Physiological and pathophysiological concentrations of fatty acids induce lipid droplet accumulation and impair functional performance of tissue engineered skeletal muscle.

Mark C Turner,Neil R.W Martin,Mark P Lewis,Rowan P Rimington,Jacob W Fleming,Leanne Hodson,Andrew J Capel

doi:10.1002/jcp.30365

Abstract

Fatty acids (FA) exert physiological and pathophysiological effects leading to changes in skeletal muscle metabolism and function, however, in vitro models to investigate these changes are limited. These experiments sought to establish the effects of physiological and pathophysiological concentrations of exogenous FA upon the function of tissue engineered skeletal muscle (TESkM). Cultured initially for 14 days, C2C12 TESkM was exposed to FA-free bovine serum albumin alone or conjugated to a FA mixture (oleic, palmitic, linoleic, and α-linoleic acids [OPLA] [ratio 45:30:24:1%]) at different concentrations (200 or 800 µM) for an additional 4 days. Subsequently, TESkM morphology, functional capacity, gene expression and insulin signaling were analyzed. There was a dose response increase in the number and size of lipid droplets within the TESkM (p < .05). Exposure to exogenous FA increased the messenger RNA expression of genes involved in lipid storage (perilipin 2 [p < .05]) and metabolism (pyruvate dehydrogenase lipoamide kinase isozyme 4 [p < .01]) in a dose dependent manner. TESkM force production was reduced (tetanic and single twitch) (p < .05) and increases in transcription of type I slow twitch fiber isoform, myosin heavy chain 7, were observed when cultured with 200 µM OPLA compared to control (p < .01). Four days of OPLA exposure results in lipid accumulation in TESkM which in turn results in changes in muscle function and metabolism; thus, providing insight ito the functional and mechanistic changes of TESkM in response to exogenous FA.

Highlights

Skeletal muscle is a major site of carbohydrate and lipid utilization, where efficient selection between these two substrates is paramount in maintaining metabolic homeostasis (Stinkens et al, 2015)
To establish the presence of lipids in tissue engineered skeletal muscle (TESkM) following exposure to OPLA, cross sections were probed with Nile Red (NR) to identify lipid droplets (LD) (Figure 2)
The average LD area was increased in a dose dependent manner, FIGURE 2 Exposure to exogenous fatty acids (FA) overload induces intracellular lipid droplet formation in 3D tissue engineered skeletal muscle

Summary

| INTRODUCTION

Skeletal muscle is a major site of carbohydrate and lipid utilization, where efficient selection between these two substrates is paramount in maintaining metabolic homeostasis (Stinkens et al, 2015). In order to offer some insight into these mechanisms, the TESkM model developed by our research group (Capel et al, 2019), was exposed to physiological and pathophysiological concentrations of FA which have previously been shown to induce lipid accumulation in non‐adipose cell lines (Green et al, 2015; Gunn et al, 2020; Hodson et al, 2008). This allowed investigation of the impact of FA overload upon LD formation, gene expression, insulin sensitivity, and functional capacity in TESkM

| METHODS

| RESULTS

| DISCUSSION