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Vitamin A-Retinoic Acid Contributes to Muscle Stem Cell and
Mitochondrial Function Loss in Old Age
Paula M. Fraczek1,2, Pamela Duran1,2, Benjamin A. Yang1,2, Valeria Ferre1,2, Leanne Alawieh1,2,
Jesus A. Castor-Macias1,2, Vivian T. Wong1,2, Steven D. Guzman1,2, Celeste Piotto1,2, Klimentini
Itsani1,2, Jacqueline Larouche1,2, Carlos A. Aguilar1,2,3,*
1Dept. of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
2Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA. 3Program in Cellular
and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA. *To whom
correspondence should be addressed: Carlos A. Aguilar, NCRC - University of Michigan, 2800
Plymouth Rd., 10-A183, Ann Arbor, Michigan 48109, USA. Phone: 734-764-8557; Email:
caguilar@umich.edu.
Summary:
"Adult stem cells decline in number and function in old age and identifying factors that can delay
or revert age-associated adult stem cell dysfunction are vital for maintaining healthy lifespan.
Here we show that Vitamin A, a micronutrient that is derived from diet and metabolized into
retinoic acid, acts as an antioxidant and transcriptional regulator in muscle stem cells. We first
show that obstruction of dietary Vitamin A in young animals drives mitochondrial and cell cycle
dysfunction in muscle stem cells that mimics old age. Next, we pharmacologically
targeted retinoic acid signaling in myoblasts and aged muscle stem cells ex vivo and in vivo and
observed reductions in oxidative damage, enhanced mitochondrial function, and improved
maintenance of quiescence through fatty acid oxidation. We next detected the receptor for
vitamin A derived retinol, stimulated by retinoic acid 6 or Stra6, was diminished with muscle
stem cell activation and in old age. To understand the relevance of Stra6 loss, we knocked
down Stra6 and observed an accumulation of mitochondrial reactive oxygen species, as
well as changes in mitochondrial morphology and respiration. These results demonstrate that
Vitamin A regulates mitochondria and metabolism in muscle stem cells and highlight a unique
mechanism connecting stem cell function with vitamin intake.
Highlights:
•Dietary loss of Vitamin A in young mice drives metabolic dysfunction of muscle stem cells
•Pharmacological targeting of retinoic acid signaling in myogenic progenitors and aged muscle
stem cells enhanced mitochondrial function and oxidative metabolism
•The vitamin A receptor, Stra6, decreases with muscle stem cell activation and aging and loss
of Stra6 perturbed mitochondrial dynamics resulting in oxidative stress
Keywords: Stra6, Oxidative Stress, Fatty Acid, Diet
Introduction
Skeletal muscle contains a small population of stem cells called muscle stem cells (MuSCs) or
satellite cells (1). MuSCs resist entry into the cell cycle via a series of mechanisms driven by β-
oxidation of fatty acids and oxidative phosphorylation (OXPHOS) resulting in low levels of
reactive oxygen species (ROS) (2, 3). Impairments in MuSC ability to maintain quiescence have
been demonstrated in old age, whereby a loss of OXPHOS, imbalances between mitochondrial
fusion and fission, and dysregulation of mitophagy (4) result in oxidative damage and stem cell
decline in number and function (5). MuSC dysfunction in old age has been shown to contribute to
reductions in regeneration leading to persistent tissue damage, and structural and functional deficits
(6–12). As such, it is critical to understand factors that promote MuSC metabolic and mitochondrial
health to prevent pathological muscle remodeling in age (3).
A potential source of deleterious behavior that occurs in old aged MuSCs is lower vitamin
consumption (13). Vitamin A (VA), or retinol, is a diet-derived antioxidant that is metabolized
into all-trans retinoic acid (RA) (14) and interacts with retinoic acid receptor (RAR)-retinoid x
receptor (RXR) transcription factors that bind at retinoic acid response elements. The interactive
output of RA+RAR/RXRs results in transcription of genes related to development, vision,
immunity, and metabolism (15, 16), and variations in RA levels have been shown to drive
alterations in spatial patterning in development, and changes in stem cell state and differentiation
(17, 18). RA has also been shown to promote quiescence in hematopoietic stem cells, and restrain
human skeletal muscle progenitors from differentiation (19–22). The lack of VA and RA has also
been associated with neuromuscular dysfunction (23, 24) and contributes to oxidative stress and
mitochondrial dysfunction in T cells (25). However, there is little information connecting MuSC
functionality with VA metabolism and in old age.
The membrane receptor Stimulated by retinoic acid gene 6 (STRA6) is the cellular gateway for
VA entry, and has been implicated in a variety of cellular functions including regulation of p53,
Wnt and mitochondrial pathways (26). STRA6 loss hinders VA homeostasis and retinoid-
dependent processes (27) but there is no information how STRA6 impacts skeletal muscle, MuSCs
or changes in old age. Given the important links between mitochondria, ROS, and MuSCs, STRA6
may be critical for regulating RA and resisting oxidative stress in age.
Herein, we investigate the role of VA and RA signaling in MuSCs through a series of in vitro and
in vivo experiments. We administered a VA-free diet to young animals and observed premature
MuSC activation, oxidative DNA damage, and mitochondrial dysfunction akin to aging. Next, we
pharmacologically upregulated RA signaling in myogenic progenitors and old aged MuSCs ex
vivo and in vivo and observed we could enhance mitochondrial function and reduce oxidative
stress. Last, we demonstrate that the VA receptor Stra6 is attenuated with activation and in old age
and that Stra6 loss perturbed mitochondrial dynamics resulting in accumulation of reactive oxygen
species and oxidative stress. These findings provide new insights into the mechanisms driving
MuSC mitochondrial health in old age with dietary derived vitamins.
Results
In Vivo Depletion of Vitamin A Drives Premature Activation of Muscle Stem Cells
To determine the impact of the lack of VA and concomitant RA signaling in MuSCs in vivo, we
administered a vitamin A (VA)-free diet ad libitum for 8 weeks to a MuSC lineage tracing mouse
model (Pax7CreERT2-Rosa26nTnG; Pax7-nTnG)(28). After 8 weeks, MuSCs were isolated from the
quadriceps and gastrocnemius muscles via using fluorescent activated cell sorting (FACS, Supp.
Fig. 1A) and profiled (Fig. 1A). Since previous studies have shown that RA restrains MuSCs from
differentiation in vitro, we first evaluated changes in MuSC activation (20, 29, 30). Freshly isolated
VA-free MuSCs displayed reductions in Pax7 (p<0.01), and increased MyoD compared to controls
(p<0.05, Figs. 1B-D). A higher overall proportion of VA-depleted MuSCs formed myogenic
colonies compared to control MuSCs (p < 0.05, Fig. 1G), suggesting an increased propensity to
enter the cell cycle. In line with these observations, we detected increases in Ki67 (p < 0.05) as
well as increased amounts of mitochondria (MitoTracker Deep Red FM; p < 0.05) in MuSCs
isolated from VA-free diet (Figs. 1E-F). To further examine if VA depletion promoted
accumulation of reactive oxygen species (ROS) and oxidative stress-related damage, we stained
for 8-hydroxyguanosine (8-OHdG), a form of DNA lesion induced by exposure to ROS (31). VA-
free MuSCs displayed increased levels of 8-OHdG when compared to controls (p < 0.05; Figs.
1H-I). These results confirm that VA depletion prompted MuSCs to shift away from quiescence
and towards premature activation.
Lack of Vitamin A Disrupts Muscle Stem Cell Metabolism
To gain further insights into changes in MuSCs from VA-free diet, we performed RNA-
Sequencing of FACS-isolated MuSCs from mice fed either a VA-free diet or control chow (Fig.
2A). We observed excellent reproducibility between libraries (Spearman ≥ 0.93) from different
conditions (Supp. Fig. 1B), and principal component analysis revealed clustering of replicates and
distinction between MuSCs from a control and VA-free diet (Supp. Fig. 1C). Differential
expression analysis revealed 4,149 genes underwent a change due to VA-free diet (Fig. 2B, Supp.
Table 1), and multiple components of the retinoic acid signaling pathway were attenuated such as
dehydrogenases that oxidize retinol into retinoic acid (e.g. Adh1, Aldh1a3) and cellular retinoic
acid binding proteins (e.g. Crabp1) that carry retinoic acid into the nucleus (Fig. 2C). Additionally,
the surface receptor that mediates cellular uptake of retinol (stimulated by retinoic acid gene 6 or
Stra6) decreased in expression with VA-free diet (Fig. 2C). We also detected several senescence-
related genes in VA-free diet MuSCs including cytokines Il-6, Il-15, and Ccl4 (Supp. Table 2).
GO term enrichment analysis of differentially expressed genes showed over-representation of
autophagy, MuSC activation and differentiation for VA-free diet MuSCs. In contrast, under-
represented terms in VA-free diet MuSCs included cell-cycle and ROS regulation, and multiple
types of mitochondrial processes including mitochondrial membrane organization, oxidative
phosphorylation, and mitochondrial ROS mitigation (Figs. 2D-E, Supp. Fig 1D).
To further probe how diet-induced changes in gene expression impacted metabolic flux, we
applied genome-scale metabolic modeling to our RNA-Seq data (32). The metabolic model
predicted VA-free diet increased flux through L-lactate dehydrogenases and retinol
dehydrogenases that contribute to NAD+/NADH balance and nucleotide synthesis as well as sense
changes in available lipids and steroids (Fig. 2F, Supp. Fig. 1E). The model also predicted
increased flux through the pentose phosphate pathway (Fig. 2F), which is consistent with
activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family and changes
in redox signaling that promotes MuSC activation (33, 34). In contrast, MuSCs from control diet
were enriched for pyruvate kinase, which has been shown to block ROS in myogenic progenitors
(35). Together, these results suggest that depleting VA promotes changes in MuSC metabolism
that reduce maintenance of quiescence and signaling through mitochondria to neutralize oxidative
stress.
Loss of Dietary Vitamin A Does Not Alter Muscle Stem Cell-Mediated Regeneration
To investigate whether dietary depletion of VA reduced the regenerative potential of MuSCs, we
administered a muscle injury via intramuscular injection with barium chloride (BaCl2). Dissection
and immunohistochemistry of tibialis anterior cross sections before injury revealed no statistically
significant variations in cross-sectional myofiber area between control or VA-free diet (Supp.
Figs. 2A-B). We also did not detect a significant change in the number of monocytes and
macrophages (CD68+) or neutrophils (Ly6G+), or in the total number of MuSCs for VA-free diet
when compared to controls (Supp. Figs. 2C-E). Seven days after injury, we detected no significant
change in myofiber size and number of regenerating myofibers measured by centrally located
nuclei (Supp. Fig. 2B). However, increases in both CD68+ and Ly6G+ cells were observed after
injury for VA-free diet, which is consistent with the known role of VA as an anti-inflammatory
micronutrient (36).
To understand if MuSC changes in regenerative potential from VA-free diet occurs over a longer
time scale, we assessed response to BaCl2 injury at 28 days after injury (Supp. Fig. 3A). In line
with previous observations, no significant differences were identified in the cross-sectional area
of all myofibers (Supp. Fig. 3B) or in the number or percentage of myofibers with MuSC derived
engraftment between control and VA-free diet (Supp. Figs. 3C-D) or centrally nucleated fibers
(Supp. Fig. 3E) was observed between groups. We also did not detect differences in the cell
density of CD68+ macrophages (Supp. Fig. 3F) or MuSCs for VA-free diet when compared to
controls (Supp. Fig. 3G). Overall, these results suggest that the lack of VA does not significantly
impact acute response to regeneration from MuSCs.
Targeting Retinoic Acid Signaling Alters Mitochondrial Function and Morphology of Muscle Stem
Cells
We have previously demonstrated that MuSCs lose RAR and RXR gene expression in old age
(37), and dietary loss of this exogenous antioxidant in young animals impinged on MuSC
mitochondria. To further understand how targeting RAR/RXRs promotes mitochondrial health in
myogenic cells, we delivered a cocktail of small molecule agonists specific to RARɣ (BMS961)
and RXR⍺ (CD3254), as well as the ligand (ATRA) to C2C12 myoblasts (Fig. 3A). We previously
observed that Rarg and Rxra are among the most significantly downregulated RA-responsive
genes in aged MuSCs, when compared to young cells(37), and Rary has been shown to be essential
in the regulation of most genes involved in retinol metabolism and retinoic acid signaling,
including Stra6 (38). We administered the small molecule cocktail to C2C12s and detected
increased expression of the RA-responsive genes Rarɣ and Rar
b
as well as Stra6 for cells treated
with the small molecule cocktail (Supp. Fig. 4A-C). We also detected that the orphan receptor
peroxisome proliferator-activated receptor gamma (Pparɣ) increased in expression, as well as the
NADH:ubiquinone oxidoreductase subunit A2 (Ndufa2), a critical catalytic component of
mitochondrial respiratory chain and OXPHOS system (Supp. Fig. 4D-E). To further validate the
delivery of the small molecule cocktail impacted mitochondrial function, we performed a Seahorse
XF Mito Stress Test assay (Fig. 3B) and observed increases in basal respiration and ATP
production as well as reductions in proton leak for treated C2C12 cells (Fig. 3C, Supp. Fig. 4F-
I). Treatment also increased coupling efficiency (rate of ATP production/rate of basal respiration)
and oxygen consumption rate / extracellular acidification rate (OCR/ECAR), indicating increased
ATP production through oxidative phosphorylation (Fig. 3D). Notably, we found that
improvements in activation restraint, mitochondria, and oxidative damage appeared strongest
when ATRA and CD3254/BMS961 were combined rather than individually (Supp. Figs. 4J-L).
These results show that increasing RA signaling in myogenic cells can promote mitochondrial
function through oxidative metabolism. To further understand how MuSC changes in
mitochondria and maintenance of quiescence that are enacted with treatment, we utilized a MuSC
reporter mouse that harbors an enhanced green fluorescent protein (EGFP) in the outer
mitochondrial membrane (OMM) of Pax7-expressing cells (Pax7CreERT2-Rosa26CAG-LSL-EGFP-3xHA-
OMM or Pax7-MitoTAG, Fig. 3E) (39). We isolated MuSCs from young muscles (4 months) and
treated cells with or without the small molecule cocktail as above. Immunostaining the cells and
analysis by 3D volume reconstruction showed reductions in Feret diameter (Fig. 3F), which is
consistent with previous observations that activated MuSCs contain larger sized mitochondriam
(4). We further confirmed treatment with the small molecule cocktail reduced activation in Pax7-
MitoTAG MuSCs by measuring EdU incorporation and detected reductions in EdU uptake for
treated cells (Fig. 3G, Supp. Fig. 4M). Combined with the results above, these findings show
increases in RA signaling reduces activation through mitochondria.
Repletion of Retinoic Acid Signaling Reduces Oxidative Stress and Promotes Metabolism
Supportive of Quiescence in Muscle Stem Cells
To determine if rescue of RA signaling in old aged MuSCs improved cellular state and
mitochondrial health, we isolated MuSCs from uninjured old aged limb muscles (24 months) and
treated old aged MuSCs ex vivo with the same small molecule cocktail. We measured
mitochondrial ROS levels by MitoTracker Orange CM-H2TMRos (40) and overall mitochondrial
density (MitoTracker Deep Red FM). We detected reductions in mitochondrial ROS and total
mitochondrial density for treated cells when compared to controls (Fig. 4A-B). To determine if
treatment reduced oxidative DNA lesions, we stained for 8-OHdG and found reductions in treated
MuSCs (Fig. 4C). As above, the combined action of ATRA and CD3254/BMS961 rather than
individual treatment resulted in strongest increases in STRA6, and reductions in MyoD,
mitochondrial ROS, and mitochondrial density (Supp. Fig. 4N-Q). These results show that the
pathological degeneration of MuSCs in old age, which is linked to dysfunctional mitochondria and
oxidative damage, can be partially alleviated by targeting RA signaling (41).
To further determine how targeting retinoic acid signaling impacted old aged MuSCs in vivo, we
intramuscularly injected same small molecule cocktail every other day for 7 days to old aged
tibialis anterior muscles (Fig. 4E). We then performed single-cell RNA sequencing (scRNA-Seq)
of mononucleated cells after therapy and generated 13,859 single cells after filtering (6,906 from
aged treated and 6,953 from aged control cells) with an average of 2,113 genes and 5,841 unique
molecular identifiers per cell (Supp. Fig. 5A). We reduced the dimensionality of the datasets using
Uniform Manifold Approximation and Projection (UMAP) and performed Louvain clustering
followed by annotation of cell types using marker genes (Fig. 4F, Supp. Fig. 5B). This analysis
revealed 12 cell types, and similar recovery of cell types with previously published single-cell
atlases from muscle (Supp. Fig. 5B) (28, 42). We detected consensus among cell types across
samples (Supp. Fig. 5C), and increased fractions of neutrophils and tenocytes for controls
compared to treated muscles (Supp. Fig. 5D). To glean insights into molecular responses of
MuSCs with treatment, we re-clustered MuSCs and found upregulated genes from treatment
included Pax7 and inhibitor of differentiation 1 and 3 (Id1, Id3), which are negative regulators of
MyoD (43, 44) (Fig. 4G, Supp. Figs. 5E-F). We also detected increased expression of sirtuin 2
(Sirt2), a fatty-acid oxidation sensitive enzyme that regulates NAD+ availability, for treated
MuSCs when compared to controls (Fig. 4H) (45, 46). Conversely, upregulated genes in untreated
cells included metallothioneins 1 and 2 (Mt1, Mt2), markers of oxidative stress, and Rock2, a
negative regulator of mitophagy (47, 48). GO Term enrichment analysis showed that treatment
reduced apoptotic pathways, cell cycle entry, and TOR signaling (Supp. Fig. 5G). To gain deeper
insights into alterations in metabolic flux from treatment, we utilized Compass, an algorithm to
determine changes in metabolism from scRNA-Seq datasets (49). Consistent with our previous
measurements, we detected increases in fatty acid oxidation and beta-oxidation of long-chain fatty
acids, as well as NAD metabolism in treated MuSCs when compared to untreated MuSCs (Fig.
4I, Supp. Fig. 5H). Combining these results further suggests targeting retinoic acid signaling
augments metabolic health and reduces oxidative stress in MuSCs.
Muscle Stem Cell Intake of Vitamin A is Mediated by Stra6, Which Decreases with Activation and
Age
The membrane receptor STRA6 is the cellular gateway for retinol entry, and our data showed
MuSCs lose Stra6 gene expression after VA-free diet (Fig. 2C). We first investigated whether
Stra6 expression changes using RT-qPCR after 3 conditions: (1) Freshly FACS-isolated MuSCs
(quiescent) from uninjured limb muscles, (2) in vitro culture in activating conditions for 3 days
(myoblast), and (3) 3 days after differentiation and fusion into myotubes (differentiated). In line
with our previous observations, we detected Stra6 expression was highest for freshly isolated
MuSCs, reduced in activated myoblasts, and lowest in differentiated myotubes (Fig. 5B). These
results suggest VA intake through STRA6 may participate in MuSC maintenance of quiescence.
Given old aged MuSCs have been demonstrated to lose quiescence and display premature
activation, we quantified protein levels of STRA6 on freshly isolated young (3-4 months) and old
aged (22 months) MuSCs from young and aged mouse hind limb muscles. We found decreases in
STRA6 levels in aged MuSCs, in accordance with our previous RNA findings where we previously
observed a loss in Stra6 gene expression from uninjured old aged MuSCs (37) (Figs. 5C-D). Since
STRA6 expression is enhanced by the binding of the RARɣ/RXR⍺ heterodimer to its promoter
(50), we treated old aged MuSCs as above, and confirmed increased Stra6 (Fig. 5E-F), which also
coincided with reductions in MyoD (Fig. 5G-H). These results suggest an attenuation of STRA6-
mediated VA transport and RA signaling occurs during MuSC activation.
Stra6 Loss Alters Mitochondrial State, Morphology, & Function
To further assess the influence of STRA6 on mitochondrial processes that assist with maintenance
of quiescence, we knocked down Stra6 using siRNAs in C2C12 myoblasts and confirmed
knockdown levels with RT-qPCR (Supp. Fig. 6A). To gain insights whether STRA6 loss disrupts
mitochondrial dynamics, we utilized multiple live-cell mitochondrial dyes following Stra6
knockdown including MitoTracker Orange CM-H2TMRos and JC-1, a mitochondrial membrane
potential probe (51). We detected an increase in mitochondrial ROS (Fig. 6C) and decrease in
mitochondrial membrane polarization (Figs. 6A-C) in Stra6 knockdown cells compared to
controls. We also observed that Stra6 knockdown increased proliferation (Ki67, Fig. 6D), as well
as lipid peroxidation detected by BODIPY 581/591 C11, a lipid peroxidation sensor (Supp. Figs.
6B-C) (52). Lastly, we found that knockdown of Stra6 triggered an upregulation of p53, which is
known to increase in scenarios of increased oxidative stress (Supp. Fig. 6D) (53).
To further determine how Stra6 knockdown impacted mitochondrial function, we utilized the
Seahorse XF Mito Stress Test assay. Stra6 knockdown resulted in increased oxygen consumption
(OCR), extracellular acidification (ECAR) rates, basal respiration, and ATP production, likely due
to the increased bioenergetic demands of their heightened proliferative state (Figs. 6E-I). Notably,
we also observed increased proton leak in Stra6 knockdown cells (Fig. 6H), which has been
similarly observed in aged mitochondria (54–56). Stra6 knockdown cells also exhibited a lower
OCR/ECAR ratio indicative of a shift toward glycolysis (Fig. 6I), which has also been observed
in proliferative and aged myoblasts (4, 57, 58).
Discussion
The pathological degeneration of MuSCs in old age is intrinsically linked to defects in
mitochondria. We detected old aged MuSCs lose Vitamin A, a powerful, exogenous antioxidant
that is metabolized into RA. To directly examine the functional effects of reduced Vitamin A-
derived RA signaling on MuSCs, we administered a Vitamin A-free diet to young animals. We
found a Vitamin A deficient diet induced premature MuSC activation and increases in redox-
dependent metabolism and mitochondrial ROS. Vitamin A free diet also was characterized by a
loss of fatty acid oxidation and OXPHOS, resulting in loss of ability to maintain quiescence.
Uniquely, the deleterious changes in MuSC mitochondria from Vitamin A free diet did not
associate with alterations in myofiber diameter, overall depletion of MuSCs or muscle
regeneration, which is consistent with previous work that showed mitochondrial dysfunction in
MuSCs do not manifest in regenerative defects in youth, but are exacerbated in age (>24 months)
(59, 60). Combining these results shows that loss of vitamin A leads to stress responses that
increase ROS and premature activation in MuSCs that in turn drive loss of ability to maintain
quiescence.
Changes in vitamins and associated metabolites have significant influence on stem cell function,
and epidemiological studies have shown that regular diet supplemented with higher intake of
antioxidant vitamins renders improvements in health during aging (13, 19). However, connecting
the functionality of adult stem cells with vitamin-derived pathways in aging remains
underexplored. VA metabolites have been shown to interact with the a-subunit of mitochondrial
ATP synthase and Peroxisome Proliferator-Activated Receptors (PPARs), which heterodimerize
with RXRs to activate the oxidoreductase acyl-CoA oxidase(61–63). Activation of mitochondrial
regulators of oxidative metabolism from vitamin A would be consistent with our data, whereby
administration of RARɣ/RXR⍺ agonists and ATRA resulted in increased expression of PPARɣ
and Ndufa2, reduced proton leak and increased ATP production through oxidative
phosphorylation. Moreover, treatment of aged MuSCs ex vivo with RARɣ/RXR⍺ agonists and
ATRA reduced mitochondrial ROS and oxidative DNA damage and promoted fatty acid oxidation
and reduced total mitochondrial density in vivo. We speculate that vitamin A-retinoic acid
activation of PPARɣ may promote fatty acid oxidation through or in synergy with Ret signaling,
which has been shown to contribute to MuSC ability to maintain quiescence (64–68). Integrating
these results show that administration of vitamin A derived retinoic acid promotes oxidative
metabolism in MuSCs, and restoration of this antioxidant augments mitochondrial function lost in
old age.
Vitamin A is an indispensable, diet-derived nutrient that is metabolized into retinol, distributed in
the bloodstream, and internalized by Stra6. We detected Stra6 expression was strongest in
quiescent MuSCs and reduced with MuSC activation, differentiation, and in old age. Reductions
in Stra6 coincided with loss of expression of multiple mitochondrial genes and likely impacts how
RAR/RXR factors interact with MyoD and MyoG (69–71). Similarly, knockdown of Stra6
increased mitochondrial ROS and fragmentation and decreased mitochondrial depolarization and
ATP production through oxidative phosphorylation. Given Stra6 has been shown to be sensitive
to Ca2+, these results suggest that retinol transport into and out of Stra6 may contribute to MuSC
mitochondrial fission and fusion dynamics through a Ca2+-mediated mechanism (72, 73).
However, future work is needed to determine changes in Ca2+ signaling with retinol levels and
mitochondria.
Limitations of Study
This work helps establish how Vitamin A derived retinoic acid and transport through Stra6 impacts
mitochondrial stress and function of MuSCs and clarifies the importance of this signaling axis in
old age. Whether Vitamin A derived retinoic acid impacts aging of human MuSCs in a similar
manner as our murine model remains to studied. Future work will address 3 key challenges. First,
our vivo delivery of ATRA and CD3254/BMS961 could be improved by encapsulation in
micro/nanoparticles, which have been shown to improve solubility, release kinetics as well as
cellular uptake(74). This would reduce the need to perform multiple injections and target MuSCs
specifically. Next, our study focused on targeting a single retinoic acid receptor (RARɣ) and
retinoid receptor (RXR⍺) in old aged MuSCs, but other retinoic acid and retinoid receptors may
also influence MuSC behavior through interaction with muscle basic helix-loop-helix transcription
factors. Since retinoic acid has been demonstrated to act as both an inducer of stemness and
differentiation, future studies may evaluate the relative contributions of each RAR and RXR to
Pax7 and MyoG-induced actions with high-throughput tools such as CRISPR/Cas9 (75, 76). Third,
our work provides a foundation for targeting STRA6 and demonstrates rescue of this receptor
positively impacts mitochondrial function. Since STRA6 also interacts with Calmodulin, future
work may evaluate the relationship between retinol, calcium and mitochondrial membrane
potential.
Methods
Sex as a biological variable
Both male and female mice were used and for experiments listed, mice were age- and sex-matched
with littermate controls used whenever possible.
Animals & Muscle Stem Cell Labeling
Male and female Pax7CreERT2-Rosa26nuclearTdTomato-nuclearGFP or Pax7-nTnG mice were obtained from
a breeding colony at the University of Michigan. Male and female Pax7CreERT2-Rosa26TdTomato or
Pax7-TdTomato were obtained from a breeding colony at the University of Michigan. Male and
female Pax7CreERT2-Rosa26CAG-LSL-EGFP-3xHA-OMM or Pax7-MitoTAG were obtained from a breeding
colony at the University of Michigan. Young and old aged C57BL/6 wild-type mice were obtained
from Jackson Laboratories or from a breeding colony at the University of Michigan (UM). To
activate Pax7-conditional reporter expression, mice were given 5 daily intraperitoneal injections
of 20 mg/mL tamoxifen in corn oil (75 mg/kg body weight) and allowed to recover 5 days before
being euthanized for experiments. All mice were housed on a 12:12 hour light-dark cycle under
UM veterinary staff supervision. All procedures were approved by the Institutional Animal Care
and Use Committee and were in accordance with the U.S. National Institute of Health (NIH).
Muscle Digestion for Muscle Stem Cell Isolations
Mice were euthanized by CO2 asphyxiation followed by cervical dislocation as a secondary
method to confirm death. The hindlimb muscles were dissected using sterile surgical tools and
muscles were kept separate by biological replicate. The dissected muscles were then minced into
fine chunks (<1 mm3) with fine surgical scissors. The minced muscle was then added into 50 mL
conical tubes containing 20 mL of digestion solution (2.5 U/mL dispase II and 0.2% mg/mL
collagenase II in DMEM). The samples were incubated at 37°C on a shaker for 30 minutes, then
mechanically dissociated by pipetting up and down several times with an FBS-coated 10 mL
serological pipette and allowed to incubate at 37°C for an additional 30 minutes. The digestion
enzymes were then quenched with 20 mL of stop solution per tube (20% heat-inactivated FBS in
Ham’s F10 nutrient mix). The samples were then passed through a 70 µm cell strainer and
centrifuged at 350g for 5 minutes at 4°C. The pellets were then washed and further processed
according to the isolation procedures described below.
FACS Isolation of Pax7CreERT2-Rosa26nuclearTdTomato-nuclearGFP Muscle Stem Cells
After muscle tissue digestion, the supernatant was aspirated and cell pellets were washed in 3-4
mL of FACS buffer (2% Heat-inactivated FBS in Hank’s Balanced Salt Solution), after which they
were centrifuged once more and resuspended in an appropriate volume of FACS buffer for sorting
(approximately 1 mL per sample). Just before sorting, the samples were incubated with DAPI at a
final concentration of 1µg/mL (protected from light) to stain for viability, and cell suspensions
were passed through a 35 µm cell strainer. Sorting was done on a Sony MA900 cell sorter, and
DAPI-/GFP+ MuSCs were collected into cold FACS buffer for immediate processing.
FACS Isolation of Wild-Type Muscle Stem Cells
After muscle tissue digestion, the supernatant was aspirated, and cell pellets were washed in 3-4
mL of FACS buffer. After centrifugation, the washed cell pellets were resuspended in 400 µl of
FACS buffer (split across two separate 5 mL FACS tube per biological replicate) containing a
mixture of primary antibodies: APC-CD31 (Biolegend #102410, 1:400), APC-CD45 (Biolegend
#103112, 1:400), APC-Sca1 (Biolegend #108112, 1:400), APC-CD11b (Biolegend #101212,
1:400), APC-TER119 (Biolegend #116212, 1:400), PE-CD29 (Biolegend #102208, 1:200),
Biotin-CXCR4 (BD Biosciences #551968, 1:200). Tubes were incubated on ice for 30 minutes,
protected from light. Samples were washed, centrifuged, and resuspended in 200 µL of FACS
buffer per tube containing PECy7-Streptavidin (eBioscience 25-4317-82, 1:100). Samples were
incubated for 20 minutes on ice, protected from light. Prior to sorting, samples were washed,
centrifuged, resuspended in 1 ml of FACS buffer per biological replicate, and passed through a 35
µm cell strainer. Propidium iodide solution (Thermo Fisher #P3566, 1:1000) was added to each
sample tube just before sorting to stain for viability. Sorting was done on a Sony MA900 cell
sorter, and PI-/APC-/PE+/PECy7+ MuSCs were collected into cold FACS buffer for immediate
processing.
Magnetic Activated Cell Sorting Isolation of Muscle Stem Cells
After digestion of muscle tissue as described above, cell pellets were treated with Miltenyi Red
Blood Cell Lysis Solution as described in the Miltenyi MACS Satellite Cell Kit isolation protocol.
After RBC lysis and centrifugation, cells were washed with MACS buffer (PBS, 0.5% bovine
serum albumin, 2 mM EDTA). Incubation with satellite cell kit microbeads and LS column
separation was performed as described by the manufacturer. Afterwards, cells were counted and
further purified using the Miltenyi Anti-Integrin Alpha 7 microbeads according to the
manufacturer’s instructions. Finally, cells were incubated in uncoated tissue culture flasks for 1
hour at 37°C and 5% CO2 as a final pre-plating step to remove remaining contaminating cell types.
The supernatants from the flasks were collected and centrifuged, and cells were counted via
hemocytometer and resuspended in appropriate volumes of media or buffer for further processing.
Myoblast Growth Medium Preparation
Muscle stem cells were cultured in myoblast growth medium consisting of Ham’s F10 nutrient
mix (Gibco #11550043), 1x Penicillin-Streptomycin (Gibco #15140122), 20% heat-inactivated
FBS (Gibco #16140071), and 0.02 µg/ml of bFGF (Gibco #PHG0263).
Vitamin A Depletion
To investigate the effect of Vitamin A depletion on muscle regeneration at the short-term
timepoint, young (9 months and younger) Pax7-nTnG mice were fed a custom Vitamin A-deficient
(VA-free) diet from Envigo Teklad (Cat. # TD.86143) ad libitum for a period of 8 weeks while
control mice continued to receive normal mouse chow. For the long-term time point, young (3-5
months) Pax7-TdTomato mice were fed the same (VA-free) diet from Envigo Teklad (n=5) as
described above, while control animals (n=3) received normal mouse chow. The VA-free chow
was replenished every 1-2 weeks. In the final week, mice received daily tamoxifen injections as
described above to label MuSCs and their progenitors after injury.
Single Cell Clonogenicity Assay
7–9-month-old Pax7-nTnG mice (2 males and 2 females per diet) were either fed VA-free or
control chow as described above. Using the Sony MA900’s single cell sorting mode and well-plate
collection adapter, GFP+ MuSCs from the gastrocnemius and quadricep muscles from were sorted
directly into 0.5% gelatin-coated 96-well plates containing 200 µL of myoblast growth media.
Single-cell wells were kept in culture at 37°C and 5% CO2 for 5 days, and media was replaced
every other day. Afterwards, the wells were fixed with 4% PFA for 10 minutes at room
temperature, washed with PBS, and labeled with DAPI (1 µg/ml in PBS) for 10 min at room
temperature. Wells were imaged using Zeiss Axio Vert.A1 inverted microscope with a Colibri 7
LED light source and an AxioCam MRm camera and myogenic colonies were counted in Fiji.
MitoTracker Deep Red, Ki67, and MyoD labeling of VA-free vs. CTRL Muscle Stem Cells
7–9-month-old Pax7-nTnG mice (2 males and 2 females per diet) were either fed VA-free or
control chow as described above. 96-well plates were coated with CellTak (Corning Cat. 354240)
with 50µL of a 1:70 dilution in PBS and allowed to incubate for 20 minutes at room temp before
aspirating and seeding cells. 2,000 nGFP+ MuSCs (isolated from the gastrocnemius and quadricep
muscles) were seeded per well. Cells were incubated in myoblast growth medium at 37°C and 5%
CO2 for 1 hour to allow the cells to settle and adhere to the CellTak. 500 nM of MitoTracker Deep
Red FM (Invitrogen Cat. M22426) was prepared in pre-warmed myoblast media. Half of the wells
were incubated with MitoTracker for 30 minutes at 37°C and 5% CO2 and then washed 3 times
with PBS before fixing the entire plate with 4% PFA (10 minutes at room temperature).
Permeabilization was performed by incubating with 0.1% Triton X-100 in PBS for 15 minutes at
room temperature, followed by 3 washes in PBST (0.1% Tween-20 in PBS). Then, the cells were
blocked with 1% BSA, 1% Goat Serum, 22.52 mg/mL of glycine in PBST for 1 hour at room
temperature. After 3 washes with PBST, the MitoTracker-labeled cells were incubated with PE-
conjugated anti-Ki67 (Santa Cruz, 1:50) and the remaining wells were incubated with Alexa Fluor
647-conjugated anti-MyoD antibody (Santa Cruz, 1:50) in 1% BSA in PBST overnight at 4°C.
Following overnight incubation with antibodies, cells were washed 3 times with PBS. Nuclei were
counterstained with DAPI (1 µg/mL) for 10 minutes at room temperature. Cells were washed in
PBS a final 3 times and left covered in 100 µL of PBS during imaging. MyoD-labeled wells were
also imaged for Pax7-nGFP fluorescence. 20x magnification images were acquired on a Zeiss Axio
Vert.A1 inverted microscope with a Colibri 7 LED light source and an AxioCam MRm camera.
Images were subsequently analyzed in Fiji.
8-OHdG Labeling of VA-free vs. CTRL Muscle Stem Cells
3-month-old Pax7-nTnG mice (2 females per diet) were either fed VA-free or control chow as
described above. 96-well plates were coated with CellTak (Corning #354240) with 50µL of a 1:70
dilution in PBS and allowed to incubate for 20 minutes at room temp before aspirating and seeding
cells. 4,000 nGFP+ MuSCs (isolated from the gastrocnemius and quadricep muscles) were seeded
per well. The plate was spun down at 50 RCF for 1 minute and then incubated in myoblast growth
medium at 37°C and 5% CO2 for 30 minutes to allow the cells to settle and adhere to the CellTak.
The plate was then fixed with 4% PFA (10 minutes at room temperature). Permeabilization and
blocking was performed as described above. After 3 washes with PBST, the wells were incubated
with Rabbit IgG anti-8-OHdG (Bioss #bs-1278R) at a dilution of 1:100 in 1% BSA in PBST
overnight at 4°C. Next, cells were washed 3 times with PBST and incubated with Alexa Fluor 647
Goat anti-Rabbit IgG (Invitrogen #a21245) in 1% BSA in PBST for 1 hour at room temperature.
Following 3 PBS washes, nuclei were counterstained with DAPI (1 µg/mL) for 10 minutes at room
temperature. Cells were washed in PBS a final 3 times and left covered in 100 µL of PBS during
imaging. 20x magnification images were acquired on a Zeiss Axio Vert.A1 inverted microscope
with a Colibri 7 LED light source and an AxioCam MRm camera. Images were subsequently
analyzed in Fiji.
RNA-Seq Library Preparation for VA-free vs. CTRL Muscle Stem Cells
5-6-month-old Pax7-nTnG mice (all males) were either fed VA-free (n=3) or control chow (n=2)
as described above. 15,000-17,000 nGFP+ MuSCs per mouse (isolated from the gastrocnemius and
quadricep muscles) were sorted on the Sony MA900 directly into 400 µL of Trizol for RNA lysis.
After sorting the tubes were promptly vortexed to homogenize the lysates and then quickly frozen
on dry ice before storing at -80°C until later processing. Trizol samples were thawed at room
temperature and the RNA-containing aqueous phase was separated by adding 0.2 mL of
chloroform per 1 mL of Trizol to each tube, vortexing for 10 seconds, and incubating at room
temperature for 2 minutes. The sample tubes were then centrifuged for 15 minutes at 12,000 RCF
at 4°C and the top aqueous phase was carefully transferred to a new tube, where it was mixed with
1.5 volumes of 100% ethanol. At this point, biological replicates were split into two technical
replicates (A & B) to account for spin column volume. The RNA was purified using the Qiagen
RNeasy Micro kit, starting at step 5 of the manufacturer’s protocol for “Purification of Total RNA
from Animal and Human Cells.” RNA was eluted into 12 µL of RNAse-free water. RNA
concentration and quality was measured using the Agilent Bioanalyzer 2100 RNA 6000 Pico kit,
following the manufacturer’s protocol. Samples with a RIN of 9 or higher and sufficient RNA
concentrations were selected for downstream library prep (VA-free Diet replicates 1B, 2A, 3A, 3B
and Control Diet replicates 1A, 1B, 2A, 2B).
The Takara Bio SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing was used for cDNA
synthesis. Following the manufacturer’s protocols A-D in section V of the kit manual, 5 ng of
input RNA was used per sample and 10 PCR cycles were run according to Table 2 of the manual.
QC and concentration measurements were performed using the Agilent Bioanalyzer 2100 High
Sensitivity DNA kit. Illumina library prep was performed with the Nextera XT DNA Library
Preparation Kit and Nextera XT Index Kit, using an input of 500 pg of full-length cDNA amplicons
per sample and following the kit protocols. Illumina libraries were then submitted to the University
of Michigan’s Advanced Genomics Core for pooling and sequencing on the Illumina NextSeq
using 75 cycles and 75 bp single-end reads.
Bulk RNA-Seq Analysis
Fastq files were tested for quality using FastQC, after which they were aligned to the Mus
musculus GRCm38 (Ensembl release 96) reference transcriptome with Kallisto (77). Abundance
matrices were imported with the tximport package into R for differential gene expression analysis
using the DESeq2 package (78). Differentially expressed genes (padj < 0.05) were selected and
genes of interest were plotted using the pheatmap package and ggplot2. Differentially expressed
genes (padj < 0.05 and log2(fold change) >1) in the VA-free samples were then analyzed for GO
term enrichment using the goseq package (79). Over- and under-represented GO terms (p < 0.05)
were selected and GO terms of interest were plotted on a bubble plot using ggplot2. Reaction
fluxes were estimated using genome-scale metabolic modeling using the RECON1 model as
previously reported (32, 37).
Barium Chloride Injury and IHC on VA-free vs. CTRL Muscle Tissue Cross Sections
3-6-month-old female Pax7-nTnG and Pax7-TdTomato were fed either a VA-free (n=3-5) or
control (n=3) for 8 weeks. After 8 weeks, mice were anesthetized with 2% isoflurane and the
tibialis anterior (TA) muscle was injured via a 40µL intramuscular injection of 1.2% barium
chloride in sterile PBS, while the left muscle served as an uninjured contralateral control or served
as a saline injection. The injuries were allowed to regenerate for 7 and 28 days, at which points
mice were euthanized and TAs were carefully dissected, embedded in OCT in a cylindrical mold,
and flash-frozen by submerging in isopentane cooled in a larger container of liquid nitrogen until
OCT was solidified. Frozen tissues were sectioned on a CryoStar NX70 Cryostat at -20°C to obtain
10µm cross sections, which were adhered to positively-charged glass slides and allowed to dry for
30 minutes before proceeding to staining.
For the uninjured controls and 7 days time point, slides were fixed in acetone at -20°C in a coplin
jar for 10 min and air-dried for 10 minutes. Then, sections were outlined with Vector Labs
ImmEdge Hydrophobic Barrier PAP pen. After outlines were dried, sections were rehydrated in
PBS for 5 minutes after room temperature. PBS was then gently aspirated off and slides were
blocked in 10% goat serum in PBS for 1 hour at room temperature in a sealed, hydrated chamber.
Blocking solution was carefully blotted off and primary antibodies were diluted in 10% goat serum
in PBS. Slides stained for CD68+ macrophages were incubated with a 1:50 dilution of Rat IgG
anti-CD68 (Bio Rad MCA1957) and a 1:500 dilution of Rabbit IgG anti-Laminin 1+2 (Abcam
ab7463). Slides stained for Ly6G+ neutrophils were incubated with a 1:50 dilution of Rat IgG anti-
Ly6G (BD Biosciences 550291) and a 1:500 dilution of Rabbit IgG anti-Laminin 1+2 (Abcam
ab7463). Slides were incubated with primary antibodies in a sealed, hydrated chamber overnight
at 4°C. Slides were then washed with PBS 3 times for 5 minutes. Secondary antibodies (Alexa
Fluor 647 Goat anti-Rat IgG, Alexa Fluor 488 Goat anti-Rabbit IgG) were diluted 1:500 and DAPI
was diluted to 1µg/ml in PBS. Slides were incubated with secondary antibodies and DAPI for 1
hour at room temperature in the dark in a sealed, hydrated chamber. Slides were then washed with
PBS 3 times for 5 minutes and coverslips were mounted with a drop of ProLong Diamond Antifade
Mountant. Coverslip mounting medium was allowed to cure overnight at room temperature before
imaging.
For the long-term time point, slides were fixed in acetone at -20°C in a coplin jar for 10 min and
air-dried for 10 minutes. Sections were then outlined with the hydrophobic pen and rehydrated in
PBS for 5 minutes. Samples were incubated with MOM blocking solution for 1 hour following
manufacturer’s instructions. After this, sections were incubated with primary antibodies overnight
at 4°C (Mouse IgG Sarcoglycan- 1:200, Leica biosystems A-SARC-L-CE, Rat IgG CD68- 1:50,
Rabbit IgG RFP- 1:50, Rockland 600-401-379). Samples were then rinsed 3x5 minutes with PBS
and incubated with secondary antibodies (Alexa Fluor 488 Goat anti-mouse-1:500, Alexa Fluor
555 Goat anti-rabbit- 1:500 and Alexa Fluor 647 Goat anti-rat-1:500) for 2 hours at room
temperature. Tissues were rinsed again 3x5 minutes with PBS and incubated with DAPI for 10
minutes, rinsed 2x5minutes and mounted with 100 µl of ProLong Diamond Antifade Mountant.
Mounting medium was allowed to cure overnight at room temperature before imaging.
Stained sections were imaged in duplicate per biological replicate at 20x on a Nikon A1si inverted
confocal microscope using ND Large Image acquisition and stitching. Image files were analyzed
in Fiji to calculate fiber area, centrally nucleated fibers, CD68+ stained area, and Ly6G+ stained
area. For the long-term time point, 3 tissue sections across the length of the muscle were imaged
per biological replicate at 20X on a Zeiss Laser Scanning Microscopy 900 with Airyscan 2. Outline
of fibers was obtained using a trained model in CellPose with the cyto3 model as a base (80).
Labels were then converted to regions of interest in Fiji to obtain fiber cross-sectional area (CSA)
(81). Positive Tdtomato+ fibers were extracted using the mean fluorescence intensity values of the
Tdtomato+ channel for each fiber. Centrally nucleated fibers and cell density of CD68+ cells and
Tdtomato+ were manually counted per tissue section.
Stra6 Expression in Freshly Isolated, Activated, and Differentiated Muscle Stem Cells
MuSCs were isolated via FACs from C57BL/6 wildtype mice (4 months old, 2 males and 1 female)
as described above and pooled together to ensure high enough yield. Cells were then split into 3
timepoints with n=2 replicates per timepoint. For the quiescent timepoint, cells were immediately
pelleted for 10 minutes at 350 RCF, lysed in 350 µL of Qiagen Buffer RLT containing 1% 2-
mercaptoethanol, and stored at -80°C. The remaining cells were cultured in a 24-well plate in
myoblast growth medium for 3 days. At 3 days, 2 of the wells were lysed directly in the plate with
350 µL of Qiagen Buffer RLT containing 1% 2-mercaptoethanol and stored at -80°C, while the
remaining wells were switched to differentiation medium (DMEM, 2% horse serum, 1%
penicillin/streptomycin). After 3 days of differentiation, the final wells were lysed as described
above. RNA isolation was performed using the Qiagen RNeasy Micro kit, following the kit’s
instructions. RNA concentrations and quality were measured using a NanoDrop
Spectrophotometer and Qubit Fluorometer using the RNA High Sensitivity Assay kit. Invitrogen’s
SuperScript III First-Strand Synthesis System was used for cDNA synthesis (using the provided
oligo dT primers), according to the manufacturer’s instructions.
Stra6 and Gapdh primer assays were purchased from IDT (Stra6: Mm.PT.58.13854804, Gapdh
Mm.PT.39a.1) and were reconstituted in the manufacturer’s recommended volume of PCR-grade
water for a 10x stock concentration. RT-qPCR master mixes were prepared for a 50 µL reaction
volume containing 25 µL of Power SYBR Green Master Mix (Applied Biosystems), 5 µL of
primers, and 0.5 ng of cDNA template diluted in PCR-grade water. RT-qPCR was performed on
the Applied Biosystems QuantStudio 3 Real-Time PCR System with 2 technical replicates per
sample and a blank water negative control to check for primer dimers. The ∆∆Ct method was then
used to find relative gene expression of Stra6 between the timepoints.
Statistical Analyses
For imaging, qPCR, and Seahorse measurements, data was imported into RStudio and statistical
tests were performed using the RStatix package. For comparisons between two groups,
comparisons were made with one-sided Student’s t-tests assuming unequal variance. For
comparisons between more than two groups, comparisons were made by one-way ANOVA
followed by post-hoc pairwise t-tests with Bonferroni correction. Data was plotted using the ggplot
package and annotated with the results of corresponding t-tests.
Study approval
All procedures were approved by the University Committee on the Use and Care of Animals at
UM and the IACUC in Ann Arbor, Michigan, USA (protocol no. PRO000010663).
Data Availability
Bulk RNA-Seq and scRNA-Seq datasets (raw files and processed matrices) are publicly available
on GEO (GSE268616 and GSE268617). Supporting data values for immunostains are provided in
the Related Data table.
Author contributions
P.M.F., P.D., V.F., L.A., J.A.C.M., V.T.W., S.D.G., C.P., K.I., J.L. performed experiments.
P.M.F., P.D., B.A.Y., S.D.G., analyzed data. P.M.F and C.A.A. designed the experiments and
wrote the manuscript with additions from other authors.
ACKNOWLEDGEMENTS
The authors thank the University of Michigan DNA Sequencing Core for assistance with
sequencing and members of the Aguilar laboratory. Research reported in this publication was
partially supported by a Genentech Research Award (CAA), the 3M Foundation (CAA), American
Federation for Aging Research Grant for Junior Faculty (CAA), the Department of Defense and
Congressionally Directed Medical Research Program W81XWH2010336 and W81XWH2110491
(CAA), a National Science Foundation CAREER award (2045977), Defense Advanced Research
Projects Agency (DARPA) “BETR” award D20AC0002 (CAA) awarded by the U.S. Department
of the Interior (DOI), Interior Business Center, Hevolution HF-AGE award (CAA), the National
Science Foundation Graduate Research Fellowship Program under DGE 1841052 (P.M.F.). The
content is solely the responsibility of the authors and does not necessarily represent the official
views of the Department of Defense or National Science Foundation, the position or the policy of
the Government, and no official endorsement should be inferred.
Competing interests
The authors declare no competing interests.
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Figure 1
A B
DAPI
CTRL
VA-Free
8-OHdG
CTRL
VA-Free
DAPI Pax7 MyoD
F HG I
D
E
8 weeks
Young Adult
Pax7-nTnG
mice
Control Diet
VA-free Diet
or
Muscle stem cell
isolation, imaging,
& clonal assay
C
Figure 1: Dietary depletion of Vitamin Ainduces premature activation and oxidative damage in
muscle stem cells.(A) Experiment schematic whereby young mice were fed either aVitamin A-deficient or
control diet for atotal of 8 weeks, after which muscle stem cells were isolated and profiled.(B)
Representative images of Pax7-nGFP and MyoD fluorescence of freshly isolated and fixed MuSCs from
CTRL diet (top row) and VA-deficient diet (bottom row).DAPI -blue, Pax7 - green, MyoD - red. Scale bar =
20 µm.(C, D) Quantification of mean fluorescence intensity of Pax7-nGFP and MyoD, respectively.
Comparisons made via t-test with n=3-4 wells per diet.(E, F) Quantification of Ki67 and MitoTracker Deep
Red (respectively) in MuSCs fixed immediately after isolation from mice receiving CTRL diet (red) or VA-
free diet (blue). Comparisons made via t-test with n=3-4 wells per diet.(G) Proportion of colony-forming
single MuSCs isolated from mice receiving CTRL diet (red) or VA-free diet (blue) after 5 days in growth
conditions. Comparisons made via t-test with n=4 mice (60 wells per mouse) for the control diet, and n=3
mice (95 wells per mouse) for VA-free diet.(H) Representative images of 8-OHdG fluorescence of freshly
isolated and fixed MuSCs from CTRL diet (top row) and VA-free diet (bottom row).DAPI –blue, 8-OHdG -
red. Scale bar =20 µm.(I) Quantification of mean fluorescence intensity of 8-OHdG in MuSCs fixed
immediately after isolation from mice receiving CTRL diet (red) or VA-free diet (blue).n=6image fields
(across 2 mice) per diet.Data are represented as average across samples with error bars showing the
S.E.M. (*:p<0.05,**:p<0.01,***:p<0.001,****: p < 0.0001 for all comparisons).
Figure 2
B C
8 weeks
Young Adult
Control Diet
VA-free Diet
or
Muscle
Stem Cell
Isolation
Gene
Expression
Metabolic
Flux
Analysis
A
E
F
D
VA-FreeCTRL Diet
Reaction flux difference z-score
Up in VA-freeUp in Control
TCA
Cycle
Glycolysis
Pentose
Phosphate
Figure 2: Vitamin Adepletion disrupts muscle stem cell metabolism, mitochondria, and cell cycle
regulation.(A) Experiment schematic: young mice were fed either aVitamin A-deficient or control diet for a
total of 8 weeks, after which muscle stem cells were isolated via FACS and RNA was extracted for RNA-
Seq,followed by differential gene expression, GO Term enrichment, and metabolic flux model analyses. (B)
Heatmap of z-scores for all differentially expressed genes with p-adjusted value<0.05 from muscle stem
cells isolated from control and vitamin-A free diet-fed young mice. (C) Dot-bar plots of selected differentially
expressed genes related to Vitamin A metabolism and RA signaling. Bars represent average across
samples with error bars showing the S.E.M. (D) Bubble plots of selected overrepresented GO terms across
differentially expressed genes.(E) Bubble plot of selected underrepresented GO terms across differentially
expressed genes.(F) Escher map of statistically significant metabolic fluxes predicted by metabolic flux
model.Red: Enriched fluxes in vitamin-A free diet, Blue: Enriched fluxes in control diet.
Agonists+ATRA DMSO
0
20
40
60
80
EdU+ cells/mm2
**
Agonists+ATRA DMSO
1.0
1.5
2.0
Ferret diameter
of mitochondria (µm)/cell
*
AB DC
E
Mitochondrial
Stress Testing
Muscle
Progenitors
Agonists
+ ATRA
Figure 3
DMSOAgo + ATRA
DAPI/SYNJ2BP
2 µm
2 µm
3D reconstruction
x
y
z
FG
Figure 3: Small molecule agonists targeting retinoic acid signaling improves mitochondrial function
and reduces reactive oxygen species.(A) Schematic depicting strategy to upregulate RA signaling by
using Rarɣ and Rxr⍺agonists (CD3254,BMS961)and ATRA as aligand (each 100nM).(B) Line graphs of
oxygen consumption rate (OCR) measured via Seahorse XFe96 Mito Stress Test in C2C12streated with
ATRA and agonists (red, n = 12 wells) and DMSO vehicle control (blue, n=12 wells) after injections of
oligomycin, FCCP, and Rotenone/Antimycin A. (C-D) Quantification of proton leak and OCR/ECAR ratio,
respectively, in C2C12streated with ATRA and agonists (red) and DMSO vehicle control (blue).
Comparisons of Seahorse Mito Stress parameters were made via t-test. (E) 3D projection and 3D
reconstruction of single MuSCs from Pax7CreERT2-Rosa26CAG-LSL-EGFP-3xHA-OMM mice showing individual
mitochondria after cellular treatment with DMSO control (top) or ATRA and agonists (bottom). Scale bar = 2
µm.(F) Quantification of three-dimensional Feret diameter between MuSCs treated with DMSO control and
ATRA and agonists groups. Comparison made via Mann-Whitney test for non-parametric distributed data
with n=4wells per treatment.Data represented as median with interquartile range. (G) Quantification of
cellular density of EdU+MuSCs between MuSCs treated with DMSO vehicle control (blue) or agonists and
ATRA (red). Comparison made via t-test with n=6wells per treatment.
E F
Single-Cell
RNA-Seq
Aged Mice (24
months)
+/-Treatment
G
HI
+
Genome-scale
single cell
metabolic flux
analysis
Control Agonist+ATRA
NAD Metabolism
NMNAT
Up in
Control
Up in Agonist
+ATRA
Fatty Acid Oxidation
Figure 4
Up in
Control
Up in Agonist
+ATRA
ADAPI CM-H2TMRos Mitotracker
Ago + ATRA DMSO
BC D
Figure 4: Repletion of Retinoic Acid Signaling Reduces Oxidative Stress and Promotes Metabolism
Supportive of Quiescence in Muscle Stem Cells.(A) Representative images of mitochondrial ROS
labeled with MitoTracker Orange CM-H2TMRos (yellow) and total mitochondria labeled with MitoTracker
Deep Red (magenta) in aged MuSCs treated with DMSO vehicle control (top) or 100nM agonists and ATRA
(bottom).DAPI counterstain is shown in blue. Scale bar =50 µm.(B-D) Quantification of MitoTracker
Orange CM-H2TMRos, MitoTracker Deep Red, and 8-OHdG mean fluorescence intensity between aged
MuSCs treated with DMSO vehicle control or agonists and ATRA.Data are represented as averages across
samples with error bars showing the S.E.M (*:p<0.05,**:p<0.01,***:p<0.001,****: p < 0.0001). (E)
Schematic depicting strategy to upregulate RA signaling in aged mice through intramuscular injections of
CD3254,BMS961,and ATRA, followed by single cell RNA-Seq of muscle cell suspensions and genome-
scale metabolic flux balance analysis of single cell transcriptomes. (F) Annotated UMAP of cell clusters
obtained in the merged scRNA-Seq dataset of treated and untreated aged cells. (G) Sub-clustering of the
MuSC subset in the treated (right) and untreated (left) samples. (H) Violin plots of selected differentially
expressed genes found via DESeq2 (pval < 0.1 & abs(log2FC) > 0.15)Top row:Pax7, Id1, Sirt2. Bottom
row: Rock2, Mt1, Mt2. (I) Volcano plot of differentially expressed metabolic fluxes in the fatty acid oxidation
and NAD metabolism subsystems determined by Compass analysis. Data are represented as averages
across samples with error bars showing the S.E.M. Statistical comparisons were made via paired t-test. (*:
p<0.05,**:p<0.01,***:p<0.001,****: p < 0.0001).
D
Figure 5
C
DAPI Stra6 Merge
Ago + ATRA DMSO
E F GH
DAPI MyoD Merge
Ago + ATRA DMSO
DAPI Stra6 Merge
Aged Young
B
A
Freshly Isolated
Myoblast
Differentiated
Stra6
Figure 5: The vitamin Areceptor Stra6is attenuated with stem cell activation and aging.(A)
Schematic depicting how Stra6 expression decreases with muscle stem cell activation and differentiation.
(B) Quantification of Stra6 expression fold change (relative to Gapdh)in freshly isolated MuSCs, activated
myoblasts (72 hours in culture with growth medium), and differentiated myotubes (an additional 72 hours in
culture with differentiation medium).qPCR was run using 2 biological replicates and 2 technical replicates
per time point.(C) Quantification of Stra6mean fluorescence intensity (MFI) between freshly isolated young
(3-4 months) and aged (22 months) MuSCs. Tissues from 2 biological replicates (C57Bl/6 females) were
used per age group and pooled during MACS isolation before seeding into a96-well plate, fixing, and
labeling for Stra6. n=8wells were imaged per age group.(D) Mean fluorescence intensity of Stra6was
averaged and compared between age groups using a t-test. (E) Representative images of Stra6
immunofluorescence staining in old aged MuSCs (pooled from n=224-month-old female C57Bl/6 mice)
treated with DMSO vehicle control (top) or agonists and ATRA (bottom) for 3 days. Stra6–green, DAPI -
blue. Scale bar =50 µm.(F) Quantification of Stra6mean fluorescence intensity between old aged MuSCs
treated with DMSO vehicle control (blue) or agonists and ATRA (red). Comparison made via t-test with n=4
wells per treatment.(G) Representative images of MyoD immunofluorescence staining in old aged MuSCs
treated with DMSO vehicle control (top) or agonists and ATRA (bottom). MyoD – yellow, DAPI -blue. Scale
bar =50 µm.(H) Quantification of MyoD mean fluorescence intensity between aged MuSCs treated with
DMSO vehicle control or agonists and ATRA. Comparison made via t-test with n=4wells per treatment.
DAPIJC1 Red
Control
Merge
Stra6 KD
DAPIJC1 Red
Merge
A
Figure 6
EFI
GH
B DC
Figure 6: Stra6loss induces mitochondrial dysfunction.(A) Schematic depicting the role of Stra6in
mitigating mitochondrial ROS and cellular functions such as transcription of retinoic acid response elements
(RAREs) and the transport of retinol.(D) Quantification of Ki67 mean fluorescence intensity (n = 6 image
fields across 2 wells per condition) after siRNA knockdown of Stra6(blue) or negative control (red).(E)
Quantification of mitochondrial ROS using MitoTracker Orange CM-H2TMRos normalized to total
mitochondrial stained by MitoTracker Deep Red (n = 3 wells per condition). (F, G) Quantification of
mitochondrial membrane depolarization via JC-1 labeling (represented as ratio of red/green fluorescence; n
= 3 wells per condition) and representative images of red JC-1 aggregates indicating healthy, polarized
mitochondria and Hoechst-counterstained nuclei (scale bars =100 µm;Stra6 knockdown cells on the right
and negative control siRNA cells on the left).(H) Line graphs of oxygen consumption rate (OCR) measured
via Seahorse XFe96 Mito Stress Test in Stra6 knockdown cells (blue line, n=11 wells) and negative control
cells (red line, n=12 wells) after injections of oligomycin, FCCP, and Rotenone/Antimycin A. (I-L)
Quantification of OCR during basal cell respiration, change in OCR related to ATP production, proton leak,
and OCR/ECAR ratio, respectively, in Stra6 knockdown cells and negative control cells. Comparisons of
Seahorse Mito Stress parameters were made via t-test.
