Young C et al.
A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor
large-pore formation and HSP90.
Supplementary Materials
Figure S1. eATP induces oxidative stress in dystrophic myoblasts. (A) Images of wild-type (Wt) and
dystrophic (Dmdmdx) myoblasts treated with 3 mM ATP for 30 min in the presence of MitoTracker
Green (200 nM) and RedoxSensor Red CC-1 (1 M); The yellow signal resulting from colocalization of
these 2 markers indicates oxidative stress in mitochondria. (B) Graphical representation of changes
in fluorescence values: Dmdmdx myoblasts exposed to eATP displayed a 2.2-fold increase in CC-1
fluorescence compared to untreated (control) cells. This change was significantly greater than the
1.3-fold increase observed in Wt myoblasts. Mean +/- SE, n=3, P<0.05* and 0.0001***. (C) The
Dmdmdx myoblasts treated with 3 mM eATP in the presence of MitoTracker Red (200 nM, membrane
potential-dependent) and MitoTracker Green (200 nM, membrane potential-independent) markers
revealed an immediate reduction in mitochondrial membrane potential (decrease in red signal), with
accompanying cell shrinkage, and alterations in mitochondrial shape.
Figure S2. The mechanism of autophagic cell death following P2RX7 LP formation in dystrophic
myoblasts is distinct from autosis. (A) Representative western blots of triplicate samples showing
LC3-II responses in dystrophic myoblasts treated with 1 mM BzATP for 30 min with or without a 30min preincubation with cardiac glycosides digoxin or digitoxigenin (10 M). Densitometric analysis of
western blot data showed that cardiac glycosides had no significant effect on the level of autophagic
flux following P2RX7 activation in dystrophic myoblasts. (B) Cardiac glycosides had no significant
effect on P2RX7-dependent LP formation in dystrophic muscle cells (left panel) or cell death (LDH
release, right panel). Mean +/- SE, n=5, P<0.001**.
1
Figure S3. P2RX7 expression levels are significantly higher in macrophages compared to dystrophic
myoblasts (Mbs). (A) Immunoblots of triplicate samples showing P2RX7 expression levels relative to
ACTB loading control in Wt myoblasts, Wt macrophages (MØ), Dmdmdx myoblasts and P2RX7transfected HEK-293 cells. (B) Macrophages were found to express significantly (over 3 fold) more
P2RX7 than Dmdmdx myoblasts. Mean +/- SE, n=3, P<0.05* and p<0.0001***.
Figure S4. Western blots demonstrating LC3-II levels in skeletal muscles of wild-type and Dmdmdx
mice. In contrast to the recently reported reduced LC3-II levels in tibialis anterior10 and increased
LC3-II levels in diaphragm muscles of the Dmdmdx mouse,31 great individual variability but no overall
significant difference in LC3-II levels was found in these muscles.
Figure S5.
Autophagy in skeletal muscle disease. Summary of studies reporting changes in
autophagy levels in models of mammalian skeletal muscle disease and how these changes affect
muscle health. Basic concept adapted from Grumati et al.14 Red and green arrows indicate
detrimental and beneficial effects on muscle health, respectively. Red squares represent studies that
have reported negative effects of autophagy in diseased versus wild-type muscles - note the
variability reported between Dmdmdx animal studies. In vivo studies (summarized above the x-axis)
have suggested that in general autophagy exists at equilibrium in normal muscle, where too little or
too much can result in atrophy.14 Specific results however, have proved conflicting. For example, in
the Dmdmdx mouse, four different studies (including this one) have shown three different LC3-II levels
in unstimulated mice compared to Wt controls.11,31,35 Moreover, the impairment of autophagy can
be muscle-type dependent35 and both autophagy augmentation10,31 and inhibition16 have been
shown to ameliorate Dmdmdx pathology. Studies carried out using cell cultures (summarized below
the x-axis) have invariably shown detrimental effects of autophagy overactivation in both myoblasts
and myotubes.4,7,8,11,18,20-23,32,36,38,39 Interestingly, in macrophages any increase in autophagy levels
can result in beneficial effects on surrounding muscle12,17 and dendritic cells33 and the opposite
2
effect is seen if the levels are decreased.28,29 Given that multiple in vivo studies have documented
beneficial effects of upregulating autophagy in skeletal muscle,10,15,31,35 it is possible that the in vivo
data reflect the tonic stimulation of autophagy leading to beneficial effects, whereas the detrimental
effects seen in culture conditions could be the result of chronic or excitotoxic stimulations. Another
explanation may involve the role of autophagy in infiltrating immune cells, which are prevalent in
diseased muscles and play a significant role in both damage and regeneration.38 Hence the interplay
between muscle and immune cells should be carefully considered when interpreting in vivo
manipulations of autophagy in muscle disease models, as it will certainly affect the derived
therapeutic benefits of such manipulations.2,5,10,31,35
SM, skeletal muscle; KO, knockout; mbs,
myoblasts; MTs, myotubes; sIBM, sporadic inclusion body myosistis.
Supplementary Video 1. Time-lapse live cell confocal microscopy images of EtBr influx, membrane
blebbing and cell death in dystrophic myoblasts exposed to 3mM eATP for 0 to 60 min. Cells were
incubated with MitoTracker Green (100 nM, green signal) and EtBr (5 M, red signal) during
stimulation with eATP. Images were taken every 5 min for a period of 1 h at 500 x magnification
using an aqueous immersion lens and 37 oC heated stage attached to a Zeiss LSM 510 Meta confocal
microscope.
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