Over the last decade, the zebrafish has entered the field of cardiovascular research as a new model organism. This is largely due to a number of highly successful small- and large-scale forward genetic screens, which have led to the identification of zebrafish mutants with cardiovascular defects. Genetic mapping and identification of the affected genes have resulted in novel insights into the molecular regulation of vertebrate cardiac development. More recently, the zebrafish has become an attractive model to study the effect of genetic variations identified in patients with cardiovascular defects by candidate gene or whole-genome-association studies. Thanks to an almost entirely sequenced genome and high conservation of gene function compared with humans, the zebrafish has proved highly informative to express and study human disease-related gene variants, providing novel insights into human cardiovascular disease mechanisms, and highlighting the suitability of the zebrafish as an excellent model to study human cardiovascular diseases. In this review, I discuss recent discoveries in the field of cardiac development and specific cases in which the zebrafish has been used to model human congenital and acquired cardiac diseases.

/pdf/zebrafish-as-a-model-to-study-cardiac-development-and-human-1kdl2kbi6b.pdf

Zebrafish as a model to study cardiac development and human cardiac disease

Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration

https://www.cell.com/trends/genetics/pdf/S0168-9525(13)00113-3.pdf

The zebrafish as a model for complex tissue regeneration

The zebrafish heart has the capacity to regenerate after ventricular resection. Although this regeneration model has proved useful for the elucidation of certain regeneration mechanisms, it is based on the removal of heart tissue rather than its damage. Here, we characterize the cellular response and regenerative capacity of the zebrafish heart after cryoinjury, an alternative procedure that more closely models the pathophysiological process undergone by the human heart after myocardial infarction (MI). Localized damage was induced in 25% of the ventricle by cryocauterization (CC). During the first 24 hours post-injury, CC leads to cardiomyocyte death within the injured area and the near coronary vasculature. Cell death is followed by a rapid proliferative response in endocardium, epicardium and myocardium. During the first 3 weeks post-injury cell debris was cleared and the injured area replaced by a massive scar. The fibrotic tissue was subsequently degraded and replaced by cardiac tissue. Although animals survived CC, their hearts showed nonhomogeneous ventricular contraction and had a thickened ventricular wall, suggesting that regeneration is associated with processes resembling mammalian ventricular remodeling after acute MI. Our results provide the first evidence that, like mammalian hearts, teleost hearts undergo massive fibrosis after cardiac damage. Unlike mammals, however, the fish heart can progressively eliminate the scar and regenerate the lost myocardium, indicating that scar formation is compatible with myocardial regeneration and the existence of endogenous mechanisms of scar regression. This finding suggests that CC-induced damage in zebrafish could provide a valuable model for the study of the mechanisms of scar removal post-MI.

/pdf/extensive-scar-formation-and-regression-during-heart-1fug9hxz61.pdf

Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish

Epithelial to mesenchymal transition (EMT) converts epithelial cells to mobile and developmentally plastic mesenchymal cells. All cells in the heart arise from one or more EMTs. Endocardial and epicardial EMTs produce most of the noncardiomyocyte lineages of the mature heart. Endocardial EMT generates valve progenitor cells and is necessary for formation of the cardiac valves and for complete cardiac septation. Epicardial EMT is required for myocardial growth and coronary vessel formation, and it generates cardiac fibroblasts, vascular smooth muscle cells, a subset of coronary endothelial cells, and possibly a subset of cardiomyocytes. Emerging studies suggest that these developmental mechanisms are redeployed in adult heart valve disease, in cardiac fibrosis, and in myocardial responses to ischemic injury. Redirection and amplification of disease-related EMTs offer potential new therapeutic strategies and approaches for treatment of heart disease. Here, we review the role and molecular regulation of endocardial and epicardial EMT in fetal heart development, and we summarize key literature implicating reactivation of endocardial and epicardial EMT in adult heart disease.

/pdf/endocardial-and-epicardial-epithelial-to-mesenchymal-1i3lvpdjii.pdf

Endocardial and Epicardial Epithelial to Mesenchymal Transitions in Heart Development and Disease

A zebrafish heart can fully regenerate after amputation of up to 20% of its ventricle. During this process, newly formed coronary blood vessels revascularize the regenerating tissue. The formation of coronary blood vessels during zebrafish heart regeneration likely recapitulates embryonic coronary vessel development, which involves the activation and proliferation of the epicardium, followed by an epithelial-to-mesenchymal transition. The molecular and cellular mechanisms underlying these processes are not well understood. We examined the role of PDGF signaling in explant-derived primary cultured epicardial cells in vitro and in regenerating zebrafish hearts in vivo. We observed that mural and mesenchymal cell markers, including pdgfrβ, are up-regulated in the regenerating hearts. Using a primary culture of epicardial cells derived from heart explants, we found that PDGF signaling is essential for epicardial cell proliferation. PDGF also induces stress fibers and loss of cell-cell contacts of epicardial cells in explant culture. This effect is mediated by Rho-associated protein kinase. Inhibition of PDGF signaling in vivo impairs epicardial cell proliferation, expression of mesenchymal and mural cell markers, and coronary blood vessel formation. Our data suggest that PDGF signaling plays important roles in epicardial function and coronary vessel formation during heart regeneration in zebrafish.

https://www.pnas.org/content/pnas/107/40/17206.full.pdf

PDGF signaling is required for epicardial function and blood vessel formation in regenerating zebrafish hearts

The zebrafish (Danio rerio) is an emerging model for cardiovascular research. The zebrafish heart regenerates after 20% ventricular amputation. However, assessment of the physiological responses during heart regeneration has been hampered by the small size of the heart and the necessity of conducting experiments in an aqueous environment. We developed a methodology to monitor a real-time surface electrocardiogram (ECG) by the use of micro-electrodes, signal amplification, and a low pass-filter at a sampling rate of 1 kHz. Wavelet transform was used to further remove ambient noises. Rather than paralyzing the fish, we performed mild sedation by placing the fish in a water bath mixed with MS-222 (tricane methanesulfonate). We recorded distinct P waves for atrial contraction, QRS complexes for ventricular depolarization, and QT intervals for ventricular repolarization prior to, and 2 and 4 days post-amputation (dpa). Sedation reduced the mean fish heart rate from 149 ± 18 to 90 ± 17 beats/min. The PR and QRS intervals remained unchanged in response to ventricular apical amputation (n = 6, p > 0.05). Corrected QT intervals (QTc) were shortened 4 dpa (n = 6, p < 0.05). In a parallel study, histology revealed that apical thrombi were replaced with fibrin clots and collagen fibers. Atrial arrhythmia was noted in response to prolonged sedation. Unlike the human counterpart, ventricular tachycardia or fibrillation was not observed in response to ventricular amputation 2 and 4 dpa. Taken together, we demonstrated a minimally invasive methodology to monitor zebrafish heart function, electrical activities, and regeneration in real-time.

/pdf/micro-electrocardiograms-to-study-post-ventricular-50fqjsc72b.pdf

Micro-Electrocardiograms to Study Post-Ventricular Amputation of Zebrafish Heart

Understanding heart regeneration in a vertebrate model system is a highly relevant public health concern Coronary heart disease is among the leading causes of disability and mortality worldwide Injured human hearts caused by myocardial infarction (MI) results in decreased cardiac performance and eventually the development of heart failure In contrast to mammals, zebrafish fully regenerate myocardium after 20% ventricular resection and provide an excellent model system to study the mechanisms of heart regeneration It has been shown that high-frequency echocardiography is potentially feasible to obtain in vivo structural, physiological and functional information of zebrafish heart [1] In this research, heart amputated fish (n=12) were allowed to regenerate for 14 days Real-time B-mode images and ventricular pulsed-wave Doppler waveforms were acquired daily for 14 days Measurement on the echocardiographic data was performed to obtain longitudinal cardiac parameters, such as scar volumes, heart rates, and E/A ratios Experimental results show a consistent decrease of the scar volume from 005plusmn0012 to 0015plusmn0004 mm3 along the days post amputation, which suggests the replacement of the blood clot by regenerated myocardium The E/A ratio display a sudden increase from 008plusmn005 to 031plusmn015 immediately after the ventricular amputation, followed by a gradual decrease to 018plusmn008 at 9dpa This indicates the continuing recovery of ventricular functions, correlating well with the structural regeneration Heart rate, however, was not observed significant change along the regenerative process

Qiong Wu

Papers

PDGF signaling is required for epicardial function and blood vessel formation in regenerating zebrafish hearts

Micro-Electrocardiograms to Study Post-Ventricular Amputation of Zebrafish Heart

Longitudinal study of adult zebrafish heart regeneration using high frequency echocardiography