Cardiac Hypertrophy Involves Both Myocyte Hypertrophy and Hyperplasia in Anemic Zebrafish

Background: An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart. Methodology/principal findings: Hearts of the tr265/tr265 Danio rerio mutant become larger than those of the sibling by week 4 post fertilization and gradually exhibit characteristics of human cardiomyopathy, such as muscular disarray, re-activated fetal gene expression, and severe arrhythmia. At the cellular level, we found both increased individual cardiomyocyte size and increased myocyte proliferation can be detected in week 4 to week 12 tr265/tr265 fish. Interestingly, all tr265/tr265 fish that survive after week-12 have many more cardiomyocytes of smaller size than those in the sibling, suggesting that myocyte hyperplasia allows the long-term survival of these fish. We also show the cardiac hypertrophy process can be recapitulated in wild-type fish using the anemia-inducing drug phenylhydrazine (PHZ). Conclusions/significance: The anemia-induced cardiac hypertrophy models reported here are the first adult zebrafish cardiac hypertrophy models characterized. Unlike mammalian models, both cardiomyocyte hypertrophy and hyperplasia contribute to the cardiac remodeling process in these models, thus allowing the effects of cardiomyocyte hyperplasia on cardiac remodeling to be studied. However, since anemia can induce effects on the heart other than biomechanical, non-anemic zebrafish cardiac hypertrophy models shall be generated and characterized

Cardiac Remodeling in Anemic Zebrafish Involves Cardiomyocyte Hypertrophy and Hyperplasia: Effects of the Wnt/Beta-Catenin Pathway

Background. Accumulating evidence is changing the paradigm that the heart is a post-mitotic organ and spurring efforts to promote cardiac regeneration as a therapeutic strategy for heart disease. As part of that effort, this thesis has established and characterized zebrafish models of cardiac remodeling to assess the therapeutic value of promoting cardiomyocyte hyperplasia and discover the mechanisms involved. Specifically, it analyzed the roles of the Wnt/beta-catenin pathway on cardiomyocyte hypertrophy and hyperplasia during cardiac remodeling and the effects this remodeling had on fish lifespan. In addition, this thesis developed assays to measure cardiac function in adult zebrafish to further increase the usefulness of adult zebrafish for heart disease research. Methodology/principle findings. Structural, cellular, and genetic analyses revealed that the Band-3, anemia zebrafish mutant tr265 exhibits hallmarks of human cardiac remodeling, such as an enlarged heart, cardiomyocyte hypertrophy, cardiomyocyte death, structural disarray, and cardiac dysfunction. However, unlike in humans, cardiomyocyte hyperplasia contributes significantly to the cardiac remodeling process of tr265. At week 6 post fertilization, tr265 has cardiomyocyte hypertrophy in the inner trabecular region and hyperplasia in the outer compact region. Modulation of the Wnt/beta-catenin pathway affects the hypertrophic response of the zebrafish trabecular cardiomyocytes, like in mammalian cardiomyocytes. Oppositely, modulation of the pathway affects cardiomyocyte hyperplasia in the compact region, demonstrating the location-dependent effects of the Wnt/beta-catenin pathway in the anemia model. Moreover, upregulation of the pathway accelerates the onset of cardiomyocyte hyperplasia in the trabecular region and improves fish survival by six weeks after modulation of the pathway, suggesting a therapeutic role of cardiomyocyte hyperplasia to the cardiac remodeling process. Two assays developed to measure cardiac function in the adult zebrafish include red blood cell flow rate and shortening fraction. The red blood cell flow rate assay can be used in most fish to measure how fast the red blood cells travel in the caudal fin as an indirect measure of cardiac function. To measure cardiac function directly via the shortening fraction assay, a semi-transparent fish that contains a red fluorescent heart (casper;Tg(cmlc2:nuDsRed) ) was created. Conclusions/significance. Cardiomyocyte hyperplasia plays a beneficial role in the cardiac remodeling process of tr265 , as demonstrated by improved fish survival after augmentation of cardiomyocyte hyperplasia via the Wnt/beta-catenin pathway. Exciting research remains to be conducted with tr265 to understand how the pathway affects the onset of cardiomyocyte hyperplasia, as well as why the pathway exhibits location-dependent effects on cardiomyocyte hypertrophy and hyperplasia. Using models such as tr265, mechanisms involved in cardiomyocyte hyperplasia can be discovered and hopefully translated into therapeutic strategies for human disease

Zebrafish functional genomics development at UW-Stout

Since the completion of the human genome sequencing project, morpholino phosphorodiamidate oligonucleotide (MO) knockdown in zebrafish has been increasingly used to elucidate human gene function. As part of the effort to expand the functional genomics screening capacity at the University of Wisconsin-Stout, this project investigated MO microinjection techniques, embryo production, and brine shrimp survival. One-to two-cell embryos injected with the pigment-inhibiting tyrosinase MO were observed at 48 hours post fertilization for pigmentation. Injection efficiency was calculated by dividing the number of zebrafish lacking pigment by the total number of injected zebrafish. To obtain sufficient embryos for MO experiments, the effects of a dry food diet and live (brine shrimp) food diet on embryo production were analyzed. Effects of a yeast diet on brine shrimp survival were also studied to provide zebrafish with healthy brine shrimp. Results included a 92% injection efficiency, greater embryo production with the dry food diet, and increased brine shrimp survival with a yeast diet. This work provided an important foundation in the development of a reverse-genetic screen for future students