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Properties of single fibers in global extraocular muscle of the cane toad, Bufo marinus

Very limited information exists on the contractile physiology of the specialized muscle fibers that make up vertebrate extraocular muscle (EOM). This study was undertaken to measure some contractile properties of fibers in the global region of EOM using the cane toad, Bufo marinus, and in particular the tonic or multiply-innervated fibers (MIFs). Fiber types were characterized using a number of methods. Tonic and twitch fibers were identified in EOM using transmission electron microscopy. Tonic fibers lacked an M-line, had sparse sarcoplasmic reticulum (SR), few mitochondria and a thick Z-line. Some twitch fibers showed the presence of lipid stores and abundant mitochondria, whereas others were distinguished more by well developed SR. In transverse section, tonic fibers showed a felderstruktur pattern of fused myofibrils and the twitch fibers displayed a fibrillenstruktur pattern of well-delineated myofibrils. The distribution and diameter of tonic and twitch fibers in the global region were characterized using histochemistry. Transverse sections stained for mATPase, α-GPDH and NADH-TR activity revealed the presence of at least three different fiber types in global EOM of Bufo marinus. Tonic fibers showed little or no activity for all three enzymes and were distributed randomly within the global region where they made up about 20% of the fibers. Tonic fibers always occurred at lower frequencies than twitch fibers at all diameter ranges analysed. Two further types of fiber were characterized by their histochemical appearance as twitch light and twitch dark. Force-pCa relationships were determined for chemically skinned single fibers by recording peak isometric force at maximal and submaximal calcium activation. Fibers were found to differ in their force response to initial and maximal calcium activation. Force-pCa data were fitted to Hill plots and Hill coefficients, n₁ and n₂, obtained for responses at high and low calcium concentrations, respectively. This information together with diameter and appearance (clear versus dark) also allowed fibers to be divided into three groups. Tonic fibers developed tension slowly and had significantly lower maximum normalized tension and n₂ values than twitch light fibers. Twitch dark and twitch light fibers developed tension rapidly in response to initial and maximal calcium activation and had similar n₂ values. However, twitch dark fibers had a significantly lower maximum normalized tension than twitch light fibers. The pCa₅₀ values did not differ between the three groups of fibers. Fiber classifications were corroborated by single fiber transmission electron microscopy (TEM). SDS-PAGE followed by silver staining was used to resolve the MLC components of single fibers for which force-pCa relationships were known. Both twitch light and twitch dark fibers had three myosin light chains, each having the same electrophoretic mobility as the light chains from the fast-twitch rabbit psoas muscle. The tonic fibers were found to lack the low molecular weight light chain, myosin light chain three (MLC3), and the regulatory light chain (MLC2) of these fibers showed reduced mobility compared to that of the twitch fibers. The finding that tonic fibers responded in a different manner to initial and maximal calcium activation from twitch fibers can be used by others to aid in their identification. The significantly different responses of twitch and tonic fibers to maximal and submaximal calcium activation highlight the extent to which these fiber types differ. Additional work using single fibers will undoubtedly reveal further differences in the composition and functioning of contractile proteins in EOM fibers.
Type of thesis
The University of Waikato
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