Miniature Hearts Beating Around the Clock

Heart attacks are not spread evenly across the day. They are more frequent in the morning hours, and the ones that strike in the early hours of the day tend to do more damage. Disrupted sleep and night shift work are also known major risk factors for heart disease. The heart attack timing throughout the day and the importance of regular sleep both connect to the function of our biological clocks, the internal timekeepers that run in every cell of the body.

Dr. Sharon Fleischer, the newest recruit to Tel Aviv University's Shmunis School of Biomedicine and Cancer Research, has recently been awarded an ERC Starting Grant to develop the tools to study this connection. Her project aims to build a miniature model of human heart muscle that keeps time, a tissue whose "time" can be read and even tweaked in the lab. In parallel, her lab is also using models of the human heart to study  a different problem, trying to understand if chronic heart diseases with no clear explanation evolve as a result of the body’s immune system attacking the heart cells.

From patches to miniatures

Fleischer did her PhD with Prof. Tal Dvir at Tel Aviv University, engineering cardiac patches: large, complex constructs of heart muscle grown in the lab and designed to be transplanted in patients whose hearts have been scarred by a heart attack – with patches of this kind now approaching the clinic. For her postdoctoral work, at Columbia University with Prof. Gordana Vunjak-Novaković, one of the founders of the field of cardiac tissue engineering, she aimed to expand her research focus beyond the generation of large-scale and complex tissues to miniature, highly controlled tissue models of the human heart.

The result is known as a heart-on-a-chip. It starts with “induced pluripotent stem cells” - ordinary adult cells, often obtained from skin or blood samples, that have been reprogrammed backward into a stem cell state and can then be guided forward again into the desired cell types. Fleischer directs them into the two main cell types of heart muscle, the contracting cardiomyocytes and the supporting fibroblasts, and seeds them into a hydrogel inside a small, custom-designed bioreactor. There the cells receive the signals they would receive in the body, above all constant electrical pacing and mechanical stimulation, and over days they stop behaving like a suspension of unconnected cells and start behaving like a tissue: aligned, coupled, and beating in unison.

The point of shrinking the heart is to be able to ask it questions. Most of what we know about cardiac biology comes from animals, and mice are not humans. Drug after drug performs well in a mouse and then fails in human trials. A functioning piece of human heart muscle, small enough to make in quantity and mature enough to mimic the human heart, opens a window on human cardiac biology directly.

The platform Fleischer built during her postdoc, called milliPillar, was designed around that goal. It was built on an earlier system developed in the lab, that had been the first to recreate an adult-like heart-on-chip - critical for studying heart diseases and drug responses in conditions that match the ages when most heart diseases develop. But that system required considerable technical expertise and expensive specialized equipment. Fleischer's milliPillar was developed to be user-friendly both in setting it up and in using it for research. The miniature hearts she developed can also be followed for weeks or months while a disease develops inside them. The platform became the central model for cardiac research in her postdoc lab at Columbia University, used in studies that ranged from building a patient-specific chip from the cells of a child with a genetic heart mutation to screen and find a medication that could help him, to testing what cosmic radiation does to the hearts of astronauts.

The heart attacked from within

Then the rheumatologists called. Systemic lupus erythematosus is an autoimmune disease, meaning the immune system produces antibodies against the patient's own proteins, called autoantibodies. Between a quarter and a half of lupus patients have some involvement of the heart muscle, sometimes without any symptoms, and in severe cases it can progress to heart failure. Why it happens to some patients and not others is one of the open questions of the field. A team at New York-Presbyterian Hospital had assembled a large cohort of lupus patients with full clinical workups, heard what the lab was doing, and asked whether the engineered tissue could tell them why some of their patients had heart disease and others did not.

The experiment was clean. The team purified the autoantibodies from each individual patient's blood and added them to the engineered human cardiac tissues for two weeks. There were no immune cells in the dish, nor any other biological machinery through which autoimmune damage is usually assumed to work. Anything that happened had to be the autoantibodies acting on the heart cells directly.

And they did. In the lupus patients with heart disease, the autoantibodies bound to the engineered muscle and damaged it. They disrupted the tissue's electrical function, they drove the fibroblasts toward fibrosis, the scarring that stiffens a failing heart, and they impaired the metabolism of the cardiomyocytes themselves. To find out which proteins the autoantibodies were grabbing, the team worked with collaborators at Harvard using a technology that profiles the full repertoire of autoantibodies carried by each individual patient. Comparing those profiles between patients revealed which proteins were behind the damage: four candidates, none of them previously linked to autoimmune heart disease.

The direction Fleischer's new lab is taking is to generalize this approach. A great deal of chronic heart damage is classified as idiopathic, a medical term that means simply that no cause has been found. Some of it may be autoantibody-driven, and an engineered human heart tissue that can be handed a patient's serum is a way to find out. Dr Fleischer, in collaboration with cardiologists From the Sourasky Medical Center (Ichilov Hospital) have started looking at a mechanism already suspected in the clinic: patients with idiopathic dilated cardiomyopathy, in which the heart muscle is expanding and weakening for unknown reasons. This project was recently funded by the Israeli Science Foundation (ISF).

Models in four dimensions

Beyond using the miniaturized hearts to decipher hidden causes for heart diseases, the flagship project in Fleischer’s new lab is to understand how a known but understudied factor in heart disease – the biological clock – actually affects heart health. Every cell in the body carries a molecular clock, a set of genes and proteins that oscillate with a period of roughly 24 hours. These internal clocks are also connected to external cycles of light, sleep and food, and they change what a cell does depending on the hour. A heart-on-a-chip is a three-dimensional model, and in ignoring the dimension of time it quietly assumes that the heart is the same organ at four in the morning and four in the afternoon. The epidemiology says it is not.

Fleischer's ERC Starting Grant project is to add the missing dimension: an engineered human heart tissue with a running biological clock, in which the tissue's hour can be controlled. Using these models, Fleischer will aim to tackle two questions that have been difficult to approach in humans. The first is why cardiac disease behaves differently by day and by night. The second is why disrupted sleep and the modern schedule, what researchers call social jet lag - the chronic mismatch between the body's clock and the hours that work and social life impose - translate into heart disease. Beyond them lies a practical one. If a drug's effect on the heart depends on when it is given, the chip could be used to work out the right hour in advance, a question clinical trials are poorly built to ask.