The coronary vasculature plays essential roles in maintaining a continuous supply of oxygen and nutrients to the heart. Coronary heart disease is a major cause of myocardial infarction and heart failure, which continues to be the leading cause of mortality worldwide. Despite its importance, the mechanisms that regulate coronary vascularization of the myocardium remain a major gap in our knowledge. We observed that during zebrafish heart development, coronary vessels form a close association with a specific subpopulation of cortical cardiomyocytes marked by the transcription factor Gata4. gata4+ cardiomyocytes follow the tracks of coronary endothelial cells when they emerge onto the surface of the juvenile zebrafish hearts. Furthermore, our preliminary data suggest that expression of the Cxcl12b chemokine, a critical angiogenic factor during zebrafish coronary vessel development, is increased in expanding gata4+ cardiomyocytes. Moreover, gata4+ cardiomyocytes fail to associate with coronary vessels in hearts lacking Cxcr4a, the receptor for Cxcl12b. Importantly, adult cxcr4a mutant zebrafish fail to regenerate after heart injury. We hypothesize that heart development occurs in two phases that are regulated by temporally distinct mechanisms: newly formed coronary endothelial cells provide instructive cues, such as paracrine factors, during the emerging phase to guide gata4+ cardiomyocytes when they emerge, while during the expanding phase, cxcl12b expression increases in gata4+ cardiomyocytes to attract coronary vessels that are essential for their morphogenesis/development and regeneration. We propose to 1) To determine how new coronary vessels guide gata4+ cardiomyocytes to populate the heart ventricle by analyzing differentially expressed candidate genes identified from RNAseq encoding secreted molecules (e.g. cxcr4, erbb4) using CRISPR mutant fish lines and a novel explant culture system; 2) To determine how coronary vessels affect morphogenesis and the regenerative capacity of gata4+ cardiomyocytes during myocardial expansion using multicolor clonal analysis and by manipulating Cxcl12-Cxcr4 chemokine signaling. We will further determine how cxcl12b expression is regulated by hypoxia and Gata4. Our proposed study will reveal potential developmental causes of coronary heart diseases. Furthermore, elucidation of the mechanisms underlying myocardial vascularization in zebrafish will shed light on potential therapeutic approaches for humans.

Public Health Relevance

Coronary heart disease is a major cause of myocardial infarction and heart failure for adults. The goal of this proposal is to determine the molecular and cellular mechanisms of myocardial vascularization during heart development in zebrafish, an organism with a natural regenerative ability. We expect the proposed research can lead to findings that will shed light on potential developmental causes of coronary heart disease and may enhance neovascularization in diseased human hearts in the future.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Cardiovascular Differentiation and Development Study Section (CDD)
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Schramm, Charlene A
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Children's Hospital of Los Angeles
Los Angeles
United States
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Patterson, Michaela; Barske, Lindsey; Van Handel, Ben et al. (2017) Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration. Nat Genet 49:1346-1353