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Morphological quantitative and ultracytochemical studies on the internal membrane systems of normal and mdx-dystrophic murine skeletal muscle fibers

University of British Columbia

Mitochondria and the sarcotubular system in skeletal muscle fibers are specialized intracellular membranes that participate in the generation of energy (ATP) and i n the regulation of contraction and relaxation. This thesis comprises four parts dealing with morphological, histochemical, stereological and biochemical approaches to study these organelles in muscle fibers of adult normal and dystrophic (mdx) mice. The purpose of this research was to test the hypothesis that the internal membrane systems of skeletal muscle fibers are altered in the mdx mouse. Mitochondrial oxidative capacities were assessed in muscle fibers of the extensor digitorum longus (EDL), soleus and diaphragm of normal and mdx mice by histochemical and microphotometric methods in Chapter 1. Relative to controls, significant shifts i n oxidative staining patterns of mdx muscle fibers were observed including a 20% overall lower oxidative capacity in the mdx EDL and soleus and a contrasting 7% higher oxidative capacity in the mdx diaphragm. Transmission electron microscopy (TEM) was combined with stereology in Chapter 2 to determine the ultrastructural arrangement and volume density of the sarcotubular system in muscle fibers in these muscles. Three fiber types (white, intermediate and red) were distinguished in the normal and mdx EDL based on mitochondrial volume in fiber cores and Z line thickness. A mixture of two fiber types, intermediate and red fibers, occupied the soleus and diaphragm. The majority of mdx muscle fibers displayed a similar ultrastructure and disposition of these organelles in comparison to their normal counterparts with nonsignificant differences in sarcotubular volume density between normal and mdx fiber types. Degenerative fibers in mdx muscles exhibited dilated SR, sarcolemmal and myofibril disruption, and mononuclear cell invasion. High-resolution scanning electron microscopy (HRSEM) was utilized in Chapter 3 to reveal the spatial distribution of mitochondria and the sarcotubular system by extracting non-membranous components of the sarcoplasm in freeze- fractured specimens. Finally, in Chapter 4, a cerium-based, ultracytochemical staining method was used for the detection and measurement of the calcium and magnesium-dependent paranitrophenylphosphatase (Ca2 +-Mg2 + pNPPase) activity associated with the sarcoplasmic reticulum (SR) membrane. By TEM, the majority of Ca2+-Mg2+pNPPase activity was localized to the terminal cisternae regions of the SR membrane with moderate phosphatase activity in mid-A band and I band regions of the SR. Comparative spectrophotometry of reaction media revealed a significant 25% higher phosphatase activity in mdx muscle tissue when compared to normal muscle suggesting that Ca2+-sequestering by the SR is enhanced in muscle fibers of the mdx-dystrophic mouse. Overall, these data suggest that mitochondria and SR are altered in the skeletal muscle fibers of mdx mice and point to specific differences in oxidative capacity and Ca2+-sequestering properties of these organelles. [Scientific formulae used in this abstract could not be reproduced.]

Thesis/Dissertation

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2009-07-15

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http://hdl.handle.net/2429/10831

Anatomy, Cell Biology and Physiology

University of British Columbia

UBCV

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