Running head: MicroRNA expression in high and low capacity runner rats Running Head: MicroRNA regulation in high and low running capacity rats

26 Impairments in mitochondrial function and substrate metabolism are implicated in the 27 etiology of obesity and type 2 diabetes. MicroRNAs (miRNAs) can degrade mRNA or 28 repress protein translation and have been implicated in the development of such disorders. 29 We used a contrasting rat model system of selectively bred high(HCR) or low(LCR) 30 intrinsic running capacity with established differences in metabolic health to investigate the 31 molecular mechanisms through which miRNAs regulate target proteins mediating 32 mitochondrial function and substrate oxidation processes. Quantification of select miRNAs 33 using the Rat miFinder miRNA PCR array revealed differential expression of 15 skeletal 34 muscle ( m. tibialis anterior ) miRNAs between HCR and LCR rats (14 with higher expression 35 in LCR; P<0.05). Ingenuity Pathway Analysis predicted these altered miRNAs to collectively 36 target multiple proteins implicated in mitochondrial dysfunction and energy substrate 37 metabolism. Total protein abundance of citrate synthase (CS; miR-19 target) and voltage38 dependent anion channel 1 ( miR-7a target) were higher in HCR compared to LCR cohorts 39 (~57 and ~26%, respectively ; P<0.05). A negative correlation was observed for miR-19a-3p 40 and CS (r =0.59, P=0.02) protein expression in LCR. To determine if miR-19a-3p can 41 regulate CS in vitro we performed luciferase reporter and transfection assays in C2C12 42 myotubes. MiR-19a-3p binding to the CS untranslated region did not change luciferase 43 reporter activity, however miR-19a-3p transfection decreased CS protein expression (~70%; 44 P<0.05). The differential miRNA expression targeting proteins implicated in mitochondrial 45 dysfunction and energy substrate metabolism may contribute to the molecular basis 46 mediating the divergent metabolic health profiles of LCR and HCR rats. 47