Immortalized human myotonic dystrophy type 1 muscle cell lines to address patient heterogeneity

Abstract

Historically, cellular models have been used as a tool to study myotonic dystrophy type 1 (DM1) and the validation of therapies in said pathology. However, there is a need for in vitro models that represent the clinical heterogeneity observed in patients with DM1 that is lacking in classical models. In this study, we immortalized three DM1 muscle lines derived from patients with different DM1 subtypes and clinical backgrounds and characterized them at the genetic, epigenetic, and molecular levels. All three cell lines display DM1 hallmarks, such as the accumulation of RNA foci, MBNL1 sequestration, splicing alterations, and reduced fusion. In addition, alterations in early myogenic markers, myotube diameter and CTCF1 DNA methylation were also found in DM1 cells. Notably, the new lines show a high level of heterogeneity in both the size of the CTG expansion and the aforementioned molecular alterations. Importantly, these immortalized cells also responded to previously tested therapeutics. Altogether, our results show that these three human DM1 cellular models are suitable to study the pathophysiological heterogeneity of DM1 and to test future therapeutic options.

Keywords: Biological sciences; Cell biology; Epigenetics; Health sciences; Molecular biology.

Graphical abstract

Figure 1

CTG instability is similar between primary and immortalized DM1 cells lines (A) Density plots showing the distribution of the CTG alleles detected in both the primary (black) and immortalized (red) myotubes derived from the three patients participating in the study. We measured between 41 and 105 alleles per sample. (B) CTG expansion ePAL, mode and instability in primary and immortalized myotubes of the three patients with DM1 participating in the study. The progenital allele length was estimated as the 10^th percentile of allele frequency distribution. The modal allele length was determined as the most frequent allele. The level of somatic instability was calculated by subtracting the 10^th percentile from the 90^th percentile. “p” and “i” before cell line name mean primary and immortalized, respectively.

Figure 2

DNA methylation levels in CTCF1 is increased in immortalized cell lines when compared to primary cell lines (A) Schematic representation of the genomic DMPK locus. (B) Methylation plotter showing the methylation status of the CTCF1 region. Each circle represents a CpG dinucleotide. The level of methylation is represented by the gray gradient. (C) Graphical representation of the methylation levels in DM1 immortalized and primary myoblasts and myotubes. (D) Relative expression of DMWD, DMPK and SIX5 genes at 5 days of differentiation. HPRT was used as housekeeping gene to normalize the data. All data are expressed as mean ± SEM. “p” and “i” before cell line name mean primary and immortalized, respectively. “mb” refers to myoblasts and “mt” to myotubes. Means were compared using unpaired two-tail Mann-Whitney test. ∗p ≤ 0.05

Figure 3

DM1 myoblasts show higher cell proliferation and reduced levels of early myogenic markers (A) Real-time impedance curves of human iCtrl (blue) and iDM1 (red) myoblasts during culture in proliferation medium (SGM) and differentiation media (bDM and cDM). (B) Proliferation of iCtrl (blue) and iDM1 (red) myoblasts was analyzed at 24 and 48 h after seeding. (C) Differentiation of iCtrl and iDM1 myoblasts was analyzed after 2 days in differentiation medium bDM (2dpd). (D) Jess Western blot analysis of Myf5, MyoD and Myogenin in iCtrl and iDM1 3 differentiating myoblasts at 4 different time-points: 0, 1, 2 and 3 dpd. Values are represented over Ctrl 0 dpd. Data information: n = 3 for iCtrl and iDM1. Dpd, days post differentiation. All data are expressed as mean ± SEM. (A–C) Dots indicate mean values of individual samples from 10 replicates. Means were compared using unpaired two-tail Student’s t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01. “i” mean immortalized cell line.

Figure 4

DM1 primary and immortalized myotubes present a lower fusion index compared to controls (A) Desmin (green) and nuclei (blue) immunofluorescence analysis performed in 5 days differentiated primary and immortalized DM1 or control myotubes. (B) Fusion index (% of nuclei in desmin positive myotubes with 2 or more nuclei) in primary and immortalized 5 days differentiated DM1 or control myotubes. (C and D) Fusion index (% of nuclei in desmin positive myotubes with 2 or more nuclei) in individualized immortalized 5 days differentiated DM1 or control myotubes D. Percentage of 5 days differentiated primary and immortalized myotubes containing 2, 3, >3 nuclei. (E) Myotube diameter (calculated using the maximum diameter value of desmin positive myotubes with 2 or more nuclei) in primary and immortalized 5 days differentiated DM1 or control myotubes. (F) Myotube diameter in individualized immortalized 5 days differentiated DM1 or control myotubes. All data are expressed as mean ± SEM. Means were compared using unpaired two-tail t-test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. “p” and “i” before cell line name mean primary and immortalized, respectively. In (B and C), dots indicate mean values of 5 individual analyzed images. In (B and D) at least 350 nuclei/cell line were analyzed. In (C) at least 850 nuclei/cell line were analyzed. In (E and F) at least 20 myotubes/cell line were analyzed.

Figure 5

DM1 immortalized myotubes present equal or higher amount of RNA foci and MBNL1 aggregates than the original primary culture (A) Foci (red), MBNL1 (green) and nuclei (blue) immunofluorescence analysis performed in 5 days differentiated primary and immortalized DM1 myotubes. (B) Number of RNA foci/nucleus in primary and immortalized 5 days differentiated DM1 myotubes. (C) MBNL1 aggregates/nucleus in primary and immortalized 5 days differentiated DM1 myotubes. (D) Comparison of the number of RNA foci/nucleus and MBNL1 aggregates/nucleus between the three immortalized DM1 cell lines. All data are expressed as mean ± SEM. Between 25 and 35 DM1 nuclei and between 20 and 25 control nuclei were analyzed per cell line. “p” and “i” before cell line name mean primary and immortalized, respectively. Means were compared using unpaired two-tail Mann-Whitney test. ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 6

Immortalized DM1 myotubes maintain the splicing defects that characterize DM1 primary myotubes (A) Exon inclusion analysis of BIN1, LDB3 and ATP2A1 in primary control and DM1 5 days differentiated myotubes. (B) Exon inclusion analysis of BIN1, MBNL1, LDB3, INSR, DMD and ATP2A1 in immortalized control and DM1 5 days differentiated myotubes. All data are expressed as mean ± SEM. 3 DM1 samples and 3 control samples were analyzed in each splicing both in primary and immortalized samples, except for ATP2A1 and LDB3 in primary samples where 2 DM1 samples and 3 controls were analyzed. Means were compared using unpaired two-tail t-test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001. “p” and “i” before cell line name mean primary and immortalized, respectively.

Figure 7

KIF13A splicing defect is heterogeneously expressed among immortalized cell lines (A) Exon inclusion analysis of KIF13A in immortalized control and DM1 5 days differentiated myotubes. (B) Exon inclusion analysis of KIF13A in immortalized 5 days differentiated myotubes derived from the three DM1 cell lines. (C) Gel analysis of KIF13A splicing analysis in control and patient DM1 myotubes. All data are expressed as mean ± SEM. n = 3 for each cell line. Means in (A) were compared using unpaired two-tail t-test and in (B) with ANOVA. ∗p ≤ 0.05, ∗∗∗p ≤ 0.001. “i” before cell line name means immortalized.

Figure 8

Immortalized DM1 myotubes respond to treatment in a similar way to primary DM1 myotubes (A) Foci (red), MBNL1 (green) and nuclei (blue) immunofluorescence analysis performed in 9 days differentiated primary and immortalized DM1 myotubes. Rows 1 and 3 correspond to non-treated cells while rows 2 and 4 correspond to 48 h ASO-treated cells. (B) Number of RNA foci/nucleus in primary and immortalized 48 h ASO-treated or non-treated 9 days differentiated DM1 myotubes. (C) Number of MBNL1 aggregates/nucleus in primary and immortalized 48 h ASO-treated or non-treated 9 days differentiated DM1 myotubes. (D) MBNL1 splicing analysis in immortalized 48 h ASO-treated, control-treated or non-treated 9 days differentiated myotubes. All data are expressed as mean ± SEM. For each cell line in (B and C), it was analyzed between 29 and 43 DM1 nuclei. In (D), 3 DM1 samples were analyzed for each condition. “p” and “i” before cell line name mean primary and immortalized, respectively. Means were compared using unpaired two-tail Mann-Whitney test. ∗p ≤ 0.05, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.