Increased gluti expression in mouse cardiomyocytes preserves mitochondrial function but is insufficient to attenuate pressure overload-induced contractile dysfunction

Lifelong over-expression of GLUT1 transporter in the heart has been shown to preserve contractile function after aortic constriction. However it is unclear whether this protective effect is due to GLUT1 overexpression itself or due to reprogramming of heart metabolism as a result of long-term increased glucose utilization. We, therefore generated mice with cardiomyocyte-restricted inducible overexpression of GLUTI(GIHA) to test if short-term GLUT1 overexpression at the onset of pressure overload hypertrophy (POH) is sufficient to prevent the progression to heart failure in a model of POH. Six-week old mice were subjected to transverse aortic constriction (TAC) to induce POH. GLUT1 transgene expression was initiated 2 days prior to TAC surgery and resulted in a 4-fold increase in cardiac GLUT1 protein levels relative to controls. Four weeks after TAC, cardiac contractile function was equivalently impaired in control and G1HA mice, as shown by increased left ventricular developed pressure and decreased in maximal dp/dt ratio. In contrast, mitochondrial function following TAC was preserved and fibrosis was significantly decreased in G1HA mice relative to control. In summary, our data suggest that a short-term increase in GLUT 1-mediated glucose uptake preserves mitochondrial function and attenuates pathological remodeling, but does not prevent cardiac dysfunction following POH.

INCREASED GLUT I EXPRESSION IN MOUSE CARDIO MYOCYTES PRESERVES MITOCHONDRIAL FUNCTION BUT IS INSUFFICIENT TO ATTENUATE PRESSURE OVERLOAD-INDUCED CONTRACTILE DYSFUNCTION by Jeffrey KaChon Lei A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Department of Bioengineenng Approved: Bradley Greger, Ph.D. Department Honors Advisor Di\ jSylvia D. Torti Dean, Honors College April 2013 ABSTRACT Lifelong over-expression of GLUT1 transporter in the heart has been shown to preserve contractile function after aortic constriction. However it is unclear whether this protective effect is due to GLUT1 overexpression itself or due to reprogramming of heart metabolism as a result of long-term increased glucose utilization. We, therefore generated mice with cardiomyocyte-restricted inducible overexpression of GLUTI(GIHA) to test if short-term GLUT1 overexpression at the onset of pressure overload hypertrophy (POH) is sufficient to prevent the progression to heart failure in a model of POH. Six-week old mice were subjected to transverse aortic constriction (TAC) to induce POH. GLUT1 transgene expression was initiated 2 days prior to TAC surgery and resulted in a 4-fold increase in cardiac GLUT1 protein levels relative to controls. Four weeks after TAC, cardiac contractile function was equivalently impaired in control and G1HA mice, as shown by increased left ventricular developed pressure and decreased in maximal dp/dt ratio. In contrast, mitochondrial function following TAC was preserved and fibrosis was significantly decreased in G1HA mice relative to control. In summary, our data suggest that a short-term increase in GLUT 1-mediated glucose uptake preserves mitochondrial function and attenuates pathological remodeling, but does not prevent cardiac dysfunction following POH. ii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 3 RESULTS 10 DISCUSSION 17 ACKNOWLEDGEMENT 20 SOURCES OF FUNDING 21 REFERENCES 22 INTRODUCTION Despite significant advancements in the treatment of cardiovascular diseases, heart failure remains the leading cause of death in the United States, affecting approximately 5 million people and causing over 287,000 deaths per year. In addition, medical costs for treating and diagnosing cardiac dysfunction are currently $147 billion 1 dollars annually, a heavy burden on the world's economy . To provide cost effective and viable solutions to addressing cardiovascular complications, including cardiac hypertrophy, the heart's energy consumption patterns and self-protecting mechanism under pressure overload hypertrophy (POH) must be further investigated. The heart is capable of metabolizing a variety of substrates to sustain its energy demands. Under physiological conditions, oxidative metabolism of fatty acids accounts 2 for more than 50% of myocardial ATP production ; however, glucose assumes greater 3 importance in many clinically relevant circumstances such as during POH . An important regulatory mechanism in cardiac glucose utilization is via regulation of glucose import, mediated by two transporters, glucose transporter 1 and glucose transporter 4 (GLUT1 and GLUT4 respectively) 4 Studies have suggested that increasing glucose transport in the heart plays a role in restoring cardiac function during POH. Specifically, lifelong overexpression of GLUT1 in transgenic mouse hearts leads to increased glucose utilization, attenuates the development of contractile dysfunction and prolongs survival 5, 6 after ascending aortic constriction . Conversely, wild type mice have a significant increase in dilation of the left ventricle, an enlargement of the left atrium and an increase in the amount of cardiac cell death. However, the possibility exists that, due to the prolonged duration of GLUT1 overexpression in these hearts, metabolic, transcriptional 1 and epigenetic reprogramming may have occurred, leading to the cardioprotective effects observed. Thus, a more clinically relevant approach would be to inducibly increase glucose utilization in the context of a hemodynamic stress such as POH. We, therefore, developed a model in which cardiomyocyte- specific GLUT1 overexpression can be induced by doxycycline injections (DOX). This model is valuable for determining whether a short-term increase in myocardial glucose uptake would still confer cardioprotection if it occurs at the onset of POH. Furthermore, the main source of energy generation in the heart is the mitochondria. Previous studies have shown that in response to POH, mitochondrial oxidative capacity is reduced in wild type mice that underwent aortic constriction . Whether increased glucose uptake and utilization in the heart can play a role in maintaining mitochondrial function in the context of POH is incompletely understood. Thus, in the present study, we also investigated the role of increased glucose uptake and utilization on preserving mitochondrial function during POH. We hypothesized that short-term inducible GLUT1 over-expression in cardiomyocytes at the onset of POH will be sufficient to retard cardiac contractile and mitochondrial dysfunction, preventing the progression to of heart failure. Should this hypothesis hold true, possible treatments involving an increased glucose delivery to the heart should be considered as tools to treat patients with pathological hypertrophy. 2 METHODS Mouse Model and Genotyping Double transgenic mice for HA tagged GLUT1 under the control of the tetracycline response element promoter (TRE-GLUT1) and the codon optimized tetracycline transactivator under control of the a-myosin heavy chain (MHC-rtTA) were generated (G1HA) in a FVB background. TRE-GLUT1 mice were generated at University of Colorado Health Science Center. To activate the TRE-GLUT1 transgene, mice were given a single injection of lOOug of doxycycline (DOX) and maintained on DOX-chow (200 mg/kg; Bio-Serv, Frenchtown, NJ) Research Diet Inc., Brunswick, NJ). Transgene induction was initiated in 6-week old mice 2 days prior to transverse aortic constriction (TAC) and resulted in a 4-fold increase in GLUT1 content in the heart. Both control and G1HA mice were injected with DOX and men kept on DOX-chow for 4 weeks. Single transgenic littermates were used as controls. The animals were housed at 22°C with a 12hour light, 12-hour dark cycle with free access to water and standard chow. The experiments were performed using exclusively male mice. All mouse experiments were approved by the Institutional Animal Care and Use Committee of the University of Utah. The following primers were used for genotyping: FABP (Cont): forward primer 5'-TGGCATGTGAGGCGGTTAGGTTATCT-3' reverse 5 primer 5'- GAGCTTTGGCCACATCACAGGTCATTC-3. GLUT1-HA: forward primer 5 '-CTCCAACTGGACCTCAAATTTC-3reverse primer 5'- CATAGTCGGGCACGT-3'. aMHC-rtTA (tON): forward primer 5>-GTCGCTAAAGAAGAAAGGGAAACAC3\ reverse primer 5 '-TTCCAAGGGCATCGGTAAACATCTG-3'. 3 VEGF (Con): forward primer 5'-AGAAACACTTGTTTGGTGTGGAGC-3reverse primer 5 '-TATCTCGTCGGGGTACTCCTGGAA-3'. Composition of Mouse Chow Mice were fed standard chow, Harland Teklad Diet 8656 (3.8Kcal/g of gross energy) that contained 65% carbohydrate (corn and soybean meal), 24.5% protein (Soy based), 4.4% fat (Soybean oil), 3.4% fiber and was supplemented with vitamins and minerals. Two days prior to TAG surgery and throughout the time-course of the experiments, both Cont and G1HA mice were kept on DOX chow diet, which consisted of 3.6% carbohydrate (corn and soybean meal), 20.8% protein, 8.7% fat (soybean oil) from Bio-Serv, Frenchtown, NJ (3.5Kcal/g of gross energy) until euthanasia (4 weeks after TAC). Surgical Procedures: Transverse aortic constriction: After the induction of the GLUT1 transgene, 6-7-week old G1HA mice were submitted to TAC or sham operations. These mice were anesthetized with a single intraperitoneal injection of 400mg chloral hydrate/kg body weight. During TAC, the aorta was accessed via a 2~3mm longitudinal cut through the sternum. Then a ligation clip applicator, calibrated to 30-G was used to place a 1/1 titanium microclip (Horizon) next to the aortic arch between the origin of the right innominate and left common carotid artery, or closed without clip application (sham operated). 4 8 Left Ventricle Catheterization: 9 Four weeks after TAC, cardiac catheterizations were performed as previously described . Mice at 6-wk of age were anesthetized (single intraperitoneal injection of 400 mg chloral hydrate / kg body weight) and placed in the supine position on a heating pad (37 °C). The right carotid artery was identified and accessed, following which a Millar Mikro-Tip catheter (1.0F; Millar Instruments, Houston TX) was then inserted into the left ventricle via the left ventricle, and hemodynamic measurements were obtained using LabChart7 Pro software (ADInstruments, Colorado Springs, CO). Isolation of Cardiac Myocytes and Determination of 2-DG Uptake Cardiomyocytes were isolated using collagenase digestion of Langendorff-perfused 10 mouse hearts as previously described by our group . This isolation procedure initially yields between 80 and 90% viable rod-shaped myocytes, the majority of which attach to laminin-coated wells and maintain their morphology for the duration of the protocol. Basal and insulin-stimulated glucose uptake was measured using 2-deoxyglucose. In brief, cardiomyocytes were prepared from mouse hearts that were obtained from heparinized mice and retrograde perfused with perfusion buffer (pH 7.3; in mmol/1): 126 NaCl, 4.4 KC1, 1.0 MgCl 4.0 NaHC0 , 10.0 HEPES, 30.0 2,3-butanedione monoxime, 2) 3 5.5 glucose, 1.8 pyruvate, and 0.025 CaCl2 supplemented with 16 U/ml type I collagenase tor 10 min. Myocytes were dispersed for 10 min at 37°C in perfusion buffer that contained 2% BSA and 0,2 mmol/1 CaCl and cultured in modified Dulbecco's modified 2 Eagle's medium (DMEM) that contained 5% FBS, 5.0 mmol/1 glucose, and 1.0 mmol/1 5 CaCl on laminin-coated plates at 37°C in humidified 95% 02-5% C0 - Cells were 2 2 allowed to attach to the wells for 60 min and were subsequently cultured for 30 min in DMEM that contained 0.1% BSA, 5.0 mmol/1 glucose, and 1.0 mmolA CaCb before measuring glucose uptake. Cells were then incubated for 40 min in the presence or absence of 0.1-10 nmol/1 insulin in glucose-free DMEM supplemented with 1 mg/ml BSA and 1 mmol/1 pyruvate. Glucose uptake was performed by adding 0.1 mmol/1 23 deoxy-d-glucose and 3.33 nCi/ml 2-[l,2- H]-deoxy-d-glucose for 30 min. Glucose transport experiments were terminated after 30 min by aspiration of the buffer followed by two washes with ice-cold PBS and then cells were lysed in 1 N NaOH for 20 min at 37°C. Nonspecific uptake was assessed in the presence of 10 umol/1 cytochalasin B and subtracted from all of the measured values. The radioactivity was counted by liquid scintillation spectroscopy using a Beckman LS 5000 TD instrument (Beckman Coulter, Fullerton, CA) and normalized to protein amount measured with a Micro BCA Protein Assay Kit (Pierce Chemical, Rockford, IL). Mouse Characteristics Right before euthanasia (4 weeks after TAC), body weights were taken. While mice were still sedated, hearts and lungs were excised and weighed. Tibiae were also removed and their lengths measured. 6 Histology and Stereology Myocardial fragments were stained by Masson's trichrome stain for visualization of fibrotic tissue. Light microscopy was performed using an Olympus TH4-100 inverted microscope that was connected to an Olympus Microfire Digital Camera (New York, NY). For quantification of fibrotic tissue, pictures from each heart section (thickness of 3 urn) were taken systematically to ensure that the entire extent of the tissue section had been covered. Five different hearts were used per group, so a total of 20 sections were utilized in the study. The micrographs were then analyzed using the Image Pro-plus software (Media Cybernetics, Silver Springs MD). The percentage of fibrotic tissue in each micrograph was measured and averaged per heart The average value for each heart was then utilized for statistical analysis. Western Blot To ensure GLUT1 expression had been induced, western blots were performed in whole heart homogenates of control and G1HA mice at basal conditions 2 days after DOX treatment. For immunoblotting analysis, ~50 mg of frozen tissue was homogenized in 200uL Lysis buffer (50mmol/L, NaCl, 10% Glycerol, 1% Triton X-100, 1.5mmol/L MgCb, 1 mmol/L EGTA, 10 mmol/L Sodium Pyrophosphate, 100 mmol/L Sodium Fluoride and 100 umol/L Sodium Vanadate, 1 mmol/L PMSF, 10 ug/ml Aprotinin, and 10 ug/ml Leupeptin) using the Tissue Lyser II (Qiagen Inc., Valencia, CA). Tissue lysates were resolved on SDS-PAGE and transferred to PVDF membranes (Millipore 7 Corp., Billerica, MA). Rabbit-GLUTl antibody was utilized (Millipore, Billerica, MA) and Alexa fluor anti-Rabbit 680 (Invitrogen, Carlsbad, CA) was used as secondary antibody. Fluorescence was quantified using the LICOR Odyssey imager (Li-COR, Lincoln, NE). Mitochondrial Function Measurement Left ventricular muscle fibers were dissected from freshly excised hearts and permeabilized with saponin. ATP synthesis and cellular respiration were measured using the substrate, palmitoyl-carnitine (20uM, PC) combined with malate (2mM). Mitochondrial oxygen consumption: the respiratory rates of cardiac fibers were measured using an oxygen sensor probe (Ocean Optics, Dunedin, FL) in 1 ml of KC1 buffer at 25°C (125mmol/L KC1, 20 mmol/L HEPES, 3mmol/L Mg-Acetate, 0.4mmol/L EGTA, 2mg/ml BSA, 5mmol/L KH P0 , 0.3mmol/L Dithiothreitol, 2mmol/L malate and 20umol/L 2 4 Palmitoyl-carnitine, pH 7.1 adjusted at 25"C) Oxygen consumption was determined under two different conditions: following ADP- stimulation (ImM; VADP) and after addition of the ATP synthase inhibitor oligomycin (lug/ml; Voii ). The respiratory control ratio g0 (RCR) is the ratio of VADP to Vou . The solubility of oxygen in KCI buffer was 246.87 go 1 nmol of 0 /mL. Oxygen consumption rates were expressed as nmol of 02*min" *mg dry 2 1 fiber weight" . Mitochondrial ATP production: ADP was added to 1ml of buffer B to a final concentration of lmmol/L. 10 ul Buffer B was then added from the respiration chamber to 190 ul DMSO every 10 seconds for a 1-min time period. A bio luminescence assay 8 based on the luciferin/luciferase reaction with the ATP assay kit (Promega Corporation, Madison, WI) was used to measure ATP generation. Statistical Analysis: Data are presented as means±SEM. RT-PCR and western blot results are presented as fold change versus Cont sham. A probability value of p<0.05 was considered significantly different. Significant differences were determined by t-Test in the experiments where we compared G1HA mice with age-matched controls, and by ANOVA followed by Tukey multiple comparison test, when we compared 4 groups (Cont sham, Cont TAC, G1HA sham and G1HA TAC). Statistical calculations were performed using the GraphPad Prism Software, Inc. (La Jolla, CA, USA). Student Participation: My participation in the GLUT1 project is as follows: 1. Maintenance of the HA-G1H colony 2. Mice genotyping (Polymerase chain reaction and gel electrophoresis) 3. Heart and lung tissue harvest 4. Cardiac hypertrophy and fibrotic tissue measurement via light microscopy 5. Protein and DNA extraction 6. Western blotting 7. Statistical analysis 9 RESULT GLUT1 Overexpression Increases 2-DG Uptake in Cardiomyocytes: Inducible over expression of GLUT1 was achieved in double transgenic mice harboring the HA tagged GLXJTl under the control of the tetracycline response element promoter (TRE-GLUT1) and the codon optimized tetracycline transactivator under control of the alpha-myosin heavy chain (MHC-rtTA) by DOX injection followed by DOX-chow feeding. Two days after DOX treatment, there was an approximate 4-fold increase in GLUT1 protein content in whole heart homogenates from G1HA mice relative to controls (Figure 1 A). As a result of increased GLUT1 content, basal and insulin-stimulated 2-DG uptake in cardiomyocytes isolated from G1HA hearts 2 days after DOX treatment were also increased when compared to control cardiomyocytes, indicating successful activation of the transgene at the time TAC surgeries were performed (Figure IB). 10 Insulin concentration Figure 1: Transgene induction. A- Protein expression of GLUT1 in the heart of G1HA and WT after 2 days of doxycycline treatment. B- Glucose uptake in isolated cardiomyocytes from G1HA and WT mice. Significant differences were determined by Student's t-test, using a significance level of p<0.05. Data are expressed as mean± SEM. n^5 per group. Cardiac Hypertrophy and Pulmonary Edema following TAC: GLUT1 overexpression did not induce hypertrophy at base line. Following TAC, heart weight to tibia length ratios were elevated in control TAC mice, but were exacerbated in G1HA mice compared to sham operated mice (Figure 2A). This elevation indicated G1HA hearts hypertrophied significantly more relative to Cont TAC. Wet lung weight to tibia length ratios were also equivalently increased in control and G1HA mice following TAC relative to shams, indicating similar degrees of pulmonary edema as a result of LV dysfunction (Figure 2B). In addition, body weight was significantly decreased in G1HA mice 4 weeks after TAC, and tibia length measurements were unchanged between groups (Table 1). 11 * [—** Sham TAC Control " r—*"—mm^m*~~ 0 Sham T A C G1HA **Mp« • Sham T A C Control • | - 1" Sham T A C G1HA Figure 2: Cardiac Hypertrophy 4 weeks after TAC. A- Heart weight to tibia length ratio of G1HA and WT B- Wet lung weight to tibia length ratio of G1HA and WT. Significant differences were determined by ANOVA, using a significance level of p<0.05. Data are expressed as mean±SEM. n~5 per group. (*) compared to sham, (#) compared to Cont TAC. rt£7 Cent sham Cont T A C 31 HA sham G1HATAC Body weight (g) 29.85*1.05 26.88*1.10 29.94±1.32 23.82*.Q57* Heart weight (mg) 123.0*4.74 198.7*12.27* 123.7*4.45 238.0*10.82*# Tibia length (mm) 16.88*0.47 17.53*0.20 17.45±0.32 17.02*0.39 Wet lung weight (mg) 173*60.38 268.5*34.71* 192.7*6.79 353.4*44.60* Table 1: Mouse characteristics 4 weeks after TAC. Significant differences were determined by ANOVA followed by Tukey multiple comparison test, using a significance level of p<0.05. n>7. Data are expressed as mean* SEM. (*) compared to sham, (#) compared to Cont TAC. 12 Short-term Increased GLUT1- mediated Glucose Uptake does not Prevent Contractile Dysfunction: Cardiac contractile function was measured by Left Ventricular Catheterization in control and G1HA mice 4 weeks after TAC. This analysis revealed similar increases in left ventricle end diastolic pressure (LVEDP), left ventricle developed pressure (LVDevP) and an equivalent decrease in peak rates of ventricular contraction (+dP/dt) between Cont and G1HA mice after TAC (Table 2), indicating similar degrees of LV contractile dysfunction in both groups. rt£8 Cont sham Cont TAC G1HA sham G1HATAC +dp/dt (mmHgteec) 9311*520.9 6638±383.7* S810±453 5501±545.4* -dp/dt (mmHg/sec) -818Q±514.3 -6764±412.1 -7361±762.3 LVDevP (mmHg) -7531±566.4 97.44±4.47 145.6±6.45* 94.76±4.26 144.4±6.24* LVEDP (mmHg) 9.65±1.74 24.29*5.0* 16.91±2.3 33.42±3.96* 440.7±16.32 436.6±15.53 441.9±7.77 413.6±19.26 Heart Rate (bpm) Table 2: Left Ventricular Catheterization parameters following pressure overload. ±dp/dt (peak rates of ventricular contraction), LVDevP (left ventricular developed pressure), LVEDP (left ventricular end diastolic pressure) and heart rate. Significant differences between groups were determined by ANOVA followed by Tukey multiple comparison test, using a significance level of p<0.05. n>7. Data are expressed as mean± SEM. (*) compared 13 to sham. GLUT1 Overexpression Attenuates Development of Cardiac Fibrotk Tissue: POH induced by 4 weeks of TAC lead to an increase in the development of cardiac fibrosis in control mice relative to shams (Figures 3A and B). In contrast, G1HA mice had a 50% reduction in cardiac fibrotic tissue relative to control mice after TAC. A Quantification of fibrosis 2,5-1 Sham TAC Control Sham T A C G1HA Figure 3: GLTJT1 short term overexpression decreases cardiac fibrotic tissues after TAC (S hearts per group). A- Mason's Trichrome staining of heart sections. BQuantification of percentage of fibrotic area. Significant differences were determined by ANOVA followed by Tukey multiple comparison test, using a significance level of p<0.05. Data are expressed as means±SEM. (*) compared to sham, (#) compared to Cont TAC. 14 GLUT1 Overexpression Preserves Mitochondrial Function and ATP Synthesis in the Heart: Afer TAC or sham surgeries, mitochondrial function was assessed in saponinpermeabilized cardiac fibers of Cont and G1HA mice. Palmitoyl-Carnitine (PC)supported maximal ADP- stimulated mitochondrial oxygen consumption (VADP) and ATP synthesis were reduced in control mice following TAC. In contrast, mitochondrial VADP and ATP synthesis in G1HA mice were maintained at levels similar to that of control sham mice, regardless of TAC operation (Figure 4A-C). 15 Sham TAC Sham Control TAC Sham G1HA TAC Control Sham TAC G1HA Figure 4. Mitochondrial function is preserved in G1HA mice after TAC (5-7 hearts per group). A- Palmitoyl- carnitine was used as substrate. A- Mitochondrial respiration, B- ATP synthesis rates, and C- ATP/O ratios were measured in saponin-permeabilized cardiac fibers. Significant differences between groups were determined by ANOVA followed by Tukey multiple comparison test, using a significance level of p<0.05. n>7. Data are expressed as mean± SEM. 16 (*) compared to sham. DISCUSSION Increased glucose uptake and utilization have been consistently observed in 11 14 hypertrophied hearts ' . Previous studies using transgenic mice with lifelong cardiac specific overexpression of GLUT1 have demonstrated that chronic increases in basal glucose uptake in adult mouse hearts delays the progression to heart failure following 15 ascending aortic constriction . However, it is unclear if the cardioprotective effects alone were the direct result of increased glucose uptake or an indirect effect due to metabolic, epigenetic and transcriptional reprogramming in the heart. Therefore, in the present study, we utilized a model of short-term inducible GLUT1 overexpression to investigate if a cardiomyocytes-specific increase in GLUT1 would still confer cardio-protection if it occurs at the onset of POH. Our data suggest that a short-term induction of GLUT1 in cardiomyocytes alone is not sufficient to prevent LV contractile dysfunction. However, they suggest a role for increased GLUT1 expression in protecting the heart against pathological remodeling. In addition, we identified an important role for increased GLUT1-mediated glucose uptake in the maintenance of mitochondrial function following POH. GLUT1 overexpression was induced successfully in our transgenic model. Two days after DOX induction, G1HA mice had increased GLUT1 protein levels in isolated cardiomyocytes, which was accompanied by increased basal and insulin-stimulated glucose uptake. However, after 4 weeks of TAC, G1HA mice had shown an augmented cardiac hypertrophy relative to Cont TAC. This suggests that GLUT1 mediated glucose uptake might amplify the hypertrophic response to TAC. Further investigation to 17 elucidate the mechanism from linking increased glucose uptake to enhance cardiac hypertrophic in response to POH is therefore necessary. Pathological hypertrophy is associated with increased interstitial fibrosis and cell 16 17 death ' . In our study, cardiac fibrosis was elevated in control mice following 4 weeks of TAC, but was significantly reduced in G1HA hearts, suggesting that GLUT1 overexpression attenuates pathological remodeling in response to TAC. In contrast, cardiac contractile function was equivalently reduced in control and G1HA mice, as evidenced by similar increases in left ventricle and diastolic pressure (LVEDP), left ventricle developed pressure (LVDevP) and decreased peak rates of ventricular contraction (+dP/dt) between Cont and GlHA mice after TAC. This suggests that short term GLUT1 over expression alone is not sufficient to attenuate heart failure. Additional studies need to be performed in the future to elucidate the glucose-independent mechanisms that contribute to heart failure in GlHA decreased fibrosis. It is widely accepted that mitochondrial dysfunction is associated with 18 pathological cardiac hypertrophy . Consistent with this notion, Cont TAC mice had impaired mitochondrial function and reduced ATP production relative to Cont sham mice, as demonstrated by reduced VADP- Conversely, GlHA mice had preserved mitochondrial function and ATP synthesis following TAC. Despite normal mitochondrial function, GlHA mouse hearts exhibit contractile dysfunction. These data therefore suggest that mitochondrial dysfunction might not mediate the transition from compensated cardiac hypertrophy to heart failure, but rather it is secondary to other pathophysiological processes that drive this transition. These data also suggest that contractile dysfunction in POH may occur independently of mitochondrial dysfunction. 18 In summary, our study identified a role for short-term induction of GLTJT1 in maintaining mitochondrial function and preventing cardiac fibrosis. Despite these beneficial effects of GLTJT1 overexpression in attenuating adverse structural and mitochondrial remodeling, they were insufficient to maintain contractile function in hearts subjected to POH. Although we conclude that increased GLTJT1-mediated glucose uptake and utilization is insufficient to preserve contractile function, the possibility remains that the reduction in LV remodeling might indicate that these hearts may exhibit more robust recovery if the hemodynamic stressor is removed. 19 ACKNOWLEDGEMENT This honors thesis would not have been possible without the support of many people. First and foremost, I would like to express my deepest gratitude to my mentors and friends—Dr. Dale Abel and his post-doc fellow Dr. Renata Alambert These individuals are extraordinary mentors that had demonstrated leadership and compassion in scientific research. In particular, their intriguing findings on cardiovascular diseases and diabetes has amplified my knowledge and academic curiosity in pursuing a career in medicine and research. Their strong work ethic had also created a very collaborative environment that helped me personally to appreciate an interdisciplinary approach to scientific discovery. I am therefore deeply grateful for their persistent guidance and friendship over the course of producing this manuscript In addition, I would also like to thank Heather Palmer and Dr. Kelly Broadhead, the undergraduate academic advisors of the Department of Bioengineering, for their support, guidance and revision on the manuscripts. I also thank Dr. Bradley Greger, the undergraduate Honor's advisor for Bioengineering, for his insightful personality and his guidance in completing my Honor's Degree in Biomedical Engineering. Lastly, I would like to express my gratitude to my beloved parents and Aunt Stella's family, for their understanding, love and support throughout the duration of my studies at the University of Utah. 20 SOURCES OF FUNDING This study was supported by National Institute of Health grant RO1DK092065 and U01 HL087947 to Dr. Dale E. Abel and the undergraduate assistantship funded by the Undergraduate Research Opportunity Program (UROP) at the University of Utah. 21 REFERENCES 1. American Heart Association. 2011. Congestive heart failure statistics, [online]. Available: http://heart.org. 2. Taegtmeyer H 1994 Energy metabolism of the heart: from basic concepts to clinical applications. Current problems in cardiology 19:59-113 3. 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