Increased expression of telomeric repeat-binding factor 2 (TRF2) helped to prolong the survival of cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) derived from patients with Duchenne muscular dystrophy (DMD), according to a study published in the Proceedings of the National Academy of Sciences (PNAS).
TRF2, a component of the shelterin complex that protects chromosome ends, can prevent the attrition of telomeres. Upregulation of TRF2 in DMD iPSC-CMs was able to halt telomere attrition which was able to remove morphological deficits found in the cells, reduce the DNA damage response, and ultimately, prolong the survival of the cells, the study authors noted.
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A previous study from the research group found that telomeres in the cardiomyocytes of patients with DMD were shorter than those found in age-matched healthy controls. These results indicated a correlation between the dilated cardiomyopathy seen in patients with DMD and telomere attrition.
“While TRF2 does not replace the function of dystrophin, TRF2 can delay the premature loss of cardiomyocytes, which sets in motion fibrosis and stiffening of the heart tissue that accelerates the development of dilated cardiomyopathy,” the authors said.
“Protection of telomeres by TRF2 can potentially be a therapeutic strategy to ameliorate other cardiovascular diseases where we and others have observed telomere attrition,” the researchers continued.
Western blot analysis showed that the DMD iPSC-CMs had reduced expressions of TRF2 compared to isogenic controls. Retroviral transduction was able to cause an upregulation of TRF2 in the DMD iPSC-CM cell lines, leading to a reduction in telomere attrition compared to the nontreated DMD iPSC-CMs.
Protection of the telomeres by TRF2 also attenuated the DNA damage response as evidenced by reduced levels of p53 and γ-H2AX. Upregulation of TRF2 also prevented premature loss of cardiomyocytes with a higher percentage of cells that survived from day 30 of differentiation until day 40.
The DMD iPSC-CMs used in the study displayed smaller nuclear size, and the overall cell size was roughly 58% the size of healthy iPSC-CMs. As measured by cTnT signal, the sarcomere density was also reduced compared to healthy cells. The smaller nuclear and cell size along with the lower sarcomere density suggest that DMD iPSC-CMs have impaired contractile ability compared to healthy cells. Upregulation of TRF2 increased the density of cTnT and improved cell and nuclear sizes.
A total of 6 cell lines were utilized in the study, including DMD19 and DMD16, derived from patients with nonsense mutations of the dystrophin gene. CRISPR-corrected versions of the cell lines, DMD19 iso, and DMD16 iso, were used in the study for comparison. Another set of cell lines included UC3.4 taken from a healthy individual, and UC1015.6, an isogenic CRISPR-induced cell line.
Reference
Eguchi A, Gonzalez AFGS, Torres-Bigio SI, et al. TRF2 rescues telomere attrition and prolongs cell survival in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs. Proc Natl Acad Sci. Published online January 31, 2023. doi:10.1073/pnas.2209967120
