Thesis

Ontogenetic changes in the skeletal muscle function of Zebrafish (Danio rerio) larvae: identification of biochemical markers for slow and fast contractile behavior

Zebrafish is an important model species to study vertebrate development. One particularly interesting aspect of fish ontogeny, which zebrafish larvae exhibit, is the high tail beat frequency up to 10 times higher than that of adult fish. Larvae therefore require a different muscle fiber type than adult fish. This study identifies the changes in muscle protein composition that might underlie this change in muscle contraction frequency. This question is interesting because contraction frequencies above 80 Hz require the so-called superfast muscles, a muscle type so far only been identified in non-locomotory muscles not required to generate significant forces. Superfast muscles are known to differ from normal fast muscles in several proteins, including myosin heavy chain. In this study we will focus on myosin, the largest protein fraction of a muscle that constitutes 40- 50% of total muscle. This study identifies and validates a protocol for myosin protein extraction and identification of myosin isoforms. Fast myosin heavy chains (210 kD) were identified in hatchlings (not yet swimming actively) and in adult Zebrafish (tail beat frequency 10 Hz). In 6-d old larvae (swimming actively, tail beat frequency 100 Hz), we found an isoform (160kD) that was identified in cat masseter muscle, which is a muscle with superfast contraction characteristics. We identified isoforms by probing Western blots with primary antibody (MYH1/2/3) against myosin heavy chains. BCA and ImageJ analysis were performed to quantify the amount of total protein and the myosin content.

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