Myofiber “Lipotoxicity” Does Not Disrupt Cellular Biophysical Properties

Introduction:: Obesity is associated with pathological changes in skeletal muscle that contribute to functional loss and disability. While it is unclear which specific factors cause this impairment in muscle performance, whole muscle and myofiber contractile dysfunction is associated with the increase in intramyocellular lipid (IMCL) within the muscle fibers, termed “lipotoxicity” (1, 2). Some studies have suggested the mechanism for this association is mitochondrial dysfunction, whereas other studies believe the increase in IMCL leads to structural changes within the muscle fibers that impact their contractile performance (3, 4). However, these hypotheses have been plagued by a paradox in which elite athletes have similar amounts of IMCL without detriments to contractile performance. This paradox could derive from specific nuances in diet-induced IMCL accumulation that are uniquely pathological, an interaction between IMCL and other disease factors (e.g., insulin resistance) or a correlative and not causative relationship between IMCL and muscle dysfunction in obesity. In this study, we used mouse models of hyperlipidemia and muscle biopsies from individuals with advanced diabetes to explore the biophysical changes to muscle fibers with pathological IMCL accumulation. Using isolated, permeabilized myofibers we studied sarcomeres in isolation from metabolic confounders and characterize contractility and passive viscoelasticity.

Materials and Methods:: Lipodystrophic (LD) mice were created by crossing adiponectin-Cre mouse and lox-stop-lox rosa diphtheria toxin (DTA) mouse as previously described (5). These mice completely lack adipose tissue and experience hyperlipidemia. Wildtype mice maintained on high fat diet (HFD) for 16 weeks were used as an additional model of hyperlipidemia (5). Human biopsies were obtained from patients undergoing below-knee amputation with diabetic peripheral neuropathy (DPN) and metabolically normal controls (MNC). All participants gave informed consent. Mouse plantar flexor muscles were dissected and portions of human tibialis anterior (TA) muscle biopsies were sectioned at 10μm, fiber typed by immunostaining and stained with Oil Red O (ORO) to visualize IMCL. The amount of lipid accumulation was quantified by fiber type by co-registration in ImageJ. Soleus (SOL) muscles and portions of biopsies were dissected, permeabilized and stored in a glycerinated storage solution at –20°C until the day of testing. Permeabilization allows experimental control of the biochemistry of the cytoplasm, isolating the physical structures of the sarcomere lattice for study. Single fibers were teased out from permeabilized muscles and biopsies and tested in a specialized system (1400A; Aurora Scientific). Physiology testing was conducted as previously described by systematically submerging fibers in solutions of varying calcium concentrations (6). Following physiology testing, fibers were subjected to an incremental stress relaxation test and elastic and viscous parameters estimated from a pseudoplastic model as previously described (7). Fibers were then subdivided by fiber type by immunostaining. Data were analyzed in Graphpad Prism by one-way ANOVA.

Results, Conclusions, and Discussions:: Co-registration of ORO and MHC staining in muscles from HFD and LD mice revealed a fiber type specific accumulation of IMCL (predominately in 2a and 2x fibers), compared with WT (Fig. 1). We confirmed that IMCL was retained in permeabilized fibers with the lipid soluble fluorescent dye BODIPY (Fig. 2A). Isolated permeabilized fibers from the SOL of LD mice did not exhibit a significant difference in peak tension compared with WT for either type 1 or 2a/2x (Fig 2B). There were also no differences between genotypes in the elastic or viscous parameters of stress relaxation for either fiber type group (instantaneous elastic modulus, relaxed elastic modulus, instantaneous viscosity, relaxed viscosity and rate of viscous recovery [7]; Fig 2C). We did find previously reported differences in elastic modulus between type 1 and 2 fibers, validating our methodology. Preliminary data from human biopsies support the mouse findings. While IMCL typically accumulates in type 1 fibers with obesity in humans, we find that in the severe metabolic challenge of advanced diabetes (DPN), it can accumulate to a larger extent in type 2a and 2x fibers (Fig 3A). Despite this high IMCL content, peak tension in DPN fibers was comparable to metabolically normal controls (MNC) (Fig. 3B). There were also no differences between MNC and DPN fibers in the elastic or viscous parameters of stress relaxation for either fiber type group (p >0.55).
Despite large quantities of IMCL filling inter-myofibrillar space, there appears to be no disruption of lateral force transmission between myofibrils which would be expected to impact both maximal active tension and passive viscoelasticity. It is important to note that the permeabilized fiber system dissociates some biochemical mediators of contraction from the biophysical interactions of proteins by controlling the available calcium and ATP for cross-bridge cycling. Thus it remains possible that IMCL impairs active force generation through mitochondrial dysfunction, but our data suggests that passive and active forces are not affected through physical disruption of the cellular architecture.


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References (Optional): : 1. Straight + J Gerontol A Biol Sci Med Sci 2019 2. Choi + The Journals of Gerontology 2015 3. Van Herpen + Physiology & Behavior 2008 4. Carter + GeroScience 2019 4. 5. Collins + PNAS 2021, 6. Roche + JOVE 2015, 7. Meyer+ J Biomech Eng 2011