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Macroautophagy(autophagy) is important in degrading damaged and dysfunction organelles andprotein aggregates accumulated in the cell, and is activated through the Ulk1pathway (Call et al., 2017; Levine and Klionsky, 2004). Garcia-Prat et al.(2016) showed that autophagy was needed for the proper function of myogenic stemcells, which are involved in skeletal muscle regeneration. Call et al.

(2017)revealed the importance of Ulk1 mediated autophagy in skeletal muscleregeneration and mitochondrial remodeling. These studies did not determine theamount of Ulk1 autophagy that occurs during skeletal muscle regeneration, therole it plays in age-related skeletal muscle regeneration, or the relationshipbetween Ulk1 mediated mitochondrial remodeling and muscle strength. The long-term goals of this study are to furthercharacterize the role of Ulk1 mediated autophagy in skeletal muscleregeneration, and use Ulk1 autophagy to increase skeletal muscle regenerationin patients with muscle injuries.

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Skeletal muscle injuries result in muscleweakness, necrosis, and fibrosis (Baoge et al., 2012; Charge and Rudnicki, 2004).Fibrosis results in scar tissue, which makes it difficult to treat skeletalmuscle injuries.

Therefore, it is important for skeletal muscle regeneration tostart quickly.                                             BackgroundAim1: Skeletal muscle is involvedin voluntary muscle movement, is multinucleate and striated; and is importantfor 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 muscleregeneration (Charge and Rudnicki, 2004). The cells are normally in the inactive(quiescent) state, but upon injury, they are activated, and they differentiate torepair and/or form skeletal muscle cells. Call et al. (2017) revealed theimportance of Ulk1 mediated autophagy in skeletal muscle regeneration.Autophagy is important in degrading damaged and dysfunctional organelles in thecell, and is initiated under conditions of nutrient deprivation and stress byAMP-activated protein kinase (AMPK; Call et al.

2017). Autophagy involves theformation of the autophagosome, which engulfs the damaged cellular components,and the autolysosome (autophagosome + lysosome), where degradation occurs (Levineand 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) andLC3-II, polyubiquitin-binding protein p62/SQSTM1, and Beclin1 (p62; Call etal., 2017). These proteins are involved in autophagosome and autolysosome formation.Call et al.

(2017) used adult (three-month old) mice to research the importanceof autophagy in muscle regeneration. However, autophagic capacity decreaseswith age, and autophagy is important in determining the quiescence ordeterioration of satellite cells (Garcia-Prat et al., 2016; Wen and Klionsky,2016). Quiescent cells can be activated for skeletal muscle regeneration, but deterioratedcells impair regeneration and lead to muscle mass loss. Therefore, it isimportant to investigate the role Ulk1 mediated autophagy plays in age-relatedskeletal muscle regeneration.

Garcia et al. (2016) investigated the effectautophagy has on satellite cells or muscle degeneration, but this study will directlydetermine the effect a declining autophagic capacity can have on skeletalmuscle regeneration. Aim2: In skeletal muscle,mitochondria, through the electron transport chain (ETC), produce the energy(ATP) required to power muscle movement (Bratic and Trifunovic, 2010). Thereare four complexes in the ETC, and one ATP synthase. Electrons travel along theETC and are used to reduce oxygen to water, and the protons, accumulated in theintramembrane space, are used by the ATP synthase to produce ATP (Bratic andTrifunovic, 2010). During mitochondrial dysfunction, energy production isimpaired, and the ETC complex IV, cytochrome c oxidase (COX IV), levels aredecreased because it is mainly encoded by mitochondrial DNA (Ross, 2011).

ComplexII, succinate dehydrogenase (SDH), levels are increased or remain the samebecause it is encoded by nuclear DNA (Ross, 2011). Thus, by measuring COX IVand SDH levels we can differentiate between functional and dysfunctional mitochondrial.Mitophagy is used to remove damaged and dysfunctional mitochondria from thecell (Call et al.

, 2017). Mitophagy is a subset of autophagy, thus, it proceedsthe same way as autophagy. Call et al. (2017) revealed that Ulk1 mediatedautophagy was important for mitochondrial remodeling after skeletal muscleinjury. Mitochondrial remodeling involves two separate processes, fission (whichmakes small mitochondria) and fusion (which makes large mitochondria; Gottlieband Bernstein, 2016).

Zane et al. (2017) revealed that mitochondrial oxidativecapacity effects muscle strength and walking performance. However, theresearchers did not investigate the effect mitochondrial remodeling, afterinjury, has on muscle strength. This is an important topic becausemitochondrial remodeling affects the metabolic capacity of mitochondria, whichcan change ATP production and impact muscle strength (Choi et al., 2015).

Thus,this study is investigating the effect of remodeled mitochondria on muscle strength. Aim3: Autophagy isimportant in skeletal muscle metabolism, forming new organelles, and cell development(Fritzen et al., 2016). Call et al., (2017) showed the importance of Ulk1mediated autophagy is skeletal muscle regeneration, but did not quantify theamount of autophagy that occurs. Instead, the researchers used LC3-II and p62 immunoblottingand protein quantification to determine whether autophagy occurs during skeletalmuscle regeneration. In the Ulk1 pathway, LC3-I combines withphosphatidylethanolamine (PE) to form LC3-II, which goes into the autophagosomeand autolysosome; p62 is responsible for recruiting damaged and dysfunctionalorganelles into the autophagosome (Call et al.

, 2017). Immunoblotting andquantification for LC3-II and p62 can be used to investigate the autophagiccapacity of the cell, but not autophagic flux (the amount of autophagyoccurring; Mizushima and Yoshimori, 2007). This is because LC3-II levels can beincreased by both increased autophagic activity and inhibited autophagicdegradation; and p62 level are not completely dependent on autophagy (Mizushimaand Yoshimori, 2007; Yoshii and Mizushima, 2017). In this study, to measure autophagicflux, I will be quantifying the LC3-II labeled puncta in autolysosomes underthe action of a lysosomal inhibitor, chloroquine.

 Questions and Previous ResearchIn our previous study, mitochondrialremodeling was measured using cytochrome c (Cyt C) and cytochrome c oxidaselevels (Call et al., 2017). In this study, ATP will be measured as an indicatorof mitochondrial remodeling and function, because it is more direct. Autophagyhas previously been quantified in cardiomyocytes and the nervous system (Castilloet al., 2013; Iwai-Kanai et al., 2008). This will be one of the first studiesthat quantifies autophagy in skeletal muscle cells. Bafilomycin, an ATPaseinhibitor, has been used in previous studies to inhibit the autophagicdegradation, however, this drug has adverse effects when used in an animalmodel (Garcia-Prat et al.

, 2016; Yoshimori et al., 1991). Therefore, in thisstudy, I will use chloroquine to inhibit autophagic degradation. All the experimentsin this study will be conducted in-vivo, in mice, because they are a good modelorganism for humans.

 Question1: Does Ulk1 mediated autophagyplay a role in age-related skeletal muscle regeneration? Call etal. (2017) revealed the importance of Ulk1 mediated autophagy in skeletalmuscle regeneration, but not the age-related effects. There is a decline inautophagic capacity with age that negatively impacts satellite cells, which areneeded for skeletal muscle regeneration (Garcia-Prat et al., 2016; Wen andKlionsky, 2016).

I hypothesize thatskeletal muscle regeneration, mediated by Ulk1 autophagy, will decrease withage. Question2: Does the degree ofUlk1 mediated mitochondrial remodeling after skeletal muscle injury affectmuscle strength? Call et al. (2017) showed that Ulk1 is involvedskeletal muscle regeneration and mitochondrial remodeling. However, the rolemitochondrial remodeling plays in skeletal muscle strength has not yet beeninvestigated.

I hypothesize that skeletalmuscle strength will be proportional to functional mitochondria. Question 3: What is the amount of Ulk1 mediatedautophagy that occurs during different times of skeletal muscle regeneration? Autophagyis important for the function of satellite cells, which are needed toregenerate skeletal muscle (Garcia-Pratet al., 2016). I hypothesize that the earlier stages ofskeletal muscle regeneration will have more autophagy. Experimental DesignAim1: To determine the role Ulk1 mediated autophagy plays in age related skeletalmuscle regeneration. Todetermine the age-related effects, I will use mice that are one, three, andtwenty months old.

I will use one month old mice as a model for young micebecause these mice are done growing, and postnatal development will not act asa confounding variable with skeletal muscle regeneration (White et al., 2010).I will use three months old mice to model adult mice based on our previousstudy (Call et al.

, 2017). I will use twenty months old mice to model old micebecause these mice are not yet undergoing sarcopenia (muscle mass loss; Rai etal., 2014). Skeletal muscle regeneration occurs differently in males versusfemales (McHale et al., 2010). Thus, to separate the age and gender relatedeffects, in this study, I will only be using male mice. In this experiment, Iwill make Ulk1 knockout (KO) and wildtype (WT) mice using the Cre/LoxP methoddescribed in a previous study (Call et al.

, 2017). Inducible Ulk1 KO (iKO) andnon-inducible Ulk1 KO (nKO) mice will be used to determine if there were any compensatoryeffects of Ulk1 KO. In iKO mice, Ulk1 will be knocked out using a previouslydescribed method (Call et al., 2017). In this experiment, each age group willhave four treatment groups: WT mice without I/R (basal control), WT mice withI/R (experimental), KO without I/R (control), KO with I/R (experimental); iKOand nKO mice will undergo the same treatments.

The left hind limb’s skeletalmuscle will be injured using ischemia reperfusion injury (I/R), this will bedone using a previously described method (Call et al., 2017). The uninjuredright hind limb will serve as an internal control.

The gastrocnemius muscle willbe used for further analysis. Muscle regeneration will be observed on Day 1(D1), D3, D7, D11, and D14 post-injury. In thisexperiment, the in-vivo maximal isometric torque, which measures skeletalmuscle strength, will be used as a proxy for skeletal muscle regeneration.

Theankle 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. Amorphological analysis will be conducted on the muscle to visually observeskeletal muscle regeneration.

The muscle tissue will be cut into sections usinga cryostat and stained in hematoxylin and eosin (allows for better tissue visualization)and non-consecutive tissue sections will be visualized, using a previouslydescribed method (Lancioni et al., 2011). Muscle regeneration will be observed andscored on a scale of 1-5, where 1 = little to no regeneration, 2-4 = increasingregeneration, 5 = regenerated and control muscle look same. This will allow acomparison between the muscle of different aged mice.Animmunoblot analysis and protein quantification will be carried out, using thegastrocnemius muscle, to test for the presence of autophagy proteins (Ulk1,beclin1, LC3, p62, LC3II), a muscle specific protein (myogenin), and controlproteins (ß-actin and GAPDH), following a previously described method (Call etal., 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), andGAPDH (ab9484, Abcam). The results of the in-vivo torque measurement,morphological analysis, and immunoblot and protein quantification will becompared between the different treatment groups and age groups to determine howUlk1 KO affects skeletal muscle regeneration in different age groups.

 Aim 2: To determine if the degree of Ulk1 mediatedmitochondrial remodeling after skeletal muscle injury affects muscle strength. Inthis aim, I will be using adult mice(three months old), Ulk1 WT and KO (iKO and nKO), that will be made using theCre/LoxP system (Call et al., 2017). The same method that was used in Aim 1will be used to induce Ulk1 KO in iKO mice. The four treatment groups in thisexperiment will be: WT micewithout cardiotoxin (Ctx; basal control), WT mice with Ctx (experimental), KOwithout Ctx (control), KO with Ctx (experimental); iKO and nKO mice willundergo the same treatments.

All treatments will use male mice. The mice willbe 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, asopposed to I/R, was used to injure the mice because it results in significantdifferences 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 bedegraded before the first mitochondrial remodeling and muscle strengthmeasurements, 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 dorsiflexorsin the left hind limb will be used for the measurement.

After isometric torqueanalysis, the mice will be sacrificed and the TA muscle will be dissected. For halfof the mice, the mitochondria will be isolated from the muscle by tissuehomogenization (Teflon Potter Elvehjem homogenizer) and centrifugation, using apreviously described method (Frezza et al., 2007). Mitochondrial function will beassessed by measuring ATP because functional, and not dysfunctional,mitochondria produce ATP (Lanza and Nair, 2009). ATP production in the isolated mitochondria will bemeasured using a previously described bioluminescent method (Lanza and Nair,2009). In this method, luciferase is used to catalyze the luciferase + ATP reaction to produceintermediate products (luciferyl adenylate + PPi), which are converted tooxyluciferin + AMP + light. Then, the amount of ATP produced by themitochondria is determined using a luminometer, which measures the bioluminescenceof the sample (Lanza and Nair,2009).

For the second half of the mice, the TA muscle will be used for animmunoblot analysis and protein quantification of proteins related to the ETC (SDH, COX IV,Cyt C) and control proteins (ß-actinand GAPDH); this will be done usinga previously described method (Call et al., 2017). The blot will serve as a proxyfor mitochondrial function because during mitochondrial dysfunction COX IVlevels decrease and SDH levels increase (Ross, 2011). The antibodies used willbe: COX IV (#4844, Cell SignalingTechnology), Cyt C (ab13575, Abcam), SDHB (ab14714, Abcam), Beta Actin(ab8226, Abcam), and GAPDH (ab9484, Abcam).

 Aim3: To use chloroquine to determine the amount of Ulk1 mediated autophagy thatoccurs during different times of skeletal muscle regeneration. In this aim, I will be using CAG-RFP-EGFP-LC3 transgenicmice that express RFP (red florescent protein) and EGFP (enhanced greenflorescent protein) under a CAG promoter/enhancer sequence (027139, Jackson Laboratory). I will be using twomonths old (oldest mice I can get) male mice. Autophagy can be tracked usingEGFP and RFP because both are present during autophagosome formation, but onlyRFP is present in the autolysosome because EGFP is degraded in acidic pHs (Zhouet al., 2012). In this experiment, there will be four treatment groups:uninjured mice without chloroquine (basal level control), uninjured mice withchloroquine (experimental), injured mice without chloroquine (control), injuredmice with chloroquine (experimental).

The mice will be injured with I/R on theleft hind limb using a previously described method; control mice will not beinjured (Call et al., 2017). In the injured mice, the uninjured right hind limbwill serve as an internal control. The gastrocnemius muscle will be selectedfor further analysis.

Chloroquine inhibits autophagic degradation by increasingthe pH of the autolysosome (to approximately 6.5), which prevents the lastphase of autophagy from occurring (Iwai-Kanai et al., 2008; Ohkuma and Poole,1978). Thus, the amount of autophagy can be determined by observing the LC3puncta that are part of the autolysosome.

A dose-response curve will be used todetermine the optimal chloroquine (C6628, Sigma-Aldrich) concentration (1mg/kg,5mg/kg, 10mg/kg, 15mg/kg, or 20mg/kg) because previous studies have used thedrug in cardiomyocytes, not skeletal muscle cells (Iwai-Kanai et al., 2008). Theoptimal concentration of chloroquine will be administered at the different timepoints that skeletal muscle regeneration will be observed. Chloroquine will beinjected intraperitoneally into the left hind limb using a previously describedmethod (Iwai-Kanai et al., 2008). Control mice will be injected with saline. Inmice treated with chloroquine, EGFP will not degrade because of the increasedautolysosomal pH (Iwai-Kanai et al., 2008).

Thus, monodansylcadaverine (MDC;D4008 Sigma-Aldrich) will be used to label autolysosomes because its functionis not dependent on pH (Iwai-Kanai et al., 2008). For consistency, all micewill have MDC (1.5mg/kg) injected intraperitoneally in left hind limb using apreviously described method (Iwai-Kanai et al., 2008). Autophagy and skeletalmuscle regeneration will be observed on D1, D3, D7, D11, and, D14 post-injury. The gastrocnemius muscle willbe dissected, and the tissue will be sectioned and prepared for widefieldflorescence microscopy using a previously described method (Iwai-Kanai et al.

,2008). Non-consecutive tissue sections will be visualized. In chloroquineuntreated 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. Animmunoblot analysis and protein quantification will be carried out on themuscle tissue for LC3, myogenin, ß-actin and GAPDH using a previously describedmethod (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 andprotein quantification will be compared between the different treatment groupsand skeletal muscle regeneration times to determine whether the amount ofautophagy differs.

 SummaryThis research will investigate theage-related effects of Ulk1 mediated autophagy in skeletal muscle regeneration.It will also determine the relationship between Ulk1 mediated mitochondrialremodeling and skeletal muscle strength. Lastly, this study will quantify theautophagy occurring during different days of skeletal muscle regeneration. Thisresearch will contribute to the long-term goal of using Ulk1 mediated autophagyto treat patients with skeletal muscle injuries and hasten their muscleregeneration. 

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