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