Efficiency of skeletal muscle decellularization methods and their effects on the extracellular matrix

Extracellular matrix (ECM) is widely considered to be integral to the function of skeletal muscle, providing mechanical support, transmitting force, and contributing to passive stiffness. Many functions and dysfunctions attributed to ECM are thought to stem from its mechanical properties, yet there are few data describing the mechanics of intact ECM. Such measurements require isolating intact ECM from the muscle cells it surrounds. The objectives of this study were to quantify the efficiency of three techniques for this purpose: Triton, Triton with sodium dodecyl sulfate, and latrunculin B; and to determine their impact on properties of the remaining ECM. Efficiency was quantified by DNA content and evaluation of western blot intensities for myosin and actin. The properties of ECM were quantified by collagen content and uniaxial tensile testing. We found that latrunculin B was the most efficient method for removing skeletal muscle cells, reducing DNA content to less than 10% of that seen in control muscles, and substantially reducing the myosin and actin to 15% and 23%, respectively; these changes were larger than for the competing methods. Collagen content after decellularization was not significantly different from control muscles for all methods. Only the stiffness of the muscles decellularized with latrunculin B differed significantly from control, having a Young's modulus reduced by 47% compared to the other methods at matched stresses. Our results suggest that latrunculin B is the most efficient method for decellularizing skeletal muscle and that the remaining ECM accounts for approximately half of the stiffness in passive muscle.