Technical Innovations for the Study of Zebrafish Heart Regeneration

斑馬魚心臟再生研究的技術創新

Student thesis: Doctoral Thesis

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Author(s)

  • Fatemeh BABAEI

Detail(s)

Awarding Institution
Supervisors/Advisors
Award date1 Sep 2015

Abstract

The striking ability of zebrafish to regenerate their heart has been a subject of intensive research. While the zebrafish is a versatile experimental model for regeneration biology, many technical limitations are restricting the scope of experiments that can be performed on this organism. As a result, although a lot is known about the molecular, cellular and anatomical events during zebrafish heart regeneration, many aspects of this complex process are still relatively uncharacterized. This PhD thesis details my efforts in addressing some of these issues, and reports a number of novel discoveries revealed as a result of these technical innovations.

In the first part of this project, I aimed to investigate the secretory factors expressed by extra-heart tissues during cardiac injury and regeneration. I hypothesized that some of these ex-situ factors maybe delivered, via blood, to the damaged heart and can influence the heart regeneration process. However, blood is one of the least investigated tissues in zebrafish, as existing methods for isolating blood from this organism are tedious, irreproducible and inefficient. To overcome this problem, I established a novel method of extracting blood from zebrafish, based on the use of low centrifugal force to collect blood from a tail wound. This method consistently recovered more blood than traditional methods. Gel electrophoresis and flow cytometry showed that the composition of blood harvested by this method is indistinguishable from traditional methods. Using this method, quantitative proteomics can be performed on the plasma collected from a single zebrafish. As a proof of concept, I performed shotgun proteomic analysis on the plasma isolated from individual male and female zebrafish, and compared the gender-specific differences in plasma proteins by the label-free relative quantitation of the mass spectrometry (MS) data.

Using this new technical paradigm, I systematically compared the plasma proteomes of zebrafish at 5 time points (1 day, 3 days, 7 days, 14 days and 21 days) after ventricular amputation and sham-operation, and in unperturbed control fish. As expected, intracellular proteins and complement components were observed to increase in the plasma of both heart-amputated and sham-operated fish, indicating the release of cellular contents and the surge of anti-microbial responses in the wake of tissue injuries. Remarkably, vitellogenin (a female-specific lipid carrying protein) was observed in the plasma of male zebrafish as soon as 1 day after heart amputation, but not in sham-operated fish. Moreover, coagulation factors were suppressed, with an induction of anti-clotting factors, in heart-injured fish. These data depict a dramatic change in plasma composition during heart regeneration, especially a shift in lipid metabolism and coagulator functions, which is not previously known to be associated with heart regeneration.

The second part of this project addresses another intrinsic limitation of zebrafish as a model organism for heart regeneration: the difficulty in controlling the size and position of cardiac injury. Standard damage models, such as amputation and cryo-damage, require the surgical removal of a part of ventricle from the beating heart of small fish. The dexterity involved in these methods leads to highly heterogeneous wounds. In addition, assessments of the wound areas, traditionally done by serial histological sections, are inaccurate and cumbersome. These two technical obstacles lead to highly inconsistent experimental data on zebrafish heart regeneration, preventing the quantitative correlation of wound size and regeneration parameters. Here, I summarize my efforts in overcoming this hurdle. First, I optimized the use of contrast enhanced micro-computed tomography (CE-microCT) technique that enables the in situ visualization of the anatomy of adult zebrafish. Heart lesions created by amputation were readily visible using this method. Since the heart was imaged without the need of dissection, its connection with the adjacent anatomical structures could be assessed. Using CE-microCT, I measured the wound area after heart amputation in different time points, and confirmed that the lesions were highly heterogeneous in size.

To remedy this problem, I created a new model of heart injury that involved the puncturing of one side of the ventricular wall by a needle. CE-microCT indicated a high degree of consistency in needle puncture wounds. Even without extensive cell death either by necrosis or apoptosis, needle puncture led to the dedifferentiation and proliferation of cardiomyocytes. Furthermore, electrophysiological measurements indicated the occurrence of signs immediately observing after needle puncture have some similarities with a condition commonly observed in myocardial ischemia in humans, suggesting that needle puncture represents a realistic model for myocardial infraction. Histological analysis showed scarless healing one month after needle puncture. However, significant electrophysiological abnormalities associated with ventricular depolarisation and repolarisation persisted, suggesting that the functional recovery of heart injuries in zebrafish lagged behind cellular regeneration. The reproducibility of needle-induced lesions enabled me to correlate the extent of heart damage and regeneration. Unexpectedly, the level of cardiomyocyte proliferation appeared to be independent of the size of the damage. The new experimental paradigm established in this study is convenient and does not require refined surgery skills and will likely enhance research on zebrafish regeneration.

The results reported in this thesis demonstrated how technical innovations can extend the scope of studies compatible with zebrafish as an experimental model for heart regeneration. Many of the methods developed in this project, such as single fish plasma proteomics and CE-microCT imaging, can be applied to other areas of zebrafish biology and to other small fish models.