Zebra fish can mend its broken heart

Watch this interesting video on how the zebrafish heart returns to nearly its original shape, size, and pumping ability after being wounded/damaged : http://www.youtube.com/watch?v=6Ak7ZUF01Ho

GFP stained zebrafish heart

A human heart does a very bad work of patching up itself after a non fatal heart attack resulting in scarred and stiff muscles (Oberpriller et al., 1973). On the contrary, our little friends, the zebrafishes have the capacity to regenerate their heart cells or the cardiomyocytes following even 20% of ventricle amputation (Poss et al, 2002). Now, what is the source of these newly cardiomyocytes in heart regeneration – dedifferentiated or  stem/precursor cells? Come on, lets find out.

Researchers, in order to address these questions established a genetic strategy to lineage trace cardiomyocytes in adult fishes. Tamoxifen (4-OTH) inducible Cre/Lox approach was used to genetically label regenerating cardiomyocytes in  the zebra fishes (Dor Y et al, 2004).

First, a series of amputation experiments were performed on adult zebrafish with GFP (green fluorescent protein) labelled cardiomyocytes. 20% of ventricle was removed and cardiac regeneration was assessed at 7, 14 and 30 days post amputation (dpa).

Fig1: Regenerated cardiomyocytes are derived from differentiated cardiomyocytes

Dashed white lines represents the plane of amputation. At 7 dpa, relatively little regeneration can be seen (a,b). Blue coloration is trichromic staining indicating fibrin clot forming adjacent to the wound. By 14 dpa, there is substantial regeneration of cardiac tissue (c,d) and at 30 dpa, heart regeneration is complete and fibrin clot has reduced in size

Hence, it was conclusive that the regenerated cardiomyocytes arise from dedifferentiated GFP positive cardiomyocytes.

In order to determine the overall contribution made by GFP positive cardiomyocytes to heart regeneration, they immunostained heart sections with anti-GFP and anti-sarcomeric myosin antibody (MF20) but were unable to detect any labeled cardiomyocytes. So it was dawning on them that the vast majority of newly formed cardiomyocytes arise from pre-existing cardiomyocytes.

Next, in order to determine if the GFP(+) cardiomyocytes had re-entered the cell cycle, they treated the adult GFP(+) transgenic zebrafish with bromodeoxyuridine (BrdU – marker of DNA synthesis) for 7 days post amputation (Poss et al, 2002). At 14 dpa, there was a increase in BrdU(+)/GFP(+) cardiomyoctyes in regeneration when compared to non amputated controls. Dedifferentiated GFP(+) cardiomyocytes have entered the cell cycle and started DNA replication. The labelled cardiomyocytes are found in areas around wound and regions far from amputation also indicating that response to injury affects the heart in a global manner.

Fig 2: Differentiated cardiomyocytes re-enter the cell cycle

Transgenic zebrafish genetically labelled at 48 hpf and grown to adulthood were amputated then treated with BrdU at 7 dpa, hearts were then isolated and processed at 14 dpa (a–f). Transgenic zebrafish genetically labelled at 48 hpfand grown to adulthood were amputated then treated with BrdU at 7 dpa, hearts were then isolated and processed at 14 dpa (a–f).

The next question was whether to determine the extent to which these cardiomyocytes dedifferentiate during heart regeneration. Using transmission electron microscopy (TEM), our dear scientists found that the uninjured hearts displayed an ordered arrangement of sarcomeres and mitochondria with clearly defined Z-lines with aligned arrays of actin and myosin filaments, whereas injured hearts showed cardi0myoctyes detaching from one another creating large intracellular spaces with disorganized actin and myosin filaments with loss of z-line structures. Dividing cardiomyocytes labeled with phosphorylated histone 3 (PH3) were observed for detachment from one another and disassembly of sarcomeric structures for cell cycle re-entry but this was not proved. Hence, the sarcomeres were disassembled in the process of cardiomyocytes division (Abbate A. et al, 2007).

Fig 3: Cardiomyocytes dedifferentiate resulting in the disassembly of sarcomeric structure and detachment

Electron microscopy (a, b) Z-lines are clearly visible. At 5 dpa many of the cardiomyocytes display a disorganised sarcomeric structure (c) along with the appearance of intercellular spaces . Loss of Z-lines (d). At 7 dpa there is a similar loss of structure and appearance of intercellular spaces (e ). (f) both longitudinal and transverse  fibres are present within the same cardiomyocyte indicating disorganised sarcomeric structure.

This brings to us to next important question : are there any specific changes in gene expression associated with the cardiomyocytes dedifferentiation?

Previous reports show that there is an increase in expression of mitotic check point kinase (mps1) and perturbation of mps1 inhibits regeneration. Similarly they examined expression of polo like kinase 1, a regulator of cell cycle progression that was found to be upregulated in previous microarray analysis of zebrafish heart regeneration (Sleep E. et al, 2010). Plk-1 transcripts and expression were upregulated as expected in regenerating hearts as detected by reverse transcriptase PCR and in situ hybridisation.

Fig 4: plk1 is necessary for cardiac regeneration

Semi-quantitative Reverse Transcriptase-PCR  of mps1 and plk1. Plk1 was upregulated and its expression was also found to be high as detected by in situ (not shown).

To further assess the role of plk-1 during heart regeneration, an embryonic model of heart regeneration was created (Good job engineers) (Curado S. et al, 2007). Removing cardiomyocytes followed by recovery in the presence and absence of plk inhibitor- cylapolin 9 was performed. Absence of inhibitor displayed 67% of heart regeneration whereas presence of inhibitor reduce this to a meager 17%. These results were confirmed in adult setting and also showed a decrease in BrdU(+)/GFP(+) cardiomyocytes in fishes treated with inhibitor. This brings us to the conclusion that plk1 is essential for heart regeneration to proceed <eureka>.

Fig 5: plk1 is necessary for cardiac regeneration

(d) 30-dpa regenerating heart (DMSO treated). The plane of amputation is indicated with a dashed line. (e) 30-dpa regenerating heart (cyclapolin9 treated).
(g) Plk1 inhibiton reduces the number of cardiomyocytes entering the cell cycle during heart regeneration. The graph indicates the average number of BrdUpos/GFP(+) cardiomyocytes/section.

Overall, these experiments show that zebrafish heart regeneration is primarily driven by pre-existing cardiomyoctyes rather than progenitor cells as suggested previously (Sorry to disappoint you guys) (Lepilina A et al, 2006). However, the possibility of stem/progenitor cells cannot be excluded but the researchers felt that it would be only marginal in the light of their results. The conclusion is that pre-existing cardiomyocytes undergo limited dedifferentiation to undergo the ones close to the wound disassemble their sarcomeric structure and detach from one another. These cardiomyocytes start expressing regulators of cell cycle progression such as plk1 and mps1 which are necessary for regeneration process to continue. It is very tempting to speculate if mammalian cardiomyocytes can undergo this phenomenal process but if it could, that will mean a whole new array of possibilities.

For more interesting information about recent advances on this topic, please view this video : Regeneration : the holy grail of transplant medicine

1 Response to Zebra fish can mend its broken heart

  1. how can Zebra fish can mend its broken heart.?

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