Zebrafish Heart Regeneration

Yes, HEART REGENERATION. Watch it for yourself!

Zebrafish, a fish of hope

Photo compliments of "Donna's Dream"

During heart regeneration in zebrafish, lost ventricular tissues is rapidly replaced. After as little as one month, most of the missing tissue has been regenerated by cardiomyocytes. Regenerated heart muscles come from proliferation of differentiated cardiomyocytes. The exact source of these new cardiomyocytes is an unanswered question. [1] How is missing tissue regenerated by cardiomyocytes?

If we are able to understand how cardiomyocytes regenerate missing tissue in zebrafish, we could then mimic this regeneration in mammalian cells and perhaps understand why regeneration does not occur in humans. [2] To address regeneration, Chris Jopling, Ph.D., a postdoctoral fellow of Izpisúa Belmonte’s at CMRB, and crew conducted a study where they developed and successfully implemented the tamoxifen (4-OHT) inducible Cre/Lox approach in zebrafish to genetically label regeneration cardiomyocytes. Through this, they discovered:

  • Regenerated heart muscles come from proliferation of differentiated cardiomyocytes
  • Differentiated cardiomyocytes re-enter the cell cycle
  • Cardiomyocytes dedifferentiate resulting in the disassembly of sarcomeric structure and detachment
  • Plk1 is necessary for cardiac regeneration

Let’s take a closer look at these discoveries.

Regenerated Cardiomyocytes are Derived From Differentiated Cardiomyocytes

Experiments were conducted on the cardiomyocytes of genetically labelled adult zebrafish 48 hours post fertilization (hpf) to determine what role, if any, differentiated cardiomyocytes played in the regeneration of heart tissue. After the removal of approximately 20% of the ventricle (amputation site signified by the white dotted line in figures below), heart regeneration was observed 7 (Fig 1a.), 14 (Fig 1d.) and 30 (Fig 1g.) days post amputation.

Fig 1a. Zebrafish heart 7dpa

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Figure 1a shows that the remaining zebrafish heart is GFPpos 7dpa. Here, the regeneration process has begun. At 7dpa, missing tissue is being replaced with a fibrin and collagen clot.

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Fig 1d. Zebrafish heart 14 dpa

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At 14dpa, it is apparent that the regeneration process is proceeding. The regenerating tissue (seen below the white dotted line) consist of GFPpos cardiomyocytes, indicating that the regenerated cardiomyocytes are derived from differentiated GFPpos cardiomyocytes.

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Fig 1g. Zebrafish heart 30dpa

Fig 1g. Zebrafish heart 30dpa

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At 30dpa, the heart is almost completely regenerated. All of the cardiomyocytes of the regenerated tissues are clearly GFPpos cardiomyocyctes.

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In comparing the zebrafish heart as days post amputation increases, we can see that the GFPpos cardiomyocyte proliferation increases leading to the regeneration of the cardiac tissues. Next, it was wondered whether or not if these GFPpos cardiomyocytes re-entered the cell cycle.

Dedifferentiated Cardiomyocytes Re-Enter the Cell Cycle

Adult GFPpos transgenic zebrafish were treated with bromodeoxyuridine (BrdU) for 7 days following amputation to determine if the cardiomyocyctes re-entered the cell cycle. At 14 dpa, there was a greater number of BrdUpos/GFPpos cardiomyocytes in regenerating hearts versus non-amputated control hearts (Figure 2g). This indicated that differentiated GFPpos cardiomyocytes re-entered the cell cycle and underwent DNA replication.

Upon further analysis of BrdU-labelled GFPposcardiomyocytes positions within the regenerating heart, it was found that a majority of BrdUpos/GFPpos labeled cardiomyocytes were concentrated around the wound, yet a proportion were also found in regions far from the site of amputation (See image: courtesy of Juan Carlos Izpisua Belmonte, Salk Institution for Biological Studies). Heart muscle cells (shown in green), regress to a more youthful state after injury, start dividing again (indicated by a red marker) to replenish lost cells and then mature a second time into cardiomyocytes. This suggests that the response to the injury affects the heart in a global manner. [2]

Fig 2g. Differentiated Cardiomyocytes Re-Enter the Cell Cycle

Image: Courtesy of Juan Carlos Izpisúa Belmonte, Salk Institute for Biological Studies

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The following inquiry was to determine to what extent cardiomyocytes dedifferentiate during zebrafish heart regeneration.

Dedifferentiation Causes Disassembly of Sarcomeric Structure and Detachment

Fig 3. Dedifferentiation Causes Disassembly of Sarcomeric Structure and Detachment

To address the extent to which cardiomyocytes dedifferentiate during zebrafish heart regeneration, regenerating hearts were analyzed using transmission electron microscopy (TEM) of a control non-amputated heart (Fig 3. a, b), a 5-dpa regenerating heart (Fig 3. c, d) and a 7-dpa regenerating heart (Fig 3. e, f). In uninjured hearts (Fig 3. a,b) cardiomyocytes are displayed in an ordered arrangement of sarcomeres and mitochondria with clearly defined z-lines (Fig 3. b). At 5 dpa, many of the cardiomyocytes displayed a disorganized sarcomeric structure (Fig 3. c). The aligned array of actin and myosin filaments became disorganized (Fig 3. d) along with the appearance of intercellular spaces (white arrows). 7 dpa (Fig 3. e,f), intercellular spaces were also readily visualized as cardiomyocytes detached from one another (Fig 3. e). Furthermore, although sarcomeric filaments were visible, there was a lack of organization leading to the presence of both transverse and longitudinal sarcomeric structures within individual cardiomyocytes (Fig 3. f).

The next question up for debate was whether cardiomyocytes dedifferentiation during heart regeneration was associated with specific changes in gene expression.

Plk1 is Necessary for Cardiac Regeneration

Fig 4a. Semi-Quantitative RT-PCR of mps1 and plk1

The expression of the polo like kinase 1 (plk1) gene, a regulator of cell cycle progression that was detected as upregulated in previous microarray analyzes of zebrafish heart regeneration was examined. The RT-PCR of upregulated plk1 in regenerating hearts at 1, 3, and 7 dpa closely resembled that of mitotic checkpoint kinase mps1, a known indicator during regeneration (Fig 4. a).

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To further assess the role of plk1 during heart regeneration, an embryonic model of heart regeneration was utilized. It was found that in the absence of the plk1 inhibitor cyclapolin 9, 67% of the embryos were able to regenerate their hearts. In the presence of the plk1 inhibitor, the number fell to 17% [1]. TUNEL labeling showed that this inhibition was not due to an increase in cardiomyocyte apoptosis.

Fig 4g. Plk1 Reduces Number of Cardiomyocytes Re-Entering Cell Cycle During Regeneration

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Furthermore, there was a significant decrease in the number of BrdUpos/GFPposcardiomyocytes in regeneration animals treated with the inhibitor (g). These results indicate that plk1 is essential for heart regeneration to proceed.

Concluding Remarks

Jopling and team found that pre-existing cardiomyocytes were the primary drivers in heart regeneration as opposed to the previously conceived progenitor cells. The regenerated cardiomyocytes were derived from differentiated cardiomyocytes. These differentiated cardiomyocytes are able to re-enter the cell cycle. Following amputation, proximal cardiomyocytes dedifferentiate, resulting in the disassembly of sarcomeric structure and detachment from one another. These proximal cardiomyocytes also express regulators, such as plk1 and mps1, which are essential to the regeneration process.

Strengths and Improvements

The paper was set up in a manner that was very easy to follow along with. The paper also made the topic of zebrafish heart regeneration seem very important and interesting despite the scientific knowledge of the audience. A lot of facts were presented without creating a dry research paper.
The paper could have done a better done explaining why certain methods were chosen. There were some technical terms that were not explained. Although this did not completely take away from the paper, deeper explanations of some scientific jargon and methods would allow for the paper to read smoother. Another specific improvement that could be made, or referenced to another paper, was why plk1 was chosen. I would have also liked to see more about the plk1 inhibitor c yclapolin 9.

Zebrafish Leading to Human Heart Regeneration Far Fetched?

Not at all. Though distantly related, human and zebrafish hearts have much in common. The zebrafish heart does have some structural differences with the human heart, having only two chambers instead of four, and having a more “spongy” composition that may facilitate clotting. [3] Scientists want to understand why fish and humans should have such differing regenerative abilities, and especially hope that understanding how the fish heart regenerates will yield clues for improving therapy after human heart attacks.If the role of important molecules like FGF1 can be determined in the zebrafish, the same molecules can then be studied in humans, and even explored as therapeutic agents. [4]

“Because zebrafish hearts are so similar to our own, scientists are using the fish to study many aspects of heart disease, particularly the role of genetic mutations that might cause heart abnormalities. One disease, called familial hypertrophic cardiomyopathy (HCM), is the leading cause of sudden death in young athletes.” [3]
Compare Different Vertebrate Circulation Patterns

Citations

[1.] Jopling C, Sleep E, Raya M, Martí M, Raya A, Izpisúa Belmonte JC. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature. 2010 Mar 25;464(7288):606-9. PubMed PMID: 20336145; PubMed Central PMCID: PMC2846535.

[2.] Salk Institute (2010, March 24). Zebrafish study with human heart implications: Cellular grown-ups outperform stem cells in cardiac repair.ScienceDaily. Retrieved March 7, 2012, from http://www.sciencedaily.com­/releases/2010/03/100324141957.htm

[3.] Exploratorium. Zebrafish: A Model For Heart Development. Microscope Imaging Station. Retrieved March 7, 2012, from http://www.exploratorium.edu/imaging_station/research/zebrafish/story_zebrafish.pdf

[4.] The 2006 Holiday Lectures Series “Potent Biology: Stem Cells, Cloning, and Regeneration.” http://www.hhmi.org/biointeractive/stemcells/zebrafish_regen.html

[5.] Wang J, Panáková D, Kikuchi K, Holdway JE, Gemberling M, Burris JS, Singh SP, Dickson AL, Lin YF, Sabeh MK, Werdich AA, Yelon D, Macrae CA, Poss KD. The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion.Development. 2011 Aug;138(16):3421-30. Epub 2011 Jul13.PubMed [citation] PMID: 21752928, PMCID: PMC3143562

[6.] Schnabel K, Wu CC, Kurth T, Weidinger G. Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation. PLoS One. 2011 Apr 12;6(4):e18503.PubMed [citation]PMID: 21533269, PMCID: PMC3075262

[7.] Lou X, Deshwar AR, Crump JG, Scott IC. Smarcd3b and Gata5 promote a cardiac progenitor fate in the zebrafish embryo. Development. 2011 Aug;138(15):3113-23. Epub 2011 Jun 29.PubMed [citation] PMID: 21715426

[8.] Kikuchi K, Gupta V, Wang J, Holdway JE, Wills AA, Fang Y, Poss KD. Tcf21+ epicardial cells adopt non-myocardial fates during zebrafish heart development and regeneration. Development. 2011 Jul;138(14):2895-902. Epub 2011 Jun 8.PubMed[citation] PMID: 21653610, PMCID: PMC3119303

[9.] González-Rosa JM, Martín V, Peralta M, Torres M, Mercader N. Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish. Development. 2011 May;138(9):1663-74. Epub 2011 Mar 23.PubMed [citation] PMID: 21429987

[10.] Kikuchi K, Holdway JE, Major RJ, Blum N, Dahn RD, Begemann G, Poss KD. Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration. Dev Cell. 2011 Mar 15;20(3):397-404.PubMed [citation] PMID: 21397850, PMCID: PMC3071981

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