Edouard de Dreuzy    - research student -

Abstract: Deficiency in membrane repair is a hallmark of dysferlin-deficient muscles. Evaluation of this deficit was usually performed using a laser-wounding assay. In this project, we investigated membrane resealing of dysferlin-deficient fibers following two different treatments: hypotonic shock and application of the bacterial toxin: Streptolysin O (SLO). It was previously shown that membrane blebbing in myotubes in response to hypotonic shock requires dysferlin 1. Here, we applied a hypotonic solution to isolated muscle fibers. Systematic blebs were observed immediately after dilution of the medium in both dysferlin-deficient and C57Bl6 fibers. It seems therefore that myofibers do not require dysferlin to generate blebs. It is also known that membrane resealing of transmembrane pore induced by SLO is a Ca2+ dependant endocytic process as it is after mechanical wounds 2. We wanted to investigate whether dysferlin is implicated in repair after pore formation. The conditions for obtaining membrane wounding by treatment with SLO were optimized to obtain an imaging kinetic of the entry of propidium iodide and FM1-43. Strong heterogeneity in the response to such treatment was obtained but no clear evidence of a significant difference between dysferlin deficient and wild type fibers was yet observed. For both assays, we could not differentiate any specific response between dysferlin-deficient and C57Bl6 fibers. Whether a difference could be obtained by exploring other technical conditions or whether this absence of difference is due to the phenotypic specificity of isolated fibers (compared to myotubes) remains to be investigated.

Abstract: Membrane tears in dysferlin deficient muscle fibers are the effects of a physiologic response to particular mechanic stress like lengthening contractions. Sarcolemmal membrane disruption can either lead to fiber necrosis or repair but little is known about the size and the kinetics of formation of the tears. To study this, we subjected dysferlin-deficient mice tibialis anterior muscle to eccentric exercise –either with downhill running on treadmill or induced lengthening contraction inspired from Bloch and al– to investigate several aspects of the formation of membrane tears. We previously estimated the size of membrane rips to be under 10µm using a size exclusion strategy based on the penetration into the damaged fibers of circulating fluorescent dextrans. In this work, we describe the frequency and location of tears in 6, 12 and 18 month old mice, as well as the muscle tissue composition transformation following exercise. To explore the spatial dimension, we attempted to couple the Large Strain Injury (LSI) device from the Bloch lab with macroconfocal imaging to observe the tearing process in real time. This set up should be helpful in following the membrane resealing after tearing in-situ muscle in a living mouse, but is limited in its spatial resolution. To overcome this limitation, a similar approach was developed in vitro for isolated extensor digitorum brevis (EDL) muscle which consists of a controlled stimulating unit enabling the muscle contraction while it is lengthening simultaneously. We hope to give new insights on the dynamics of the mechanisms of generation and resealing of membrane tears in a dysferlin deficient context.