Figure 5. Calcium influx into the cell as measured by Fluo-4 relative fluorescence shows membrane repair is a much faster process than as previously indicated by FM1-43 wounding studies.

Isolated wild-type C57BL/6 muscle fibers were loaded with 3μM Fluo-4 for 1hr and then wounded with a high powered laser, representative images shown (top A). Calcium increases at the wound site were quantified, described by the white box in B, which showed that wounding causes a rapid increase in calcium at the wound site (one representative trace shown B, summary data from one representative experiment C). Calcium increases at the wound are followed by an increase in cytoplasmic calcium at sites distal to the wound (red box in B, and magenta open circles C). Calcium at the wound peaks and reverses at the wound at approximately 40s post wound (solid line B, summary data in D), but cytoplasmic calcium reversal is significantly delayed to approximately 60s (D) A bolus of 10mM caffeine to wounded fibers, one representative trace shown, stimulates calcium release from the SR that exceeds peak calcium after wounding (E). Removing calcium from extracellular media and chelating any trace calcium with 1mM EGTA prevents the increase in cytosolic calcium following wounding (representative images A bottom, quantified F). While calcium flux reverses within the first minute following wounding, the concentration of FM1-43 rises in the cell for more than 2 minutes, and then continues to increase at a constant rate even after 2 minutes (G). Statistical compaisons were performed using an unpaired, two-tailed Student’s t-test. Statistical significance (p<0.05) is denoted by (*). Error bars represent the standard error of the mean. One representative experiment of three independent replicates is shown.