different muscles in normal muscles
22 Proteome e Proteome- Set of proteins that are expressed from a genome at a given time and tissue. The proteome is highly dynamic and varies depending on the different states and environment in the cell or a particular subcellular compartment.
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24 Hierarchical Clustering This method is based on a statistical algorithm that allows grouping of samples that share a particular expression pattern (mrna, protein ).
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28 Our proteomic studies have shown a protein expression pattern common to all the muscular dystrophies studied and different from the controls (E.g. FHL1, Triosephosphate Isomerase). The increased levels of Ankrd2 observed in dysferlin myopathy suggest that there is indeed an active switch to type I fibers rather than a loss of type II fibers.
29 Proteomic analysis Same muscle (triceps) in controls and different muscular dystrophies: calpainopathy, FSH and dysferlin myopathy. Different muscles in normal subjects
30 Muscles selected for this study Vastus Intermedius Adductor brevis Adductor Longus Gracilis Semitendinous Soleus
31 Protein expression maps Gracilis Semitendinous Adductor Longus Adductor Brevis Vastus medius Soleus
32 Hierarchical clustering of the muscles studied
33 S G S S G S Gracilis and semitendinous display a protein expression pattern that could be protective.
34 Differential expression of proteins involved in muscle contraction Spot Nº Identificación # acces Función Gracilis/Semitendinoso Otros Músculos Myosin Light chain 1, skeletal lmuscle MLE1_HUMAN Aumentado Disminuidoi id Troponin T, fast skeletal muscle Q6FH29 Aumentado Disminuido HUMTROPA 3 M19714 Aumentado Disminuido Tropomyosin beta, chain, TPM2 TPM2_HUMAN Aumentado Disminuido Troponin T, Slow skeletal muscle TNNT1_HUMAN Aumentado Disminuido Actin alpha, skeletal muscle A24904 Disminuido Aumentado Tropomyosin alpha-3 chain TPM3_HUMAN Aumentado Disminuido Myosin light polipeptide 3 P08590 Disminuido Aumentado Troponin T, Slow skeletal muscle TNNT1_HUMAN Contractíl Aumentado Disminuido Tropomyosin beta, chain, TPM2 TPM2_HUMAN Aumentado Disminuido Myosin light polipeptide 3 P08590 Disminuido Aumentado Myosin light polipeptide 3 P08590 Disminuido Aumentado Troponin T, Fast skeletal muscle* P48788 Aumentado Disminuido Troponin I, Fast skeletal muscle P45378 Aumentado Disminuido Actin, alpha skeletal muscle ACTS_HUMAN Aumentado Disminuido 477 Myosin regulatory light chain 2* MLRS_HUMAN Aumentado Disminuido 1225 Myosin regulatory light chain 2* MLRV_HUMAN Aumentado Disminuido Actin-related protein 2/3 complex subunit 2 ARPC2_HUMAN Aumentado Disminuido 14 proteins upregulated and 4 downregulated
35 Differential expression of proteins involved in muscle metabolism Spot Nº Identificación # acces Función Gracilis/Semitendinoso Otros Músculos aldolase A X06352 Disminuido Aumentado Fatty acid-binding protein, heart FABPH_HUMAN Aumentado Disminuido Creatinine Kinase M-type KCRM_HUMAN Aumentado Disminuido Alpha - Crystallin chain B A35332 Disminuido Aumentado Triosephosphate Isomerase 1 TPIS_HUMAN Aumentado Disminuido Triosephosphate Isomerase TPIS_HUMAN Aumentado Disminuido Adenilate Kinase 1* AL Aumentado Disminuido enolase 3* BC Metabolismo Aumentado Disminuido Creatinine Kinase M-type KCRM_HUMAN Disminuido Aumentado 2306 Beta-enolase ENOB_HUMAN Aumentado Disminuido 191 Beta-enolase ENOB_HUMAN Aumentado Disminuido 893 Pyruvate dehydrogenase [lipoamide]]-phosphatase PDP1_HUMAN Aumentado Disminuido 86 Beta-enolase ENOB_HUMAN Aumentado Disminuido Malate dehydrogenase,mitochondrial precursor MDHM_HUMAN Aumentado Disminuido 1269 Heat shock protein beta-1, Mezcla HSPB1_HUMAN Aumentado Disminuido 1269 Enoyl-CoA hydratase, mitochondrial precursor, Me ECHM_HUMAN Aumentado Disminuido 13 proteins upregulated and 3 downregulated
36 Conclusions 2-D gel analysis is useful to stablish protein expression patterns specific for each muscle. Gracilis and semitendinous muscles display a similar protein expression pattern in comparison with the other muscles studied. d Their particular protein expression profile could explain why they spared or mildly affected in the majority of muscular dystrophies.
37 Future To unravel the patterns of molecules implicated in muscle degeneration and membrane regeneration would allow therapeutical interventions. To know the differential pattern of protein expression present in specific muscles (spared in diseases, resistant to injury,..) could be used to increase efficacy in muscle repair, or to avoid muscle fibrosis,...
38 Thank you for your attention Moltes gràcies per la seva atenció Interested?
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