Comparison of Fatty Acid and Gene Profiles in Skeletal Muscle in Normal and Obese C57BL/6J Mice before and after Blunt Muscle Injury

Abstract

Injury and obesity are two major health burdens affecting millions of people worldwide. Obesity is recognized as a state of chronic inflammation accompanied by various co-morbidities like T2D or cardiovascular diseases. There is increasing evidence that obesity impairs muscle regeneration, which is mainly due to chronic inflammation and to excessive accumulation of lipids in adipose and non-adipose tissue. To compare fatty acid profiles and changes in gene expression at different time points after muscle injury, we used an established drop tower-based model with a defined force input to damage the extensor iliotibialis anticus on the left hind limb of female C57BL/6J mice of normal weight and obese mice. Although most changes in fatty acid content in muscle tissue are diet related, levels of eicosaenoic (normal weight) and DHG-linolenic acid (obese) in the phospholipid and docosahexaenoic acid (normal weight) in the triglyceride fraction are altered after injury. Furthermore, changes in gene transcription were detected in 3829 genes in muscles of normal weight mice, whereas only 287 genes were altered in muscles of obese mice after trauma. Alterations were found within several pathways, among them notch-signaling, insulin-signaling, sonic hedgehog-signaling, apoptosis related pathways, fat metabolism related cholesterol homeostasis, fatty acid biosynthetic process, fatty acid elongation, and acyl-CoA metabolic process. We could show that genes involved in fat metabolism are affected 3 days after trauma induction mostly in normal weight but not in obese mice. The strongest effects were observed in normal weight mice for Alox5ap, the activating protein for leukotriene synthesis, and Apobec1, an enzyme substantial for LDL synthesis. In summary, we show that obesity changes the fat content of skeletal muscle and generally shows a negative impact upon blunt muscle injury on various cellular processes, among them fatty acid related metabolism, notch-, insulin-, sonic hedgehog-signaling, and apoptosis.

Diet-Induced Obesity Affects Muscle Regeneration After Murine Blunt Muscle Trauma—A Broad Spectrum Analysis

“…For this purpose, female C57BL/6J litter animals were randomly grouped into sham and treated groups. Induction of injury to extensor iliotibialis anticus muscle of the left hind leg was carried out by using a physical trauma model previously described (Claes et al, 2006 ; Weckbach et al, 2013 ; Wanner et al, 2017 ; Werner et al, 2018 ). Briefly, anesthesia was carried out with 2.5 vol% sevoflurane (Sevorane™ Abbott, Wiesbaden, Germany) mixed with 97.5 vol% oxygen.…”

“…Muscle tissue from female normal weight ( n = 3) and obese mice ( n = 3) was collected before and 1, 6, 24 h, 3, 8, and 21 days after trauma induction and subsequently embedded in paraffin. 5 μm thick tissue-sections were deparaffinized, rehydrated, and stained by HE as described previously (Werner et al, 2018 ).…”

“…Harvesting time points were 1, 6, 24 h, 3, and 8 days to cover the early and middle stage regeneration process. Isolation of total RNA, microarray analysis and evaluation method were described previously (Werner et al, 2018 ). Briefly, CEL-files of trauma vs. control were compared per time point and diet, respectively.…”

“…Here, we continued to further analyze the role of obesity on muscle regeneration using our drop-tower based model (Werner et al, 2018 ) for induction of a blunt trauma to the extensor iliotibialis anticus muscle of normal weight and obese mice. Imaging the trauma induction and regeneration process by magnetic resonance imaging (MRI) clearly showed edema formation and hemorrhage exudate in muscles of normal weight and obese mice.…”

“…Regeneration was delayed in muscles of obese mice, with a higher incidence of fibrosis formation due to hampered expression levels of underlying genes. To further analyze previously obtained microarray data (Werner et al, 2018 ) a time lapse heatmap for an unbiased evaluation of striking candidates was generated. With this approach, we identified 81 differentially expressed genes (DEG) associated, but not limited to, five pathways, which play crucial roles during regeneration.…”