About 2–3% of the general population has atrial fibrillation (AF), abnormal heart rhythm in the atrial chambers of the heart. It was previously believed to be primarily associated with age. However, a new method of studying the heart rhythm of zebrafish reveals that AF may actually be caused by a congenital disorder that occurs already at the embryonic stage. The researchers could also rectify the heart disorder in the zebrafish and now hope that this new knowledge can counteract AF causing some people’s hearts to gradually wear out and eventually fail.
People who have AF have a four-fold risk of death – primarily because their risk of a fatal stroke or sudden cardiac death increases. Despite the clinical importance of AF, the mechanisms underlying its initiation and pathogenesis remain poorly understood. AF interferes with the ventricular contraction of the heart and thus its pumping function. However, a new model system has now enabled researchers to investigate the causes of this severe disorder.
“Perhaps we should also consider a heart with AF as defective – and not just as experiencing electrical instability. We studied the hearts of zebrafish, which are very similar to those of humans, and found some genetic changes that lead to serious structural changes in the heart early in the zebrafish’s life. By influencing these changes early with antioxidants, we rectified the disorder. We hope we can transfer this knowledge to humans, so that we can identify and protect these affected hearts in time,” explains Pia Rengtved Lundegaard, Assistant Professor, Department of Biomedical Sciences, University of Copenhagen, a main author of the article in the Proceedings of the National Academy of Sciences of the United States of America.
Surviving with a heart disorder
The major breakthrough in this research is using zebrafish to study heart function combined with assessing genetic changes. Zebrafish have been used for genetic studies for many years; one reason is that they are transparent, which enables researchers to study DNA and genetic changes by using dyes. Zebrafish now prove to be uniquely suitable for studying heart function at both the larval and the adult stages.
“Compared with rodents, many aspects of the cardiac electrophysiology of zebrafish are more closely aligned with those of humans, including heart rhythm, developmental physiology and cardiac action potential characteristics. Indeed, modelling human arrhythmia in zebrafish previously provided significant insight into the mechanisms of conduction defects. This combination of similarity and the opportunity to alter and study genetic changes makes zebrafish unique for studying the development of the heart and heart disorders,” says Pia Rengtved Lundegaard.
In a study published in Nature Communications 2 years ago, the researchers used CRISPR-Cas9 editing to discover that zebrafish with a truncated version of the muscle protein titin had a strongly increased risk of arrhythmia. This time, the researchers investigated Pitx2c, a protein that had previously been shown to cause AF among humans if it was defective.
“The previous experiments were performed on mice, and if the protein was defective, the mice died during pregnancy. Zebrafish embryos can survive, since they get oxygen through diffusion, so they can easily survive to adulthood even if they have heart disorders. This enabled us to study how the heart developed with defective Pitx2c protein and thereby also learn how AF occurs as a result of a defect in protein biogenesis,” explains Pia Rengtved Lundegaard.
Antioxidants rectify defects
To analyse how genetic changes affected the hearts of zebrafish, researchers used confocal microscopy to record films and thus could evaluate the heart function of the zebrafish at the larval and the adult stages. Since the focus was on AF, the researchers initially looked for the defects in the ion channel genes that modulate electrical conduction between the heart’s muscle cells. However, they were in for a surprise.
“Contrary to our expectations, we found defects in the structure of the heart. Usually, longitudinal sections of sarcomeres – muscle fibres – have a very fine grid structure. But these zebrafish had clearly disorganized and irregularly spaced muscle fibres from a very early stage,” says Pia Rengtved Lundegaard.
In addition to the defects in the structure of the heart muscle, the researchers also found defects in the mitochondria – the muscle cells’ power plants. The researchers used a marker that changes colour during high production of reactive oxygen species – a by-product of the mitochondria – and found some defective mitochondria, with many defects already in the larval stage that became exponentially worse with age.
“The mitochondria increase the level of oxidative stress and thereby create an unhealthy environment in the cell. We therefore tried to treat the zebrafish larvae with N-acetylcysteine, a potent scavenger of reactive oxygen species, and it worked by counteracting the stress and the heart disorder,” explains Pia Rengtved Lundegaard.
No reason to hoard antioxidants
The experiments with the zebrafish thus indicate that AF is not merely electrical instability but also definitely has genetic and muscular causes and that the heart tries to compensate for the defective muscle fibres.
“This suggests that the heart has a structural defect and that an increased number of mitochondria appear to aggravate the negative spiral, which over time might augment a rhythm disorder, so the arrhythmia itself is secondary to the actual problem,” says Pia Rengtved Lundegaard.
However, Pia Lundegaard emphasizes that the gene studied is probably just one of many possible factors behind AFMany studies have previously shown that AF is strongly influenced by a person’s lifestyle, but the new finding provides important new insight into why some medicine intended to counteract AF does not always work optimally.
“We want to understand even better how AF occurs and how it can be treated. So we will investigate other genes associated with AF through the new model, but we will also try to treat adult zebrafish with antioxidants to determine whether they have an acute function and treat the larvae at different times to determine how late they can be treated and still save their hearts,” explains Pia Rengtved Lundegaard.
The major scientific breakthrough and the reason why this research ended up in one of the world’s leading journals is the development of a model to study arrhythmia in zebrafish in one gene associated with AF in humans. Ultimately, however, Pia Lundegaard naturally hopes that the knowledge about AF will benefit patients.
“People with heart disorders have no reason to hoard antioxidants such as N-acetylcysteine, but the new knowledge about which genes can cause AF will help us to identify people at higher risk when they are young and perhaps help them to reduce the oxidative stress in their heart. So we hope that we can contribute to developing some better procedures in the future that counteract the long-term wear and tear and eventual failure of some people’s hearts,” concludes Pia Rengtved Lundegaard.
“Early sarcomere and metabolic defects in a zebrafish pitx2c cardiac arrhythmia model” has been published in the Proceedings of the National Academy of Sciences of the United States of America. The Novo Nordisk Foundation awarded grants to co-authors Pia Rengtved Lundegaard and Morten Olesen, including for Nationwide Study of the Genetics behind Atrial Fibrillation.