Exercise-Induced Sirtuin Expression in Blood Vessels and Skeletal Muscles of Rats

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Undergraduate Honors Theses Theses, Dissertations, & Master Projects

2013

Rebecka M. Hoffman College of William and Mary

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Recommended Citation Hoffman, Rebecka M., "Exercise-Induced Sirtuin Expression in Blood Vessels and Skeletal Muscles of Rats" (2013). Undergraduate Honors Theses. Paper 636. https://scholarworks.wm.edu/honorstheses/636

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Exercise-induced sirtuin expression in blood vessels and skeletal muscles of rats

A thesis submitted in partial fulfillment of the requirement for the degree of Bachelors of Science in Kinesiology and Health Sciences from The College of William & Mary

Rebecka M. Hoffman

Accepted for Honors ______

______Michael Brennan Harris, PhD, Director

______Randolph A. Coleman, PhD

______Scott Ickes, PhD

Williamsburg, VA April 12, 2013

Table of Contents

Title Page…………………………………………………………………………………………………………….1

Abstract……………….………………………………………………………………………………………………4

Background…………………………...…………………………………………………………………………….5

Cardiovascular Disease………………………………………………………………………………5

Endothelial Nitric Oxide Synthase………………………………………………………………6

Shear Stress, Exercise, and eNOS………………………………………………………………..9

Oxidative Stress………………………………………………………………………………………10

Sirtuins…………..……………………………………………………………….………………………11

SIRT1……………..…………………………………………………………………………………….…12

SIRT3………………………...……………………………………………………………………………15

Study 1………………………………………………………………………………………………………………16

Methods………………………………………………………………………………………………….17

Results……………………………………………………………………………………………………20

Discussion……………….………………………………………………………………………………23

Study 2……………………………………………………………………………………………………………...27

Methods………………………………………………………………………………………………….27

Results……………………………………………………………………………………………………29

Discussion………….……………………………………………………………………………………30

Acknowledgements……………………………………………………………………………………………32

Figure Captions.…………………………………………………………………………………………………34

Figures..…….………………………………………………………………………………………………………39

Figure 1…….……………………………………………………………………………………………39

Figure 2…….……………………………………………………………………………………………40

Figure 3…….……………………………………………………………………………………………41

Figure 4…….……………………………………………………………………………………………42

Figure 5…….……………………………………………………………………………………………43

Figure 6…….……………………………………………………………………………………………44

Figure 7…….……………………………………………………………………………………………45

Figure 8…….……………………………………………………………………………………………46

Figure 9…….……………………………………………………………………………………………47

Figure 10.….……………………………………………………………………………………………48

Figure 11.….……………………………………………………………………………………………49

Figure 12.….……………………………………………………………………………………………50

Tables....…….………………………………………………………………………………………………………51

Table 1…….………………………..…………………………………………………………………….51

Table 2…….………………………..…………………………………………………………………….52

Works Cited………………………………………………………………………………………………………53

Abstract

Cardiovascular disease (CVD) is the leading cause of death in United States and worldwide. Exercise has been shown to improve endothelial function and provide protection against cardiovascular diseases. Endothelial nitric oxide synthase, or eNOS, is an enzyme that produces the potent vasodilator nitric oxide

(NO). Interruptions in NO bioavailability and oxidative stress are commonly linked to CVD. SIRT1, a NAD-dependent deacetylase, is a regulator of eNOS and endothelial function. The mechanism by which SIRT1 is involved in endothelium dependent vasodilation is poorly understood. SIRT3, another NAD-dependent deacetylase of the same family as SIRT1, is implicated in oxidative stress response. Exercise has been shown to enhance both SIRT1 and SIRT3 expression, however, changes in expression across an exercise-training program has yet to be determined.

The first study examines endothelial function with or without SIRT1 inhibition and SIRT1 and eNOS protein expression with or without Vitamin E supplementation (a known reducer of oxidative stress) after different durations of training in rat aortas. The purpose of the second study is to determine SIRT1 and

SIRT3 protein expression in skeletal muscles (soleus, plantaris, and extensor digitorum longus (EDL)) and heart muscles. These results demonstrate that mechanisms regulating vascular reactivity during the course of an exercise-training program involve dynamic changes in SIRT1 and eNOS and exercise also induces changes in skeletal muscle expression of SIRT1 and SIRT3.

Background

Cardiovascular Disease

Cardiovascular disease (CVD), disease of the heart and blood vessels, is the leading cause of death worldwide, accounting for 30% of all deaths globally (The world health organization: Cardiovascular disease.2013). With 35.3% of American adults dealing with at least one type of CVD such as hypertension and coronary heart disease, the promotion of cardiovascular health is of intense interest (Go et al.,

2013). Behavioral risks such as unhealthy diet, physical inactivity, alcohol, and tobacco are responsible for about 80% of coronary heart disease, as well as cerebrovascular disease. Regular exercise significantly reduces the risk of CVD through such effects as a reduction in diastolic blood pressure, triglycerides and fasting glucose levels (The world health organization: Cardiovascular disease.2013;

Go et al., 2013).

The etiology of most CVD can be traced to endothelial dysfunction, or dysfunction of the epithelial cells that line the vessels (Golbidi & Laher, 2012). It has been shown that endothelial dysfunction is a precursor to atherosclerosis, and those with endothelial dysfunction have a three- to four-fold higher risk of myocardial infarction or ischemic stroke (Golbidi & Laher, 2012). The endothelium regulates vascular tone by secreting substances that act on the smooth muscle cells, causing vasoconstriction or vasodilation (Levick, 2009). This critical role for vascular homeostasis is important for cardiovascular function and health. Various vasoactive agents have been discovered: nitric oxide, endothelium-derived

6 hyperpolarizing factor (EDHF), prostacyclin, and bradykinin are vasodilators, whereas, angiotensin II and endothelin are examples of vasoconstrictors (Levick,

2009; Ruschitzka, Corti, Noll, & Lüscher, 1999). NO, in particular, is of interest because of its cardioprotective roles in the vasculature: it promotes vasodilation, inhibits vascular smooth muscle contraction (Boulanger & Lüscher, 1990) and proliferation (Scott-Burden, Schini, Elizondo, Junquero, & Vanhoutte, 1992), suppresses platelet aggregation (Radomski, Palmer, & Moncada, 1990), and inhibits leukocyte adhesion to the endothelium (Kubes, Suzuki, & Granger, 1991). Exercise has been shown to increase NO bioavailability and subsequently increase endothelial vasodilation (Gielen, Schuler, & Adams, 2010).

Endothelial Nitric Oxide Synthase (eNOS)

There are three isoforms of nitric oxide synthase, the enzyme that produces nitric oxide. Endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) are constitutively expressed and are largely activated in a calcium- dependent manner. In contrast, inducible nitric oxide synthase (iNOS), is neither consitutively expressed nor dependent on calcium for activation. High expression levels of iNOS exist after induction by cytokines or other inflammatory factors. nNOS has important functions in signaling and eNOS is necessary for endothelium- derived vasodilation. (Andrew & Mayer, 1999) All isoforms of NOS produce nitric oxide (NO) by oxidizing L-arginine to NO and L-citrulline (Andrew & Mayer, 1999;

Fleming & Busse, 2003). NOS is a dimer containing an oxygenase domain and a reductase domain. After the dimerization of eNOS, NADPH is able to bind to the

7 reductase domain and transfer electrons to the oxygenase domain, allowing it to oxidize L-arginine (Fleming & Busse, 2003; Sessa, 2004). The coupling of NOS is not only important for NO production but eNOS, in its uncoupled form, produces superoxide instead (Jacobson et al., 2007). This rise in reactive oxygen species

(ROS) leads to endothelial dysfunction (Yang, Huang, Kaley, & Sun, 2009)

Schematic 1: eNOS structure and regulation. Adapted from(Sessa, 2004).

As previously stated, proper vasodilation is essential for blood flow and cardiovascular health. eNOS is strongly implicated in endothelial dysfunction and the resulting CVD. A potential mechanism of endothelial dysfunction is a decrease in eNOS substrate and cofactor availability, limiting its ability to produce NO. Another mechanism is a decrease in the activity of eNOS and an inactivation of NO by ROS.

Finally, it has been proposed that vascular regeneration by endothelial progenitor cells (EPCs) could cause endothelial dysfunction. (Gielen et al., 2010)

Originally thought to be only a calmodulin (CaM) regulated protein, eNOS

2+ 2+ activity has been shown to increase with intracellular calcium ([Ca ]i) by the Ca -

CaM complex to the CaM binding domain of the enzyme. Research has now elucidated that calcium is not necessary for eNOS activation and it is regulated by post-translational lipid modifications, phosphorylation, and protein-protein interactions (Sessa, 2004). Caveolin-1 is an inhibitor of eNOS activity by interfering with it’s ability to interact with Ca2+-CaM (Ju, Zou, Venema, & Venema, 1997) The binding of eNOS to activated Hsp90 causes enhanced eNOS activity and increased

NO production. Heat shock protein 90 (Hsp90) is activated by eNOS agonists

(bradykinin, vascular endothelial growth factor (VEGF), shear stress, and histamine)

(Harris, Ju, Venema, Blackstone, & Venema, 2000).

Other stimuli regulate eNOS through phosphorylation by activating signaling cascades. The six known phosphorylation sites on eNOS are Tyr83, Ser116, Thr497,

Ser617, Ser645, and Ser179, as shown on Schematic 1. All phosphorylation sites enhance eNOS activity except for Thr497. eNOS agonists such as vascular endothelial growth factor (VEGF) and bradykinin activate the phosphoinoside 3-kinase (PI3K) and adenylate cyclase (AC) pathways, leading to the activation of the serine kinases

Akt (by PI3K) and protein kinase A (PKA, by AC) (Sessa, 2004). Akt phosphorylates

Ser617 and Ser1179 while PKA phosphorylates Ser635 and Ser1179 (Dimmeler et al.,

1999; Fisslthaler, Dimmeler, Hermann, Busse, & Fleming, 2000; Sessa, 2004)

Furthermore, they activate phospholipase C- γ (PLC- γ), causing an increase in

9 cytoplasmic calcium levels and an increase in the eNOS activator Ca2+-CaM. PLC- γ also increases diacylglycerol (DAG) levels which activates protein kinase C (PKC) which in turn phosphorylated eNOS at Thr497 (Sessa, 2004). Shear stress is another activator of the PI3K and AC pathways.

Shear Stress, Exercise, and eNOS

Normal cellular levels of calcium are sufficient for eNOS activation and thus an increase in calcium is not always necessary for eNOS activation. Shear stress can activate eNOS in the absence of calcium increase. In a healthy state, normal blood flow through the vasculature produces shear stress against the walls of epithelial cells. Turbulent flow can lead to endothelial dysfunction by activation of inflammatory and proliferative signaling cascades and by increasing ROS (Stein &

Matter, 2011). Shear stress operates through various G-proteins to activate PI3K and AC pathways previously described. With aging, the shear-stress induced NO production decreases which research suggests is due to a variety of mechanisms: an increased production of superoxide with age, a reduced activity of superoxide dismutase (SOD), and an impaired activation of eNOS by shear stress (Sun et al.,

2004).

Vascular shear stress is increased during exercise as working muscles have a higher oxygen demand, requiring an increased cardiac output and heart rate to increase the blood flow through the vasculature. Exercise has been shown to increase eNOS phosphorylation at Ser1179, previously described to be a serine residue phosphorylated by both Akt and PKA, and in turn an increase in eNOS

10 activity (Gielen et al., 2010). By increasing eNOS phosphorylation, eNOS activity is increased, and more NO is improved. Thus, exercise provides a mechanism for enhancing the cardioprotective role of NO.

Oxidative Stress

Oxidative stress is the result of a disturbed redox state, which can result from an increase in ROS and/or a decrease in the ability to detoxify the ROS. ROS themselves do not signal a distressed state as they are the byproducts of aerobic metabolism; however, the imbalance of ROS and a cell’s ability to cope can lead to damage and, as it pertains to cardiovascular disease, endothelial dysfunction.

(Jacobson et al., 2007)

Antioxidants, frequently reducing agents, are in place to control ROS production and propagation (Golbidi & Laher, 2012). Exercise increases antioxidant enzymes like SOD, catalase, and glutathione peroxidase (Gielen et al.,

2010). ROS associated with chronic exercise enhance the antioxidant defense mechanisms by acting as signaling molecules for transcriptional upregulation of the antioxidant systems (Gomes, Silva, & de Oliveira, 2012). Vitamin E, another antioxidant, has been shown to significantly decrease the oxidative stress associated with exercise in the endothelium (Marsh, Laursen, Pat, Gobe, & Coombes, 2005).

.- Superoxide (O2 ) production is a major cause of CVD and increases with aging (Jacobson et al., 2007). Superoxide decreases the bioavailability of NO, damaging the epithelial and leading to a reduction in vasodilation. A decrease in NO and an increase in superoxide mediate the impaired endothelium-dependent

11 vasodilation seen in hypertension and aging (Jacobson et al., 2007). Addition of mnTBAP, a superoxide scavenger, is sufficient to restore endothelial function in aged rats (Andersson et al., 2006).

Superoxide can be produced in the endothelium by several mechanisms. The enzymes NADPH oxidase, xanathine oxidase, and myeloperoxidase all produce superoxide. Furthermore, eNOS itself produces superoxide in its uncoupled, monomer form. The uncoupling of the oxygenase and reductase domains can result from reduced availability of the cofactor tetrahydrobiopterin (BH4) (Guzik et al.,

2002). In this instance, the oyxgenase domain is able to oxidize NADPH but the electrons are unable to transfer to the reductase domain for L-arginine reduction.

Instead, the electrons reduce oxygen to superoxide. Superoxide is a scavenger for

NO and together they form the reactive nitrogen species peroxynitrite (ONOO-) and in doing so, reduces the bioavailability of NO. (Jacobson et al., 2007; Schiffrin, 2008;

Yang et al., 2009)

Sirtuins

The sirtuins are a family of NAD+-dependent deacetylases and ADP- ribosyltransferases. They share a conserved catalytic core and the diversity lies in the N and C termini (Haigas & Sinclair, 2010; Michan & Sinclair, 2007). The name sirtuin is derived from the classification silent mating type information regulation.

The NAD+ requirement implicates the sirtuins as sensors for the metabolic and oxidative status of cells (Imaai, Armstrong, Kaeberlein, & Giaremte, 2000). The deacetylation reaction catalyzed by sirtuins requires the hydrolysis of one NAD+ to

12 one ADP-ribose and one nicotinamide (NAM) for each acetyl group removed

(Landry, Slama, & Sternglanz, 2000). Humans possess seven sirtuins, SIRT1-7, which are then subdivided into classes and subclasses based on phylogenetic differences. These sirtuins vary in their function and subcellular localization. For example, SIRT1 is found in the nucleus and the cytoplasm, whereas SIRT3 is a mitochondrial sirtuin (Haigas & Sinclair, 2010; Onyango, Celic, McCaffery, Boeke, &

Feinberg, 2002; Tanno, Sakamoto, Miura, Shimamoto, & Horio, 2007).

The sirtuins were first implicated in aging earning them the classification as

“anti-aging” genes. This anti-aging role was first seen in 1999 in the budding yeast

Saccharomyces cerevisiae’s Sir2 protein, a homolog for human sirtuins. The addition of an extra copy of the SIR2 gene increased the replicative lifespan by about

30% by suppressing rDNA recombination (Kaeberlein, McVey, & Guarente, 1999), a process previously shown to be a major source of aging in the species (Sinclair &

Guarente, 1997).

SIRT1

SIRT1’s, a class Ia of the sirtuins, cellular functions include gene silencing, cell cycle, stress resistance, inflammation, and metabolic diseases (Michan & Sinclair,

2007). It targets histone, illustrating its role in gene silencing, and non-histone proteins (Michan & Sinclair, 2007; Vaquero et al., 2004). SIRT1’s deacetylation activity depends on the NAD+ availability in the cell, tying SIRT1’s activity closely to the redox and metabolic status of the cell (Fulco et al., 2003). Primarily found in the nucleus, SIRT1 has also has been shown to shuttle to the cytoplasm (Tanno et al.,

2007). One instance of this is when it colocalizes with eNOS in the cytoplasm

(Mattagajasingh et al., 2007).

Caloric restriction is a dietary regime where fewer calories are consumed than would be ad libitum and consistently increases maximal life span in mammals

(Michan & Sinclair, 2007). Research shows it decreases age-related physical decline as well as reducing the incidence and delaying the onset of age-related diseases such as cancers (Barger, Walford, & Weindruch, 2003). Caloric restriction increases

SIRT1 expression and its activity. There is evidence that this relationship is mediated by NO activating its secondary messenger cGMP (3’,5’-cyclic guanosine monophosphate) which increases mitochondrial biogenesis and respiration (Nisoli et al., 2005). To date, one paper by Mattagajasingh et al. (Mattagajasingh et al.,

2007) suggests that SIRT1 increases eNOS activity and thus increases endothelial- derived nitric oxide by deacetylating eNOS at the lysine residues Lys496 and Lys506 in the calmodulin-binding domain. SIRT1 inhibition leads to increased acetylation of eNOS and endothelial dysfunction (Mattagajasingh et al., 2007). Taken together, there is strong evidence for SIRT1’s role in promoting vascular homeostasis and cardiovascular health through eNOS enhancement.

Changes in metabolic and redox status from caloric restriction change SIRT1 expression and activity and so it has been hypothesized that similar changes resulting from exercise could have the same effect. Several studies have shown that

SIRT1 increases with exercise training (Civitarese et al., 2007; Fulco et al., 2003;

Lagouge et al., 2006; Suwa, Nakano, Radak, & Kumagai, 2008), however, there have been differing results showing no change in SIRT1 expression with exercise and

14 caloric restriction (Huffman et al., 2008; Philp et al., 2011). Amongst these studies there is variation in exercise training duration, methods of SIRT1 measurement, and tissues studied.

Suwa et al. (Suwa et al., 2008) conducted the most recent study on SIRT1 expression in skeletal muscle tissue following acute (one instance of exercise to exhaustion) exercise, low intensity endurance (14 days) exercise and high intensity endurance exercise. Acute exercise showed an increase in soleus muscle SIRT1 expression but no change in plantaris muscle SIRT1. Low intensity exercise (rats ran on a treadmill at a speed of 20m/min at an 18.5% incline for 90min/day) saw an increase in SIRT1 expression in soleus but not plantaris muscle. High intensity exercise (30m/min, 18.5%, 60min/day) increased SIRT1 expression in both muscle types. This suggests that SIRT1 expression is higher in the red, slow-twitch, oxidative muscles versus the white, fast-twitch, glycolytic muscles and that SIRT1 responds faster to exercise in the slow-twitch, oxidative muscles. (Suwa et al.,

2008) In similar studies, soleus muscle is frequently used as an example of red, slow-twitch, oxidative muscle and extensor digitorum longus (EDL) and plantaris muscles are used as examples of white, fast-twitch, glycolytic muscles.

Most studies on exercise’s effects on SIRT1 expression and activity look at skeletal muscle and there currently appears to be only one study that has examined vascular tissues. Cacicedo et al. reported an increase in SIRT1 and eNOS in mouse aortas following acute exercise (Cacicedo et al., 2011). It appears that there are currently no published studies looking at SIRT1 expression in vascular tissues with chronic endurance exercise.

SIRT3

Similar to SIRT1, SIRT3 is a NAD+-dependent deacetylase that is sensitive to the metabolic and redox state of the cell (Onyango et al., 2002; Sack, 2011) and has enhanced activity with exercise and caloric restriction (Palacios et al., 2009). It is ubiquitously expressed, with greatest expression in metabolically active tissues like the kidney, brown adipose tissue, and the heart, and is localized to the cristae of the mitochondria(Onyango et al., 2002). While most research has been done on SIRT1, in recent years SIRT3 has been growing in interest because of genomic studies linking it to longevity in humans (Bellizzi et al., 2005).

SIRT3 has important roles in mitochondrial metabolism and respiration by targeting both transcription factors and metabolic enzymes (Hallows, Lee, & Denu,

2006). It has been shown to increase cellular respiration, deacetylate and activate acetyl-CoA synthetase (Shi, Wang, Stieren, & Tong, 2005), and regulates fatty acid oxidation(Hirschey et al., 2010).

SIRT3 also plays important roles in oxidative stress response. Recent research shows SIRT3 directly affecting antioxidant function by deacetylating mitochondria manganese superoxide dismutase (SOD2) at the lysine residues

Lys68(Chen et al., 2011) and Lys122(Tao et al., 2010) and indirectly by deacetylating and activating the Foxo3a transcription factor which in turn enhances MnSOD and catalase activity (Sundaresan et al., 2009). It is also strongly implicated as having a cardioprotective role by blocking cardiac hypertrophic, or thickening of the myocardium, response(Sundaresan et al., 2009) and having an anti-apoptotic

16 function in cardiomyocytes (Sundaresan, Samant, Pillai, Rajamohan, & Gupta,

2008).

To date, there appears to be only two studies that have looked at the effect of exercise on SIRT3 expression. Hokari et al. (Hokari et al., 2010) reported increases in SIRT3 expression in both soleus and plantaris muscle following a four-week treadmill exercise regime at 20m/min, 60min/day but no changes following acute, one-day exercise. Palacios et al. (Palacios et al., 2009) looked at SIRT3 expression after a voluntary exercise regime where mice had access to a rodent running wheel.

They reported higher SIRT3 expression in the slow-twitch, oxidative soleus versus the fast-twitch, glycolytic EDL and gastrocnemius (Palacios et al., 2009). These findings are consistent with the higher mitochondrial content of the soleus muscle and it’s main use of oxygen as an energy source. Furthermore, this mimics the expression pattern seen in SIRT1 (Suwa et al., 2008).

Study One

The purpose was to evaluate the role of SIRT1 in exercise-induced changes in vascular function. Exercise has been shown to affect vascular function but it is unknown whether SIRT1 plays a role in these changes. Male, Wistar rats (8-9 months old) were divided into four groups based on length of training: sedentary

(SED, n=11), 1 day acute exercise (1D, n=10), 2 week endurance exercise (2WK, n=12), and 6 week endurance exercise (6WK, n=8). The vascular tissues chosen to study were the aorta as it is a tissue frequently used in research to investigate

17 vascular function and the femoral artery as it is a major vessel supplying the exercising muscles. Vascular reactivity was examined by constructing dose response curves on a wire myograph to phenylephrine (PE, 10-7-10-4 M), sodium nitroprusside (SNP, 10-8-10-5 M), and acetylcholine (Ach, 10-7-3x10-5 M) in the presence or absence of the sirtuin inhibitor salermide. Exercise-induced changes in

SIRT1 and eNOS protein expression were determined by immunoblotting. The aim of study 1 was to determine if exercise increases SIRT1 expression in vascular tissues and whether this is implicated in endothelium-dependent vasodilation.

The next part of study one was to test the hypothesis that exercise-induced

SIRT1 expression in vascular tissues is dependent on oxidative stress. Additional groups of rats underwent the same exercise-training program but will receive a diet with Vitamin E (1000 IU/ kg diet) and alpha-lipoic acid (1.6 g/kg diet) supplementation, a diet previously shown to significantly reduce oxidative stress in the endothelium of exercise trained rats (Marsh et al., 2005). The supplemented- diet groups are: 1 day acute exercise (1E, n=12), 2 week endurance exercise (2E, n=12), and 6 week endurance exercise (6E, n=12. As in the control diet, exercised induced changes in SIRT1 and eNOS expression was determined by immunoblotting.

Methods

Animals

Experiments were approved by the Institutional Animal Care and Use

Committee of The College of William & Mary and adhered to the Guide for the Care

18 and Use of Laboratory Animals published by the U.S. National Institutes of Health. 8-

9 month old male Wistar rats were divided into seven groups based on length of training and diet fed (control or supplemented): Sedentary (S, n= 11), 1 day acute exercise (1DAY, n=10), 1 day acute exercise with Vitamin E diet (1DAY + VitE, n=12),

2 weeks (2WK n=12), 2 weeks Vitamin E diet (2WK + VitE, n=12), 6 weeks (6WK, n=8), and 6 weeks Vitamin E diet (6WK + VitE, n=12). Those on the Vitamin E diet received Vitamin E (1000 IU/kg diet) and alpha-lipoic acid (1.6g/kg diet) supplementation.

Training

Exercise consisted of running on a motor-driven treadmill. One day runners completed one run at 15 m/min, 15% grade, for 40 min. Animals which continued to train gradually increased the duration and intensity of the training over 2 weeks until reaching 30 m/min, 18.5% grade for 60 min which was then maintained for an additional 4 weeks for a total of 6 weeks of training.

Vascular Reactivity

Twenty-four hours following the last exercise bout, animals were anesthetized with sodium pentobarbital, which was administered intraperitoneally at a dose of 50 mg/kg of body weight. The aortas and femoral arteries were removed and placed in a physiological salt solution (PSS) consisting of: 130 mM

NaCl, 4.7 mM KCl, 1.18 mM KH2PO4, 1.17 mM MgSO4·7H2O, 1.6 CaCl2, 14.9 mM

NaHCO3, 5.5 mM dextrose, and 0.03 mM Na2EDTA. Vessels were cleaned of

19 adherent fat and connective tissue and cut into two, ~4 mm rings. The remainder of the aorta was frozen in liquid nitrogen and used for immunoblotting. The rings were mounted using small wires on stainless steel holders in muscle baths on a

DMT610 myograph for isometric force recording. Muscle baths were filled with PSS heated to 37°C and aerated with 95% O2/5% CO2. The rings were stretched to an optimal resting tension (1.5 g) and allowed to stabilize for 30 min to 1 h. After equilibration one of the two rings was then pre-treated with 30 µm salermide for 1 hour. Vasoconstrictor responses to phenylephrine were determined using cumulative doses (10−7 to 10−4 M). Relaxation responses to cumulative doses of acetylcholine (Ach, 10−7 to 10−4 M) and sodium nitroprusside (SNP, 10−9 to

3 × 10−4 M) were determined following preconstriction with 10−5 M phenylephrine with ~15-30 min of recovery between each drug until the resting tension stabilized.

Immunoblotting

Portions of the aortic tissues frozen in liquid nitrogen were homogenized in ice-cold lysis buffer (50 mM Tris-HCl, 0.1 mM EDTA, 0.1 mM EGTA, 1% TX-100, 1% sodium deoxycholate, 1mM PMSF, 1 mM Na3VO4, and protease inhibitors) using a glass-glass homogenizer. Samples were then centrifuged at 10,000xg for 10 min at

4oC and divided for analysis.

Protein concentrations of the homogenized aorta samples were determined using the Bio-Rad detergent-compatible protein assay (Bio-Rad, Hercules, CA) .

Volumes were adjusted to equalize the concentration of the samples.

Approximately 70 μg of each sample were then loaded and run on a 10%

20 polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Bio-

Rad), which was subsequently immunoblotted using an anti-mouse WesternDot kit

((W10132, Invitrogen) according to the manufacturer’s instruction using the following primary antibodies: SIRT1 anti-mouse (74465, Santa Cruz Biotechnology; dilution 1:300), GAPDH anti-mouse (MAB374, Millipore; dilution 1:1000), eNOS anti-mouse (610297, BD Transduction Laboratories; dilution 1:400). Results from immunoblotting were determined by densitometric analysis of the chemiluninescent bands using a BioRad GelDocXR imager. Densities for each sample were normalized to a percentage of an aorta standard loaded on each blot and compared to GAPDH as a loading control.

Statistical Analysis

Sigmoidal dose response curves with variable slopes were analyzed using non-linear regression. Relaxation responses in aortic and femoral artery rings were

−7 calculated as percent relaxation from 10 M phenylephrine contraction. EC50 was determined using a non-linear regression (P < 0.05). A two-way ANOVA with a

Bonferonni post hoc test (P < 0.05) was used for multiple comparisons. Animals whose aortas and femoral arteries were non-responsive to acetylcholine-induced relaxation were eliminated from the analysis as the endothelium was damaged during the surgery. Results from the densitometric analysis of the immunoblots were analyzed using a one-way ANOVA with Dunnet’s post-hoc tests (P < 0.05).

Results

Vascular Reactivity in Aortas

Cumulative doses of phenylephrine (PE) induced vasoconstrictor responses in all exercise groups (SED, 1DAY, 2WK, and 6WK) as observed in Figure 1A.

Constriction was significantly greater (P<0.05) at higher doses (3x10-6-10-5). The

EC50 was not significantly different between SED, 1DAY, 2WK, and 6WK (Table 1).

As shown in Figure 1B, there were no significant differences in contractile responses to PE following pretreatment with the sirtuin inhibitor salermide for any group

(SED, 1DAY, 2WK, or 6WK).

Figure 2 indicates significant relaxation in all groups (SED, 1DAY, 2WK, and

6WK) following cumulative doses of acetylcholine (ACh). ACh-induced vasorelaxation was not significantly different between SED, 2WK, and 6WK groups, however, ACh-induced relaxation (Fig. 2) as well as the EC50 (Table 2) was significantly impaired (P<0.05) in 1DAY versus SED, 2WK, and 6WK. Furthermore, the EC50 was significantly less (P<0.05) for 2WK versus SED, 1DAY, and 6WK.

Pretreatment with salermide resulted in significantly (P<0.05) reduced ACh- induced vasorelaxation in SED, 1DAY, and 2WK but not 6WK groups (Fig. 3B).

Furthermore, pretreatment with salermide resulted in a significantly greater EC50 in all groups (Table 2).

Figure 3 A and B illustrates that cumulative doses of sodium nitroprusside

(SNP) with or without salermide pretreatment produced no significant differences in vasodilation in all groups (SED, 1DAY, 2WK, and 6WK).

Vitamin E supplementation, as seen in figure 5, significantly (P<0.05)

22 increased ACh-dependent vasorelaxation in the 1DAY group compared to the control diet at the highest dosage. Viamin E supplementation had no significant differences in ACh-induced vasorelaxation in the 2WK or 6WK groups.

Immunoblotting

Analysis of immunoblotting results using one-way ANOVA with Dunnet’s post-hoc test revealed a significant (P<0.05) increase in eNOS protein expression in the aortas of the exercise groups (1DAY, 2WK, and 6WK) versus SED (Fig. 4A).

SIRT1 protein expression, however, was only significantly (P<0.05) increased in the aortas of the 1DAY and 6WK exercise groups. Additional post-hoc analysis revealed no significant differences in eNOS protein expression or SIRT1 protein expression in aortas between exercise groups.

Vascular Reactivity in Femoral Arteries

Cumulative doses of phenylephrine (PE) induced vasoconstrictor responses in all exercise groups (SED, 1DAY, 2WK, and 6WK) and there were no significant differences amongst groups (Fig 6A). Pretreatment with salermide produced no significant differences in PE-induced vasoconstriction for any group (SED, 1DAY,

2WK, or 6WK) (Fig 6B).

As indicated by figure 7 A and B, cumulative doses of acetylcholine (ACh) with or without salermide pretreatment produced no significant differences in vasorelaxation in all groups (SED, 1DAY, 2WK, and 6WK).

Cumulative doses of sodium nitroprusside (SNP) produced no significant

23 differences in vasorelaxation in all groups (SED, 1DAY, 2WK, and 6WK) (Fig 8A) and no significant differences in SNP-induced vasorelaxation following pretreatment with salermide (8B).

Discussion

Several studies have looked at SIRT1 expression in the active skeletal muscle following endurance exercise training (Civitarese et al., 2007; Fulco et al., 2003;

Huffman et al., 2008; Lagouge et al., 2006; Philp et al., 2011; Suwa et al., 2008), however, there appears to be only one published study on SIRT1 expression in vascular tissues following exercise(Cacicedo et al., 2011). Cacicedo et. al. reported an increase in SIRT1 protein expression and eNOS protein expression in mouse aorta following acute (one bout of exhaustive exercise). To date, there seems to be no study that has investigated SIRT1 expression in vascular tissues across an endurance exercise program. Furthermore, this study adds the variable of an antioxidant defense affecting SIRT1’s role on endothelium function and is the first study to look at the femoral artery, the major vessel to the exercising tissues.

The role of SIRT1 in the vascular reactivity of rat aortas was investigated using preconstruction with phenylephrine followed by acetylcholine-induced relaxation and sodium nitroprusside-induced relaxation. Acetylcholine induces NO production and thus acts on the endothelium. Sodium nitroprusside relaxes the vessels at the smooth muscle level and thus produces endothelium-independent vasodilation(Levick, 2009). The novel pharmacological sirtuin inhibitor salermide was also used. Salermide had been developed as a cancer drug due to its high

24 cancer cell specificity and subsequent induction of apoptosis and has been shown to be a more potent inhibitor of SIRT1 than previous mechanisms(Lara et al., 2009).

Mattagajasingh et al. (Mattagajasingh et al., 2007) appear to have conducted the only other study on the role of SIRT1 in vascular reactivity. In their study, SIRT1 was inhibited in rat aortas using a dominant negative mutant of SIRT1 and SIRT1 inhibition led to an inhibiton of acetylcholine-induced, or endothelium-dependent, vasorelxation (Mattagajasingh et al., 2007).

There were no significant differences in PE-induced vasoconstriction and sodium nitroprusside-induced vasorelaxation observed among the exercise groups suggesting that exercise training had no apparent effect on smooth muscle function

(Fig 1A and Fig 3A). Furthermore, there were no differences in vasorelaxtion following cumulative doses of sodium nitroprusside with or without SIRT1 inhibition; however, there are differences in acetylcholine-induced vasorelaxation with or without SIRT1 inhibition, implying that SIRT1 acts on the endothelium. This study has the novel finding that SIRT1 plays a significant role in endothelium- dependent vasorelaxation but this role is dependent on duration of exercise. Based on the vascular reactivity data, SIRT1 appears to be especially important in endothelium-dependent vasorelaxation following acute exercise but this role is diminished as exercise continues up to 6 weeks (Fig 2).

Research indicates that acute exercise may be detrimental to vascular function whereas chronic exercise improves vascular function, partly due to an increase in antioxidant defenses(Gomes et al., 2012). This could explain the diminished role of SIRT1 in endothelium-induced vasorelaxation with chronic

25 exercise. The considerable oxidative stress associated with acute exercise could explain the importance of SIRT1 in this duration of exercise. The apparent adaption of SIRT1 to chronic exercise could be related to an adaptation to oxidative stress.

Based on the observations of changes in vascular reactivity in aortas with exercise, we attempted to determine whether these changes in vascular function were the results of changes in SIRT1 or eNOS expression. This study confirms the results of Cacicedo et al. (Cacicedo et al., 2011) as well as other studies that reported increases in vascular eNOS expression with exercise (Gielen et al., 2010).

However, the increased expression of eNOS protein did not correlate with enhanced endothelial-dependent vasodilation. There were no significant differences of eNOS protein expression among exercise groups (acute and endurance execise) (Fig 4A) and yet there were significant differences in acetylcholine-induced vasorelaxation

(Fig 2). Thus, adaptations in endothelial function due to exercise appears to be independent of an increase in eNOS expression.

As with eNOS, SIRT1 protein expression increased in the aorta in acute exercise and remained elevated throughout chronic exercise (Fig 4B). The supports the findings of Cacideo et al. (Cacicedo et al., 2011) who looked at acute exercise but this study is the first to extend the findings to chronic exercise.

Changes in eNOS and SIRT1 protein expression in the rat aorta do not adequately account for the observed differences in vascular reactivity following acute and chronic exercise. This suggests that there are dynamic mechanisms for the adaptation of endothelium function to chronic exercise. The reduction in SIRT1 regulation of endothelial-dependent relaxation and the adaptation of vascular

26 function to exercise could be because of increases in vascular antioxidant capacity, which would increase NO bioavailability.

Supplementation of the diet with Vitamin E and alpha-lipoic acid, a diet shown to significantly decrease the oxidative stress associated with exercise in the endothelium (Marsh et al., 2005), was meant to provide an alternative antioxidant defense. The Vitamin E diet improved endothelium-dependent vasorelaxation only in acute exercise (Fig 5A). This could be because the alternative antioxidant defense is only necessary in the vascular tissues before they achieve the proposed adaptive response of chronic exercise. Thus, once chronic exercise improves the oxidative capacity of the tissue, the Vitamin E diet’s antioxidant capabilities are made redundant.

Finally, SIRT1’s role in the endothelium function of the femoral artery, a vessel supplying the exercising tissues, was investigated. SIRT1 shows no significant role in the vascular reactivity of femoral arteries in sedentary or exercised rats. Phenylephrine-induced vasoconstriction, acetylcholine-induced vasorelaxation, and sodium nitroprusside-induced vasodilation in all exercise groups showed no significant difference with or without SIRT1 inhibition (Fig 6, Fig

7, Fig 8). NO has a more important role in the larger vessels, such as the aorta, than the smaller vessels, such as the femoral artery (Andrew & Mayer, 1999). Thus, the diminished role of SIRT1 in the vascular reactivity of femoral arteries as compared to aortas could be due to the diminished role of eNOS-produced NO.

Study Two

The goal of study two is to look at exercise-induced changes in SIRT1 and

SIRT3 protein expression in muscle tissue. Soleus, plantaris, EDL, and heart tissues were saved from the rats used in the previous study. This study compared changes between sedentary (SED, n=9) and 6 week endurance exercise (6WK, n=9) male,

Wistar rats. The purpose of examining SIRT1 and SIRT3 in skeletal muscles is to examine the role of metabolic stress of SIRT expression. Exercise results in significant metabolic stress (and adaptation) and this is dependent on muscle fiber type. The soleus was chosen as an example of a red, slow-twitch, oxidative muscle, whereas the plantaris is a white, fast-twitch, glycolytic muscle.

Methods

Animals

Experiments were approved by the Institutional Animal Care and Use

Committee of The College of William & Mary and adhered to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. 8-

9 month old male Wistar rats were divided between sedentary (SED, n=9) and a 6- week endurance exercise program (6WK, n=9).

Training

Exercise consisted of running on a motor-driven treadmill. 6WK runners gradually increased the duration and intensity of the training over 2 weeks until

28 reaching 30 m/min, 18.5% grade for 60 min which was then maintained for an additional 4 weeks for a total of 6 weeks of training.

Immunoblotting

A portion of the soleus, plantaris, EDL, and heart tissues frozen in liquid nitrogen were homogenized in ice-cold lysis buffer (50 mM Tris-HCl, 0.1 mM EDTA,

0.1 mM EGTA, 1% TX-100, 1% sodium deoxycholate, 1mM PMSF, 1 mM Na3VO4, and protease inhibitors) using a glass-glass homogenizer. Samples were then centrifuged at 10,000xg for 10 min at 4oC and divided for analysis.

Protein concentrations of the homogenized aorta samples were determined using the Bio-Rad detergent-compatible protein assay (Bio-Rad, Hercules, CA) .

Volumes were adjusted to equalize the concentration of the samples.

Approximately 70 μg of each sample were then loaded and run on a 10% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Bio-

Rad), which was subsequently immunoblotted using an anti-mouse WesternDot kit

1:300), β-actin anti-mouse (47778, Santa Cruz Biotechnology; dilution 1:1000).

Results from immunoblotting were determined by densitometric analysis of the chemiluninescent bands using a BioRad GelDocXR imager. Densities for each sample were normalized to a percentage of a heart standard loaded and compared to β-

29 actin as a loading control comparing tissue types and compared to total protein concentration as determined by protein assay when comparing exercise training.

Statistical Analysis

Results from the densitometric analysis of the immunoblots were analyzed using a one-way ANOVA with Turkey’s post-hoc tests (comparing tissues) or t-tests

(SED vs. 6WK) (P < 0.05).

Results

Immunoblotting

Analysis of immunoblotting results using one-way ANOVA with Turkey’s post-hoc test revealed a significant (P<0.05) increase in both SIRT1 protein expression (Fig 9) and SIRT3 protein expression (Fig 11) in the skeletal muscles

(SOL, PLT, and EDL) versus the heart (HRT) in both sedentary (Fig 9A and 11A) and

6 week trained rats (Fig 9B and 11B).

Analysis of immunoblotting results comparing sedentary and 6 week rats within each muscle group was completed with one-way ANOVAs and t-tests. SIRT1 protein expression significantly (P<0.05) increased in the soleus of sedentary versus

6 week trained rats (Fig 10A). SIRT1 protein expression significantly (P<0.05) decreased in the plantaris of sedentary versus 6 week trained rats (Fig 10B). There were no significant differences in SIRT1 protein expression in sedentary versus 6 week trained rats in the EDL or the heart (Fig 10 C and D).

SIRT3 protein expression significantly (P<0.05) decreased in the EDL of sedentary versus 6 week trained rats (Fig 12C). There were no significant differences in SIRT protein expression in the soleus, plantaris, or heart of sedentary versus 6 week trained rats (Fig 12A, B, and D).

Discussion

SIRT1 and SIRT3 are both sensitive to the redox and metabolic status of the cell (Fulco et al., 2003; Hebert et al., 2013; Onyango et al., 2002; Sack, 2011).

Exercise induces these changes in cellular status and because these changes with caloric restriction influence SIRT1 and SIRT3 expression, it has been hypothesized that exercise could have a similar effect. Both SIRT1(Suwa et al., 2008) and SIRT3

(Chen et al., 2011; Tao et al., 2010) have mitochondrial functions and oxidative stress responses. In addition, studies show that SIRT3 is increased in metabolically active tissues (Palacios et al., 2009) The relationship between the redox and metabolic status of the cell to SIRT1 and SIRT3 suggests that these proteins would have higher expression in the red, slow-twitch, oxidative muscles such as the soleus than the white, fast-twitch, glycolytic muscles such as the plantaris and extensor digitorum longus (EDL).

The most recent study on SIRT1 expression in skeletal muscle by Suwa et al.

(Suwa et al., 2008)reported an increase in SIRT1 expression in the soleus muscle following acute exercise and the soleus and plantaris muscles following high intensity (30m/min, 18.5%, 60min/day for 14 days) endurance exercise. Hokari et al. (Hokari et al., 2010)examined exercise’s effect on SIRT3 expression. They

31 described an increase in SIRT3 expression in both soleus and plantaris muscle following a four-week treadmill exercise regime at 20m/min, 60min/day but no changes following acute, one-day exercise (Hokari et al., 2010) Both studies support the hypothesis that SIRT1 and SIRT3 are increased in the muscles with higher oxidative demands and mitochondrial content.

Investigation of SIRT1 and SIRT3 expression in sedentary rats and in 6 week trained rats indicated the highest expression in the most metabolically active tissue, the heart, in both groups (Fig 9 and Fig 11); however, there was no significant

(P>0.05) change in heart SIRT3 expression from exercise training. This supports the results of Palacios et al. (Palacios et al., 2009) who reported no changes in

SIRT3 expression in cardiac tissue in rats training in a voluntary 6-week wheel- caged system but that heart tissue expressed the most SIRT3 protein..

Our data supports the findings of Suwa et al.(Suwa et al., 2008)in that the soleus muscle was the only muscle with a significantly (P>0.05) increased SIRT1 expression from sedentary rats to 6 week trained rats. SIRT1 expression in the plantaris was decreased with endurance exercise. However, our results conflict with those of Hokari et al.(Hokari et al., 2010)in that there was no significant

(P<0.05) difference in SIRT3 expression in either the soleus or plantaris muscle (Fig

12A and B). EDL, however, significantly (P<0.05) decreased with endurance exercise (Fig 12C). The conflicting results could be the result of a lower intensity of exercise in this study. Also, the longer duration of 6 weeks of endurance training versus the 4 weeks in the study by Hokari et al. (Hokari et al., 2010) may have allowed additional adaptation and resulted in a return of baseline levels.

Acknowledgements

I would like to thank the William & Mary Charles Center for granting me an honors fellowship. Their support and the generous support of family, friends, and

William & Mary alumni who funded my fellowship not only allowed for me to complete my research, but allowed me to go to Boston, MA for the Experimental

Biology conference where I gained experience and ideas on how to continue my project. I would also like to thank the College of William & Mary Undergraduate

Science Education and Research Program, funded by a Howard Hughes Medical

Institute grant, for granting me the Student Conference Travel Award allowing me to attend the Experimental Biology conference in San Diego, CA in 2012. Finally, thank you to the Jeffress Memorial Trust for funding this work and my advisor, Dr. Michael

Brennan Harris, for allowing me to use it for this project. Without their generosity, it would not have been possible.

My defense committee, Professor Harris, Professor Coleman, and Professor

Ickes, also deserve to be recognized. I am incredibly grateful for their support and participation in my thesis. Their unique and different scientific backgrounds drove me to think about my project in different ways. I could not have had a more knowledgeable and experienced committee.

The Kinesiology & Health Sciences department has been supportive through this entire process. Without them, I would not have been able to undertake this demanding project. My lab mates in the Molecular and Cardiovascular Lab have also shown their support. Matthew Olesiak deserves a special thank you. He was my mentor when I entered the lab and did critical work before I joined in on the project,

33 including all of the surgeries, without of which none of this would have been possible. He spent hours working with me and introducing me to laboratory techniques and physiology background.

Finally, my advisor, Professor Harris, has my unyielding gratitude. He has spent countless hours over the past four years giving me counsel, in and out of the lab. I thank him for allowing me to take on this project. His easygoing support has been critical in this process as a complement to my intensity. I feel very lucky to have signed up for his Freshman Seminar and to join the Molecular and

Cardiovascular Physiology lab. My experience working for him has defined my undergraduate career and he has given me tools and support that will surely help me in my next chapter of life.

Figure Captions

Figure 1. (A) Phenylephrine-induced vasoconstriction in aortas from sedentary

(SED), 1-day (1DAY), 2 week (2WK), or 6 week (6WK) treadmill trained rats (n=8-

10/group). (B) Comparison of phenylephrine-induced vasoconstriction in aortas with or without pre-treatment with 30 µM salermide for 1 hour in sedentary (SED),

1-day (1DAY), 2 weeks (2WK), and 6 week (6WK) trained rats. Data represents the absolute change in tension from rest and is expressed as mean±SE. No significant

(P>0.05) differences in phenylephrine-induced vasoconstriction were observed.

Figure 2. (A) Acetylcholine-induced vasorelaxation in aortas from sedentary (SED),

1-day (1DAY), 2 week (2WK), or 6 week (6WK) treadmill trained rats (n=6-

11/group). (B) Comparison of acetylcholine-induced vasoconstriction in aortas with or without pre-treatment with 30 µM salermide for 1 hour in sedentary (SED),

1-day (1DAY), 2 weeks (2WK), and 6 week (6WK) trained rats. Data represents percent relaxation from preconstruction to baseline tension and is expressed as mean±SE. *Significant difference between salermide treatment and no treatment at the specified dose (P<0.05).

Figure 3. (A) Sodium nitroprusside-induced vasorelaxation in aortas from sedentary (SED), 1-day (1DAY), 2 week (2WK), or 6 week (6WK) treadmill trained rats (n=6-9/group). (B) Comparison of sodium nitroprusside-induced vasorelaxation in aortas with or without pre-treatment with 30 µM salermide for 1

35 hour in sedentary (SED), 1-day (1DAY), 2 weeks (2WK), and 6 week (6WK) trained rats. Data represents percent relaxation from preconstruction to baseline tension and is expressed as mean±SE. No significant (P>0.05) differences in sodium nitroprusside-induced vasoconstriction were observed.

Figure 4. (A) eNOS expression relative to GAPDH in aortas from sedentary (SED), 1- day (1DAY), 2 week (2WK), or 6 week (6WK) treadmill trained rats (n=7-10/group).

*Significantly different from SED (P<0.05). (B) SIRT1 expression relative to GAPDH in aortas from sedentary (SED), 1-day (1DAY), 2 week (2WK), or 6 week (6WK) treadmill trained rats (n=10-12/group). All data is expressed as mean±SE.

*Significantly different from SED (P<0.05).

Figure 5. (A) Comparison of acetylcholine-induced vasorelaxation in aortas from 1- day with control diet (1DAY) and 1 day Vitamin E supplemented diet (1DAY + VitE) treadmill trained rats (n=6-11/group). *Significant difference between Vitamin E diet and control diet at the specified dose (P<0.05). (B) Comparison of acetylcholine-induced vasoconstriction in aortas with or without Vitamin E supplementation in 2 weeks (2WK) and 6 week (6WK) trained rats. Data represents percent relaxation from preconstruction to baseline tension and is expressed as mean±SE.

Figure 6. (A) Phenylephrine-induced vasoconstriction in femoral arteries from sedentary (SED), 1-day (1DAY), 2 week (2WK), or 6 week (6WK) treadmill trained

36 rats (n=5-10/group). (B) Comparison of phenylephrine-induced vasoconstriction in aortas with or without pre-treatment with 30 µM salermide for 1 hour in sedentary

(SED), 1-day (1DAY), 2 weeks (2WK), and 6 week (6WK) trained rats. Data represents the absolute change in tension from rest and is expressed as mean±SE.

No significant (P>0.05) differences in phenylephrine-induced vasoconstriction were observed.

Figure 7. (A) Acetylcholine-induced vasorelaxation in femoral arteries from sedentary (SED), 1-day (1DAY), 2 week (2WK), or 6 week (6WK) treadmill trained rats (n=5-10/group). (B) Comparison of acetylcholine-induced vasoconstriction in aortas with or without pre-treatment with 30 µM salermide for 1 hour in sedentary

(SED), 1-day (1DAY), 2 weeks (2WK), and 6 week (6WK) trained rats. Data represents percent relaxation from preconstruction to baseline tension and is expressed as mean±SE. No significant (P>0.05) differences in acetylcholine-induced vasorelaxation were observed.

Figure 8. (A) Sodium nitroprusside-induced vasorelaxation in femoral arteries from sedentary (SED), 1-day (1DAY), 2 week (2WK), or 6 week (6WK) treadmill trained rats (n=5-10/group). (B) Comparison of sodium nitroprusside-induced vasorelaxation in aortas with or without pre-treatment with 30 µM salermide for 1 hour in sedentary (SED), 1-day (1DAY), 2 weeks (2WK), and 6 week (6WK) trained rats. Data represents percent relaxation from preconstruction to baseline tension

37 and is expressed as mean±SE. No significant (P>0.05) differences in sodium nitroprusside-induced vasorelaxation were observed.

Figure 9. (A) SIRT1 expression relative to B-actin and standardized to heart in soleus (SOL), plantaris (PLT), extensor digitorum longus (EDL), and heart tissues

(HRT) of sedentary (n=9) rats. *Significantly different from HRT (P<0.05). (B) SIRT1 expression relative to B-actin and standardized to heart in soleus (SOL), plantaris

(PLT), extensor digitorum longus (EDL), and heart tissues (HRT) of 6 week treadmill trained rats (n=8). *Significantly different from HRT (P<0.05).

Figure 10. (A) SIRT1 expression relative to total protein concentration and standardized to heart in soleus tissue (SOL) of sedentary (SED) and 6 week (6WK) treadmill trained rats (n=9/group). *Significant difference between SED and 6WK

(P<0.05). (B) SIRT1 expression relative to total protein concentration and standardized to heart in plantaris tissue (PLT) of sedentary (SED) and 6 week

(6WK) treadmill trained rats (n=9/group). *Significant difference between SED and

6WK (P<0.05). (C) SIRT1 expression relative to total protein concentration and standardized to heart in extensor digitorum longus tissue (EDL) of sedentary (SED) and 6 week (6WK) treadmill trained rats (n=9/group). (D) SIRT1 expression relative to total protein concentration and standardized to heart in hearts (HRT) of sedentary (SED) and 6 week (6WK) treadmill trained rats (n=9/group).

Figure 11. (A) SIRT3 expression relative to B-actin and standardized to heart in soleus (SOL), plantaris (PLT), extensor digitorum longus (EDL), and heart tissues

(HRT) of sedentary (n=9) rats. *Significantly different from HRT (P<0.05). (B) SIRT3 expression relative to B-actin and standardized to heart in soleus (SOL), plantaris

(PLT), extensor digitorum longus (EDL), and heart tissues (HRT) of 6 week treadmill trained rats (n=8). *Significantly different from HRT (P<0.05).

Figure 12. (A) SIRT13 expression relative to total protein concentration and standardized to heart in soleus tissue (SOL) of sedentary (SED) and 6 week (6WK) treadmill trained rats (n=9/group). (B) SIRT3 expression relative to total protein concentration and standardized to heart in plantaris tissue (PLT) of sedentary (SED) and 6 week (6WK) treadmill trained rats (n=9/group). (C) SIRT3 expression relative to total protein concentration and standardized to heart in extensor digitorum longus tissue (EDL) of sedentary (SED) and 6 week (6WK) treadmill trained rats (n=9/group). (D) SIRT3 expression relative to total protein concentration and standardized to heart in hearts (HRT) of sedentary (SED) and 6 week (6WK) treadmill trained rats (n=9/group). *Significant difference between

SED and 6WK (P<0.05).

Figures

Figure 1

45 40 SED 35 1D 30 2WK 25 6WK 20 15 tension, mN tension,

Δ 10 5 0 -8 -7 -6 -5 -4 -3 phenylephrine, log [mol/L]

Figure 2

40 SED 60 1DAY 2WK

% relaxation 80 6WK 100

-10 -9 -8 -7 -6 -5 acetycholine, log [mol/L]

Figure 3

50 SED 1D 75 2WK relaxation, % relaxation, 6WK 100

-10 -9 -8 -7 -6 -5 sodium nitroprusside, log [mol/L]

Figure 4

eNOS

GAPDH

SED 1D 2WK 6WK

SIRT1

GAPDH

SED 1D 2WK 6WK

Figure 5

20 * * 40 ** 60 SED

% relaxation 80 1DAY 100 1DAY + VitE -10 -9 -8 -7 -6 -5 acetycholine, log [mol/L]

0 0

20 20

40 40 SED 60 SED 60 2WK 6WK

% relaxation 80 % relaxation 80 2WK + VitE 6WK + VitE 100 100

-10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5 acetycholine, log [mol/L] acetycholine, log [mol/L]

Figure 6

20 SED 15 1 DAY 2WK

6WK 10

5 B.

Developed Tension, mN Developed 0 -8 -7 -6 -5 -4 -3 phenylephrine, [log M]

17.5 14 13 15.0 SED 12 1D 11 12.5 SED+SM 10 1D+SM 9 10.0 8 7 7.5 6 5

tension, mN tension, 5.0 mN tension, 4 Δ Δ 3 2.5 2 1 0.0 0 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 phenylephrine, log [mol\L] phenylephrine, log [mol/L]

20 20 2WK 6WK 15 2WK+SM 15 6WK+SM

10 10

tension, mN tension, mN tension,

Δ 5 Δ 5

0 0 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 phenylephrine, log [mol/L] phenylephrine, log [mol/L]

Figure 7

A. SED 1DAY 2WK 0 6WK

% relaxation 40

50 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 acetycholine, log [mol/L]

0 0

25 10

50 20 SED 1DAY

% relaxation SED + SM 75 % relaxation 30 1DAY + SM

100 40 -10 -9 -8 -7 -6 -5 -8 -7 -6 -5 -4 -3 acetycholine, log [mol/L] acetycholine, log [mol/L]

6WK 0 0 10 6WK + SM 20 10 30 20 40 50 2WK 30

% relaxation 2WK + SM 60 % relaxation 40 70 80 50 -8 -7 -6 -5 -4 -8 -7 -6 -5 -4 acetycholine, log [mol/L] acetycholine, log [mol/L]

Figure 8

50 SED 75 1D relaxation, % relaxation, 2WK 100 6WK -10 -9 -8 -7 -6 -5 sodium nitroprusside, log [mol/L]

0 0

25 25 50 50 75 75 1D SED % relaxation, relaxation, % relaxation, 100 SED + SM 1D+SM 100 125 -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5 sodium nitroprusside, log [mol\L] sodium nitroprusside, log [mol\L] 0 0 25 25 50 50 75 75 6WK 2WK % relaxation, relaxation, % relaxation, 100 2WK+SM 6WK+SM 100 125 -10 -9 -8 -7 -6 -5 -10 -9 -8 -7 -6 -5 sodium nitroprusside, log [mol\L] sodium nitroprusside, log [mol\L]

Figure 9

A. SIRT1 expression in tissues from sedentary rats 100

% of HRT * * 25 *

0 SOL PLT EDL HRT tissue

SIRT1

β -actin

SOL PLT EDL HRT B. SIRT1 expression in tissues from 6 week trained rats 250

200

150

100 * % of HRT

50 * *

0 SOL PLT EDL HRT tissue

SIRT1

β-actin

SOL PLT EDL HRT

Figure 10

A. B. SIRT1 expression in soleus SIRT1 expression in plantaris

35 * 110 30 100 90 25 80 70 20 60 15 50 *

% of HRT 40 % of HRT 10 30 5 20 10 0 0 SED 6WK SED 6WK

C. D.

SIRT1 expression in EDL SIRT1 expression in HRT

14 250 13 12 11 200 10 9 8 150 7 6 100 5 % of HRT % of HRT 4 3 50 2 1 0 0 SED 6WK SED 6WK

Figure 11.

A. SIRT3 expression in tissues from sedentary rats 100

50 % of HRT 25 * * *

0 SOL PLT EDL HRT tissue

SIRT3

β-actin

SOL PLT EDL HRT

B. SIRT3 expression in tissues from 6WK trained rats 130 120 110 100 90 80 70 60 50 % of HRT 40 * 30 20 * * 10 0 SOL PLT EDL HRT tissue

SIRT3

β-actin SOL PLT EDL HRT

Figure 12.

A. B. SIRT3 expression in soleus SIRT3 expression in plantaris

12 11 11 10 10 9 9 8 8 7 7 6 6 5 5 4 % of HRT % of HRT 4 3 3 2 2 1 1 0 0 SED 6WK SED 6WK

C. D.

SIRT3 expression in EDL SIRT3 expression in HRT

30 140 130 120 110 100 20 90 80 70 60

% of HRT 50 10 * % of HRT 40 30 20 10 0 0 SED 6WK SED 6WK

Tables

Table 1.

CTRL +Salermide

phenyleprine, log[mol/L] GROUP expressed as mean±SE

SED -7.351±0.638 -7.351±0.638

1DAY -11.33±65.66 -11.24±82.28

2WK -6.565±0.051 -6.747±0.149

6WK -5.909±0.059 -6.030±0.083

Table 2.

(*P<0.05, CTRL vs Salermide)

(†P<0.05, 1DAY vs SED, 2WK, 6WK)

(‡P<0.05, 2WK vs SED, 1DAY, 6WK)

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