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Scientists around the world have attempted to replace damaged heart tissue with cardiac cells (cardiomyocytes) made in the laboratory, either by injecting them into the heart or by applying cell-bound patches. But the results so far have been disappointing.
"If you make cardiomyocytes in a dish from pluripotent stem cells, they will graft into the heart and form muscles," said William Pu, director of basic and translational cardiovascular research at Boston Children's Hospital. "But the muscle does not work very well because the myocytes are stuck in an immature stage."
The way cardiomyocytes that we begin to develop before birth contract vigorously and have not been well understood.
"I think there are specific signaling pathways that control this process, which we have not been able to replicate in a dish," Pu said.
Selective deletions
The study of cardiomyocyte maturation in live mice was equally difficult. Previous studies have tried to genetically manipulate mice with different "destroyed" genes in their heart muscle to determine which ones are required. However, complete loss of a gene often results in dysfunction of the entire heart, which can hinder maturation. In addition, the multiplication of multiple "knockout" mouse strains with different deleted genes is an expensive and time-consuming procedure.
In a new study published in Nature CommunicationsPu and his colleagues have bypassed these obstacles. Using live mice and a CRISPR / Cas9 gene editing technology, they discovered a key factor involved in the maturation of the heart muscle in a new and original way.
The beauty of their approach is twofold. First of all, it only took a single strain of mice that expresses the enzyme Cas9, coupled with a virus that directs Cas9 to suppress a specific gene. To target different genes, the researchers simply made small changes to the virus.
"We were able to test 10 potential interest genes in 10 weeks," Pu said.
Second, rather than suppressing the gene in each cell, the researchers injected the virus to affect about one in ten cardiac muscle cells. This "mosaic" distribution scheme allowed the unaffected cardiac muscle to continue functioning. The team could then study mutated cardiac muscle cells separately.
Regulation of cardiac muscle development
A factor, called serum response factor (SRF), has emerged as a key regulator of cardiomyocyte maturation. When the SRF gene was mosaic suppressed in the heart muscle of newborn mice, many aspects of maturation were disrupted.
In normal mature myocytes, the basic contractile structures, called sarcomeres, are highly organized. But in mutant myocytes, the sarcomeres became disorganized. In addition, normal cells develop transverse tubules, important structures that help coordinate sarcomere contraction. These too have been greatly disturbed in mutant myocytes. Finally, mitochondria, which provide energy and are normally placed next to sarcomeres in the same way, have been considerably reduced in size and number.
In other experiments, the team has shown that SRF regulates the activity of several other genes, including critical sarcomeric genes, in immature cardiomyocytes, but not in mature cardiomyocytes. They think that SRF receives signals from the rest of the cell that give it the signal to run a main program of organization during maturation.
Finally, Pu and his colleagues have shown that the sarcomeres themselves must be properly assembled so that other aspects of maturation occur.
"We have learned that sarcomeres are not only important for contraction, but probably lead to the maturation program," Pu said.
Grow heart cells
Overall, the results would seem to suggest that if you could simply find a way to stimulate SRF, you could grow laboratory-made heart muscle cells. But it was not so simple: when SRF was overexpressed, the cells appeared to be identical to those without SRF. And that defines the next goal of the lab.
"The cells seem very sensitive at the level of SRF," Pu said. "You probably can not handle SRF itself – you have to understand what controls it up front."
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