Optimizing the Approach for Maintaining Single Muscle Fibers in Culture

Title: Optimizing the Approach for Maintaining Single Muscle Fibers in Culture
Authors: Hind, Albadrani
Date: 2014
Abstract: The skeletal muscle is a dynamic tissue that has the ability to change and modify itself to fit the level of required activity; a phenomenon called muscle plasticity. Most studies of muscle plasticity are carried out in situ, a condition for which it is difficult to study and discern between the intrinsic properties of skeletal muscle, the myokines released by muscle fibers and the neurotrophic factors released by neurons innervating skeletal muscles that play various roles in the mechanisms of muscle plasticity. Another approach is to study the morphological and contractile properties of single adult muscle fibers under culture conditions for which one can fully control the level of activity and exogenous factors affecting muscle plasticity. However, the survival of single muscle fiber in culture is very low as most fibers degenerated or supercontracted within 5-7 days. The first objective of this study was to optimize fiber survival in culture. The application of chronic stimulation and beta-adrenergic agonists are two major factors that prevent muscle atrophy and loss of force in denervated muscles in situ. So, objective two was to determine if chronically stimulated single fibers in culture also improve fiber survival and contractile characteristic under culture conditions. The third objective was the same for salbutamol, a beta 2-adrenergic agonist. In regard to the optimization of fiber survival, the Minimum Essential Medium (MEM) was a better medium than Dulbecco’s Modified Eagle Medium (DMEM), changing 50% of the culture medium every two days also improved fiber survival compared to changing the medium every day. Interestingly, inhibiting the proliferation of satellite cells with AraC largely improved fiber survival when fibers were kept under resting conditions, but not when they were chronically stimulated. Finally, under conditions in which proliferation of satellite cells was inhibited, the use of a collagen/laminin mixture as adhering substrate to improve fiber adhesion to glass coverslip gave rise to a better fiber survival than Matrigel that contains not only collagen and laminin but several growth factors. The results suggest i) that when satellite cells (or fibroblasts) are allowed to proliferate they appear to contribute to the degeneration of fibers under resting conditions and ii) that the release of myokines by skeletal muscle fibers (or cytokines by other cells) likely play a role in fiber survival. Contrary to the situation in situ, neither the chronic stimulation nor salbutamol improved fiber survival and contractile characteristics of muscle fibers in culture suggesting that some important factors in culture are missing to allow chronic stimulation and salbutamol to reduce muscle atrophy and loss of force.
URL: http://hdl.handle.net/10393/31900
CollectionThèses, 2011 - // Theses, 2011 -