Zebrafish Heart Regeneration: Pune Study Shows How the ccn2a Gene Helps

Along the ridges of paddy fields, in the ditches, canals, beels and ponds of Bangladesh and north-eastern India, lives a finger-sized fish with dark stripes that most people pass without a second look. It is the zebrafish, Danio rerio, and in laboratories around the world it is famous for something no human can do: when part of its heart muscle is destroyed, it grows new muscle and the heart works again.

In January 2021 a team of ten researchers, led from the Agharkar Research Institute (ARI) in Pune, India, published a study in the British journal Development showing how one gene helps the injured zebrafish heart rebuild itself. The gene is ccn2a, short for cellular communication network factor 2a, which was earlier called ctgfa, the zebrafish form of the connective tissue growth factor gene. This report explains what the zebrafish is, why scientists rely on it, what the Pune team found, and what the finding does and does not mean for people with heart disease.

The zebrafish is native to fresh water in South Asia: India, Pakistan, Bangladesh, Nepal and Bhutan. Its range is concentrated in the Ganges and Brahmaputra river basins, and the species was first described from the Kosi river in the lower Ganges basin. A 2008 review in Biological Reviews by Spence and colleagues at the University of Leicester describes it as a small shoaling member of the carp family, native to the flood plains of the Indian subcontinent, where it lives in shallow, slow-flowing water.

Bangladesh has a special place in what is known about the fish in the wild. In a 2006 doctoral study at the University of Leicester, Spence reported a field survey in Bangladesh which found zebrafish most abundant in shallow lakes, ponds and ditches, often linked with rice cultivation. Length measurements showed that in the wild it is an annual fish whose young appear with the monsoon, and gut contents showed a diet mainly of zooplankton and insects.

A field study in north-eastern India by Engeszer and colleagues of the University of Washington, published in the journal Zebrafish in 2007, found the fish particularly abundant in silt-bottomed, well-vegetated pools and in rice paddies next to slow-moving streams. The same study recorded substantial loss and degradation of those habitats.

The fish is named for the five horizontal blue stripes along its body, which run to the end of the tail fin. Wild fish are typically 1.8 to 3.7 centimetres long, and the largest reach 4 to 5 centimetres. The male is slim and torpedo-shaped with gold stripes between the blue ones; the female has a larger, whitish belly. Zebrafish spawn in groups and scatter their eggs, and the species is sold in the aquarium trade as the zebra danio.

In captivity a zebrafish usually lives about two to three years, and it takes about three months to grow from egg to a breeding adult. That short generation time suits laboratories.

The zebrafish's rise as a genetic model began in the late 1960s, when George Streisinger and Charline Walker at the University of Oregon in the United States brought fish from a local pet shop into their laboratory.

The turning point came in 1981, when Streisinger and colleagues reported in the journal Nature that they had produced homozygous diploid zebrafish on a large scale with simple physical treatments, and then whole clones of such fish, which opened the way to genetic analysis of a backboned animal.

A 2013 study in the journal Zebrafish by Kinth and colleagues of India's National Institute of Science Communication and Information Resources analysed 17,151 records from the Zebrafish Information Network (ZFIN) and the Web of Science. The earliest work in those databases went back to 1951; output rose from 226 publications in 1996 to 1,929 in 2012, and the United States was the most productive country with 8,196 articles.

The eggs are fertilised outside the mother's body, and the embryos are almost transparent, so scientists can watch organs form under a simple microscope. Precursors of all major organs appear within 36 hours of fertilisation. For 10 to 15 days after birth, the original report notes, the heart, liver, pancreas and other organs can be watched developing from outside, and even the formation of the heart valves can be followed down to the level of single cells.

Genetics is the other reason. In 2013 a large international team, Howe and colleagues, published the reference sequence of the zebrafish genome in Nature. It found more than 26,000 protein-coding genes, the largest gene set of any vertebrate sequenced up to then, and showed that about 70 percent of human genes have at least one obvious counterpart in the zebrafish.

The zebrafish's ability to repair organs is not limited to the heart. A 2013 review in Trends in Genetics by Gemberling, Poss and colleagues described a fish able to regrow amputated fins as well as a damaged brain, retina, spinal cord and heart and other tissues.

The heart story began in December 2002, when Kenneth D. Poss and his co-authors Wilson and Keating reported in Science that zebrafish fully regenerate their hearts within two months after 20 percent of the ventricle, the main pumping chamber, is cut away. The new muscle came from heart muscle cells dividing at the edge of the wound. Fish carrying a mutation in a cell-division control gene called Mps1 failed to regenerate and formed scars instead. The authors noted that heart injury in mammals and amphibians typically ends in scarring.

Cutting away tissue is not what happens in a human heart attack, where muscle dies but stays in place. In 2011 two groups therefore introduced cryoinjury, in which a chilled probe is pressed on the ventricle to kill a patch of muscle. Chablais and colleagues at the University of Fribourg in Switzerland reported in BMC Developmental Biology that freezing killed cells in about 20 percent of the ventricular wall, much as in a mammalian infarct. At first the fish heart behaved like a mammal's, with clotting and collagen being laid down. Over the next two months, however, the scar was gradually removed and replaced by new heart muscle, and electrocardiograms showed the heart's function restored.

González-Rosa and colleagues at Spain's National Centre for Cardiovascular Research (CNIC) in Madrid reported in Development the same year that cryoinjury of about 25 percent of the ventricle produced a massive scar during the first three weeks. The scar was later broken down and replaced by heart tissue, although the regenerated hearts showed uneven contraction and a thickened wall. A fish heart, in other words, can form a scar and still remove it.

A direct forerunner of the Pune study appeared in Science in November 2016. A team of six, Mokalled, Patra, Dickson, Endo, Stainier and Poss, found that ctgfa, the same gene under its older name, is switched on around the glial cells that form a bridge across a cut spinal cord in zebrafish. Fish lacking a working ctgfa repaired poorly, while extra ctgfa or human CTGF protein sped up repair. Patra, a co-author of that paper, later led the heart study at ARI.

The Pune paper opens by noting that heart disease is the leading cause of death in industrialised nations and that the adult mammalian heart fails to regenerate after damage, usually leaving a lasting scar. The Swiss group put the human side plainly: in a myocardial infarction, heart tissue is lost for good and replaced with a fibrous scar.

Human heart muscle cells are not entirely fixed. In 2009 Bergmann and colleagues used carbon-14 left in the air by Cold War nuclear bomb tests to date the DNA of heart cells. They reported in Science that heart muscle cells do renew, but slowly: about 1 percent are replaced each year at the age of 25, falling to 0.45 percent at 75, and fewer than half are exchanged during a normal life span.

Newborn mammals have a brief window. In 2011 Porrello, Olson, Sadek and colleagues reported in Science that the hearts of one-day-old mice can regrow after part of the heart is removed, but that this ability is lost by seven days of age.

For Bangladesh, the World Health Organization's Noncommunicable Diseases Country Profiles 2018 estimated that Bangladesh, with a population of 163 million, recorded 856,000 deaths in 2016. Noncommunicable diseases accounted for an estimated 67 percent of them, and cardiovascular diseases alone for 30 percent of all deaths, the largest single group.

The Agharkar Research Institute in Pune, Maharashtra, is an autonomous, grant-in-aid research institute of the Department of Science and Technology of the Government of India. It was founded in 1946 by the Maharashtra Association for the Cultivation of Science as the MACS Research Institute and renamed in 1992 in memory of its founder director, Professor Shankar Purushottam Agharkar. It works in animal, microbial and plant sciences.

The heart study was carried out in ARI's Department of Developmental Biology. Its first author, Debanjan Mukherjee, worked under the supervision of Chinmoy Patra, the corresponding author, in that department. Two other authors were affiliated both with ARI and with Savitribai Phule Pune University.

Two foreign laboratories took part. From the United States came researchers of Regeneration Next and the Department of Cell Biology at Duke University Medical Center in Durham, North Carolina, including Poss, whose 2002 paper first showed zebrafish heart regeneration. From Germany came the Max Planck Institute for Heart and Lung Research in Bad Nauheim, through its Department of Developmental Genetics, where Stainier works, and its bioinformatics and deep sequencing platform. The research record lists grants from the National Heart, Lung, and Blood Institute of the US National Institutes of Health and from the Max Planck Society.

The paper, titled "Ccn2a is an injury-induced matricellular factor that promotes cardiac regeneration in zebrafish", was received by Development on 24 May 2020, accepted on 6 November 2020 and published on 18 January 2021 in volume 148, issue 2. Its publisher is The Company of Biologists in Cambridge, United Kingdom.

Ccn2a belongs to a small family of genes called CCN. In 2003 a group of researchers endorsed by the International CCN Society proposed one naming system for the family: CCN1 (also called CYR61), CCN2 (CTGF, connective tissue growth factor), CCN3 (NOV), and CCN4, CCN5 and CCN6 (the WISP proteins). They described the CCN proteins as matricellular proteins, released outside cells, that help control how cells attach, move, multiply, survive and specialise.

The space between cells is filled with the extracellular matrix, a mesh of proteins such as collagen and fibronectin. The Pune paper notes that this matrix was long seen as an inert scaffold but is now known to send signals that shape the development, upkeep, disease and repair of organs.

CCN2 has a mixed reputation. The paper reviews studies linking it with scarring, or fibrosis, in the liver, kidney, skin, lung and heart of mammals. In the mouse heart the evidence was contradictory: one study found that extra CCN2 partly protected the heart from scarring, while another found that changing CCN2 levels did not affect scarring in stressed adult hearts. Other work had linked it with repair of ligaments in rabbits and of the spinal cord in zebrafish.

Zebrafish carry two copies of the gene, ccn2a and ccn2b. To find matrix genes that might drive repair, the Pune team screened for those switched on four days after cryoinjury and found nine whose activity rose significantly. Both ccn2 copies rose after injury, but ccn2a rose far more, so the team concentrated on it.

The researchers injured adult zebrafish hearts by cryoinjury and by cutting away part of the ventricle. According to the original Agro News report, the fish used were at least six months old, already adult, because heart attacks are mainly a problem of adults. They built a reporter fish line in which the cells that switch on ccn2a glow green. The gene was switched on in the endocardium, the thin inner lining of the heart, in the injured area.

To test whether the gene is needed, they used mutant fish whose ccn2a gene does not work. The paper reports these results:

• At 60 days after cryoinjury, about 60 percent of normal fish showed vigorous regeneration, while all fish with two faulty copies of ccn2a, and about half of those with one faulty copy, showed minimal regeneration.
• Even 150 days after injury, a lasting scar remained in about 60 percent of the mutant fish.
• In normal fish, new heart muscle grew along the new blood vessels entering the wound; seven days after injury about 88 percent of the heart muscle cells there were lined up with new vessels. In mutants this movement was impaired, although the blood vessels themselves grew back normally.
• Division of heart muscle cells fell by about 75, 90 and 60 percent in the mutants at 4, 7 and 12 days after injury.

The team then did the opposite. In fish carrying an extra copy of the gene that switches on with a short daily heat treatment, more ccn2a raised the division of heart muscle cells by about 45 percent and helped clear the temporary collagen laid down in the wound.

The experiments also pointed to how the gene works. Ccn2a raised the activity of matrix genes that favour repair, among them the fibronectin gene fn1a and the collagen genes col1a1a and col2a1a, and held down a gene called cxcr3.1, which carries signals for immune cells and was about 50 percent more active in mutants. Both effects ran through the Tgf-beta signalling pathway: in mutants, cells carrying the active signal protein pSmad3 in their nucleus fell by about 62 to 71 percent, and blocking the pathway with a laboratory drug named SB431542 confirmed the link. The authors concluded that Ccn2a positively modulates the adult zebrafish heart's own regenerative response.

The study explains part of why the zebrafish heart can be damaged and still heal. The authors set their finding against the hypothesis that adult mammals, including humans, whose hearts have limited power to regenerate, might be induced to regenerate heart tissue if the right regenerative genes are identified and activated.

This is basic science, not a treatment. All the experiments were done in zebrafish; no medicine, injection or gene therapy based on ccn2a was tested in people, and the earlier mouse studies of CCN2 in the heart gave mixed results. The study does not show that a damaged human heart can be made new. People with heart disease should keep to the treatment and advice of their doctors.

Photo: Soulkeeper / Wikimedia Commons (Public domain)

Sources: The Agro News (Staff Correspondent), Mukherjee D, Patra C and colleagues, Development 148(2), dev193219 (published 18 January 2021), Poss KD and colleagues, Science (2002), Chablais F and colleagues, BMC Developmental Biology (2011), González-Rosa JM and colleagues, Development (2011), Mokalled MH and colleagues, Science (2016), Bergmann O and colleagues, Science (2009), Porrello ER and colleagues, Science (2011), Howe K and colleagues, Nature (2013), Streisinger G and colleagues, Nature (1981), Spence R and colleagues, Biological Reviews (2008), Spence R, University of Leicester thesis (2006), Engeszer RE and colleagues, Zebrafish (2007), Gemberling M and colleagues, Trends in Genetics (2013), Kinth P and colleagues, Zebrafish (2013), Brigstock DR and colleagues, Molecular Pathology (2003), World Health Organization, Noncommunicable Diseases Country Profiles 2018 (Bangladesh), University of Oregon, Wikipedia (Zebrafish, Agharkar Research Institute)