Changes in Skeletal Muscle Proteolytic Gene Expression After Prophylactic Supplementation of EGCG and NAC and Eccentric Damage

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PURPOSE: To investigate the impact of prophylactic supplementation of N-acetylcysteine (NAC) and epigallocatechin gallate (EGCG) after eccentric muscle contractions on mRNA expression of genes involved in skeletal muscle proteolysis. METHODS: Thirty healthy, active males (20.0±1.8 years, 160±7.1cm, 167.4±37.3 kg) ingested on a daily basis either 1,800 mg of NAC, 1,200 mg of EGCG, or a placebo (PLC) in a double blind, prophylactic fashion for 14 days. Subjects then completed one eccentric exercise bout (100 repetitions @ 30°•s-1) of the dominant knee extensors. Percutaneous skeletal muscle biopsies were collected before, 6 and 24 h after exercise. Total RNA content was isolated from each muscle sample and reverse transcribed into cDNA. Forward and reverse gene primers were developed using Beacon Designer software and published nucleotide sequences and commercially produced. Gene transcription was quantified by real-time RT-PCR using a MyiQ thermal cycler (Bio- Rad, Hercules, CA). To correct for changes in primer efficiency, the Pfaffl method (i.e. 2-ΔΔCT adjusted for primer binding efficiency) was used to calculate fold-change in gene expression within each condition 6 and 24 hours (h) after exercise. Separate repeated measures ANOVA were used for all statistical analysis at a probability level of 0.05. All data is presented as fold increases (mean ± SE) from baseline. RESULTS: No significant group x time interactive effects were found (p>0.05). Significant main effects were found for MuRF (6 h: 5.3±10.8 and 24 h: 1.3±1.2; p=0.041) and the E3 ubiquitin ligase (6 h: 5.3±7.7 and 24 h: 3.3±4.5; p=0.004). Overall main effects tended to increase for the HC2 20S proteosome subunit (6 h 1.9±2.4 and 24 h: 1.6±1.9; p=0.097) and calpain-2 (p=0.073), while one-way ANOVA revealed significant increases at 6 h for calpain-2 (2.7±4.4; p=0.045) and a tendency to increase at 24 h (1.8±2.3; p=0.084). No changes (p>0.05) were found for atrogin-1, the HC3 20S proteosome subunit and calpain-1. CONCLUSION: Prophylactic supplementation of NAC and EGCG had no impact over changes in skeletal muscle proteolytic gene expression after damaging eccentric exercise. Eccentric muscle contractions did result in significant increases in MuRF and E3 ubiquitin ligase mRNA while the HC2 20S proteosome subunit and calpain-2 mRNA tended to increase.