Direct Assessment of Skeletal Muscle Contractile Strength in Live Wildtype and Rbfox Morphant Zebrafish Larvae

Abstract

The accessible genetics and abundant skeletal musculature of the zebrafish make it an ideal model for studies of muscle contraction. Current functional assays for quantifying the effect of experimental modification of zebrafish muscle are indirect and observational, or they underestimate contractile force. Therefore, we developed an in vivo assay for measuring muscle contractile force. As proof of concept, we have measured contractile strength of skeletal muscle in wildtype larvae and in a morphant model from 2-5 days post fertilization (dpf). Mean maximum tetanic whole-body forces produced by wildtype 2, 3, 4, and 5 dpf larvae amounted to 3.03 mN, 7.31 mN, 8.67 mN, and 10.94 mN, respectively. Mean twitch forces produced by larvae were 0.90 mN, 5.58 mN, 7.08 mN, and 9.08 mN for 2, 3, 4, and 5 dpf respectively. The morphants we have analyzed are knockdowns of two zebrafish paralogs of rbfox, rbfox1l and rbfox2, which regulate muscle-specific splicing programs. We assessed the contractile force developed during contraction and found that our assay is clearly able to differentiate between morphant phenotypes. rbfox2 morphants appear to produce maximal tetanic forces similar to wildtype larvae, while rbfox1l morphants demonstrate significantly impaired function by producing decreased forces at the same developmental time points. This supports the conclusion that rbfox1l regulates the majority of splicing events in larval skeletal muscle. rbfox1l/rbfox2 morphants are paralyzed and their lack of significant contractile force production in our assay indicated a muscle-specific defect, not just a motoneural defect, causing the characteristic paralysis. We have also developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle in order quantify muscle-specific force per cross-sectional area. These functional results quantify muscle-specific phenotypes sans neural input.