Activation of NF-kB in Pericytes of Human Skeletal Muscle Following Eccentric Exercise-Induced Damage

Abstract

Eccentric contractions (ECC) can result in ultra-structural and histological damage to skeletal muscle. The damage incurred following ECC is typically followed by a subsequent regenerative and adaptive response. The specific mechanisms that drive this response, particularly in human muscle, are not well understood. PURPOSE: The objective of this study was to characterize the early molecular response in skeletal muscle following ECC in humans. METHODS: We used an Agilent whole human genome microarray to assess global gene expression in male subjects (N=35) at 3 hours post-100 eccentric contractions of the knee extensors. ANCOVA (age and BMI covariates) was used to compare mRNA expression between the ECC and non-exercised legs of each subject. These data were then further examined using Ingenuity Pathway Analysis (IPA) software to ascertain functional biological relationships. Novel transcripts from IPA identified networks were confirmed with quantitative real-time (qRT)-PCR. Immunohistochemistry, confocal microscopy and a transcription factor ELISA were used in a subset of 15 randomly selected subjects to test hypotheses generated from gene expression data. RESULTS: ECC resulted in muscle damage as indicated by decreased strength, soreness and elevated serum creatine kinase. Network analysis (IPA) highlighted the relationships of several differentially regulated transcripts to the activation of the NF-kB transcription factor complex. qRT-PCR analysis of 3 of these transcripts (IkBα, TNFRSF1A and ICAM-1) confirmed changes observed in the microarray analysis. ECC resulted in a 1.3 fold (p=0.018) increase in NF-kB binding activity and a 2.8 fold (p=0.008) increase in p65 nuclear localization of non-myonuclei. Subsequent immunostaining revealed that 74% and 69% of p65+ nuclei co-localized with the pericyte markers NG2 proteoglycan and alkaline phosphatase, respectively. CONCLUSIONS: We show for the first time that NF-kB is activated in human skeletal muscle shortly following a damaging exercise and that its activity is primarily associated with a distinct population of pericyte cells. Future work will be important in determining the functional significance of pericyte specific NF-kB activation. This study was funded by Interleukin Genetics; the authors would like to thank Dr. Kenneth Kornman, Dr. Valerie Greger, Dr. Prakash Prabhakar and Karen Shaver of Interleukin Genetics for their contribution to the study.