Assessment of Skeletal Muscle Nicotinamide Adenine Dinucleotide Concentrations in Rat Strains Exhibiting Different Levels of Play Behavior

Abstract

Relative to Sprague‐Dawley (SD) and Lewis (LEW) rats, the Fischer 344 (F344) strain displays reduced play behavior. While neurobiological explanations for these behavioral differences have been explored, we investigated whether impaired skeletal muscle mitochondrial function may contribute to these observed variations.Nicotinamide adenine dinucleotide (NAD) is an important cofactor in protein deacetylase reactions regulating mitochondrial content and activity. We hypothesized that there would be strain‐dependent differences in skeletal muscle NAD concentrations ([NAD]) matching differing levels of play behavior in these strains. We also hypothesized that F344 rats would show reduced protein expression of nicotinamide phosphoribosyl transferase (Nampt), a key enzyme in intracellular NAD recycling, and sirtuin 3 (SIRT3), an NAD‐dependent protein deacetylase governing mitochondrial content and function.Rat soleus muscles were obtained from anesthetized SD (n = 10), LEW (n = 7), and F344 (n = 7) rats and processed for NAD measurements. Skeletal muscle tissue samples were homogenized in perchloric acid. Intracellular NAD was quantified via HPLC elution and UV‐visible spectroscopy detection at 261 nm. NAD quantification via HPLC was validated through the analysis of NAD standards and spike‐recovery samples. Tissue harvesting and processing methods for HPLC analysis were optimized to account for the effects of tissue hypoxia, sample storage temperature, and the number of freeze‐thaw cycles on [NAD]. In addition, relative amounts of Nampt and SIRT3 protein expression were determined via immunoblots.NAD could be detected reliably and reproducibly by HPLC. Tissues exposed to hypoxic conditions during dissection and collection showed sensitivity to NAD degradation relative to controls. Sample [NAD] were stable at or below 4°C for at least 1 week.Average NAD concentrations were not statistically significantly different between the three rat strains (SD, 13.2 ± 0.89 [mean ± S.E.M] μM/mg tissue, LEW, 14.4 ± 0.81 μM/mg, F344, 13.9 ± 1.21 μM/mg; p = 0.66). No significant difference in Nampt expression was found between the three strains (p = 0.56). However, SIRT3 expression differed statistically significantly between the three strains (SD 0.97 ± 0.076 arbitrary units (AU), LEW 1.26 ± 0.085 AU, F344 1.72 ± 0.187 AU; p = 0.0006). Post‐hoc testing revealed that SIRT3 expression was significantly lower in SD (p < 0.01) and LEW (p < 0.05) strains compared to F344 rats. There was no statistically significant difference in SIRT3 expression between the SD and LEW strains (p = 0.50). No significant correlations between skeletal muscle [NAD], Nampt expression, or SIRT3 expression were detected.In conclusion, F344 rats exhibiting reduced playfulness are characterized by a marked increase in skeletal muscle SIRT3 protein expression despite unaltered intracellular NAD compared to both SD and LEW rats. While intracellular [NAD] are stable between strains, different NAD/SIRT3 ratios suggest altered dynamics of NAD‐dependent mitochondrial protein deacetylases.Support or Funding InformationThis work was supported in part by a grant to Gettysburg College from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program, and by Gettysburg College.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.