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Mend After All
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In Memoriam
Unexpected Tragedy in a Little
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Broken Hearts May Mend After All
Combo Therapy Coaxes Heart Cells to Multiply
Of all the organs in the body, the brain and the heart are the ones most often seen as the seats of identity. Perhaps this is a scientific insight since the cells in these organs remain largely intact after development, unlike the constant tearing down and building up of many other tissues. People have known for a long time that you can die of a broken heart because adult cardiac muscle cells refuse to divide and renew after injury from heart failure, the primary cause of death in the developed world. But in the May 15 issue of Genes and Development, a team led by Mark T. Keating and Felix Engel announces it has accomplished a long-sought goal: coaxing adult mammalian cardiomyocytes to divide and proliferate. The achievement is a first step in trying to accomplish the same feat in a living animal and suggests a molecular pathway for repairing a damaged heart after injury.
Photo by Graham Ramsay
Felix Engel (left) and Mark T. Keating believe that delivering the right molecule or combination of molecules as a drug could someday nudge heart muscle cells to divide after an injury.
Though stem cells have been detected in the heart, the role they play in tissue repair is unclear. “The heart does not regenerate after it’s injured,” said Keating, a Howard Hughes investigator and HMS professor of cell biology at Children’s Hospital Boston. “The reason is not entirely known, but cardiomyocytes in adults are generally thought to be incapable of cell proliferation.” After it has differentiated, the adult muscle cell settles into an inflexible, striated block that Keating compares to a muscle-bound oaf who can hardly move. Yet in fetal development, these same cells must temporarily loosen this structure to divide. Keating’s lab found that a similar phenomenon takes place in lower vertebrates: zebrafish can lose 20 percent of their hearts and regrow the tissue in a couple of months without the scarring seen in mammals. And they seem to accomplish this not through stem cells but by proliferation of existing cardiomyocytes.
A Great
Divide
Lead author Engel, an HMS research fellow in Keating’s lab, set out
three years ago to accomplish the same in mammals. “I wanted to show
that these cells can divide,” he said. After studying properties of
mammalian cardiomyocytes in his PhD work in Germany, he was convinced it
was possible. Keating’s
lab, which had recently announced they coaxed skeletal muscle cells to divide,
seemed like the best place to do it.
Engel first identified a growth factor, FGF1, that could specifically stimulate cardiomyocytes. Adding the factor to cells increased DNA synthesis by up to 30 percent, a starting point for cell replication, but it could not nudge the cells toward division. To probe its activity further, Engel began testing the factor in combination with inhibitors of signaling pathways known to lie downstream. Originally he used an inhibitor of the enzyme p38 MAP kinase as a control, but the rise in DNA synthesis in these control cells leaped to 80 percent, and the cell populations looked denser. Could it be that this combination was the magic formula? “At this moment, we started a rigorous analysis to demonstrate that differentiated mammalian cardiomyocytes are able to proliferate,” Engel said.
The team then began to study p38’s natural role in cardiomyocyte development. They examined hearts taken from rats at different developmental stages and found that p38 activity was lowest at times when the heart was growing and highest when the muscle cells grew the slowest and in adulthood when growth had stopped. The team performed DNA microarray analysis on cells treated with FGF1 and p38 alone and in combination. Treatment with both factors stimulated a set of genes that had not been expressed by either alone—genes involved in cell cycle progression.
The team then used immunofluorescent stains to prove their cells had undergone three necessary stages of cell division: DNA synthesis, mitosis, and cell separation (the latter test is useful because heart muscle cells often duplicate their nuclei without truly dividing). When the cells were then counted, their numbers had significantly increased after one treatment, and with continuous treatment, the cells underwent many rounds of cell division.
A protein involved in cell contraction was also diminished, suggesting that the stiff fibers were loosening to allow the cells to divide. The mechanism for the effect is still unknown. P38 seems to act as a brake on cell division, but releasing the brake is not enough: a growth factor is needed to put on the gas. “Alone, both do nothing,” Engel said. “But when you put the two together, suddenly this combination causes the cells to really respond and divide.” Although the team had proof of p38’s role in vitro, they teamed with researchers at UCLA to begin testing its role in vivo using a p38-knockout mouse. Mice that lacked p38 showed a 92 percent increase in cardiomyocyte mitosis during development.
Rousing the Heart
When the team set out to prove that adult cardiomyoctytes could divide,
the field was abuzz with the notion that stem cells injected into
the heart might be able to repair tissue after injury. So far, several
clinical trials
have explored cell-based therapies in humans, and results have been
mixed. Keating and Engel believe that their approach may be the more
elegant
one since it would ultimately involve delivering only small molecules
or proteins
to rouse existing heart cells from their slumber.
Keating does not believe that p38 is the sole switch. “There are probably several ways to induce cardiomyocytes to proliferate,” he said. But finding them will still be challenging. “There are infinite ways to fail, and only a few ways to succeed,” he added. Ideally, researchers could isolate a single factor that would persuade cells to divide. In the meantime, Keating and Engel are administering the combination of a p38 inhibitor and growth factor to animals that have undergone a myocardial infarction, to see if their approach to spur cell division can actually fix a broken heart in vivo.