MiPsummer Programme pdf - Mitochondrial Physiology Society

51
Abstract # 23
Developing models for the study of Periodic Paralysis
N. Amior 12 , M. Duchen 1 and M. Hanna 2
1 Department of Cell and Developmental Biology; University College London
2 Institute of Neurology; University College London
Periodic Paralysis is caused by mutations of skeletal voltage gated sodium and calcium channels.
The direct effects of these mutations are well characterised, leading to an understanding of the
initial disease phenotype (attacks of paralysis). However, many patients develop a progressive
untreatable myopathy that may render them wheelchair-bound. The pathophysiological mechanisms
that link muscle membrane ion channel dysfunction to myopathy remain obscure. We propose that
dysregulation of cellular calcium signaling and downstream effects on mitochondrial biology play a
central role. Study of these pathways requires the direct analysis of functional muscle cells carrying
the mutations. Patient muscle biopsies are not available for the study of such diseases and to this
end three alternative models are being developed.
1: Myotubes generated from patient and control fibroblasts. The fibroblasts are transduced to
myoblasts by lentiviral delivery of MyoD to the cells. Culture of these cells in low serum medium
in a matrigel matrix with addition of Agrin promotes differentiation into myotubes.
2: An immortalised myoblast cell line. A similar differentiation protocol gives rise to myotubes in
which control or mutant sodium channels will be over-expressed.
3: Muscle is dissected from mutant and control mice. Single fibres are released from the Flexor
Digitorum Brevis for confocal imaging and fibre bundles from the Soleus for analysis of oxygen
consumption.
In each case, dynamic live cell imaging is used to characterise changes in intracellular calcium
signaling and mitochondrial function while respirometry is used to assess the activity of
mitochondrial complexes. This will help to determine the impact of mutations on intracellular
calcium handling and mitochondrial structure and function.