Premature senescence in primary muscle cultures of myotonic dystrophy type 2 is not associated with p16 induction

Abstract

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are multisystemic disorders linked to two different genetic loci and characterized by several features including myotonia, muscle weakness and atrophy, cardiac dysfunctions, cataracts and insulin-resistance. In both forms, expanded nucleotide sequences cause the accumulation of mutant transcripts in the nucleus deregulating the activity of some RNAbinding proteins and providing an explanation for the multisystemic phenotype of DM patients. However this pathogenetic mechanism does not explain some histopathological features of DM skeletal muscle like muscle atrophy. It has been observed that DM muscle shares similarities with the ageing muscle, where the progressive muscle weakness and atrophy is accompanied by a lower regenerative capacity possibly due to the failure in satellite cells activation. The aim of our study is to investigate if DM2 satellite cell derived myoblasts exhibit a premature senescence as reported for DM1 and if alterations in their proliferation potential and differentiation capabilities might contribute to some of the histopathological features observed in DM2 muscles. Our results indicate that DM myoblasts have lower proliferative capability than control myoblasts and reach in vitro senescence earlier than controls. Differentely from DM1, the p16 pathway is not responsible for the premature growth arrest observed in DM2 myoblasts which stop dividing with telomeres shorter than controls. During in vitro senescence, a progressive decrease in fusion index is observable in both DM and control myotubes with no significant differences between groups. Moreover, myotubes obtained from senescent myoblasts appear to be smaller than those from young myoblasts. Taken together, our data indicate a possible role of DM2 premature myoblast senescence in skeletal muscle histopathological alterations i.e., dystrophic changes and type 2 fibre atrophy.

Figure 1.

Cross-sections of skeletal muscle from DM1 (A) and DM2 (B) patient after immunostaining for fast myosin. Note the presence of type 1 (white arrowhead) and type 2 (black arrowhead) atrophic fibers in DM1 section (A), while in DM2 section (B) type 2 fibre atrophy (black arrowhead) is evident. Black arrows indicate fast positive nuclear clumps. Relative hypertrophy factor (HF) and atrophy factor (AF) of each DM1 and DM2 patients (C) and mean values ± standard error of the mean (SEM) of HF and AF for each patient group (D). The results are based on the morphometric analysis of sections immunostained for MHC fast or slow myosin.

Figure 2.

A) Representative western blot analysis of MyoD expression in myoblasts at early (e) and late (l) passages of proliferative lifespan obtained from biceps brachii muscle samples from healthy (CTR), DM1 and DM2 patients; density of the bands has been normalized to β-tubulin expression used as internal control. B) Histograms representing mean values of MyoD protein expression analysed by densitometry of CTR (n=4), DM1 (n=3) and DM2 (n=4) patients; scale bars are for SEM. MyoD expression is similar in young and senescent cells indicating that the myogenic purity persists to be high throughout the experiment. C) Lifespan plots of control and DM myoblasts. D) Mean of max number of divisions of CTR (n=4), DM1 (n=3) and DM2 (n=4) patients; scale bars represent SEM; the average proliferative lifespan of the DM myoblasts is reduced as compared to that of control cells. E) Representative western blot analyses of PCNA in the early (e) and late (l) stages of myoblast proliferative lifespan; the results have been normalized to the expression of β-tubulin. F) Histograms representing mean values of PCNA protein expression analyzed by densitometry of CTR (n=4), DM1 (n=3) and DM2 (n=4) patients; scale bars represent SEM; levels of PCNA are significantly decreased in both DM and control senescent cells compared to young cells. *P<0.05, **P<0.01.

Figure 3.

Representative images of CTR (A,B), DM1 (C,D) and DM2 (E,F) myoblasts stained for the senescence-associated β-galactosidase (SA-βGal) activity. At the proliferative (early) stages, very few cells were positive (A,C,E). At the high cell passage (late stages), myoblasts appear large, with the characteristic cytoplasmic blue staining typical of senescence (B,D,F). G) Quantification of the amount of SA-βGal positive cells shows a dramatic increase in the percentage of positive cells at the late stages; scale bars represent SEM.

Figure 4.

Representative images of CTR (A,B), DM1 (C,D) and DM2 (E,F) myoblasts immunostained for the myogenic marker, desmin. Both DM and control myoblasts at early stages of proliferation were relatively small and elongated (A,C,E), while at the late stages of proliferation they showed a flattened morphology with enlarged cytoplasm and extended cytosolic processes (B,D,F). Nuclei have been visualized with DAPI (blue).

Figure 5.

DM premature senescence pathway. A) Representative western blot analyses of p16 in the early (e) and late (l) stages of myoblast proliferative lifespan; the results have been normalized to the expression of β-tubulin. B) Histograms representing mean values of p16 protein expression analysed by densitometry in healthy (CTR; n=4), DM1 (n=3) and DM2 (n=4) patients; at both stages analyzed, p16 was more expressed in DM1 cells as compared to the controls while the expression was similar in DM2 and control myoblasts; scale bars represent SEM. C) Mean length of telomeric restriction fragments (TRF) measured on DM2 (n=4) and control (n=4) myoblasts at proliferative and senescent stage; senescent DM2 cells had shorter telomeres than the proliferating cells (*P<0.05); scale bars represent SEM. D) Mean length (in bps) of telomeric DNA lost per division in control and DM2 myoblasts; DM2 cells lost more bps per division than control cells; scale bars represent SEM.

Figure 6.

Differentiation capability of young and senescent myoblasts. A-F) Fast myosin immunofluorescence (green) of control (A,B), DM1 (C,D) and DM2 (E,F) myotubes from myoblasts at early and late stages of their proliferative lifespan, after 5 days of differentiation (T5). Nuclei have been visualized with DAPI (blue). Young myoblasts generate well-differentiated and cross-striated (C,E) myotubes larger than those formed by the senescent cells. G) Fusion index, i.e. the number of nuclei incorporated into myotubes as a percentage of the total number of nuclei, has been calculated in young and senescent myoblasts after 5 days of differentiation; a significant reduction of fusion index is observable in both DM and control myotubes obtained from senescent cells (*P<0.05, **P<0.01). H) Histograms represents mean values of myogenin protein expression analysed by densitometry of control (n=4), DM1 (n=3) and DM2 (n=4) patients; a reduced myogenin expression is observed in myotubes from senescent cells as compared to those obtained from young cells; scale bars represent SEM. I) Representative western blot analyses of myogenin in myotubes obtained from myoblasts at the early (e) and late (l) stages of their proliferative lifespan; the results have been normalized to the expression of β-tubulin.