Striated Preferentially Expressed Protein Kinase (SPEG)-Deficient Skeletal Muscles Display Fewer Satellite Cells with Reduced Proliferation and Delayed Differentiation

Abstract

Centronuclear myopathies (CNMs) are a subtype of congenital myopathies characterized by skeletal muscle weakness and an increase in the number of central myonuclei. SPEG (striated preferentially expressed protein kinase) has been identified as the sixth gene associated with CNM, and it has been shown that striated muscle-specific Speg-knockout (KO) mice have defective triad formation, abnormal excitation-contraction coupling, and calcium mishandling. The impact of SPEG deficiency on the survival and function of myogenic cells remains to be deciphered. In this study, the authors examined the overall population, proliferation, and differentiation of myogenic cells obtained from striated muscle-specific Speg-KO mice and compared them with wild-type (WT) controls. SPEG-deficient skeletal muscles contained fewer myogenic cells, which on further study demonstrated reduced proliferation and delayed differentiation compared with those from WT muscles. Regenerative response to skeletal muscle injury in Speg-KO mice was compared with that of WT mice, leading to the identification of similar abnormalities including fewer satellite cells, fewer dividing cells, and an increase in apoptotic cells in KO mice. Overall, these results reveal specific abnormalities in myogenic cell number and behavior associated with SPEG deficiency. Similar satellite cell defects have been reported in mouse models of MTM1- and DNM2-associated CNM, suggestive of shared underlying pathways.

Figure 1

FACS profiles of quadruple negative prospective myogenic cell (QNMC) and satellite cell (SC) populations in the skeletal muscles of wild-type (WT) and Speg knockout (KO) mice. The digested tissue from limb muscles of 1-month–old animals were stained with propidium iodide (PI), Alexa Fluor 488 anti-mouse CD31, Alexa Fluor 488 anti-mouse CD45, Alexa Fluor 488 anti-mouse Ly-6A/E (Sca1), and APC anti-mouse CD106 (VCAM1). A: WT profiles. B: KO profiles. The boxed areas in the P1 plots excludes cell debris; the boxed areas in both the P2 and P3 plots exclude the multiplets and doublets to collect single cells; cells in the boxed areas of the P4 and P5 plots are QNMCs and SCs, respectively.C: The population hierarchy of cells from WT and KO mice. D: The percentage of QNMCs in KO mice was comparable to that of WT. E: The percentage of SCs was markedly reduced in KO mice compared with WT n = 6 mice per genotype. ∗P < 0.05. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area; SSC-H, side scatter height; SSC-W, side scatter width.

Figure 2

Pax7 expression in wild-type (WT) and Speg knockout (KO) skeletal muscle. A: Immunofluorescence results for Pax7 staining in both WT and KO quadricep muscles at 30 days of life. White arrows indicate Pax7-positive nuclei in WT and KO animals. B: Quantification of Pax7-positive nuclei per fiber shows a reduction in the satellite cell population in the KO mice. C: Western blot analyses performed on tissue lysates from quadriceps muscle reveal decreased levels of Pax7 in KO animals. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a control. n = 4 mice per genotype. ∗P ˂ 0.05. Scale bars = 20 μm.

Figure 3

Reduced proliferation of SPEG-deficient quadruple negative prospective myogenic cells (QNMCs) in vitro. A: Phase contrast images of representative clones of wild-type (WT) (top row) and Speg knockout (KO) QNMCs (bottom row) in growth medium over a 10-day period. B: Proliferation rate is significantly lower in freshly isolated KO QNMCs compared with cells from WT littermates after 7 days in culture. n = 6 mice per genotype. ∗P < 0.05. Scale bars = 100 μm. abs, absorption; D, day.

Figure 4

Delayed differentiation of SPEG-deficient quadruple negative prospective myogenic cells (QNMCs) in vitro. A: Representative phase contrast images of wild-type (WT) and Speg knockout (KO) QNMC clones in differentiation medium over a 7-day period. B: Representative images of immunofluorescence staining for desmin (red) after 5 days of differentiation. DAPI (blue) was used for nuclear staining. White arrows indicate fused nuclei within myotubes. C: Fusion assay reveals a lower fusion index for Speg-KO QNMCs after 5 days in culture. n = 6 mice per genotype. ∗P < 0.05 versus KO. Scale bars: 100 μm (A); 20 μm (B). D, day.

Figure 5

Reduced proliferation and increased apoptosis in SPEG-deficient cells after muscle injury in vivo. A: At 5 to 6 days after muscle injury by cardiotoxin injections, hematoxylin and eosin staining of the injured transverse abdominal (TA) muscles show signs of muscle regeneration, including numerous centrally nucleated myofibers. The cross-sectional area (CSA) of Speg-KO TA muscles is significant less than that of wild-type (WT) (over 100 fibers were analyzed from each group). B: Immunofluorescence staining of myofibers using antibodies against Pax7 (green) for satellite cells, Ki-67 (red) for proliferating cells, and caspase 3 (red) for apoptotic cells, show evidence of reduced proliferation and increased apoptosis in knockout (KO) mice. C: This was further confirmed by quantification. n = 3 mice per genotype. ∗P < 0.05. Scale bars = 100 μm (A and B).

Figure 6

Fewer activated satellite cells in Speg knockout (KO) skeletal muscle before and after muscle injury in vivo. A: Immunofluorescence staining of myofibers using antibodies against Pax7 (red) and Ki-67 (green) shows fewer activated satellite cells in KO mice in both uninjured and injured states. Pax7 stains all satellite cells. Pax7 and Ki-67 together stain only the activated ones. Arrows indicate Pax7-positive nuclei; arrowheads indicate both Pax7- and Ki-67–positive nuclei. B: This was further confirmed by quantification. n = 3 mice per genotype. ∗P < 0.05, ∗∗P < 0.01. Scale bars = 20 μm. SC, satellite cell; WT, wild type.

Supplemental Figure S1

Publicly available single-cell RNA-seq (scRNA-seq) data from mouse skeletal muscles (Tabula Muris, https://tabula-muris.ds.czbiohub.org, last accessed June 6, 2020). A: Graph-based clustering of fluorescence-activated cell sorting (FACS)-isolated single cells identifies distinct clusters corresponding to different cell populations. B: Speg expression in distinct cell clusters. C: Variable levels of Speg expression among different clusters. CPM, counts per million mapped reads.

Supplemental Figure S2

SPEG (green) colocalizes with Pax7 (red) in wild-type mouse skeletal muscles. White arrows indicate both SPEG- and Pax7-positive nuclei. Boxed areas are shown at higher magnification in the bottom row. Scale bars = 20 μm.

Supplemental Figure S3

Apoptosis assay of wild-type (WT) and Speg knockout (KO) quadruple negative prospective myogenic cells at 0, 2, 4, 7, and 10 days in culture. n = 4 per genotype, each sample was tested in triplicate. Abs, absorption.

Supplemental Figure S4

Publicly available single-cell RNA-seq (scRNA-seq) data from in vitro hPSC-derived muscle cells (UCSC Cell Browser; https://skeletal-muscle.cells.ucsc.edu/; last accessed August 6, 2020). A: SPEG expression in myogenic subset. B: CKM expression in myogenic subset. C: Variable levels of SPEG expression among different cell muscle groups. D: Variable levels of CKM expression among different cell muscle groups. CKM, creatine kinase, muscle; hPSC, human pluripotent stem cell; MB, myoblasts; MC, myocytes; MP, myogenic progenitors; SkM, skeletal muscle.