Membrane depolarization is the initial action of crotoxin on isolated murine skeletal muscle

Although much is known about the pathogenesis of crotoxin-induced muscle damage, the initial site and action of the toxin is still not clear. In this study we used an electrochromic fluorescent dye, Di-4-ANEPPS, to measure the changes in membrane potential of isolated murine omohyoid muscle to determine if depolarization could be one of the initial effects of crotoxin. Omohyoid isolates were pre-loaded with 1 microM Di-4-ANEPPS, exposed to various crotoxin treatments, and the change in fluorescence was recorded using either a dual-wavelength spectrofluorometer or digital imaging. Spectrofluorometry indicated that crotoxin depolarized isolated omohyoid muscles within 4 min as indicated by an increase in fluorescence to 122% of control values. Crotoxin also induced depolarization of extensor digitorum longus and soleus muscles as indicated by an increase in fluorescence of 140 and 110% of the control, respectively. Fluorescent images obtained from omohyoid muscle preparations exposed to crotoxin and Di-4-ANEPPS revealed localized areas of increased fluorescence, muscle contractions, derangement of myofibrils, and differing sensitivity to crotoxin of different muscle cells. Light microscopy results confirmed this variable disruption of muscle cell integrity and differing sensitivity to crotoxin. An increase in creatine kinase release rates confirmed damage to the plasma membrane. We conclude that plasma membrane depolarization is most likely the earliest indicator of cell damage from crotoxin and is quickly followed by hypercontraction of myofilaments, disruption of the plasma membrane, release of creatine kinase and necrosis.