Cardiovascular disease is the #1 cause of death in the world [1]. Cardiovascular disease results from the deterioration of heart tissue – an irreversible disease due to our inability to regenerate our heart tissue. There are model organisms used in science with regenerative capabilities, but they have some limitations. Mice have cardiac regenerative abilities one day after birth but lose it at seven days [5]. Adult zebrafish and salamanders can regenerate their hearts as adults but are considered lower vertebrates [6]. Humans are higher vertebrates and have more complex systems, so research on lower vertebrates can only tell us so much. In an article titled “Fosl1 is vital to heart regeneration upon apex resection in adult Xenopus tropicalis,” Hai-Yan Wu and Yi-Min Zhou from Jinan University focus on the X. tropicalis, or the western clawed frog, a vertebrate with a closer evolutionary distance to mammals [3,7]. Previous data demonstrated that one-year-old X. tropicalis could regenerate their hearts but did not dive into the mechanisms [3]. This more recent paper uses fully developed frogs matured at six months to show that the protein Fosl1 plays an important role in heart regeneration in vertebrates. Additionally, the authors propose X. tropicalis as an ideal model organism for heart regeneration.

WORD BANK:

α-actinin+: positive expression of protein that contributes to the formation of cardiac muscle filaments

cardiomyocytes (CMs): Cells specific to the heart

cTnT+: Cardiac Troponin T; used as a marker for acute myocardial infarction 

EdU: 5-Ethynyl-2′-deoxyuridine (EdU) ; incorporated into DNA to label dividing cells and is commonly used in pulse-chase techniques.

in vivo: Experiments and tests conducted in an entire living organism

Fosl1 vs. Fosl1: It is important to distinguish between the formatting of gene and protein names. Proteins and their encoding gene share the same name. To tell the difference, gene names are italicized while protein names are not. In this case, Fosl1 is the protein and Fosl1 is the gene.

Ki67+: Positive expression indicating the cell is actively dividing

luciferase assay: Determines if a protein can turn on or off the expression of a target gene. If the protein can increase expression, cells will express luciferase and glow, if the protein decreases expression, cells express less luciferase than normal.

myocardial infarction (MI):  Heart attack

pH3+: ositive expression of dividing cells

pulse-chase: Technique to analyze cellular processes over time. Cells are exposed to a labelled compound (pulse) that is incorporated during DNA synthesis. The pulse is “chased” by the same compound, but unlabeled. The compounds are present to track the replication of cellular DNA.This is monitored to see how long the labeled compound lasts in cells.

Quantitative polymerase chain reaction (qPCR): Measures expression of DNA

RNA-seq analysis: Sequencing technique that analyzes RNA to determine what RNAs are present within a sample, which is used to conclude what genes are expressed or suppressed.

Frog heart tissue can regenerate!

The researchers’ first goal was to compare X. tropicalis with another species X. laevis since previous work showed the latter lost cardiac regenerative capability after 6 months [4]. After cutting the tip of the heart in a surgical process called apex resection, tissue staining showed 6-month X. tropicalis can regenerate heart tissue, with full regeneration 30 days post resection. They also confirmed that there was no significant difference in the heart weight or surface area between the resected hearts and sham (control) hearts. Successful regeneration was characterized by contraction at 30 days post resection.

Heart regeneration is successful through cardiomyocyte division

Next, the scientists wanted to rule out the possibility that the cardiomyocytes (CMs), the cells that make the heart contract, were simply getting bigger as opposed to actually dividing. They did this by staining the heart tissue with pH3+ and α-actinin+. They found that there was an increase in dividing cells 1 to 14 days post resection. They again confirmed this by measuring the uptake of EdU, a DNA synthesis marker. If a cell were simply expanding, the DNA would not need to divide and no EdU would be incorporated into the cells. However, they saw a significant increase in EdU, confirming the cells were, in fact, dividing. They show a significant increase in EdU+ ɑ-actinin cells 3 to 14 days post resection. Finally, the researchers used another method to confirm proliferation using another DNA synthesis label, proliferation cell nuclear antigen (PCNA). While EdU specifically labels the S stage where chromosomes are duplicating, PCNA is expressed in all stages of cell division. The S stage is only the replication of DNA, so using PCNA as well ensures the increase is in cell division and not simply an increase of DNA in S stage.

Now that it is known the CMs are dividing, the researchers asked if the new heart tip was made of newly formed CMs. They prove this using a pulse-chase experiment.  The CMs  are exposed to EdU for a brief amount of time (pulse) which is incorporated into cells. There is a limited amount of  EdU given so when the cells are “chased” by unlabeled compounds. This is monitored to see how long the labeled compound lasted in the cells. EdU+ CMs were seen in the new tip 30 days post resection but are absent elsewhere. This shows that regenerated heart tissue is made up of dividing and newly formed CMs. If there were EdU+ CMs in tissues other than the tip, that could suggest that CMs have a broader role and are not the main cells responsible for heart regeneration. 

Fosl1 gene is necessary for cardiomyocyte division 

Now the authors want to find target genes involved in heart regeneration. They collected cut heart ventricles and did RNA-seq analysis (technique that analyzes RNA to determine which genes are turned on or off and by how much). They found that genes related to cell growth and cell cycle regulation were increased in cut hearts versus the controls. The control frogs underwent the same surgical procedure of the incision but their ventricles were not cut. The gene Fosl1 has known roles in cell proliferation. qPCR showed an increase in Fosl1 after the heart tip was cut. To determine what type of cells expressed Fosl1 during heart regeneration, they examined the pattern of Fosl1 in CMs and non-CMs. They showed Fosl1 expression levels were higher in CMs compared to non-CMs, indicating CMs are responsible for the increase. 

To determine target genes or genes affected by the protein Fosl1 during heart regeneration, they used qPCR and screened 7 targets. Using a luciferase assay, they show Fosl1 interacts with JunB, another player in cell proliferation, to activate all 7 genes. One gene, ccnt1, was shown to be directly regulated by Fosl1. This makes sense because the ccnt1 pathway is known to be related to heart injury and regeneration. These results suggest that Fosl1 promotes CM division by increasing ccnt1 expression by interacting with JunB, contributing to heart regeneration. 

Fosl1 protein is necessary for cardiomycote division 

Researchers asked next if the protein Fosl1 influences the proliferation of CMs. Using a rat CM cell line, H9c2, they transfected the cells with a virus that overexpresses Fosl1 protein, meaning that Fos11 will be expressed more than the natural amount. After a cell count and EdU incorporation assay, they found overexpression of Fosl1 promoted cell growth. Short interfering RNA (siRNA) is very specific and is used to cut out instructions for a gene, thereby “silencing” it. When they silenced Fosl1 protein using siRNA they saw a decrease in proliferation (decrease in cell division), as they did when they used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology to knockdown the Fosl1 gene. siRNA silenced Fosl1 by generating knockdowns at the mRNA level, while CRISPR generated knockouts at the DNA level. The researchers concluded that Fosl1 protein is necessary for proliferation of CMs. 

To see if the gene, Fosl1, influences proliferation, the authors isolated CMs in neonatal mice which lose heart regenerative abilities 7 days after birth. They compared proliferation between mice with and without Fosl1 silencing. Silencing Fosl1 suppressed the amount of  Ki67+ cTnT+  cells. Ki67+ is the textbook marker for cell proliferation and has been heavily researched and PCNA is a newer cell proliferation marker [2]. The reduction in PCNA in the active phase of the cell cycle suggests Fosl1 is extremely important for the proliferation of primary CMs. 

To look at the in vivo Fosl1 function in X. tropicalis, a dominant-negative transgenic line of  Fosl1 (dnFosl1) was made. This specifically blocks the function of Fosl1 while leaving other Fos members intact. After cutting the tip and looking at CM proliferation 3 days post resection, they saw that the percentage of EdU+ α-actinin+ cells (proliferating heart cells) were significantly lower in dnFosl1 hearts compared to the controls. They also used pH3/α-actinin staining to show CM proliferation was significantly lower in dnFosl1 hearts compared to controls. This data shows that loss of function of Fosl1 causes a defect in CM proliferation during heart regeneration in X. tropicalis. 

Loss of Fosl1 function decreases neonatal mice cardiomyocyte division 

The authors were able to show similar results in neonatal mice and used a virus to silence Fosl1 as opposed to creating a transgenic line. They repeated this in mice because they are mammals and more closely related to humans. If the mechanism wasn’t the same in mice, studying heart regeneration in frogs would not be very beneficial for human therapeutic use. Suppression of CM proliferation following Fosl1 silencing is shown by a decrease in EdU+ cTnT+ cells. 

Fosl1 improves heart repair in adult mice!

Lastly, the researchers wanted to determine if Fosl1 had any roles in heart repair in a non-regenerative model. They used adult mice to observe the expression pattern of Fosl1 after a myocardial infarction (MI). They saw Fosl1 expression decreased in hearts up to 3 days after the MI. They next wanted to see if overexpressing Fosl1 in these adult mice’s hearts could improve heart repair after an MI. Mice were injected with AAV9-Fosl1 for 30 days to overexpress Fosl1 and then had surgery to induce a heart attack. AAV9 is a virus that has been repurposed to be like a delivery vehicle. Instead of the original viral DNA, the authors replaced it with Fosl1 and used the machinery already in the virus to make abundant copies of Fosl1, leading to overexpression. Overexpression of Fos11 significantly improved cardiac function, which was confirmed by an increase in how much blood could be pumped out of the heart, increased change in the diameter of the left ventricle, and reduced scarring. 

It is important to do cardiac regeneration research on model organisms so that we can develop therapies for heart damage in humans. While there are several model organisms with better regenerative capability than X. tropicalis, they are farther away evolutionary-wise to humans. X. tropicalis has the ability to regenerate spinal cord, tail, eye and limbs. This lab has successfully proved that adult western clawed frogs can regenerate their hearts at 6-months. In the past, the authors proved this with 1 year-old frogs. Decreasing the experiment timeline by half a year can be useful in the future for testing therapeutic treatments quickly and speeding up the process of clinical trials. This paper shows Fosl1 plays an essential role in CM proliferation and heart regeneration in vertebrates. This includes interacting with JunB – a gene activity regulator – to increase expression of cell cycle regulators such as Ccnt1. Ultimately, the authors show 6-month X. tropicalis is an ideal model organism for studying heart regeneration in vertebrates. This puts us closer to fully understanding the mechanisms of vertebral heart regeneration, which will allow us to develop therapies to treat cardiovascular disease. 

References:

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