Scientists at Boston Children’s Hospital have made important steps towards overcoming the hurdles that stand in the way of getting heart muscle to regenerate following engraftment with lab-made heart muscle cells (cardiomyocytes).
A mutant heart muscle cell (in green) surrounded by normal cells. The mutant cell lacks Srf, a master maturation gene. It is unable to grow in size and lacks the fine membrane invaginations that help coordinate muscle contractions (appearing as vertical striations in the normal cells). Credit Guo Y; et al. / Boston Children's Hospital
So far, engrafting the heart cells either by injecting them or applying patches coated with the cells has led to disappointing results.
If you make cardiomyocytes in a dish from pluripotent stem cells, they will engraft in the heart and form muscle. But the muscle doesn't work very well because the myocytes are stuck in an immature stage,”
William Pu, director of Basic and Translational Cardiovascular Research at the hospital.
Scientists are unclear on how the cardiomyocytes we develop before birth develop into strong, contracting, mature heart muscle cells.
Studying the process in mice has also been difficult. Researchers have looked at “knock-out” models where genes are deleted in order to work out which ones are required. However, deleting a gene completely often causes the heart to malfunction and this disrupts the maturation process anyway.
Now, Pu and team have used the CRISPR/Cas9 gene editing tool to eliminate a key factor in heart muscle cell maturation in a new way.
Rather than the target gene being deleted in every cell, only one in every 10 cells were affected. This “mosaic” pattern of deletion meant the heart muscle could still function, while the team analysed the mutated cells independently of this.
As reported in the journal Nature Communications, when a gene called Srf that encodes for serum response factor (SRF) was deleted in this mosaic pattern, maturation of the heart muscle cells was disrupted. Contractile structures called sarcomeres became disorganized and structures called transverse tubules that aid the contraction of sarcomeres were disrupted. The mutated cells also had significantly less mitochondria, which are needed to provide energy.
Further studies showed that SRF regulates many other genes, including sarcomere genes that are active in immature cardiomyocytes, but not in mature ones.
The team suggests that SRF receives signals that instruct it to oversee organization as a cell matures. The researchers then showed that sarcomeres themselves need to be properly arranged before other aspects of maturation can take place.
"We learned that sarcomeres are not just important for contraction, but are probably driving the maturation program," says Pu.
The study seemed to suggest that if SRF could be boosted, it may be possible to make lab-grown heart muscle cells mature properly. However, this was contradicted by the fact that when SRF was overexpressed, the heart muscle cells appeared to be no different to the cells without any SRF.
This finding leads the team onto their next challenge:
"Cells seem very sensitive to the level of SRF. You probably can't manipulate SRF itself. You have to understand what's controlling it upstream,” says Pu.