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P34 Exploring IGFN1 in cardiac muscle
S14
Abstracts, UK Neuromuscular Translational Research Conference 2010 Posters / Neuromuscular Disorders 20S1 (2010) S5–S30
P33 Poster Analysis of small fibre function in the C3 mouse, a model of CMT1A A. Clark1 . 1 MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK CMT1A neuropathy caused by duplication of the PMP22 gene is generally thought to affect large myelinated fibres. It is however reported that elevated thermal thresholds signalled by thin myelinated and unmyelinated fibres are commonly found in these patients. We therefore analysed the properties of sensory neurons in the C3 mouse, a novel mouse model of CMT1A with 3 or 4 copies of PMP22. Using the skin-saphenous nerve-preparation in vitro we found a reduction in the conduction velocity and amplitude of A-fibres. To further investigate the properties of distal A- and C-fibre terminals we used immunohistochemistry to study intraepidermal nerve fibre density and Merkel cell distribution and their innervations by myelinated fibres. We found a significant reduction in intra-epidermal nerve fibre density indicating that there is a distal loss of unmyelinated fibres. Futhermore, there is a trend towards a reduced number of Merkel cells in the glabrous skin indicative of Abeta fibre innervations. In addition, we investigated whether the functional properties of the cell body of sensory neurons were affected. DRG neurons from all spinal levels were dissociated and kept in short term culture. There was no change in cell size distribution indicating that there is not a selective loss of sensory neurons. However, there was a significant reduction of neurons stained with isolectin B4, a marker for non-peptidergic unmyelinated fibres and a significant increase in cells binding the Cholera toxin B subunit. These changes in unmyelinated fibres qualitatively resemble those after axotomy. Using Fura ratiometric calcium imaging we studied the functional properties of neurons to TRP channel agonists which are selectively expressed in unmyelinated and thin myelinated fibres. There was no significant change in the percentage of neurons responding to the TRPM8 agonist menthol, or the TRPA1 ligand mustard oil. However there was a significant reduction in the percentage of neurons responding to the TRPV1 agonist capsaicin expressed on heat sensitive neurons. P34 Exploring IGFN1 in cardiac muscle
Poster
G. Riley1 , J. Baker1 , R. Romero1 , H. Hilton1 , G. Blanco1 . 1 Mammalian Genetics Unit, MRC Harwell, Science and Innovation Campus, Oxfordshire, OX11 0RD, UK IGFN1 was identified as an interacting partner of the sarcomeric protein, KY, the loss of which underlies a form of postural musclespecific muscular dystrophy in the mouse (kyphoscoliotic mutant). IGFN1 has a predicted domain composition comparable to that of other sarcomeric support proteins including filamin C, myosin binding protein C and titin. Disruption within these genes is associated with muscular dystrophies and cardiomyopathies hence, IGFN1 may also contribute to the degenerative molecular pathways common to these striated muscle-specific diseases. In this work, northern analysis revealed that Igfn1 is exclusively expressed in skeletal muscle, with smaller isoforms expressed in both skeletal muscle and heart. Several proteins have been identified as putative cardiac IGFN1 products with molecular weights of 189 kDa, 135 kDa and 64 kDa, the 135 kDa isoform being the most predominantly expressed form. Antibodies recognising C-terminal epitopes of IGFN1 localise to the intercalated disk in heart and primary cultured murine cardiomyocytes. Ultrastructural analysis refined this localisation to the adherens junction. Furthermore, peptide mass fingerprint analysis and liquid chromatography followed by tandem mass spectroscopy of IGFN1 immunoprecipitated complexes identified N-cadherin, beta-catenin and plakoglobin, three well-characterised proteins of the adherens junction, as interacting partners of the 135 kDa IGFN1 variant. The molecular roles of IGFN1 remain to be established hence
a conditional targeting vector has been generated and used to generate chimeras; matings to confirm germline transmission of the targeting sequence are currently underway. Once available, this knockout will help clarify the role of IGFN1 at the intercalated disk. Funded by the British Heart Foundation.
Muscle Satellite Cells P35 Poster Immortalisation and characterisation of muscle stem cells M. Ritso1 , L. Jørgensen1 , S. Laval1 , V. Straub1 , K. Bushby1 , 1 1 H. Lochmuller ¨ . Institute of Human Genetics, Newcastle University, UK SJL-Dysf and mdx mice are well-established models for investigating dysferlin-deficient and dystrophin-deficient muscular dystrophies, respectively. In vivo mouse studies are important research methods, yet time-consuming and costly. Establishing primary myoblast cell lines from these disease-model and control mice – here C57BL/10 – will provide a valuable tool to investigate cellular and molecular paths and mechanisms involved in muscular dystrophies. Potential treatments can also be tested, allowing efficient analysis and screening strategies without the use of animals. Myoblast cell lines were derived from C57BL/10 control, mdx and C57BL/10.SJL-Dysf mice. These mice had been crossed into the background of the “Immorto-mouse”, which express a genetic construct constituting a thermolabile large T antigen controlled by the IFNg-inducible H2Kb promoter. Hence, permissive growth conditions were used throughout with IFNg in growth medium and a set temperature of 33°C. Myoblast isolation was achieved successfully using an adaptation of a standard enzymatic digestion protocol. The subsequent derivation of single-cell clones was optimised to large area plating rather than employing the conventional limiting dilution method. The obtained cell colonies were induced to differentiate for testing the ability to fuse and form myotubes. Additionally the cells were analysed with immunofluorescence for expression of Desmin and Myogenin, both well-known myoblast markers. The presence of these markers in several populations of C57BL/10-Immorto control as well as mdx-Immorto and C57BL/10.SJL-Dysf -Immorto confirmed their myogenic nature. Despite the expression of orthodox markers determining these clones as myoblasts, it will still have to be demonstrated that these cell lines will be proliferating after multiple passages and will not become senescent. P36 Poster Phenotypic separation of satellite stem cells using flow cytometry D. Briggs1 , L. Boldrin1 , J.E. Morgan1 . 1 The Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, UK Satellite cells (SCs) are the principal muscle stem cells. They are defined by their anatomical location under the basal lamina of myofibres [1] and are physiologically quiescent. Upon activation SCs proliferate to form a pool of muscle precursor cells, express myogenic regulatory factors and differentiate to repair or replace damaged or lost muscle fibres [2]. It is known that SCs are a heterogeneous population on the basis of marker expression, such as CD34 and M-cadherin [3]. Furthermore, only a small proportion of satellite cells regenerate skeletal muscle [4] and these are likely to be satellite stem cells. We have been fractionating either freshly isolated or cultured SCs by flow cytometric sorting on the basis of different phenotypes, including proliferative state, expression of low or high reactive oxygen species and different cell surface markers. Here we report limitations and advantages of the separation criteria used so far. In vitro characterization of the different sorted SC sub-populations is extending our knowledge
Abstracts, UK Neuromuscular Translational Research Conference 2010 Posters / Neuromuscular Disorders 20S1 (2010) S5–S30
P33 Poster Analysis of small fibre function in the C3 mouse, a model of CMT1A A. Clark1 . 1 MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK CMT1A neuropathy caused by duplication of the PMP22 gene is generally thought to affect large myelinated fibres. It is however reported that elevated thermal thresholds signalled by thin myelinated and unmyelinated fibres are commonly found in these patients. We therefore analysed the properties of sensory neurons in the C3 mouse, a novel mouse model of CMT1A with 3 or 4 copies of PMP22. Using the skin-saphenous nerve-preparation in vitro we found a reduction in the conduction velocity and amplitude of A-fibres. To further investigate the properties of distal A- and C-fibre terminals we used immunohistochemistry to study intraepidermal nerve fibre density and Merkel cell distribution and their innervations by myelinated fibres. We found a significant reduction in intra-epidermal nerve fibre density indicating that there is a distal loss of unmyelinated fibres. Futhermore, there is a trend towards a reduced number of Merkel cells in the glabrous skin indicative of Abeta fibre innervations. In addition, we investigated whether the functional properties of the cell body of sensory neurons were affected. DRG neurons from all spinal levels were dissociated and kept in short term culture. There was no change in cell size distribution indicating that there is not a selective loss of sensory neurons. However, there was a significant reduction of neurons stained with isolectin B4, a marker for non-peptidergic unmyelinated fibres and a significant increase in cells binding the Cholera toxin B subunit. These changes in unmyelinated fibres qualitatively resemble those after axotomy. Using Fura ratiometric calcium imaging we studied the functional properties of neurons to TRP channel agonists which are selectively expressed in unmyelinated and thin myelinated fibres. There was no significant change in the percentage of neurons responding to the TRPM8 agonist menthol, or the TRPA1 ligand mustard oil. However there was a significant reduction in the percentage of neurons responding to the TRPV1 agonist capsaicin expressed on heat sensitive neurons. P34 Exploring IGFN1 in cardiac muscle
Poster
G. Riley1 , J. Baker1 , R. Romero1 , H. Hilton1 , G. Blanco1 . 1 Mammalian Genetics Unit, MRC Harwell, Science and Innovation Campus, Oxfordshire, OX11 0RD, UK IGFN1 was identified as an interacting partner of the sarcomeric protein, KY, the loss of which underlies a form of postural musclespecific muscular dystrophy in the mouse (kyphoscoliotic mutant). IGFN1 has a predicted domain composition comparable to that of other sarcomeric support proteins including filamin C, myosin binding protein C and titin. Disruption within these genes is associated with muscular dystrophies and cardiomyopathies hence, IGFN1 may also contribute to the degenerative molecular pathways common to these striated muscle-specific diseases. In this work, northern analysis revealed that Igfn1 is exclusively expressed in skeletal muscle, with smaller isoforms expressed in both skeletal muscle and heart. Several proteins have been identified as putative cardiac IGFN1 products with molecular weights of 189 kDa, 135 kDa and 64 kDa, the 135 kDa isoform being the most predominantly expressed form. Antibodies recognising C-terminal epitopes of IGFN1 localise to the intercalated disk in heart and primary cultured murine cardiomyocytes. Ultrastructural analysis refined this localisation to the adherens junction. Furthermore, peptide mass fingerprint analysis and liquid chromatography followed by tandem mass spectroscopy of IGFN1 immunoprecipitated complexes identified N-cadherin, beta-catenin and plakoglobin, three well-characterised proteins of the adherens junction, as interacting partners of the 135 kDa IGFN1 variant. The molecular roles of IGFN1 remain to be established hence
a conditional targeting vector has been generated and used to generate chimeras; matings to confirm germline transmission of the targeting sequence are currently underway. Once available, this knockout will help clarify the role of IGFN1 at the intercalated disk. Funded by the British Heart Foundation.
Muscle Satellite Cells P35 Poster Immortalisation and characterisation of muscle stem cells M. Ritso1 , L. Jørgensen1 , S. Laval1 , V. Straub1 , K. Bushby1 , 1 1 H. Lochmuller ¨ . Institute of Human Genetics, Newcastle University, UK SJL-Dysf and mdx mice are well-established models for investigating dysferlin-deficient and dystrophin-deficient muscular dystrophies, respectively. In vivo mouse studies are important research methods, yet time-consuming and costly. Establishing primary myoblast cell lines from these disease-model and control mice – here C57BL/10 – will provide a valuable tool to investigate cellular and molecular paths and mechanisms involved in muscular dystrophies. Potential treatments can also be tested, allowing efficient analysis and screening strategies without the use of animals. Myoblast cell lines were derived from C57BL/10 control, mdx and C57BL/10.SJL-Dysf mice. These mice had been crossed into the background of the “Immorto-mouse”, which express a genetic construct constituting a thermolabile large T antigen controlled by the IFNg-inducible H2Kb promoter. Hence, permissive growth conditions were used throughout with IFNg in growth medium and a set temperature of 33°C. Myoblast isolation was achieved successfully using an adaptation of a standard enzymatic digestion protocol. The subsequent derivation of single-cell clones was optimised to large area plating rather than employing the conventional limiting dilution method. The obtained cell colonies were induced to differentiate for testing the ability to fuse and form myotubes. Additionally the cells were analysed with immunofluorescence for expression of Desmin and Myogenin, both well-known myoblast markers. The presence of these markers in several populations of C57BL/10-Immorto control as well as mdx-Immorto and C57BL/10.SJL-Dysf -Immorto confirmed their myogenic nature. Despite the expression of orthodox markers determining these clones as myoblasts, it will still have to be demonstrated that these cell lines will be proliferating after multiple passages and will not become senescent. P36 Poster Phenotypic separation of satellite stem cells using flow cytometry D. Briggs1 , L. Boldrin1 , J.E. Morgan1 . 1 The Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, UK Satellite cells (SCs) are the principal muscle stem cells. They are defined by their anatomical location under the basal lamina of myofibres [1] and are physiologically quiescent. Upon activation SCs proliferate to form a pool of muscle precursor cells, express myogenic regulatory factors and differentiate to repair or replace damaged or lost muscle fibres [2]. It is known that SCs are a heterogeneous population on the basis of marker expression, such as CD34 and M-cadherin [3]. Furthermore, only a small proportion of satellite cells regenerate skeletal muscle [4] and these are likely to be satellite stem cells. We have been fractionating either freshly isolated or cultured SCs by flow cytometric sorting on the basis of different phenotypes, including proliferative state, expression of low or high reactive oxygen species and different cell surface markers. Here we report limitations and advantages of the separation criteria used so far. In vitro characterization of the different sorted SC sub-populations is extending our knowledge