Contribution of Social Isolation, Restraint, and Hindlimb Unloading to Changes in Hemodynamic Parameters and Motion Activity in Rats

The most accepted animal model for simulation of the physiological and morphological consequences of microgravity on the cardiovascular system is one of head-down hindlimb unloading. Experimental conditions surrounding this model include not only head-down tilting of rats, but also social and restraint stresses that have their own influences on cardiovascular system function. Here, we studied levels of spontaneous locomotor activity, blood pressure, and heart rate during 14 days under the following experimental conditions: cage control, social isolation in standard rat housing, social isolation in special cages for hindlimb unloading, horizontal attachment (restraint), and head-down hindlimb unloading. General activity and hemodynamic parameters were continuously monitored in conscious rats by telemetry. Heart rate and blood pressure were both evaluated during treadmill running to reveal cardiovascular deconditioning development as a result of unloading. The main findings of our work are that: social isolation and restraint induced persistent physical inactivity, while unloading in rats resulted in initial inactivity followed by normalization and increased locomotion after one week. Moreover, 14 days of hindlimb unloading showed significant elevation of blood pressure and slight elevation of heart rate. Hemodynamic changes in isolated and restrained rats largely reproduced the trends observed during unloading. Finally, we detected no augmentation of tachycardia during moderate exercise in rats after 14 days of unloading. Thus, we concluded that both social isolation and restraint, as an integral part of the model conditions, contribute essentially to cardiovascular reactions during head-down hindlimb unloading, compared to the little changes in the hydrostatic gradient

MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION INITIATION FACTOR PROTEIN EXPRESSION ENHANCEMENT THROUGH HIGH-VOLUME RESISTANCE TRAINING

David E. Lee1, James D. Fluckey2, Mats I. Nilsson2, Lemuel A.Brown1, Kevin L. Shimkus2, Tyrone A. Washington1, Stephen F.Crouse, FACSM3, and Nicholas P.Greene123 1Human Performance Laboratory, Department of Health, Human Performance and Recreation, University of Arkansas, Fayetteville, AR, 2Muscle Biology Laboratory, Department of Health & Kinesiology, Texas A&M University, College Station, TX, 3Applied Exercise Science Laboratory, Department of Health & Kinesiology, Texas A&M University, College Station, TX Exercise is known to stimulate muscle protein synthesis through transcription and translation of nuclear DNA leading to muscular hypertrophy and mitochondrial biogenesis. To our knowledge, the effect of exercise on mitochondrial translation has yet to be tested even though proteins coded by the mitochondria are paramount to metabolic health. PURPOSE: This investigation aims to determine if transcription factor A of the mitochondria (TFAM) and mitochondrial translation initiation factor 2 (mtIF2) protein expression is enhanced following high-volume resistance training and see if any difference exists in obesity compared to lean controls. METHODS: Zucker Rats (N=30, 16 lean, 14 obese) engaged in either a resistance training protocol or remained sedentary. The exercised rats (n = 8 lean and 8 obese) undertook a resistance exercise protocol involving a hind limb ‘squat-like’ exercise while the remainder (n=8 lean and 6 obese) were sedentary. Sixteen hours following the final bout of resistance exercise, the animals were euthanized and mixed fiber gastrocnemius muscles were removed and immediately frozen in liquid nitrogen. Samples were later analyzed for TFAM and mtIF2 protein expression via Western blot analysis. Data were analyzed using a 2x2 ANOVA (exercise vs. sedentary X lean vs. obese); α was set at p≤0.05. RESULTS: TFAM protein content increased by 54.62%(p0.05). No significant difference in TFAM and mtIF2 was observed between lean and obese responses to exercise. CONCLUSION: Resistance exercise increased the expression of mitochondrial transcription factors and appears to increase expression of mitochondrial translation factors in lean and obese animals indicating enhanced capacity for transcription and translation of mitochondrially encoded genes with high volume exercise training. These data suggest that resistance training could lead to increased metabolic proteins derived from the mitochondrial DNA in mammals. Enhanced mitochondrial protein synthesis would likely lead to increased oxidative phosphorylation enzymes such as those used in the electron transport system indicating a possible pathway for resistance training improvements on whole organism metabolic health. This investigation was funded by the Sydney & J.L. Huffines Institue for Sports Medicine & Human Performance at Texas A&M University

DIFFERENTIAL RESPONSES OF SKELETAL MUSCLE MICRORNAS TO RESISTANCE EXERCISE TRAINING IN DIABETES

David E. Lee1, Jacob L. Brown1, Megan E. Rosa1, Lemuel A. Brown1, Richard A. Perry, Jr.1, Mats I. Nilsson2, Tyrone A. Washington1, James D. Fluckey2 & Nicholas P. Greene1 1University of Arkansas, Fayetteville, Arkansas; 2Texas A&M University, College Station, Texas MicroRNAs are an emerging post-transcriptional modifier of protein expression. Some microRNAs are tissue specific including miRNA-1, -133, and -206, which are skeletal muscle specific. Skeletal muscle protein synthesis and metabolic health are vital in situations of atrophy and insulin resistance as seen with sarcopenic obesity and type II diabetes, respectively. PURPOSE: The purpose of this study was to investigate the changes in muscle specific microRNAs in healthy and diabetic rats and their responses to resistance exercise training (RE) METHODS: Zucker rats (16 healthy, 14 diabetic) were sedentary (sed, 8 healthy, 6 diabetic) subjected to a RE protocol (8 healthy, 8 diabetic) involving a high-volume, weighted squat-jump-like movement. After 4 progressive bouts, animals were euthanized and gastrocnemus muscles were removed and later processed for microRNA and mRNA gene targets, various protein contents and protein fractional synthetic rates (FSR). Data were analyzed by 2X2 ANOVA (phenotype [healthy vs diabetic] vs. exercise [sed vs. RE]), correlations were assessed using Pearson’s product moment, α = 0.05. RESULTS: miRNA-1 significantly increased in the diabetic condition ~4-fold (pp\u3c0.05) compared to healthy sed. In healthy groups, no difference was seen with RE on miRNA-133a levels while in the diabetic phenotype miRNA-133a content in RE was 113% greater than sed (pp\u3c0.05) compared to healthy sed. No significant differences were seen in levels of microRNA-206. miRNA-1 was negatively correlated with FSR in RE groups but not sed. miRNA-133b showed a negative correlation with mitochondrial FSR in healthy but not diabetic animals. CONCLUSION: Control of protein synthesis and mitochondrial health is under complex control involving many factors. The alteration of muscle specific microRNAs in diabetes and RE and the relationships to targeted mRNAs suggests that the control of protein expression by microRNAs is at least partially responsible for protein synthetic and metabolic dysfunctions. Further research into the direct mechanisms and microRNA targets is warranted. The investigation was funded, in part, by the University of Arkansas, and the Sydney and J. L. Huffines Institute of Texas A&M Universit

MITOCHONDRIAL BIOGENESIS AFTER REPEATED BOUTS OF DISUSE

Jacob L. Brown1, Richard A. Perry, Jr1., Kevin L. Shimkus2,David E. Lee1, Megan E. Rosa1, Jessica M. Cardin2, Lemuel A. Brown1, Elizabeth K. McBee1, Yasaman Sharazi-Fard2, Harry Hogan2, James D. Fluckey2, Tyrone A. Washington1 & Nicholas P.Greene1,2 1University of Arkansas, Fayetteville, Arkansas. 2Texas A&M University, College Station, Texas Muscular disuse affects a great number of people have sedentary lifestyles and/or chronic disease. Disuse has been shown to cause severe muscular atrophy and to disrupt mitochondrial quality. PURPOSE: To examine if disuse affects mitochondrial biogenesis, and if resistance exercise following bouts of unloading can promote biogenesis. METHODS: Sprague-Dawley rats were subjected to chronic disuse atrophy by hindlimb unloading (28-d, 1HU) followed by ambulatory recovery (56-d) with (1HU+EX) and without (1HU+REC) resistance exercise. To mimic repeated bouts of disuse animals were subjected to a second bout of HU (28-d, 2HU) and again allowed ambulatory recovery with (2HU+EX) or without (2HU+REC) resistance exercise. Control (CON) animals were allowed normal cage activity throughout. Samples were analyzed for Pgc-1α, Tfam, Nrf2 and Pparα gene expression by real time RT-PCR. To test if disuse impacted mitochondrial biogenesis regulators a T-Test was performed between CON and 1HU groups, to test impact of reloading and exercise data were analyzed by one-way ANOVA across all groups with α set at PRESULTS: Pgc-1α expression decreased by 59% (p=0.042) and Nrf2 by 75% (p=0.047) following disuse (1HU) compared to CON. 1HU+Ex showed a 280% increase in Pparα expression (p=0.005) as well as a 278% increase in Tfam expression (p=0.013) compared to CON. Pgc-1α, Pparα, and Tfam displayed a greater increase in expression with exercise recovery (1HU+Ex) than without (1HU+Rec). Pgc-1α showed an 80% increase in expression (p=0.05), Pparα showed a 208% increase in expression (p=0.01), and Tfam showed a 195% increase in expression (p=0.01) when comparing 1HU+Ex and 1HU+Rec. Nrf2 decreased by 61% (p=0.008) with 2HU. Expression of other biogenesis markers was not changed in the 2HU group. Neither 2HU+Ex nor 2HU+Rec were able to attenuate the loss of Nrf2 expression. CONCLUSION: A single bout of disuse significantly decreases the expression of Pgc-1α and Nrf2. 1HU+Ex promotes mitochondrial biogenesis more than 1HU+Rec. Multiple bouts of disuse decreases the expression of Nrf2. 2HU+Ex and 2HU+Rec does not attenuate the loss of Nrf2 expression. More research needs to be conducted to examine other aspects of mitochondrial quality such as mitochondrial dynamics and autophagy. Funded By National Space Biomedical Research Institut

AUTOPHAGY FOLLOWING MULTIPLE BOUTS OF HINDLIMB UNLOADING

Elizabeth K. McBee1, Jacob L. Brown1, Richard A. Perry, Jr1, Kevin L. Shimkus2,David E. Lee1, Megan E. Rosa1, Jessica M. Cardin2, Lemuel A. Brown1, Yasaman Shirazi-Fard2, Harry A. Hogan2, James D. Fluckey2, Tyrone A. Washington1, Nicholas P. Greene1,2 1University of Arkansas, Fayetteville, AR. 2Texas A&M University, College Station, TX; e-mail: [email protected] Muscular disuse affects an abundance of people with sedentary lifestyles and/or chronic diseases. Disuse has proven to cause severe muscular atrophy and disrupt mitochondrial quality. Autophagy is a cellular mechanism used to remove wasteful or damaged materials, mitophagy is the process of autophagically removing presumably damaged mitochondria. Beclin1 and Atg7 are machinery for autophagy, while Bnip3 specifically regulates mitophagy. Any dysregulation of autophagic processes may significantly impair cellular health following muscle disuse. PURPOSE: The purpose of this study was to investigate whether multiple bouts of muscle disuse (hindlimb unloading, HU) affect the expression of markers for autophagy. METHODS: Sprague-Dawley rats were subjected to chronic disuse atrophy by hindlimb unloading (28-d, 1HU) followed by ambulatory recovery (56-d) (1HU+REC). To mimic repeated bouts of disuse, the animals were subjected to a second bout of HU (28-d, 2HU) and then allowed ambulatory recovery (2HU+REC). Control (CON) animals were allowed normal cage activity throughout. Samples were analyzed for Beclin1, Atg7, and Bnip3 mRNA content by real time RT-PCR. To verify if HU impacted autophagy markers pre-planned T-tests were performed comparing CON and 1HU. To test if multiple bouts of disuse and reloading impacted regulators of autophagy, a one-way ANOVA across all groups was employed with α set at p\u3c0.05. RESULTS: Beclin1, Atg7, and Bnip3 mRNA contents were not different, 270% greater, and ~50% lower following 1HU compared to CON, respectively. Across all conditions, Beclin1 was unchanged. Atg7 was not different from CON in 1HU+REC, 2HU, and 2HU+REC groups. Atg7 was ~30% of 1HU in 1HU+REC and 2HU groups. Bnip3 showed no further differences among conditions. CONCLUSION: A single bout of HU enhanced the capacity for general autophagy, while diminishing the capacity for mitophagy. Recovery from repeated bouts of HU did not alter mRNA levels of autophagic markers relative to CON. These data show that autophagic markers are dysregulated with HU, and that animals adapted to multiple bouts of HU, which dampened the impact of subsequent HU on autophagic markers. Funding provided by NASA Grant Number NNX08AQ35

Hypoxia refines plasticity of mitochondrial respiration to repeated muscle work

PURPOSE: We explored whether altered expression of factors tuning mitochondrial metabolism contributes to muscular adaptations with endurance training in the condition of lowered ambient oxygen concentration (hypoxia) and whether these adaptations relate to oxygen transfer as reflected by subsarcolemmal mitochondria and oxygen metabolism in muscle. METHODS: Male volunteers completed 30 bicycle exercise sessions in normoxia or normobaric hypoxia (4,000 m above sea level) at 65 % of the respective peak aerobic power output. Myoglobin content, basal oxygen consumption, and re-oxygenation rates upon reperfusion after 8 min of arterial occlusion were measured in vastus muscles by magnetic resonance spectroscopy. Biopsies from vastus lateralis muscle, collected pre and post a single exercise bout, and training, were assessed for levels of transcripts and proteins being associated with mitochondrial metabolism. RESULTS: Hypoxia specifically lowered the training-induced expression of markers of respiratory complex II and IV (i.e. SDHA and isoform 1 of COX-4; COX4I1) and preserved fibre cross-sectional area. Concomitantly, trends (p < 0.10) were found for a hypoxia-specific reduction in the basal oxygen consumption rate, and improvements in oxygen repletion, and aerobic performance in hypoxia. Repeated exercise in hypoxia promoted the biogenesis of subsarcolemmal mitochondria and this was co-related to expression of isoform 2 of COX-4 with higher oxygen affinity after single exercise, de-oxygenation time and myoglobin content (r ≥ 0.75). Conversely, expression in COX4I1 with training correlated negatively with changes of subsarcolemmal mitochondria (r < -0.82). CONCLUSION: Hypoxia-modulated adjustments of aerobic performance with repeated muscle work are reflected by expressional adaptations within the respiratory chain and modified muscle oxygen metabolism