Poster AA18 - In Vitro Toxicity Assays Using Engineered Human Skeletal Muscle Myobundles

Introduction: The health effects of most chemicals released into the environment are not well studied or only partially understood. There is ongoing pressure to shift to in vitro assays for many reasons including to reduce animal testing and detect effects at lower exposure levels as chemicals may alter function and gene expression at levels below which they cause toxicity. Two-dimensional cell culture often does not provide accurate measures of function, while more accurate animal models can be costly, and differences in animal metabolism may make results less translatable to humans. High throughput screening assays can identify specific molecular pathways affected but do not provide tissue-level functional information which is affected by interactions among different molecular pathways and various cell types. In contrast, human microphysiological systems (MPS) provide molecular and tissue level response of organs and organ systems in the presence of environmental pollutants, although throughput can be modest. In this study, we investigated the impact of a set of candidate toxicants upon contractile and energetic function of engineered human skeletal muscle myobundles. Primary measurements include contractile force and fatigue which provide an overall indicator of skeletal muscle function.

Materials and

Methods: All human subject procedures were reviewed and approved by the Duke University Institutional Review Board. Human skeletal muscle myobundles are prepared with primary myoblasts encapsulated in a fibrinogen and matrigel mixture. Each nylon frame is attached to two myobundles within PDMS molds. After polymerization, myobundles are cultured in growth media for 4 days for myoblast proliferation (Figure 1). Myobundles are then removed from molds and cultured in serum free differentiation media for 7 days to induce myotube formation and maturation. Then, myobundles are cultured with compounds for 4 days at specific concentrations along with media and vehicle control. If a decline in force is not observed after 4 days, the experiment is repeated with a 7-day compound treatment.
For contractility, twitch and tetanus force, and percent fatigue are obtained from the myobundles using a custom-made force measurement setup. In a 37°C bath, one end of the myobundle is immobilized, and the other end is connected to the force transducer. The sides of the frame are cut and the myobundle is stretched by 15% of its original length. Electrical stimulation at 1 Hz for 1 second is applied to obtain twitch force, at 20 Hz for 1 second to obtain tetanus force, and at 20 Hz for 30 seconds to obtain fatigue force. Force data are analyzed with MATLAB. One way analysis of variance (ANOVA) was performed with JMP Pro 17.2.0.

Results, Conclusions, and Discussions: As the compounds were dissolved in DMSO, tolerance of myobundles to different vehicle concentrations were tested to ensure minimal influence on myobundle function. We determined that 0.3% or less DMSO did not significantly impact the force of myobundles, therefore, 0.3% DMSO was used in culture media. Initial studies examined triclosan, ephedra, didecyldimethylammonium chloride (DDAC), and monocrotaline toxicity. Triclosan is absorbed through the skin and oral mucosa and targets the ryanodine receptor in skeletal muscle to cause muscle weakness. Ephedra is a botanical product that stimulates the heart, lung, and nervous system. Ephedra activates AMP-activated protein kinase (AMPK) and reduces lipid accumulation in fat-fed mice. Monocrotaline is a pyrrolizidine alkaloid that causes vascular abnormality to influence heart and pulmonary function. DDAC is a biocide used widely for surgical instrument and surface disinfection.
We measured the force of myobundles exposed to 0.3 µM and 3 µM of each compound for 4 days. Initial results are shown in Figure 2. A 4-day exposure of ephedra at 0.3 µM and 3µM caused significant twitch and tetanus force reduction compared to vehicle control and increased fatigue at 3 µM. In contrast, a 4-day exposure of triclosan, DDAC, and monocrotaline did not cause significant change in the contractile force profiles compared to the vehicle control.
Planned studies involve exposing myobundles to triclosan, DDAC, and monocrotaline at 30 μM for 4 days and extend the treatment to 7 days if none of the 4-day treatment showed significant effects on skeletal muscle contraction. We will continue contractile force analysis of the myobundles exposed to multiple concentrations of candidate compounds to evaluate their direct influences on myobundle contractility. To assess mechanism of action, we will measure oxygen consumption and calcium release to determine the effect of the compounds on mitochondrial function and contractility. The work to date suggests that the human skeletal muscle myobundle system can detect functional changes induced by environmental pollutants.

Acknowledgements (Optional): This work was supported by contract 75N96023P00240 from the National Institute of Environmental Health Sciences.