(882.6) Enhanced Isolation of Single Myofibers in Flexor Digitorum Brevis Dissociation

Poster Board Number: E289

Logan Moore (University of Texas at Arlington), Kamal Awad (University of Texas at Arlington), Chenglin Mo (University of Texas at Arlington), Jian Huang (University of Texas at Arlington), Zhiyang Wang (University of Texas at Arlington), Leticia Brotto (University of Texas at Arlington), Marco Brotto (University of Texas at Arlington)

Introduction:

 The isolation of single myofibers from the Flexor Digitorum Brevis (FBD) has been a well-established model in skeletal muscle physiology research. The use of isolated single-fibers has led to advancements in the understating of the excitation – contraction (E-C) coupling mechanisms in skeletal muscle, calcium transients, the role of mitochondria, and much more. However, these experiments rely on the quality of the dissociation of myofibers and their attachment to the imaging dish which can be difficult and time exhaustive. The purpose of this study was to build upon previously established methodologies to improve the yield of intact-alive myofibers in a time efficient manner.

Methods and Materials:

 Euthanasia and experimental protocols were approved by the University of Texas at Arlington’s IACUC. Based upon previously established dissociation techniques (1–3), we modified the protocol as follows: the intact FDB was dissected out and placed in 2 mL of Ringer’s solution (142 mM NaCl2, 5 mM KCl, 1.8 mM MgCl2, 10 mM Hepes, 10 mM Glucose). Under the dissection microscope the whole muscle was separated into 4 muscle bundles; separated by the distal tendons. The muscle bundles were transferred to the digestion media (DMEM with glutamine, 2% sterile filtered fetal bovine serum (FBS), 0.1% gentamycin, and 4 mg ml-1 Collagenase A). The muscle bundles were incubated for 30-45 minutes at 37° C in 5% CO2.  Following incubation, the bundles were transferred to 1mL of Ringer’s solution in a 24-well plate and gently titrated with the cut end of a P1000 pipette tip. The solution was transferred to laminin coated glass bottom dishes and incubated for 15-30 minutes for fiber attachment.  Following fiber attachment, the fibers were either stained with the LIVE/DEAD™ Viability/Cytoxicity kit or loaded with Fura-2 AM fluorescent dye for measurement of maximal calcium depolarization response induced with 100mM Potassium Chloride (KCl) using a PTI spectrophotometry  system.

Results and

Conclusion:

We found that using an increased concentration of collagenase and a reduced time of digestion successfully increased the number of fibers in the field of vision.. Laminin coated glass bottom dishes produced quick and strong attachment, allowing for optimal stimulation conditions. The use of cell culture media with FBS protected the sarcolemma increasing the viability of the fibers. This is shown in both the LIVE/DEAD imaging and the response to KCl stimulation. This study demonstrates that the change of dissociation media and the decrease in dissociation time allow for protection of the myofiber for an increased yield and viability. This protocol will allow for improved experimental outcomes in which mature myofibers are subjected to electrical or chemical stimulations.

References:

1.        Calderón et al. JMRCM doi: 10.1007/s10974-009-9181-1.

2.        Capote et al. JP doi: 10.1113/jphysiol.2004.081034.

3.        Yi et al. JBC doi: 10.1074/jbc.M110.217711.

The authors want to thank the National Institutes of Health (1R03DE023872-01, 1R56DE027964-01A1-01, NIH S10OD025230) and the National Institutes of Aging (NIA 7-PO1AG039355; 5R01AG056504, 5R01AG060341) for their generous support for this study.



Figure 1: A) Live fibers stained in calcein-AM (Green). B) Dead cells stained in ethidium homodimer-1 (Red). C) Fura-2 loaded fiber at rest D) Fiber after stimulated with KCl. E) Graph showing the Ca2+ transient response after KCl treatment.