In 2015, a team of inquisitive young scientists, absorbed in the study of metabolism, regeneration, and biological development, set out to answer a question. Could the way cells produce energy help explain why certain organisms have an envious ability to regenerate heart tissue after injury? And, if so, could that knowledge be used to help heal human hearts? The results of that inquiry have now been revealed in a paper that could have broad implications for regenerative medicine research, beginning with the heart.
Nearly 18 million people die each year from heart disease, making it the leading cause of death in the world. In the United States alone, the annual economic impact of heart disease exceeds $200 billion, a figure that is expected to rise dramatically. Patients with heart disease experience progressive, significant declines in quality of life marked by reduced activity and higher hospitalization rates. While lifestyle changes and medical treatments are available to slow the progression to end-stage heart failure, there are no lifestyle changes or currently approved medical treatments that have the ability to restore normal heart functioning.
At the Institute for Stem Cell and Regenerative Medicine (ISCRM), researchers in multiple labs are pursuing novel approaches that can potentially cure rather than manage heart disease. In 2018, a study led by ISCRM Director Dr. Charles Murry demonstrated that stem cell-derived cardiomyocytes have the potential to regenerate heart tissue in large non-human primates, a major step toward human clinical trials. In another investigation, ISCRM faculty members Jen Davis, PhD and Farid Moussavi-Harami, MD are developing new tools to help cardiologists design personalized treatments for certain heart diseases.
Jason Miklas, PhD, now a postdoctoral researcher at Stanford University, is the lead author of the long-term study, which was published this week in the journal iScience. When the effort began, Miklas was a PhD student in the lab of Hannele Ruohola-Baker, PhD, Professor of Biochemistry and an Associate Director of ISCRM. His early collaborators were members of labs led by Murry, ISCRM co-founder Randy Moon, PhD, and ISCRM faculty member Rong Tian, MD, PhD.
“Our idea was to take a new look at how to regenerate the heart,” says Miklas. “We wanted to study how metabolism dictates cell fate, potentially helping cells to return to an undifferentiated state and eventually regenerate the heart.”
For clues, the researchers focused on two animals that have an ability to regenerate lost or damaged heart tissue. Their first model was the zebrafish. Peter Hofsteen, PhD, then a postdoc in the Murry Lab, had a knack for modeling heart regeneration in zebrafish. Hofsteen also worked closely with the Moon Lab, which was exploring the means by which heart amputation in zebrafish switched on the Wnt pathway, a driver of cell development and proliferation. That research would help point Miklas and Hofsteen in the right direction.
Operating in the ISCRM Aquatics Core, Hofsteen induced heart injuries in genetically-modified zebrafish. Laura Abell, at the time a graduate student in the Tian Lab, performed the detailed metabolomic analysis of the data gathered from the zebrafish experiments. Miklas explains what the researchers discovered. “We found a metabolite signature that was present in uninjured zebrafish hearts and that changed during regeneration. Specifically, we identified two amino acids that played an important role in the process that made heart regeneration possible. One was glutamine. The other was leucine.”
The second model was a mammalian heart closer to the human heart, a mouse heart. Just like humans, adult mice are not able to regenerate their heart tissue after injury. However, curiously during the first week of life, neonatal mice are able to regenerate their heart. The Ruohola-Baker team leveraged this knowledge to find similarities between the regenerative state of infant mice and adult zebrafish while contrasting what was different or lost once the mice became adults and could not regenerate further.
As they examined the data, the team noticed that, in a pre-injury state, the hearts of zebrafish and newborn mice display high levels of glutamine. In the first week after injury – a period in which regeneration occurs – levels of glutamine were depleted while levels of leucine increased. Because these amino acids also regulate the mTOR signaling pathway, which, in conjunction with Wnt, is crucial to cell growth, survival, and proliferation, the researchers hypothesized that this connection could be the “metabolic ticket” to regenerating heart tissue.
In other words, the researchers believed that an injury to the heart changes the expression of these amino acids, which, in turn, triggers the mTOR signaling pathway and, subsequently jump starts regeneration of the heart.
To test this hunch, the investigators used a stem cell model to show that disrupting mTOR signaling by manipulating glutamine and leucine levels resulted in reduced proliferation (and therefore, limited regeneration). The researchers saw the same amino acid profile in mouse hearts, indicating that mammalian heart cells can also be primed to enter a cell cycle that represents an important early step toward heart regeneration.
Hannele Ruohola-Baker is the lead investigator on the paper published in iScience. She credits the initiative of the postdocs and graduate students who helped launch the study. “The whole start of this research was born in the collaborative atmosphere of ISCRM,” says Ruohola-Baker. “Jason, Peter, Shiri, and their peers came up with the idea of doing metabolomics in organisms that regenerate the heart. And in true ISCRM fashion, it involved senior faculty and students from multiple labs working together to produce the findings.”
Shiri Levy, an Acting Instructor in the Department of Biochemistry, is one of the young scientists praised by Ruohola-Baker. Levy contributed to the research in multiple ways, including as a mentor to undergraduate students Diego Ic-Mex and Gargi Sivaram, who played a key part in the collection and the quantification of the data.
With guidance from Levy, Diego overcame at least two major obstacles in the fish experiments. First, he developed a technique to cut, section, and fix zebrafish tissue in a way that preserved the morphology of the samples even in extremely thin slices. This is a key reason the team had such detailed images of zebrafish hearts to analyze. Second, he identified two antibodies that are indicators of mTOR activity, helping the researchers prove that mTOR was contributing to heart cell proliferation.
“I definitely appreciated how much the team trusted me,” says Diego, who is now part of a cancer research team at a local biotech firm. “It gave me confidence to handle the zebrafish challenges, collect the data, and help create the figures. The opportunity to learn and work in this environment was amazing.”
Miklas speaks to what this finding could mean for human health. “It’s very exciting to see that changing levels of amino acids in cells can kick off so many biochemical processes and actually bring about proliferation and regeneration. We don’t know the very early events of heart regeneration, but this amino acid signaling is close to it. It also opens the door to asking more questions about how cell-to-cell connections cause these events and how we can optimally design synthetic proteins capable of promoting regeneration and wound healing in multiple organs.”
Levy imagines a future where physicians use amino acids as medicine for heart patients. “There is definitely therapeutic potential for this discovery. Wouldn’t it be amazing if during a heart attack, a heart could be primed with the right combination of amino acids to prevent scar tissue?” The researchers emphasize an approach like this would likely be used in combination with other interventions.