R23 - Investigation of Environmental Toxicants Mechanism of Action Using Engineered Human Skeletal Muscle Myobundles

Introduction: : The impact of many pollutants in the environment on human health is poorly studied. To understand the effect and mechanism of action of human skeletal muscle in response to environmental toxicants, we examined 20 selected compounds using engineered myobundles. We have studied the impact of the toxicants at different concentrations on the myobundle contractile forces [BMES 2024]. From these results, we identified compounds that are more potent to human skeletal muscle and carried out further investigation of their mechanisms leading to the change in force we have observed.

Fungicides such as Triphenyltin hydroxide (TPTH), Pyraclostrobin, and Azoxystrobin work by inhibiting mitochondrial function of fungal cells and restricting ATP synthesis. Antiseptic agents such as Benzethonium Chloride (BEC), Didethydimethylammounium Chloride (DDAC), and Triclosan (TCS) can disrupt microbial lipid membranes or inhibit bacterial fatty acid synthesis, which might target the mitochondrial membrane. Therefore, we performed Seahorse Cell Mito Stress test to study the influence of these compounds on respirational function of the myobundles. Deltamethrin (DLM) is an insecticide that attacks the nervous system and interferes with ion channels. Malathion is an insecticide that inhibits acetylcholinesterase and shows no change in force. We measured calcium transients to understand if DLM affects the calcium channels in the muscle and interferes with force production while using Malathion as a negative control. In addition, we treated myobundles with a live/dead assay kit to detect toxicant concentrations that caused cell death. Collectively, these data provide a better perspective of how toxic compounds may act on human skeletal muscle.

Materials and

Methods: : Human skeletal muscle myobundles were prepared with myoblasts and a fibrin gel mixture that were attached to nylon frames within polydimethylsiloxane molds following gel polymerization. The myobundles were exposed to growth media for 4 days and differentiation media for 7 days. After maturation, myobundles were treated with vehicle control or compounds at 0.3, 3 or 30 µM for 4 days, followed by end point testing.

The Cell Mito Stress Test was performed using Seahorse XFe24 analyzer. Oxygen consumption rate (OCR) was normalized against the total amount of protein per myobundle obtained through a BCA assay (Sigma Aldrich). Myobundles were lysed with mammalian cell lysis buffer containing protease inhibitor, and the supernatant was collected for protein quantification.

Myobundles were transduced with MHCK7-GCaMP6 lentivirus for calcium transient measurements. Ca2+ transients were recorded from myobundles within a 37°C live-imaging chamber in Tyrode's solution on a Nikon microscope using an Electron Multiplying Charge-Coupled Device camera (iXon Ultra 897, Andor). Tissues were field stimulated at 1 and 20Hz to obtain twitch and tetanus ΔF/F measurements. Ca2+ transient amplitudes were calculated as the maximum relative change in fluorescence signal.

Myobundle live/dead staining was measured using the Cyto3D® Live-Dead Assay Kit. Images were obtained with Zeiss 880 Airyscan Inverted Confocal. Mean fluorescence intensity (MFI) of dead cells was calculated by dividing the total dead cell grayscale intensity by the number of cell pixels (Fiji/ImageJ).

One way analysis of variance was performed with JMP Pro 17.2.0 followed by a post hoc Tukey HSD test, if p < 0.05.

Results, Conclusions, and Discussions:: Contractile forces of myobundles treated with TPTH at 3 µM and 30 µM were eliminated and were significantly decreased at 0.3 µM (Figure 1a). MFI of dead cells indicated that there was significant cell death following treatment with TPTH at 3 µM and 30 µM but not at 0.3 µM (Figure 1b). There was a significant decrease in respirational capacity of myobundles treated with TPTH at all concentrations (Figure 1c-d). The elimination of contractile forces from the higher doses can be explained by the large number of dead cells. The reduction in force by TPTH at 0.3 µM was likely due to impaired mitochondrial function, leading to the myobundles being unable to generate enough ATP for contraction.

Similar experiments and analyses were done for the selected compounds. To summarize, Pyraclostrobin, DDAC, and BEC at 0.3 µM, and TCS and Azoxystrobin at 0.3 and 3 µM, showed minimal impact on muscle function, while Pyraclostrobin, DDAC, and BEC at 30 µM killed the myobundles. Force reduction induced by BEC at 3 µM was likely due to limited ATP production as cell death was minimal. There was a decrease in respiration and a moderate amount of cell death for myobundles treated with Pyraclostrobin and DDAC at 3 µM and TCS at 30 µM, and their reduced contractile forces could be caused by a combination of dead cells and incomplete mitochondrial function. Azoxystrobin at 30 µM reduced respiration and slightly increased the amount of dead cells, but the reason for eliminated contractile forces is unclear.

Calcium transient measurements in general followed the trend of contractile forces. DLM showed a dose dependent reduction of both force and calcium transient but did not alter the kinetics (Figure 2). Malathion did not demonstrate statistically significant changes in Ca2+ transient in comparison to the vehicle control (Figure 2c-d).

Looking beyond the function versus cell death, we will examine structural changes of actin fibers in the skeletal muscle caused by the treatments through staining. In conclusion, we were able to investigate the effect and mechanism of selected environmental toxicants on human skeletal muscle using in vitro myobundle assays.

Acknowledgements and/or References (Optional):: This work was supported [in part] by the Intramural Research Program of the NIH (ES103382) and by contract 75N96023P00240 with NIEHS.