A Novel Assay of Muscle Energy Dynamics in Mechanically Loaded Enzymatically Isolated Adult Mammalian Skeletal Myocytes

Abstract

Current methods and tools available to study bioenergetics in adult mammalian skeletal muscle are mostly limited to whole organ or fiber bundle preparations. However, single cell preparations remain attractive for studying mechanics and bioenergetics, because they are free from connective tissue and endothelium, allow for consistent and uniform oxygen and substrate availability and can be investigated using real-time imaging methods at high temporal and spatial resolution. The majority of research on single muscle cells is conducted in mechanically unloaded conditions in which the rate of ATP consumption is low. ATP production is generally matched to consumption and due to both cytosolic glycolysis and mitochondrial oxidative phosphorylation. Investigations of mechanically loaded muscle cells that may consume ATP at “normal” rates provide us with a preparation that enables us to explore issues related to mitochondrial function, ROS production as will as functional Ca2+ signaling and contractile performance. However, doing experiments with mechanically loaded single cells requires technically challenging manually dissection methods. We have recently developed an improvement that enables us to use enzymatically dissociated skeletal myocytes. Using MyoTak, a tenacious biological adhesive, we have been able to attach dissociated skeletal myocytes to both force transducer and length controller units. The myocytes thus prepared can be readily imaged using an inverted microscope fluorescence imaging system in confocal, widefield and multiphoton systems. We will show results that use this system to examine the force produced in enzymatically isolated mammalian skeletal myocytes with isometric contractions elicited by field stimulation, NADPH auto-fluorescence, ROS (reactive oxygen species) production during contraction.