Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Apr;96(4):208-17.
doi: 10.1016/j.ymgme.2008.12.012. Epub 2009 Jan 22.

Murine muscle cell models for Pompe disease and their use in studying therapeutic approaches

Shoichi Takikita  1 , Rachel Myerowitz, Kristien Zaal, Nina Raben, Paul H Plotz
Affiliations

Murine muscle cell models for Pompe disease and their use in studying therapeutic approaches

Shoichi Takikita et al. Mol Genet Metab. 2009 Apr.

Abstract

Lysosomes filled with glycogen are a major pathologic feature of Pompe disease, a fatal myopathy and cardiomyopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme, acid alpha-glucosidase (GAA). To facilitate studies germane to this genetic disorder, we developed two in vitro Pompe models: myotubes derived from cultured primary myoblasts isolated from Pompe (GAA KO) mice, and myotubes derived from primary myoblasts of the same genotype that had been transduced with cyclin-dependent kinase 4 (CDK4). This latter model is endowed with extended proliferative capacity. Both models showed extremely large alkalinized, glycogen-filled lysosomes as well as impaired trafficking to lysosomes. Although both Pompe tissue culture models were derived from fast muscles and were fast myosin positive, they strongly resemble slow fibers in terms of their pathologic phenotype and their response to therapy with recombinant human GAA (rhGAA). Autophagic buildup, a hallmark of Pompe disease in fast muscle fibers, was absent, but basal autophagy was functional. To evaluate substrate deprivation as a strategy to prevent the accumulation of lysosomal glycogen, we knocked down Atg7, a gene essential for autophagosome formation, via siRNA, but we observed no effect on the extent of glycogen accumulation, thus confirming our recent observation in autophagy-deficient Pompe mice [N. Raben, V. Hill, L. Shea, S. Takikita, R. Baum, N. Mizushima, E. Ralston, P. Plotz, Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease, Hum. Mol. Genet. 17 (2008) 3897-3908] that macroautophagy is not the major route of glycogen transport to lysosomes. The in vitro Pompe models should be useful in addressing fundamental questions regarding the pathway of glycogen to the lysosomes and testing panels of small molecules that could affect glycogen biosynthesis or speed delivery of the replacement enzyme to affected lysosomes.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Characterization of lysosomes in myotubes cultured from GAA KO primary mouse myoblasts. (A) PAS stained WT and GAA KO myotubes. (B) LAMP1 stained myotubes cultured from WT and GAA KO primary myoblasts. (C) LAMP1 staining of GAA KO myotubes in DIC image where vacuoles are often recognized as lucent structures. (D) Myotubes were randomly selected from 4 week old WT and GAA KO cultures and areas of largest LAMP1 stained vacuoles in these myotubes were measured (n=16 for WT myotubes and n=40 for GAA KO myotubes, p=0.0003). (E) Fast myosin immunostained myotubes cultured from WT and GAA KO primary myoblasts.
Fig. 2
Fig. 2
Analysis of pH of expanded lysosomes. (A) AO stained live cultured myotubes derived form WT and GAA KO primary myoblasts. AO shows red fluorescence (650 nm) in an acidic environment and green fluorescence (526 nm) in a neutral environment. Expanded lysosomes in GAA KO myotubes show green fluorescence (arrows). Small structures clustered around the expanded alkalinized lysosomes are acidic vesicles. (B) Live imaging of GAA KO myotubes loaded with AO (left panel) followed by fixation and LAMP1 staining of the same myotubes. (C) LysoSensor DND-153 (pH ≤7.5) fluoresces green in large lysosomes of GAA KO (left panel) but LysoSensor DND-189 (pH ≤5.2) does not (right panel). Green fluorescence is seen in WT with both dyes. (D) AO stained live fibers isolated from white gastrocnemius muscle (predominantly fast Type II) of WT and GAA KO mice. Arrows show alkalinized lysosomes (green fluorescence) in GAA KO fibers. In WT fibers, small red vesicles (normal sized lysosomes) are scattered. Arrowheads point to area of autophagic buildup. (E) Live GAA KO myofibers stained with LysoSensors. LysoSensor DND-153 (pH≤7.5) fluoresces green in large lysosomes (arrows) but the lysosomes do not stain with LysoSensor DND-189 (pH ≤5.2). The lysosomes in the WT fibers stain with both LysoSensors.
Fig. 3
Fig. 3
Analysis of myotubes for cathepsin D and B. WT and GAA KO myotubes were doubly immunostained for LAMP1 and either cathepsin D or B. In WT myotubes, the majority of the LAMP1 positive vacuoles contain cathepsin D. No cathepsin D staining is observed in expanded GAA KO lysosomes. A similar result is evident for cathepsin B. Small structures clustered around the expanded lysosomes (similar to those shown in Fig. 2) are cathepsin D and B positive.
Fig. 4
Fig. 4
Characterization of lysosomes in myotubes cultured from CDK4 transduced GAA KO primary myoblasts. (A) As in myotubes derived from GAA KO primary myoblasts, enlarged vesicles (arrows) are observed in CDK4 transduced cells (left, DIC). These vesicles are positive for LAMP1 (right, red). (B) PAS staining for glycogen is positive in expanded lysosomes.
Fig. 5
Fig. 5
Examination of trafficking in cultured myotubes. (A) WT and GAA KO cultured myotubes incubated with 1mg/ml TMR-dextran (red) overnight followed by a two-hour wash in medium lacking labeled dextran. Small lysosomes (LAMP1 positive green vacuoles) in WT are positive for dextran but enlarged lysosomes in GAA KO show weak staining. (B) GAA KO expanded lysosomes (LAMP1-positive structures) show strong staining for dextran 48 hours after removal of labeled dextran. (C) Quantification of fluid phase endocytosis in cultured myotubes loaded with Alexa Fluor 546-labeled dextran. Left panel: histograms of the pixel values of 8-13 myotubes show a shift in the distribution of the pixel values between 3h and 48 h in the GAA KO but not in the WT. Right panel: analysis of the fluorescent signal in the cross-section of individual lysosomes. WT lysosomes (the majority of which is ∼1 μm2) change very little in the fluorescence signal between 3h and 48 h after washout of the dye. In contrast, GAA KO lysosomes, especially the large ones (> 5 μm2), increased in fluorescence between 3h and 48 h after washout of the dye, indicating that trafficking of labeled dextran had continued after it had ceased in WT. The table shows that the total lysosomal area in KO myotubes is significantly larger than in WT and that the summed fluorescence of the KO lysosomes doubled between 3h and 48h, while there was a slight decrease in the WT lysosomes fluorescence. (D) WT and GAA KO myotubes were incubated in the presence of Alexa-Fluor 546 rhGAA (red) followed by a two-hour wash. Lysosomes of WT myotubes are positive. Small lysosomes but not the large lysosomes in GAA KO contain labeled rhGAA. (E) TMR-dextran was microinjected into myotubes. Twenty four hours after injection, dextran is localized in small vesicles in both WT and GAA KO; small structures clustered around the expanded lysosomes in the GAA KO (similar to those shown in Fig. 2 & 3) are dextran positive. Three days after injection, expanded vacuoles (LAMP1 positive) stained strongly for dextran in GAA KO myotubes and no background dextran is seen in the cytoplasm. The image of the LAMP1 staining (inset) was collected from the same myotubes that were analyzed in live cultures.
Fig. 6
Fig. 6
Analysis of autophagy in in vitro Pompe models. (A) Western blotting of lysates obtained from WT and GAA KO myotubes using LC3 antibodies. (B) Following transfection of CS1 cells with GFP-LC3, green fluorescence is observed in structures similar in appearance to expanded lysosomes. (C) Post-transfection immunostaining using anti-GFP and anti- LAMP1 reveals the presence of a doubly-labeled vacuole, an autophagolysosome (arrow). Note arrowhead which shows a vacuole that stains only for LAMP1.
Fig. 7
Fig. 7
Lysosomes in autophagy deficient myotubes. Myotubes were prepared from primary myoblasts of skeletal muscle-specific autophagy-deficient GAA KO mice (DKO) [1]. (A) Absence of autophagy is confirmed by western blotting using LC3 antibody. (B) Enlarged lysosomes are shown by LAMP1 staining of DKO myotubes. (C) The difference between the size of the LAMP-I positive vacuoles in DKO and GAA KO mice is statistically insignificant (n=36 myotubes for GAA KO and n=41 myotubes for DKO; p =0.6). (D) Glycogen accumulation in DKO myotubes is shown by PAS staining.
Fig. 8
Fig. 8
Inhibition of macroautophagy with Atg7 siRNA in GAA KO myotubes. (A) GAA KO myoblasts (CS1 cells) were treated with Atg7 siRNA or negative control siRNA. Macroautophagy is successfully inhibited by Atg7 siRNA as shown by the absence of LC3-II signal in western blotting. (B) Atg7 siRNA treated cells contain large LAMP1 positive lysosomes. (C) The difference between the area of swollen lysosomes in the control and Atg7 siRNA treated cells is statistically insignificant (n=30 for control siRNA and n=35 for Atg7 siRNA; p=0.66). (D) Glycogen accumulation in Atg7 siRNA treated CS1 cells is confirmed by PAS staining.
Fig. 9
Fig. 9
Effect of the therapeutic enzyme (rhGAA) in GAA KO myotubes. (A) GAA KO myotubes were treated with rhGAA. After 4 days of incubation, LAMP1 positive structures shrink in a dose dependent manner. (B) Graphical presentation of data in A. Myotubes analyzed: n=26 for GAA KO (control); n=23 for GAA KO myotubes treated with 0.5 uM rhGAA; n=19 for GAA KO myotubes treated with 5.0μM rhGAA; n=8 for WT myotubes; n=9 for WT myotubes treated with 5.0μM rhGAA. *p=0.00003.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Raben N, Hill V, Shea L, Takikita S, Baum R, Mizushima N, Ralston E, Plotz P. Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease. Hum Mol Genet. 2008;17:3897–3908. - PMC - PubMed
    1. Hirschhorn R, Reuser AJ. Glycogen storage disease type II; acid alpha-glucosidase (acid maltase) deficiency. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular basis of inherited disease. McGraw-Hill; New York: 2001. pp. 3389–3420.
    1. Fukuda T, Roberts A, Plotz P, Raben N. Acid alpha-glucosidase deficiency (Pompe disease) Curr Neurol Neurosci Rep. 2007;7:71–77. - PubMed
    1. van der Ploeg A, Reuser A. Pompe's disease. Lancet. 2008;372:1342–1353. - PubMed
    1. Engel AG, Hirschhorn R, Huie ML. Acid Maltase Deficiency. In: Engel AG, Franzini-Armstrong C, editors. Myology. McGraw-Hill; New York: 2004. pp. 1559–1586.

Publication types

MeSH terms