Ultrastructural study of gap filaments, endomysium and intermyofibrillar bridges

Abstract

Super-stretched muscle fiber bundles (taken within ten minutes postmortem) were incubated at one of four combinations of temperature and pH (pH 5.5, 37 °C; pH 5.5, 0-4 °C; pH 7.4, 37 °C; pH 7.4, 0-4 °C) for one hour prior to fixation and embedding for transmission electron microscopy. These tissues were compared to controls which were fixed immediately after stretching. Disruption of gap filament ultrastructure was greatest in those muscle bundles incubated at pH 5.5 and 37 °C. It was determined that pH had greater influence upon gap filament disruption than did temperature and that pH 5.5 caused more gap filament disruption than did pH 7.4. Super-stretched muscle bundles taken within ten minutes postmortem were incubated for five hours in one of six treatments: ( 1 ) 0.1M KCl, 0.02M imidazole, 15mM EDTA, 1mM NaN3, pH 7.4; ( 2 ) same as ( 1 ) , plus 10mM CaCl2; ( 3 ) same as ( 1 ), plus CaCl2 , minus EDTA; ( 4 ) same as ( 1 ) , minus EDTA; ( 5 ) 0.1M KCl, 0.02M citrate, 1mM NaN3, pH 5.5, and ( 6 ) same as ( 5 ) , plus EDTA. Gap filaments were not visible in all pH 5.5 treatments and in those pH 7.4 treatments not containing EDTA, indicating that gap filaments were degraded by both calcium dependent neutral and acid proteases. Bovine sternomandibularis and rabbit semimembranosus muscles were fixed in buffered hypertonic glutaraldehyde fixative. Freeze substitution methods were utilized to minimize ice crystal damage, and samples were further prepared for scanning electron microscopy (SEM) using traditional methods. Regularly spaced filaments were found connecting neighboring myofibrils within both rabbit and bovine muscle. Moreover, bridges between myofibrils at or near the Z - lines were observed in transmission electron micrographs of conventionally prepared bovine sternomandibularis muscle. The endomysium was observed to contain rows of reticular fibers arranged either obliquely or perpendicularly to the longitudinal axis of myofibers, depending upon the degree of muscle stretch.