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Zebrafish as Novel Model for Exercise-enhanced Skeletal and Cardiac Muscle Growth and Immune Functioning

Final Report Summary - SWIMFIT (Zebrafish as Novel Model for Exercise-enhanced Skeletal and Cardiac Muscle Growth and Immune Functioning)

Summary description of the project objectives
SWIMFIT had the objective to demonstrate the functional mechanisms behind the beneficial effects of swimming using all the advantages that zebrafish offers as a model fish for exercise-enhanced skeletal and cardiac muscle growth and immune functioning. Within the borders of the SWIMFIT project, swimming experiments, RNAseq, microarray analyses, Q-PCR and histochemistry were performed to demonstrate the functional mechanisms behind (1) exercise-enhanced skeletal muscle growth; (2) cardiac muscle growth, and (3) immune functioning. Additionally, insights were provided into the role of the stress axis on the basis of the effects of exercise.

Description of the work performed and main results achieved
During RP1 and RP2, Dr. Palstra has closely collaborated with Dr. Josep Planas (University of Barcelona) and his PhD student Mireia Rovira. Dr. Palstra has supervised MSc student Silvia Mendez from Planas’ group who was awarded with an Erasmus grant to perform a practical internship at IMARES. Dr. Palstra has also closely collaborated with Drs. Herman Spaink and Marcel Schaaf (Leiden University), in fact since March 2014 he holds a guest worker position in the group of Dr. Spaink. Together with Dr. Schaaf, several Marie Curie IEF proposals were written as well as two VIDI proposals, and Dr. Schaaf co-supervised MSc student Silvia Mendez with the lab analyses performed in WP4 at the Leiden University. Four large swimming tunnels (collaboration with Dr. G. van den Thillart, Leiden University) were transferred to the IMARES facilities and tested for zebrafish swimming at various speeds. An aquarium set-up was built for housing experimental zebrafish. Set-ups were used for performing experiments in WPs 1-4:

1. exercise-enhanced skeletal muscle growth: Two swimming experiments have been performed following the protocol that was developed by Palstra et al. (2010) which lead to swimming-enhanced growth. A significant increase in fibre cross-sectional area (1,665 ± 106 vs. 1,290 ± 88 μm2) and vascularization (458 ± 38 vs. 298 ± 23 capillaries/mm2) was found in exercised over non-exercised fish. Gene expression profiling by microarray analysis evidenced the activation of a series of complex transcriptional networks of extracellular and intracellular signaling molecules and pathways involved in the regulation of muscle mass (e.g. IGF-1/PI3K/mTOR, BMP, MSTN), myogenesis and satellite cell activation (e.g. PAX3, FGF, Notch, Wnt, MEF2, Hh, EphrinB2) and angiogenesis (e.g. VEGF, HIF, Notch, EphrinB2, KLF2), some of which had not been previously associated with exercise-induced contractile activity. The results from the present study show that exercise-induced contractile activity in adult zebrafish promotes a coordinated adaptive response in fast muscle that leads to increased muscle mass by hypertrophy and increased vascularization by angiogenesis. We propose that these phenotypic adaptations are the result of extensive transcriptional changes induced by exercise. Analysis of the transcriptional networks that are activated in response to exercise in the adult zebrafish fast muscle resulted in the identification of key signaling pathways and factors for the regulation of skeletal muscle mass, myogenesis and angiogenesis that have been remarkably conserved during evolution from fish to mammals. These results further support the validity of the adult zebrafish as an exercise model to decipher the complex molecular and cellular mechanisms governing skeletal muscle mass and function in vertebrates. This study was published in BMC Genomics.
2. exercise-enhanced cardiac muscle growth: Cardiovascular research using the zebrafish (Danio rerio) as model species has made important contributions to cardiac cell specification, regeneration and function over the last decade. However, the response of the adult zebrafish heart to exercise, known in mammals to elicit important adaptations in this tissue, has not been evaluated to date. We subjected adult zebrafish to 20 days of swim-training and analyzed the transcriptomic response in the zebrafish heart by microarray analysis. We identified more than seven hundred differentially expressed genes involved in processes such as cell cycle and proliferation, extracellular matrix and cytoskeleton, muscle contraction, growth factors/signalling pathways and metabolism. These results provide insights into the molecular adaptative mechanisms taking place in the zebrafish heart in response to swimming-induced activity. A book chapter and a draft paper on this study have now been written.
3. exercise-enhanced immune functioning: Results show that exercised and non-exercised zebrafish are completely different in their molecular regulation of processes in the white muscle. Zebrafish that were exercised or that rested were injected with the vehicle (PBS) or lipopolysaccharide to mimic a bacterial infection. At 72 h after the injection, fish were sacrificed and total RNA from white muscle was used for single color microarray analyses. A total of 2,283 genes were differentially expressed between LPS- and PBS-injected swimmers, with 1,379 genes upregulated and 904 downregulated in the LPS-injected fish. Furthermore, 621 genes were differentially expressed between resters injected with LPS and their controls, with 260 genes upregulated and 361 genes downregulated in response to LPS in the resters. Interestingly, only 40 differentially expressed genes were common between LPS-injected swimmers and LPS-injected resters, which is less than 2 % of the total number of differentially expressed genes. Exercised fish are thus physiologically different in their molecular response to bacterial infection in the white muscle. Gene ontology analysis showed that developmental processes in the white muscle of resters were downregulated which was not the case in swimmers. This leads to the conclusion that while resters switch off developmental processes in response to an immune challenge in the form of administered LPS, swimmers do not. A draft paper that includes these results has now been written.
4. exercise-enhanced cortisol stress response: A central role in regulating the effects of exercise is expected for the stress axis or hypothalamic-pituitary-interrrenal (HPI) axis acting through the steroid hormone cortisol. To explore the mechanism, we first subjected wild-type zebrafish to an exercise protocol and showed that exercised fish were indeed heavier and had higher cortisol levels than the non-exercised controls. Second, we subjected wild-type zebrafish and zebrafish with a functional knockout for the gene encoding the glucocorticoid receptor to exercise at optimal, suboptimal and super-optimal speeds and compared them with non-exercised controls. Exercised fish showed growth enhancement which was highest at optimal speeds and lower at both sub and super-optimal speeds indicating that swimming at optimal speed represents maximal eustress. In mutants, exercise resulted in an even larger growth enhancement as compared to wild type fish. Apparently, cortisol brakes exercise-enhanced growth in wild type fish. The molecular mechanism has been studied by transcriptome analysis using RNA sequencing. A paper will be written.

Within RP1 and RP2 of SWIMFIT: one paper has been published and three are on the way representing all four work packages; a book has been edited and published by Springer incl. one chapter on exercising zebrafish and we have been invited to work on a 2nd edition; a special issue of Frontiers has been edited (16,821 views); sixteen congress abstracts were published; two popular scientific papers were written on the swimming physiology of fish, and a youtube movie and the website were created; three symposia on swimming physiology were moderated; two invited lectures, seven other oral presentations and four posters were presented, and the project raised significant publicity a.o. three times in national newspapers. Additionally, SWIMFIT lead to the approved COST Action FA1304 FITFISH (

Final results and their potential impact and use
SWIMFIT has further launched zebrafish as exercise model. SWIMFIT has provided new insights on the functional mechanisms of exercise effects on skeletal and cardiac muscle growth, immune functioning and the cortisol stress response. The mechanisms and the actors involved are probably universal and understanding will add to an improved approach towards both medical biology as well as sustainable aquaculture. SWIMFIT has enabled the development of a novel research line on exercise integrating Dr. Palstra’s existing expertise on exercise physiology with applying high throughput genomic methodology thereby creating a new scientific niche. Dr. Palstra postulates a permanent position as senior researcher at Wageningen University and Research.