Survival motor neuron protein deficiency impairs myotube formation by altering myogenic gene expression and focal adhesion dynamics

Abstract

While spinal muscular atrophy (SMA) is characterized by motor neuron degeneration, it is unclear whether and how much survival motor neuron (SMN) protein deficiency in muscle contributes to the pathophysiology of the disease. There is increasing evidence from patients and SMA model organisms that SMN deficiency causes intrinsic muscle defects. Here we investigated the role of SMN in muscle development using muscle cell lines and primary myoblasts. Formation of multinucleate myotubes by SMN-deficient muscle cells is inhibited at a stage preceding plasma membrane fusion. We found increased expression and reduced induction of key muscle development factors, such as MyoD and myogenin, with differentiation of SMN-deficient cells. In addition, SMN-deficient muscle cells had impaired cell migration and altered organization of focal adhesions and the actin cytoskeleton. Partially restoring SMN inhibited the premature expression of muscle differentiation markers, corrected the cytoskeletal abnormalities and improved myoblast fusion. These findings are consistent with a role for SMN in myotube formation through effects on muscle differentiation and cell motility.

Figure 1.

SMN-deficient muscle cells have a fusion deficit. Muscle cells were differentiated for 6 days and stained for sarcomeric myosin (green) and desmin (red). DAPI (blue) was added to label nuclei. Representative images are shown in (A). Scale bar, 100 µm. Arrows indicate myotube nuclei in each myotube. The number of nuclei in myotubes divided by the total number of nuclei in myoblasts and myotubes, the fusion index, is quantified in A. Myotubes were defined as cells positive for desmin and MF-20 with two or more nuclei. The percentage of myotubes with 2–3, 4–7 and 8 or more nuclei are quantified in (B). Nikon Elements software was used to count cells and nuclei. Six experiments were done per cell line. The values represent mean ± SEM (*P < 0.05, **P < 0.01, ****P < 0.001).

Figure 2.

Restoration of SMN rescues the fusion deficit in SMN-deficient muscle cells. Electroporation was used to introduce either human SMN or empty vector. Muscle cells were allowed to recover in proliferation media for 6 h and then switched to differentiation conditions. The cells were differentiated for 6 days and stained for sarcomeric myosin (green) and desmin (red). DAPI (blue) was added to label nuclei. Representative images are shown in (A). Scale bar, 100 µm. The number of nuclei in myotubes divided by the total number of nuclei in myoblasts and myotubes, the fusion index, is quantified in A. Myotubes were defined as cells positive for desmin and MF-20 with two or more nuclei. The percentage of myotubes with 2–3, 4–7 and 8 or more nuclei are quantified in (B). Nikon Elements software was used to count cells and nuclei. Six experiments each were done for WT, SMN-deficient and SMN-deficient + SMN. Myoblasts were isolated from three mice each for WT, SMA and SMA + MyoD^Cre and differentiated to myotubes for 4 days. Myotubes were stained for sarcomeric myosin (red). DAPI (blue) was added to label nuclei. Representative images are shown in (C). Scale bar, 100 µm. The percentage of total nuclei in myotubes is quantified in C. Myotubes were defined as cells positive for MF-20 with two or more nuclei. The percentage of myotubes with 2–3, 4–7 and 8 or more nuclei were counted by a blinded investigator and quantified in (D). Values represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3.

SMN deficiency inhibits both lipid mixing (cell-to-cell redistribution of membrane probes) and syncytium (myotube) formation. The syncytium formation assay reports only completed fusion events. In contrast, an appearance of co-labeled cells after co-incubation of differently labeled cells reveals even cell fusion events stalled upstream of generation of multinucleated cells. (A) Left panel, fluorescence microscopy images of the wild-type (WT) myoblasts (top) and SMN-deficient (SMN-deficient) myoblasts (bottom): DiI (red) and DiO (green). Arrowheads mark cells co-labeled with both membrane probes. A characteristic example of double-labeled cell is also shown in the 2-fold enlargement of boxed region (left top corner of the panel). Note that by the time we scored fusion, DiI and DiO incorporated into the plasma membrane are mostly internalized and label intracellular membranes. Right panel, phase contrast with nuclear staining (blue) images of the same cells. Scale bar, 40 µm. (B) Fusion extents for the cells expressing SMN and for the SMN-deficient cells assayed as lipid mixing (red) and as syncytium formation (green). All results are shown as mean ± SEM (n > 3) (**P < 0.01). (C) Schematic of the sequence of events leading to myotube formation. SMN-deficient cells do not reach the third stage of the myogenesis.

Figure 4.

SMN-deficient muscle cells have increased expression of differentiation markers. (A) Proliferating muscle cells were collected and RNA isolated for qRT-PCR following transfection with SMN or empty vector. (B) qRT-PCR analysis of miR-206 and -486 expression in proliferating wild-type and SMN-deficient myoblast. (C) qRT-PCR following transfection with SMN or empty vector. Five time courses per genotype were run. The data are represented as the fold change compared with wild type (black bars). Four time courses per genotype were run. Values represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5.

SMN-deficient muscle cells have altered motility and actin fiber organization. (A) After 1 day in differentiation conditions, confluent muscle cells were scraped off with a cell scraper and the media was replaced. The number of cells that moved into the denuded area after 24 h was counted. The data represent the average of at least three fields per experiment from four experiments. The data are shown as percentages of the wild-type average. Representative images are shown at left. (B) Representative images of undifferentiated muscle cells stained for F-actin (red) showing protrusions (yellow arrows) and stress fibers (white arrows). Muscle cells were transfected with GFP-SMN (green) and stained for F-actin (red). At least 50 cells per condition were counted for the presence of central stress fibers and percent cells with at least one central fiber calculated and graphed. (C) Representative images of undifferentiated muscle cells were stained for vinculin (green). Smaller panels are magnified images of adhesions. The values represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6.

SMN-deficient muscle cells have decreased cleavage and increased retention of talin. (A) Undifferentiated muscled cells were collected and cell lysates prepared for western blot. Data are quantified as the ratio of cleaved to full-length talin. (B) Undifferentiated muscle cells were electroporated with human GFP-talin construct and were analyzed by time-lapse microscopy. Duration measurements were made by counting the time elapsed between the first and last frames in which an adhesion was observed. (C) Quantification of data in B. At least 20 adhesions were counted for each genotype. Values represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001). (D) Schematic representation of proposed focal adhesion dynamics in SMN-deficient cells.