Effect of endurance exercise on microRNAs in myositis skeletal muscle-A randomized controlled study

Abstract

Objective: To identify changes in skeletal muscle microRNA expression after endurance exercise and associate the identified microRNAs with mRNA and protein expression to disease-specific pathways in polymyositis (PM) and dermatomyositis (DM) patients.

Methods: Following a parallel clinical trial design, patients with probable PM or DM, exercising less than once a week, and on stable medication for at least one month were randomized into two groups at Karolinska University Hospital: a 12-week endurance exercise group (n = 12) or a non-exercised control group (n = 11). Using an Affymetrix microarray, microRNA expression was determined in paired muscle biopsies taken before and after the exercise intervention from 3 patients in each group. Ingenuity pathway analysis with a microRNA target filter was used to identify microRNA transcript targets. These targets were investigated at the mRNA (microarray) and protein (mass spectrometry) levels in patients.

Results: Endurance exercise altered 39 microRNAs. The microRNAs with increased expression were predicted to target transcripts involved in inflammatory processes, metabolism, and muscle atrophy. Further, these target transcripts had an associated decrease in mRNA expression in exercised patients. In particular, a decrease in the NF-κB regulator IKBKB was associated with an increase in its target microRNA (miR-196b). At the protein level, there was an increase in mitochondrial proteins (AK3, HIBADH), which were associated with a decrease in microRNAs that were predicted to regulate their expression.

Conclusion: Improvement in disease phenotype after exercise is associated with increasing microRNAs that target and downregulate immune processes at the transcript level, as well as decreasing microRNAs that target and upregulate mitochondrial content at the protein level. Therefore, microRNAs may improve disease by decreasing immune responses and increasing mitochondrial biogenesis.

Trial registration: ClinicalTrials.gov NCT01184625.

Fig 1. CONSORT 2010 flow diagram.

The flow chart depicts the numbers of participants who were randomly assigned, exercised, and were analyzed for study.

Fig 2. Overview of the data analysis of exercised and non-exercised myositis patients.

The above work plan was used to the identify exercised-induced microRNA interactions in myositis patients. (A) Total RNA was extracted from baseline (pre) and exercised (post) muscle biopsies from the exercise group and the control group. (B) After pre values were subtracted from post values as an internal control measure, a total of 39 microRNAs were identified. Ingenutiy Pathway Analysis (IPA) MicroRNA Target Filter identified that 8 of these microRNAs had predicted transcript targets. (C) A gene expression microarray was run to identify if any of these transcript targets were altered in the patients after exercise. (D) Expression pairing between the microRNA and mRNA data sets was performed using IPA to determine biological relevance. (E) Protein was extracted from pre and post exercised muscle for SuperSILAC mass spectrometry. Expression pairing was again performed with microRNA and protein data sets since microRNAs are known to inhibit translation.

Fig 3. Validation of miR-196b expression after exercise and its associated effect on classical NF-κB signaling.

(A) RT-qPCR validated that miR-196b was increased after exercise. For normalization, pre Ct values were first subtracted from post Ct values from both miR-196b and the housekeeper, U47. Double delta Ct method was then used to calculate the average fold change (2^-((Normalized Target-Housekeeper)-(Average(Normalized Control-Housekeeper)). (B) Western blot in pre-and post exercised skeletal muscle shows an average increase of 42% in total IκBα protein after exercise.

Fig 4. Exercise induces microRNAs that target transcripts and proteins important for muscle and immune response.

Exercise-induced microRNAs help improve disease outcomes in myositis by modulating transcripts and proteins important for aerobic metabolism, immune response, and muscle atrophy.