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Kinesiology Purpose: In response to resistance exercise training, it has been shown that individuals with a previous training history acquire muscle volume at an accelerated rate. This phenomenon may be attributed, in part, to the myonuclear enrichment resulting from the proliferation of muscle progenitor cells, which promotes essential protein synthesis following subsequent muscle training. As a highly energy demand tissue, the successful hypertrophy of muscle fiber depends on mitochondrial biogenic progression. Moreover, the majority of genes that encode mitochondrial proteins are within nuclear genome. Therefore, in this study, we investigated the effect of increased number of myonuclei in response to the previous resistance exercise preconditioning on mitochondrial adaptations to subsequent resistance training. Our central hypothesis was that pre-trained muscles would show an accelerated acquisition of training-induced mitochondrial function leading to a greater skeletal muscle hypertrophy compared to previously non-trained muscles and this may be associated with increased number of myonuclei in the pre trained muscles. Methods: Thirty-two Sprague-Dawley rats were randomly assigned to four groups (n=8 per group) which include control, pre-training, training, and retraining group. Resistance exercise training was carried out by ladder climbing with weights attached to the tail at ages of either 8- (pre-training) and 36-week-old (training), or both (retraining). Each training session consisted of 3 sets of 5 repetitions, and the training protocol was performed every third day for 8 weeks. At 44 weeks of age, specific muscle groups were carefully collected and stored at -80 °C until further analyses. 4', 6-Diamidino-2-phenylindole staining, hematoxylin & eosin staining, cytochrome c oxidase and succinate dehydrogenase staining were performed. Western blotting and immunohistochemstry were performed to assess the abundance of mitochondrial regulatory proteins and the mitochondrial content. In complementary in vitro studies, confluent L6 myoblast cells were further grown in differentiation media for 4 days with or without insulin-like growth factor 1 (50 ng/ml) supplementation. Mitochondrial gene expression levels and mitochondrial respiratory function were assessed after 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM), a 5' AMP-activated protein kinase activator, treatment. Results: Myonuclei numbers were higher in training and retraining groups than control group (all, p < 0.05), suggesting that ladder climbing training protocol increased myonuclei number. There was a significantly higher level of myonuclei number in pretraining group compared to the control group indicating that the acquired myonuclei during exercise preconditioning were retained over the 20-week detraining period. Muscle cross-sectional area, mitochondrial content and mitochondrial enzymatic activities (COX and SDH) were significantly greater in retraining group compared to training group (p < 0.01, p < 0.01 and p < 0.05,… Advisors/Committee Members: Park, Joon-Young;, Park, Joon-Young, Tierney, Ryan T, Hudson, Matthew B, Chen, Xiongwen;.