A novel D2O tracer method to quantify RNA turnover as a biomarker of de novo ribosomal biogenesis, in vitro, in animal models, and in human skeletal muscle

Abstract

Current methods to quantify in vivo RNA dynamics are limited. Here, we developed a novel stable isotope (D₂O) methodology to quantify RNA synthesis (i.e., ribosomal biogenesis) in cells, animal models, and humans. First, proliferating C2C12 cells were incubated in D₂O-enriched media and myotubes ±50 ng/ml IGF-I. Second, rat quadriceps (untrained, n = 9; 7-wk interval-"like" training, n = 13) were collected after ~3-wk D₂O (70 atom %) administration, with body-water enrichment monitored via blood sampling. Finally, 10 (23 ± 1 yr) men consumed 150-ml D₂O followed by 50 ml/wk and undertook 6-wk resistance exercise (6 × 8 repetitions, 75% 1-repetition maximum 3/wk) with body-water enrichment monitored by saliva sampling and muscle biopsies (for determination of RNA synthesis) at 0, 3, and 6 wk. Ribose mole percent excess (r-MPE) from purine nucleotides was analyzed via GC-MS/MS. Proliferating C2C12 cell r-MPE exhibited a rise to plateau, whereas IGF-I increased myotube RNA from 76 ± 3 to 123 ± 3 ng/μl and r-MPE by 0.39 ± 0.1% (both P < 0.01). After 3 wk, rat quadriceps r-MPE had increased to 0.25 ± 0.01% (P < 0.01) and was greater with running exercise (0.36 ± 0.02%; P < 0.01). Human muscle r-MPE increased to 0.06 ± 0.01 and 0.13 ± 0.02% at 3/6 wk, respectively, equating to synthesis rates of ~0.8%/day, increasing with resistance exercise to 1.7 ± 0.3%/day (P < 0.01) and 1.2 ± 0.1%/day (P < 0.05) at 3/6 wk, respectively. Therefore, we have developed and physiologically validated a novel technique to explore ribosomal biogenesis in a multimodal fashion.

Keywords: D2O; RNA synthesis; muscle; ribosomal biogenesis.

Fig. 1.

A: structure and mass of the ribose benzylhydroxylamine tetra-acetate derivative. B: typical GC-MS/MS chromatogram of the ribose derivative on a DB-17 column for the SRM transitions of 273.1–111.1. Higher line represents the M, and lower line the M⁺¹. C: standard curve of 0.02, 0.05, 0.1, and 0.5 [1-¹³C]ribose. D: measurement of the +5 isotopomer of RNA bound ribose from C2C12 myotubes incubated in [U-¹³C]glucose enriched media.

Fig. 2.

A: MPE of RNA from maximally labeled C2C12s vs. D₂O media MPE. B: the amplification of deuterium into PBMCs from human subjects. ***Significantly different from baseline, P < 0.001.

Fig. 3.

Time course of cell number (A) and MPE of RNA (B) in proliferating C2C12s. Time courses in the concentration of RNA (C) and MPE of RNA (D) in nontreated condition (Control) and in responses to IGF-I are shown. Dotted line represents plateau enrichment. Significantly different from baseline, *P < 0.05, ***P < 0.001, ****P < 0.0001. §Significantly different from control at that time point, P < 0.01.

Fig. 4.

A: MPE of RNA bound ribose from control and exercised rat quadriceps. B: FSR of RNA from control and exercised rat quadriceps. C: correlation between quadriceps in MPS%/day and RNA in FSR%/day. Dotted line represents plateau enrichment. ***Significantly different from control, P < 0.001. d, Day.

Fig. 5.

A: time course of body-water enrichment measured through saliva samples over the 6 wk of labeling. B: MPE of RNA bound ribose from human VL in rest and RET legs. Dotted line represents the average plateau PBMC enrichment. C: FSR of RNA bound ribose from human VL in rest and RET legs. D: correlation between VL MPS%/day and RNA FSR %/day. ***Significantly different from baseline, P < 0.001. §Significantly different from control at that time point, P < 0.05.