TRF2 rescues telomere attrition and prolongs cell survival in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs

Abstract

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart but do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that telomere shortening is a hallmark of DMD cardiomyocytes. Here, we test whether prevention of telomere attrition is possible in cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSC-CMs) and if preventing telomere shortening impacts cardiomyocyte function. We observe reduced cell size, nuclear size, and sarcomere density in DMD iPSC-CMs compared with healthy isogenic controls. We find that expression of just one telomere-binding protein, telomeric repeat-binding factor 2 (TRF2), a core component of the shelterin complex, prevents telomere attrition and rescues deficiencies in cell size as well as sarcomere density. We employ a bioengineered platform to micropattern cardiomyocytes for calcium imaging and perform Southern blots of telomere restriction fragments, the gold standard for telomere length assessments. Importantly, preservation of telomere lengths in DMD cardiomyocytes improves their viability. These data provide evidence that preventing telomere attrition ameliorates deficits in cell morphology, activation of the DNA damage response, and premature cell death, suggesting that TRF2 is a key player in DMD-associated cardiac failure.

Keywords: Duchenne muscular dystrophy; cardiomyocytes; induced pluripotent stem cells; telomere.

Fig. 1.

Cardiomyocytes differentiated from dystrophin-deficient iPSCs. (A) Schematic of dystrophin gene and the mutations corresponding to the cell lines. The UC1015.6 line harbors a CRISPR-induced mutation that results in expression of a truncated dystrophin missing the N terminus. DMD19 and DMD16 are patient-derived iPSCs that have nonsense mutations. (B) cTnT and dystrophin immunostaining in day 30 iPSC-CMs. The UC lines were stained with the MANEX1A antibody that recognizes the N terminus of dystrophin. The DMD19 and DMD16 lines were stained with the ab15277 antibody that recognizes the C terminus. DAPI in blue marks nuclei. Scale bar, 100 µm.

Fig. 2.

DMD iPSC-CMs exhibit deficits in cell size, nuclear size, and sarcomere density on day 30 of differentiation. cTnT immunostaining and DAPI staining for nuclei in (A) UC3.4 and UC1015.6 iPSC-CMs, (B) DMD19 iso and DMD19 iPSC-CMs, and (C) DMD16 iso and DMD16 iPSC-CMs. Scale bar, 50 µm. Area of cells for (D) UC3.4 and UC1015.6 iPSC-CMs, (E) DMD19 iso and DMD19 iPSC-CMs, and (F) DMD16 iso and DMD16 iPSC-CMs. Nuclear size for (G) UC3.4 and UC1015.6 iPSC-CMs, (H) DMD19 iso and DMD19 iPSC-CMs, and (I) DMD16 iso and DMD16 iPSC-CMs. Sarcomere density as measured by cTnT signal over cell area for (J) UC3.4 and UC1015.6 iPSC-CMs, (K) DMD19 iso and DMD19 iPSC-CMs, and (L) DMD16 iso and DMD16 iPSC-CMs. Cells were scored from three differentiation experiments. N = 97 to 205 cells. Data shown are mean ± SEM. One-way ANOVA and Mann–Whitney test for post hoc comparison was used to determine significance. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3.

TRF2 overexpression rescues telomere attrition. (A) The shelterin complex is made up of six subunits. TRF1 and TRF2 directly bind telomere sequences. (B) On day 10 of differentiation to cardiomyocytes from iPSCs, cells were transduced with an empty retroviral vector with no open reading frame (ev) or TRF2. Assays were performed on day 30 of differentiations. Southern blot of telomere restriction fragments of day 30 iPSC-CMs from (C) UC iPSC-CMs, (D) DMD19 iPSC-CMs, and (E) DMD16 iPSC-CMs. Signal distribution of telomere lengths from the Southern blots in arbitrary units (AU) for (F) UC iPSC-CMs, (G) DMD19 iPSC-CMs, and (H) DMD16 iPSC-CMs.

Fig. 4.

TRF2 attenuates ATM-mediated DNA damage response and prolongs cell survival. Cells were transduced with ev or TRF2 on day 10 and assayed on day 30. (A) Western blot of TRF2 levels with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. TRF2 signal normalized to GAPDH signal in AU. The expected sizes are 65 kDa for TRF2 and 35 kDa for GAPDH. (B) Western blot of P53 normalized to GAPDH signal in AU. The expected size for P53 is 50 kDa. (C) Western blot of gH2AX normalized to GAPDH signal in AU. The expected size for gH2AX is 17 kDa. (D) Western blot of CHK2 phosphorylated at threonine 68 and total CHK2. Signals are normalized to GAPDH signal in AU. The expected size of phosphor-CHK2 is 62 kDa. Percentage of cells that survived on day 40 when compared with day 30 of differentiation for (E) UC iPSC-CMs, (F) DMD19 iPSC-CMs, and (G) DMD16 iPSC-CMs. Survival was scored from three to five differentiation experiments. N = 375 to 12,036 cells on day 30. Data shown are mean ± SEM. One-way ANOVA and Tukey test for post hoc comparison were used to calculate significance. ****P < 0.0001.

Fig. 5.

TRF2 rescues deficits in cell size, nuclear size, and sarcomere density. Cells were transduced with ev or TRF2 on day 10 and assayed on day 30. cTnT immunostaining and DAPI staining for nuclei in (A) UC iPSC-CMs, (B) DMD19 iPSC-CMs, and (C) DMD16 iPSC-CMs. Scale bar, 20 µm. Area of cells for (D) UC iPSC-CMs, (E) DMD19 iPSC-CMs, and (F) DMD16 iPSC-CMs. Nuclear size for (G) UC iPSC-CMs, (H) DMD19 iPSC-CMs, and (I) DMD16 iPSC-CMs. Sarcomere density as measured by cTnT signal over cell area for (J) UC iPSC-CMs, (K) DMD19 iPSC-CMs, and (L) DMD16 iPSC-CMs. Cells were scored from three differentiation experiments. N = 90 to 230 cells. Data shown are mean ± SEM. One-way ANOVA and Tukey test for post hoc comparison were used to determine significance. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 6.

Model for protection of telomeres by TRF2. Due to the absence of dystrophin, reactive oxygen species in DMD iPSC-CMs lead to 8-oxoguanine lesions and abasic sites. Uncapped telomeres become subject to attrition through DNA damage and activity of nucleases, triggering cell death. Upregulation of TRF2 occludes the binding of DNA damage factors and promotes cell survival.