Macroautophagy regeneration, and use Ulk1 autophagy to increase skeletal

Macroautophagy
(autophagy) is important in degrading damaged and dysfunction organelles and
protein aggregates accumulated in the cell, and is activated through the Ulk1
pathway (Call et al., 2017; Levine and Klionsky, 2004). Garcia-Prat et al.

(2016) showed that autophagy was needed for the proper function of myogenic stem
cells, which are involved in skeletal muscle regeneration. Call et al. (2017)
revealed the importance of Ulk1 mediated autophagy in skeletal muscle
regeneration and mitochondrial remodeling. These studies did not determine the
amount of Ulk1 autophagy that occurs during skeletal muscle regeneration, the
role it plays in age-related skeletal muscle regeneration, or the relationship
between Ulk1 mediated mitochondrial remodeling and muscle strength. The long-term goals of this study are to further
characterize the role of Ulk1 mediated autophagy in skeletal muscle
regeneration, and use Ulk1 autophagy to increase skeletal muscle regeneration
in patients with muscle injuries. Skeletal muscle injuries result in muscle
weakness, necrosis, and fibrosis (Baoge et al., 2012; Charge and Rudnicki, 2004).

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Fibrosis results in scar tissue, which makes it difficult to treat skeletal
muscle injuries. Therefore, it is important for skeletal muscle regeneration to
start quickly.

                                            

Background

Aim
1: Skeletal muscle is involved
in voluntary muscle movement, is multinucleate and striated; and is important
for maintaining posture and thermoregulation (Charge and Rudnicki, 2004).

Skeletal muscle injuries can occur due to trauma, strenuous exercise, ischemia-reperfusion,
or cardiotoxins. Myogenic stem cells (satellite cells) are responsible for muscle
regeneration (Charge and Rudnicki, 2004). The cells are normally in the inactive
(quiescent) state, but upon injury, they are activated, and they differentiate to
repair and/or form skeletal muscle cells. Call et al. (2017) revealed the
importance of Ulk1 mediated autophagy in skeletal muscle regeneration.

Autophagy is important in degrading damaged and dysfunctional organelles in the
cell, and is initiated under conditions of nutrient deprivation and stress by
AMP-activated protein kinase (AMPK; Call et al. 2017). Autophagy involves the
formation of the autophagosome, which engulfs the damaged cellular components,
and the autolysosome (autophagosome + lysosome), where degradation occurs (Levine
and Klionsky, 2004). AMPK activates the Unc-51-like kinase 1 (Ulk1) pathway,
which involves the autophagy proteins, 1A/1B light chain 3A I (LC3-I) and
LC3-II, polyubiquitin-binding protein p62/SQSTM1, and Beclin1 (p62; Call et
al., 2017). These proteins are involved in autophagosome and autolysosome formation.

Call et al. (2017) used adult (three-month old) mice to research the importance
of autophagy in muscle regeneration. However, autophagic capacity decreases
with age, and autophagy is important in determining the quiescence or
deterioration of satellite cells (Garcia-Prat et al., 2016; Wen and Klionsky,
2016). Quiescent cells can be activated for skeletal muscle regeneration, but deteriorated
cells impair regeneration and lead to muscle mass loss. Therefore, it is
important to investigate the role Ulk1 mediated autophagy plays in age-related
skeletal muscle regeneration. Garcia et al. (2016) investigated the effect
autophagy has on satellite cells or muscle degeneration, but this study will directly
determine the effect a declining autophagic capacity can have on skeletal
muscle regeneration.

 

Aim
2: In skeletal muscle,
mitochondria, through the electron transport chain (ETC), produce the energy
(ATP) required to power muscle movement (Bratic and Trifunovic, 2010). There
are four complexes in the ETC, and one ATP synthase. Electrons travel along the
ETC and are used to reduce oxygen to water, and the protons, accumulated in the
intramembrane space, are used by the ATP synthase to produce ATP (Bratic and
Trifunovic, 2010). During mitochondrial dysfunction, energy production is
impaired, and the ETC complex IV, cytochrome c oxidase (COX IV), levels are
decreased because it is mainly encoded by mitochondrial DNA (Ross, 2011). Complex
II, succinate dehydrogenase (SDH), levels are increased or remain the same
because it is encoded by nuclear DNA (Ross, 2011). Thus, by measuring COX IV
and SDH levels we can differentiate between functional and dysfunctional mitochondrial.

Mitophagy is used to remove damaged and dysfunctional mitochondria from the
cell (Call et al., 2017). Mitophagy is a subset of autophagy, thus, it proceeds
the same way as autophagy. Call et al. (2017) revealed that Ulk1 mediated
autophagy was important for mitochondrial remodeling after skeletal muscle
injury. Mitochondrial remodeling involves two separate processes, fission (which
makes small mitochondria) and fusion (which makes large mitochondria; Gottlieb
and Bernstein, 2016). Zane et al. (2017) revealed that mitochondrial oxidative
capacity effects muscle strength and walking performance. However, the
researchers did not investigate the effect mitochondrial remodeling, after
injury, has on muscle strength. This is an important topic because
mitochondrial remodeling affects the metabolic capacity of mitochondria, which
can change ATP production and impact muscle strength (Choi et al., 2015). Thus,
this study is investigating the effect of remodeled mitochondria on muscle strength.

 

Aim
3: Autophagy is
important in skeletal muscle metabolism, forming new organelles, and cell development
(Fritzen et al., 2016). Call et al., (2017) showed the importance of Ulk1
mediated autophagy is skeletal muscle regeneration, but did not quantify the
amount of autophagy that occurs. Instead, the researchers used LC3-II and p62 immunoblotting
and protein quantification to determine whether autophagy occurs during skeletal
muscle regeneration. In the Ulk1 pathway, LC3-I combines with
phosphatidylethanolamine (PE) to form LC3-II, which goes into the autophagosome
and autolysosome; p62 is responsible for recruiting damaged and dysfunctional
organelles into the autophagosome (Call et al., 2017). Immunoblotting and
quantification for LC3-II and p62 can be used to investigate the autophagic
capacity of the cell, but not autophagic flux (the amount of autophagy
occurring; Mizushima and Yoshimori, 2007). This is because LC3-II levels can be
increased by both increased autophagic activity and inhibited autophagic
degradation; and p62 level are not completely dependent on autophagy (Mizushima
and Yoshimori, 2007; Yoshii and Mizushima, 2017). In this study, to measure autophagic
flux, I will be quantifying the LC3-II labeled puncta in autolysosomes under
the action of a lysosomal inhibitor, chloroquine.

 

Questions and Previous Research

In our previous study, mitochondrial
remodeling was measured using cytochrome c (Cyt C) and cytochrome c oxidase
levels (Call et al., 2017). In this study, ATP will be measured as an indicator
of mitochondrial remodeling and function, because it is more direct. Autophagy
has previously been quantified in cardiomyocytes and the nervous system (Castillo
et al., 2013; Iwai-Kanai et al., 2008). This will be one of the first studies
that quantifies autophagy in skeletal muscle cells. Bafilomycin, an ATPase
inhibitor, has been used in previous studies to inhibit the autophagic
degradation, however, this drug has adverse effects when used in an animal
model (Garcia-Prat et al., 2016; Yoshimori et al., 1991). Therefore, in this
study, I will use chloroquine to inhibit autophagic degradation. All the experiments
in this study will be conducted in-vivo, in mice, because they are a good model
organism for humans.

 

Question
1: Does Ulk1 mediated autophagy
play a role in age-related skeletal muscle regeneration? Call et
al. (2017) revealed the importance of Ulk1 mediated autophagy in skeletal
muscle regeneration, but not the age-related effects. There is a decline in
autophagic capacity with age that negatively impacts satellite cells, which are
needed for skeletal muscle regeneration (Garcia-Prat et al., 2016; Wen and
Klionsky, 2016). I hypothesize that
skeletal muscle regeneration, mediated by Ulk1 autophagy, will decrease with
age.

 

Question
2: Does the degree of
Ulk1 mediated mitochondrial remodeling after skeletal muscle injury affect
muscle strength? Call et al. (2017) showed that Ulk1 is involved
skeletal muscle regeneration and mitochondrial remodeling. However, the role
mitochondrial remodeling plays in skeletal muscle strength has not yet been
investigated. I hypothesize that skeletal
muscle strength will be proportional to functional mitochondria.

 

Question 3: What is the amount of Ulk1 mediated
autophagy that occurs during different times of skeletal muscle regeneration? Autophagy
is important for the function of satellite cells, which are needed to
regenerate skeletal muscle (Garcia-Prat
et al., 2016). I hypothesize that the earlier stages of
skeletal muscle regeneration will have more autophagy.

 

Experimental Design

Aim
1: To determine the role Ulk1 mediated autophagy plays in age related skeletal
muscle regeneration. To
determine the age-related effects, I will use mice that are one, three, and
twenty months old. I will use one month old mice as a model for young mice
because these mice are done growing, and postnatal development will not act as
a confounding variable with skeletal muscle regeneration (White et al., 2010).

I will use three months old mice to model adult mice based on our previous
study (Call et al., 2017). I will use twenty months old mice to model old mice
because these mice are not yet undergoing sarcopenia (muscle mass loss; Rai et
al., 2014). Skeletal muscle regeneration occurs differently in males versus
females (McHale et al., 2010). Thus, to separate the age and gender related
effects, in this study, I will only be using male mice. In this experiment, I
will make Ulk1 knockout (KO) and wildtype (WT) mice using the Cre/LoxP method
described in a previous study (Call et al., 2017). Inducible Ulk1 KO (iKO) and
non-inducible Ulk1 KO (nKO) mice will be used to determine if there were any compensatory
effects of Ulk1 KO. In iKO mice, Ulk1 will be knocked out using a previously
described method (Call et al., 2017). In this experiment, each age group will
have four treatment groups: WT mice without I/R (basal control), WT mice with
I/R (experimental), KO without I/R (control), KO with I/R (experimental); iKO
and nKO mice will undergo the same treatments. The left hind limb’s skeletal
muscle will be injured using ischemia reperfusion injury (I/R), this will be
done using a previously described method (Call et al., 2017). The uninjured
right hind limb will serve as an internal control. The gastrocnemius muscle will
be used for further analysis. Muscle regeneration will be observed on Day 1
(D1), D3, D7, D11, and D14 post-injury.

In this
experiment, the in-vivo maximal isometric torque, which measures skeletal
muscle strength, will be used as a proxy for skeletal muscle regeneration. The
ankle plantarflexors in the left hind limb will be used for the measurement,
which will be conducted using a previously described method (Call et al.,
2017). After isometric torque analysis, the mice will be sacrificed. A
morphological analysis will be conducted on the muscle to visually observe
skeletal muscle regeneration. The muscle tissue will be cut into sections using
a cryostat and stained in hematoxylin and eosin (allows for better tissue visualization)
and non-consecutive tissue sections will be visualized, using a previously
described method (Lancioni et al., 2011). Muscle regeneration will be observed and
scored on a scale of 1-5, where 1 = little to no regeneration, 2-4 = increasing
regeneration, 5 = regenerated and control muscle look same. This will allow a
comparison between the muscle of different aged mice.

An
immunoblot analysis and protein quantification will be carried out, using the
gastrocnemius muscle, to test for the presence of autophagy proteins (Ulk1,
beclin1, LC3, p62, LC3II), a muscle specific protein (myogenin), and control
proteins (ß-actin and GAPDH), following a previously described method (Call et
al., 2017). The antibodies used will be: Ulk1 (ab128859, Abcam), Beclin1
(ab62557, Abcam), LC3C (ab167421, Abcam), SQSTM1/p62 (ab227207, Abcam), LC3B
(ab192890, Abcam), Myogenin (ab1835, Abcam), Beta Actin (ab8226, Abcam), and
GAPDH (ab9484, Abcam). The results of the in-vivo torque measurement,
morphological analysis, and immunoblot and protein quantification will be
compared between the different treatment groups and age groups to determine how
Ulk1 KO affects skeletal muscle regeneration in different age groups.

 

Aim 2: To determine if the degree of Ulk1 mediated
mitochondrial remodeling after skeletal muscle injury affects muscle strength. In
this aim, I will be using adult mice
(three months old), Ulk1 WT and KO (iKO and nKO), that will be made using the
Cre/LoxP system (Call et al., 2017). The same method that was used in Aim 1
will be used to induce Ulk1 KO in iKO mice. The four treatment groups in this
experiment will be: WT mice
without cardiotoxin (Ctx; basal control), WT mice with Ctx (experimental), KO
without Ctx (control), KO with Ctx (experimental); iKO and nKO mice will
undergo the same treatments. All treatments will use male mice. The mice will
be injured on the left tibialis anterior (TA) with Ctx (0.071 mg/ml;
Calbiochem) using a previously described method (Call et al.,
2017). The left TA of the uninjured mice will be injected with saline. Ctx, as
opposed to I/R, was used to injure the mice because it results in significant
differences in mitochondrial remodeling between days 1-14 (Call et al., 2017).

Mitochondrial remodeling and muscle strength will be observed on D1, D3, D7,
D11, and D14 post-injury. Ctx has a half-life of 13.6 hours, thus, it will be
degraded before the first mitochondrial remodeling and muscle strength
measurements, and will not cause further injury (Yap et al., 2014).

An in-vivo maximal isometric torque analysis will be used to measure muscle strength,
using a previously described method (Call et al., 2017). The ankle dorsiflexors
in the left hind limb will be used for the measurement. After isometric torque
analysis, the mice will be sacrificed and the TA muscle will be dissected. For half
of the mice, the mitochondria will be isolated from the muscle by tissue
homogenization (Teflon Potter Elvehjem homogenizer) and centrifugation, using a
previously described method (Frezza et al., 2007). Mitochondrial function will be
assessed by measuring ATP because functional, and not dysfunctional,
mitochondria produce ATP (Lanza and Nair, 2009). ATP production in the isolated mitochondria will be
measured using a previously described bioluminescent method (Lanza and Nair,
2009). In this method, luciferase is used to catalyze the luciferase + ATP reaction to produce
intermediate products (luciferyl adenylate + PPi), which are converted to
oxyluciferin + AMP + light. Then, the amount of ATP produced by the
mitochondria is determined using a luminometer, which measures the bioluminescence
of the sample (Lanza and Nair,
2009). For the second half of the mice, the TA muscle will be used for an
immunoblot analysis and protein quantification of proteins related to the ETC (SDH, COX IV,
Cyt C) and control proteins (ß-actin
and GAPDH); this will be done using
a previously described method (Call et al., 2017). The blot will serve as a proxy
for mitochondrial function because during mitochondrial dysfunction COX IV
levels decrease and SDH levels increase (Ross, 2011). The antibodies used will
be: COX IV (#4844, Cell Signaling
Technology), Cyt C (ab13575, Abcam), SDHB (ab14714, Abcam), Beta Actin
(ab8226, Abcam), and GAPDH (ab9484, Abcam).

 

Aim
3: To use chloroquine to determine the amount of Ulk1 mediated autophagy that
occurs during different times of skeletal muscle regeneration. In this aim, I will be using CAG-RFP-EGFP-LC3 transgenic
mice that express RFP (red florescent protein) and EGFP (enhanced green
florescent protein) under a CAG promoter/enhancer sequence (027139, Jackson Laboratory). I will be using two
months old (oldest mice I can get) male mice. Autophagy can be tracked using
EGFP and RFP because both are present during autophagosome formation, but only
RFP is present in the autolysosome because EGFP is degraded in acidic pHs (Zhou
et al., 2012). In this experiment, there will be four treatment groups:
uninjured mice without chloroquine (basal level control), uninjured mice with
chloroquine (experimental), injured mice without chloroquine (control), injured
mice with chloroquine (experimental). The mice will be injured with I/R on the
left hind limb using a previously described method; control mice will not be
injured (Call et al., 2017). In the injured mice, the uninjured right hind limb
will serve as an internal control. The gastrocnemius muscle will be selected
for further analysis. Chloroquine inhibits autophagic degradation by increasing
the pH of the autolysosome (to approximately 6.5), which prevents the last
phase of autophagy from occurring (Iwai-Kanai et al., 2008; Ohkuma and Poole,
1978). Thus, the amount of autophagy can be determined by observing the LC3
puncta that are part of the autolysosome. A dose-response curve will be used to
determine the optimal chloroquine (C6628, Sigma-Aldrich) concentration (1mg/kg,
5mg/kg, 10mg/kg, 15mg/kg, or 20mg/kg) because previous studies have used the
drug in cardiomyocytes, not skeletal muscle cells (Iwai-Kanai et al., 2008). The
optimal concentration of chloroquine will be administered at the different time
points that skeletal muscle regeneration will be observed. Chloroquine will be
injected intraperitoneally into the left hind limb using a previously described
method (Iwai-Kanai et al., 2008). Control mice will be injected with saline. In
mice treated with chloroquine, EGFP will not degrade because of the increased
autolysosomal pH (Iwai-Kanai et al., 2008). Thus, monodansylcadaverine (MDC;
D4008 Sigma-Aldrich) will be used to label autolysosomes because its function
is not dependent on pH (Iwai-Kanai et al., 2008). For consistency, all mice
will have MDC (1.5mg/kg) injected intraperitoneally in left hind limb using a
previously described method (Iwai-Kanai et al., 2008). Autophagy and skeletal
muscle regeneration will be observed on D1, D3, D7, D11, and, D14 post-injury.

The gastrocnemius muscle will
be dissected, and the tissue will be sectioned and prepared for widefield
florescence microscopy using a previously described method (Iwai-Kanai et al.,
2008). Non-consecutive tissue sections will be visualized. In chloroquine
untreated and treated tissue, RFP-LC3 or EGFP-RFP-LC3 puncta, respectively,
co-localized with MDC will be observed and counted as a measure of autophagy. An
immunoblot analysis and protein quantification will be carried out on the
muscle tissue for LC3, myogenin, ß-actin and GAPDH using a previously described
method (Call et al., 2017). The antibodies used will be: LC3B (ab192890,
Abcam), Myogenin (ab1835, Abcam), Beta Actin (ab8226, Abcam), and GAPDH
(ab9484, Abcam). The results of the fluorescence microscopy, immunoblot and
protein quantification will be compared between the different treatment groups
and skeletal muscle regeneration times to determine whether the amount of
autophagy differs.

 

Summary

This research will investigate the
age-related effects of Ulk1 mediated autophagy in skeletal muscle regeneration.

It will also determine the relationship between Ulk1 mediated mitochondrial
remodeling and skeletal muscle strength. Lastly, this study will quantify the
autophagy occurring during different days of skeletal muscle regeneration. This
research will contribute to the long-term goal of using Ulk1 mediated autophagy
to treat patients with skeletal muscle injuries and hasten their muscle
regeneration.