Enzymatic Dissociation Makes Skeletal Muscle Fibers Susceptible to Osmotic Stress and More Prone to Mitochondrial Calcium Uptake

Abstract

Introduction: Enzymatic dissociation using collagenases is a frequently used method when studying function in individual adult skeletal muscles fibers. A potential problem with this method is that enzymatic disruption of the extracellular matrix may alter the anchorage of the intracellular cytoskeleton to the cell membrane and hence affect cellular structure and function.Methods: We used single mouse flexor digitorum brevis (FDB) fibers isolated either by enzymatic dissociation with collagenase or by mechanical dissection, which leaves the immediate extracellular matrix intact. The gross structure of isolated fibers was assessed with Second Harmonic Generation (SHG) microscopy. Cytosolic and mitochondrial free [Ca2+] were measured with fluo-3 and rhod-2, respectively.Results: In comparison to dissected fibers, enzymatically dissociated fibers show: (1) less elaborate gross structure; (2) increased susceptibility to develop defective intracellular Ca2+ handling (increased basal cytosolic [Ca2+], spontaneous Ca2+ waves and Ca2+ sparks) in response to a hypo-osmotic shock (3 min exposure to 50% of the normal NaCl concentration); (3) increased mitochondrial Ca2+ uptake during repeated tetanic contractions.Conclusion: Disruption of the extracellular matrix results in marked changes in muscle fiber structure and function. This raises concerns when interpreting results obtained in experiments performed with enzymatically dissociated fibers. Moreover, our results support a role of altered cellular Ca2+ homeostasis in the disease process of muscle dystrophies caused by mutations in extracellular matrix proteins.