Human, Tissue-Engineered, Skeletal Muscle Myobundles to Measure Oxygen Uptake and Assess Mitochondrial Toxicity

Abstract

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.

Neuromuscular disease modeling on a chip

“…Over the past two decades, approaches for engineering 3D muscle bundles have been advanced and refined. Several different types of culture chambers with anchor points have been fabricated ( Costantini et al, 2017a ; Smith et al, 2016 ), including Velcro and nylon frames ( Davis et al, 2017 , 2019 ; Madden et al, 2015 ; Rao et al, 2018 ; Smith et al, 2016 ; Zhang et al, 2018 ) and microfabricated chambers with pillars ( Osaki et al, 2018 , 2020 ; Uzel et al, 2016 ). Testing of multiple ECM solutions has also revealed that fibrin hydrogels are optimal for encapsulating myotubes due to their strength ( Hinds et al, 2011 ; Pollot et al, 2018 ), although these hydrogel compositions are not necessarily physiological.…”

In Vitro Tissue‐Engineered Skeletal Muscle Models for Studying Muscle Physiology and Disease

“…[9,255] Unforeseen mitochondrial toxicity is the most common cause for failure of candidate drugs identified from in vitro drug screens in preclinical animal models. [256] This can be attributed to the Crabtree effect, [257] where in vitro cultured cells typically utilize glycolysis to generate ATP despite the presence of abundant oxygen and functional mitochondria.…”

Modeling the Effect of TNF-α upon Drug-Induced Toxicity in Human, Tissue-Engineered Myobundles

“…Muscle biopsies were obtained from discarded surgical waste with Duke IRB approved protocols. Human skeletal muscle myoblasts were isolated according to previously described methods 12 . Briefly, muscle samples were minced, washed in phosphate buffered saline (PBS), and enzymatically digested in 0.05% trypsin for 30 minutes.…”

“…The myobundle assembly procedure was used as previously described 9,12,29 . Briefly, cells were dissociated in 0.025% trypsin-EDTA to a single cell solution then encapsulated in a fibrinogen (Akron, Boca Raton, FL) and matrigel solution on laser cut Cerex frames within PDMS molds at 7.5 × 10 5 cells/myobundle.…”