Heart scar tissue may take active role in promoting deadly arrhythmias

Susan Thompson, PhD student in biomedical engineering, and Craig Copeland, PhD student in physics and astronomy, observe a single non-beating heart cell called a myofibroblast growing on a micropost device. (Photo Jay VanRensselaer)

Johns Hopkins University biomedical engineers and physicists affiliated with the Institute for NanoBioTechnology have completed a study that suggests that mechanical forces exerted by cells that build scar tissue following a heart attack may later disrupt rhythms of beating heart cells and trigger deadly arrhythmias. Their findings, published in a recent issue of the journal Circulation, could result in a new target for heart disease therapies.

Principal investigator Leslie Tung, a School of Medicine professor in the department of biomedical engineering, led a team that looked at how heart cells that beat (called “cardiomyocytes”) were affected by the non-beating cells (called “myofibroblasts”). Myofibroblasts are called to arms at the site of injury following a heart attack.

“The role of the myofibroblast (non-beating cells) is to make the injured area as small as possible. Through contraction, the myofibroblasts close the wound and lay down a protein matrix to reduce the scar area,” said lead investigator Susan Thompson, a pre-doctoral fellow in Tung’s Cardiac Bioelectric Systems Laboratory. “In doing so, the myofibroblasts pull on the membranes of adjacent cardiomyocytes. We found that these forces were strong enough to decrease the electrical activity of the working heart cells through mechanical coupling.”

Thompson electrically stimulated cultures containing both the beating and non-beating cells growing together, and found that when the electrical impulses occurred, the non-beating myofibroblasts pulled on the membranes of beating cardiomyocytes and disturbed their electrical rhythm. Before this study, scientists were aware that myofibroblasts influenced the function of cardiomyocytes by depositing scar tissue, which produces regions of poor or no conductivity in healing cardiac tissue. But the “pulling” scenario described by Tung’s group indicates that myofibroblasts play a more active role than previously realized, Thompson said.

Biomedical engineering professor Leslie Tung collaborated with physics professor Daniel Reich to understand how heart scar tissue actively contributes to deadly arrhythmias. (Photo by Jay VanRensselaer)

In fact, images created using a voltage-sensitive dye showed that the spread of electrical waves was greatly impaired in the cultures with the most non-beating cells. Electrical conduction improved significantly, however, when drugs were added that inhibited contraction or that blocked so called “mechano-sensitive” channels.

“This is a truly exciting discovery because it radically affects our way of thinking about how cardiac arrhythmias might arise,” Tung said.

Tung and Thompson wanted to find out how strong the forces exerted by the myofibroblasts were and whether they changed when certain drugs were added. So they turned for answers to Daniel Reich, professor and chair of the Henry A. Rowland Department of Physics and Astronomy in the Krieger School of Arts and Sciences, and his pre-doctoral student Craig Copeland.

To measure the strength of the contractile forces of the myofibroblasts, the team used a device made up of a platform comprising an array of flexible “microposts.” The array resembled a carpet with widely spaced fibers upon which single cells can grow. As the cells responded to their environment, they pulled on the posts. How much the posts bent provided data about the direction and strength of forces exerted. Single layers of myofibroblasts were grown on the micropost device and tested in the presence of the same compounds Thompson used in her conductivity experiments.

“Imagine gripping a basketball with one hand, palm facing downward,” Copeland said. “The forces you apply to the ball with your fingertips to keep it suspended are similar to the forces cells exert on their environment. If you were to place your hand on a bed of rubber nails and apply the same gripping force with your fingertips as you did with the basketball, the nails would bend and their tips be deflected. This is exactly what happens with cells cultured on the post arrays.”

Thompson also explained that scientists previously thought that non-beating cells affected the beating cells simply through openings called “gap junctions,” where the two cells came into physical contact. The greater electrical charge of the myofibroblasts would flow passively downhill through the gap junctions toward the cardiomyocytes and disrupt their rhythms.

Photo by Jay VanRensselaer

The group’s new hypothesis suggests another type of membrane channel opened by physical force—the mechano-sensitive channels—may be more important in regulating electrical activity of the cardiomyocytes than mere junctions connecting membranes.

The results of both the conductivity and the micropost experiments fully support this new hypothesis, the team said. Although they acknowledge that both the passive gap channels and the active pulling forces can explain how myofibroblasts affect the electrical activity of cardiomyocytes, the researchers believe the pulling forces could be more relevant to the development of deadly arrhythmias.

“We are not ruling out the current theory,” Thompson said. “But we are saying there is something else we should be looking at, and we think the pulling forces are a major component. This could provide another lane of therapeutic investigation, especially if drugs could be targeted specifically to the contraction of the myofibroblasts.”

The next step in the project will be to combine the micropost device with electrical experiments on cultures containing cardiomyocyte and myofibroblast cell pairs.

“Although technically quite challenging, it will allow us to unravel how pulling forces applied by the myofibroblast to the cardiomyocyte affects the cardiomyocyte’s electrical activity,” said Tung.

Both Tung and Reich are affiliated faculty members of Johns Hopkins Institute for NanoBioTechnology. Thompson and Copeland are INBT fellows in the institute’s Integrative Graduated Education and Research Traineeship (IGERT), funded by the National Science Foundation (NSF). The National Institutes of Health, American Heart Association and the NSF IGERT funded their work. Findings were published in the May 17, 2011 issue of the journal Circulation.

Story by Mary Spiro

Photos by Jay VanRenesselaer/Homewood Photography

On new lab chip, heart cells display a behavior-guiding ‘nanosense’

Johns Hopkins biomedical engineers, working with colleagues in Korea, have produced a laboratory chip with nanoscopic grooves and ridges capable of growing cardiac tissue that more closely resembles natural heart muscle. Surprisingly, heart cells cultured in this way used a “nanosense” to collect instructions for growth and function solely from the physical patterns on the nanotextured chip and did not require any special chemical cues to steer the tissue development in distinct ways. The scientists say this tool could be used to design new therapies or diagnostic tests for cardiac disease.

Leslie Tung, left, and Andre Levchenko, right, both of the Department of Biomedical Engineering, with Deok-Ho Kim, a doctoral student in Levchenko’s lab, who holds a nanopatterned chip able to cue heart cells to behave like natural heart tissue. Photo: Will Kirk/homewoodphoto.jhu.edu

Leslie Tung, left, and Andre Levchenko, right, both of the Department of Biomedical Engineering, with Deok-Ho Kim, a doctoral student in Levchenko’s lab, who holds a nanopatterned chip able to cue heart cells to behave like natural heart tissue. Photo: Will Kirk/homewoodphoto.jhu.edu

The device and experiments using it are described in this week’s online Early Edition issue of Proceedings of the National Academy of Sciences. The work, a collaboration with Seoul National University, represents an important advance for researchers who grow cells in the lab to learn more about cardiac disorders and possible remedies.

“Heart muscle cells grown on the smooth surface of a Petri dish would possess some, but never all, of the same physiological characteristics of an actual heart in a living organism,” said Andre Levchenko, an associate professor of biomedical engineering in Johns Hopkins’ Whiting School of Engineering. “That’s because heart muscle cells—cardiomyocytes—take cues from the highly structured extracellular matrix, or ECM, which is a scaffold made of fibers that supports all tissue growth in mammals. These cues from the ECM influence tissue structure and function, but when you grow cells on a smooth surface in the lab, the physical signals can be missing. To address this, we developed a chip whose surface and softness mimic the ECM. The result was lab-grown heart tissue that more closely resembles the real thing.”

Levchenko said that when he and his colleagues examined the natural heart tissue taken from a living animal, they “immediately noticed that the cell layer closest to the extracellular matrix grew in a highly elongated and linear fashion. The cells orient with the direction of the fibers in the matrix, which suggests that ECM fibers give structural or functional instructions to the myocardium, a general term for the heart muscle.” These instructions, Levchenko said, are delivered on the nanoscale—activity at the scale of one-billionth of a meter and a thousand times smaller than the width of a human hair.

Levchenko and his Korean colleagues, working with Deok-Ho Kim, a biomedical engineering doctoral student in Levchenko’s lab and the lead author of the PNAS article, developed a two-dimensional hydrogel surface simulating the rigidity, size and shape of the fibers found throughout a natural ECM network. This biofriendly surface made of nontoxic polyethylene glycol displays an array of long ridges resembling the folded pattern of corrugated cardboard. The ridged hydrogel sits upon a glass slide about the size of a U.S. dollar coin. The team made a variety of chips with ridge widths spanning from 150 to 800 nanometers, groove widths ranging from 50 to 800 nanometers and ridge heights varying from 200 to 500 nanometers. This allowed researchers to control the surface texture over more than five orders of magnitude of length.

“We were pleased to find that within just two days the cells became longer and grew along the ridges on the surface of the slide,” Kim said. Furthermore, the researchers found improved coupling between adjacent cells, an arrangement that more closely resembled the architecture found in natural layers of heart muscle tissue. Cells grown on smooth, unpatterned hydrogels, however, remained smaller and less organized, with poorer cell-to-cell coupling between layers. “It was very exciting to observe engineered heart cells behave on a tiny chip in two dimensions like they would in the native heart in three dimensions,” Kim said.

Collaborating with Leslie Tung, a professor of biomedical engineering in the Johns Hopkins School of Medicine, the researchers found that after a few more days of growth, cells on the nanopatterned surface began to conduct electric waves and contract strongly in a specific direction, as intact heart muscle would. “Perhaps most surprisingly, these tissue functions and the structure of the engineered heart tissue could be controlled by simply altering the nanoscale properties of the scaffold. That shows us that heart cells have an acute ‘nanosense,’” Levchenko said.

Johns Hopkins researchers developed this chip to culture heart cells that more closely resemble natural cardiac tissue. Photo: Will Kirk/homewoodphoto.jhu.edu

Johns Hopkins researchers developed this chip to culture heart cells that more closely resemble natural cardiac tissue. Photo: Will Kirk/homewoodphoto.jhu.edu

“This nanoscale sensitivity was due to the ability of cells to deform in sticking to the crevices in the nanotextured surface and probably not because of the presence of any molecular cue,” Levchenko said. “These results show that the ECM serves as a powerful cue for cell growth, as well as a supporting structure, and that it can control heart cell function on the nanoscale separately in different parts of this vital organ. By mimicking this ECM property, we could start designing better-engineered heart tissue.”

Looking ahead, Levchenko said that he anticipates that engineering surfaces with similar nanoscale features in three dimensions, instead of just two, could provide an even more potent way to control the structure and function of cultured cardiac tissue.

In addition to Kim, Levchenko and Tung, authors on this paper are postdoctoral fellow Elizabeth A. Lipke and doctoral students Raymond Cheong and Susan Edmonds Thompson, all from the Johns Hopkins School of Medicine Department of Biomedical Engineering; Michael Delannoy, assistant director of the Johns Hopkins School of Medicine Microscope Facility Center; and Pilnam Kim and Kahp-Yang Suh, both of Seoul National University.

Tung and Levchenko are affiliated faculty members of the Johns Hopkins Institute for NanoBioTechnology. Thompson is a member of INBT’s Integrative Graduate Education and Research Traineeship in nanobiotechnology. Funding for this research was provided by the National Institutes of Health and the American Heart Association.

Related Web sites

Andre Levchenko’s Lab

Leslie Tung’s Lab

Johns Hopkins Institute for NanoBioTechnology

Story by Mary Spiro