Analysis of Titin in Red and White Muscles: Crucial Role on Muscle Contractions Using a Fish Model

Figures

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  Figure 1. Red muscle (R) contains more lipids than white muscle (W). (a) The dotted line shows a cross-section near the cloaca of Tilapia. (b) Cross-section; red muscle is located on the lateral sides of the trunk (arrows), while white muscle is located on the back and ventral parts of the fish. (c) Oil-red O staining of a frozen section; red muscle contains more lipids than does white muscle. (d) More intracellular lipid droplets were seen in red muscle.

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  Figure 2. Negative ion TOF-SIMS mass spectrum. (a) Partial mass spectrum obtained from a region of interest containing red and white muscles in a fish muscle section in the m/z range of 0~300. (b) Values of m/z of identified peak areas are labeled in the TOF-SIMS spectrum.

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  Figure 3. TOF-SIMS images of negative ion and fatty acid compositions of red (R) and white (W) muscles. (a) TOF-SIMS ion image showing the distribution of phosphate (PO3−) m/z 79. (b) TOF-SIMS ion image showing the distribution of palmitic acid m/z 255. (c) TOF-SIMS ion image showing the distribution of oleic acid m/z 281. (d) Bright-field standard. Bar: 100 μm. Image size 500 × 500 μm (256 × 256 pixels).

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  Figure 4. Elastica-van Gieson (EVG) and Mito-Tracker Green FM staining to, respectively, detect connective tissues and mitochondria. (a) and (b) EVG staining labeled connective tissue between muscle fibers. Collagen fibers are red, and elastic fibers are purple. There was more connective tissue (arrowheads) between muscle fibers in red muscle (R) than white muscle (W). In addition, the size of red muscle fibers was smaller than that of white muscle fibers. (c–f) Mito-Tracker Green FM staining to label intracellular mitochondria. (c) and (e) show the mitochondrial distribution images of red muscle. (d) and (f) show the mitochondrial distribution images of white muscle. There was a high density of green fluorescence surrounding each muscle fiber in both muscles, indicating that abundant mitochondria were located beneath the sarcolemma. In addition, more green dots or trabecula (arrows) were found in red muscle fibers than in white muscle fibers ((e) and (f)), indicating that more-abundant mitochondria were located between myofibrils in red muscle than in white muscle.

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  Figure 5. Electron microscopic (EM) ultrastructural images of red (R) and white (W) muscle fibers. (a) Several mitochondria are located near the sarcolemma. Lipid droplets can be seen between the myofibrils in red muscle fibers. (b) EM image indicating less cytoplasm and fewer mitochondria between myofibrils in white muscle fibers. (c) A longitudinal section displayed the well-organized sarcomere in red muscle fibers. (d) Well-organized sarcomeres were also observed in white muscle fibers. The sarcomere length (double-headed arrow) is the distance between two Z-lines. (e) The sarcomere length was shorter in white muscle fibers. (f) The Z-line width was thinner in white muscle fibers. (g) The myofibril width was larger in white muscle fibers. ∗ p < 0.001. L, lipid droplet; M, mitochondria; SL, sarcomere length; Z, Z-line. Bar: 0.5 μm.

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  Figure 6. SDS-PAGE protein analysis of red (R) and white (W) muscles. (a) After loading equal concentrations (2~10 μg) of total proteins, white muscle displayed more myofibrillar proteins than did red muscle. Titin, myosin heavy chain (MHC), and actin were the three major myofibrillar proteins. (b) Ratios of titin, MHC, and actin in red muscle to white muscle. (c) Mean ratios of titin/MHC in red and white muscles. # p < 0.01, ∗ p < 0.001.