Mitochondrial dysfunction is responsible for the toxicity of a number of drugs. Current isolated mitochondria or cellular monoculture mitochondrial respiration measurement systems lack physiological relevance. Using a tissue engineering rather than cell- or mitochondria-based approach enables a more physiologically relevant detection of drug-induced mitochondrial impairment. To probe oxygen consumption and mitochondrial health, we assayed the bioenergetic profile of engineered three-dimensional human skeletal muscle myobundles derived from primary myoblasts. Through experimental and computational techniques, we did not find external or internal oxygen transport limiting the engineered myobundles in the commercial O2k system to measure oxygen consumption. In response to the complex I inhibitor rotenone, myobundle basal respiration decreased dose dependently with an IC of 9.24 ± 0.03 nM. At a 20 nM concentration of rotenone, myobundle maximal respiration decreased by 44.4% ± 9.8%. Respiratory depression by rotenone suggests that cultured myobundles rely heavily on the complex I pathway for ATP synthesis during times of both basal and increased energy demand. To address whether these decrements in mitochondrial function corresponded to alterations in physiological muscle function, we determined fatigue susceptibility that revealed a 46.0% ± 7.4% depression at 20 nM rotenone. The bioenergetic health index, which is a measure of normal oxidative mitochondrial function, was inversely correlated with the extent of fatigue. The human myobundles reproduce normal muscle metabolism under both basal and maximal energy demand conditions enabling the detection of drug-induced mitochondrial toxicity.

A number of significant muscle diseases, such as cachexia, sarcopenia, systemic chronic inflammation, along with inflammatory myopathies share TNF-α-dominated inflammation in their pathogenesis. In addition, inflammatory episodes may increase susceptibility to drug toxicity. To assess the effect of TNF-α-induced inflammation on drug responses, we engineered 3D, human skeletal myobundles, chronically exposed them to TNF-α during maturation, and measured the combined response of TNF-α and the chemotherapeutic doxorubicin on muscle function. First, the myobundle inflammatory environment was characterized by assessing the effects of TNF-α on 2D human skeletal muscle cultures and 3D human myobundles. High doses of TNF-α inhibited maturation in human 2D cultures and maturation and function in 3D myobundles. Then, a tetanus force dose-response curve was constructed to characterize doxorubicin's effects on function alone. The combination of TNF-α and 10nM doxorubicin exhibited a synergistic effect on both twitch and tetanus force production. Overall, the results demonstrated that inflammation of a 3D, human skeletal muscle inflammatory system alters the response to doxorubicin.

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