Properties of single FDB fibers following a collagenase digestion for studying contractility, fatigue, and pCa-sarcomere shortening relationship.

The objective of this study was to optimize the approach to obtain viable single flexor digitorum brevis (FDB) fibers following a collagenase digestion. A first aim was to determine the culture medium conditions for the collagenase digestion. The MEM yielded better fibers in terms of morphology and contractility than the DMEM. The addition of FBS to culture media was crucial to prevent fiber supercontraction. The addition of FBS to the physiological solution used during an experiment was also beneficial, especially during fatigue. Optimum FBS concentration in MEM was 10% (vol/vol), and for the physiological solution, it ranged between 0.2 and 1.0%. A second aim was to document the stability of single FDB fibers. If tested the day of the preparation, most fibers (∼80%) had stable contractions for up to 3 h, normal stimulus duration strength to elicit contractions, and normal and stable resting membrane potential during prolonged microelectrode penetration. A third aim was to document their fatigue kinetics. Major differences in fatigue resistance were observed between fibers as expected from the FDB fiber-type composition. All sarcoplasmic [Ca(2+)] and sarcomere length parameters returned to their prefatigue levels after a short recovery. The pCa-sarcomere shortening relationship of unfatigued fibers is very similar to the pCa-force curve reported in other studies. The pCa-sarcomere shortening from fatigue data is complicated by large decreases in sarcomere length between contractions. It is concluded that isolation of single fibers by a collagenase digestion is a viable preparation to study contractility and fatigue kinetics.

Changes in myoplasmic Ca2+ during fatigue differ between FDB fibers, between glibenclamide-exposed and Kir6.2-/- fibers and are further modulated by verapamil.

One objective of this study was to document how individual FDB muscle fibers depend on the myoprotection of KATP channels during fatigue. Verapamil, a CaV1.1 channel blocker, prevents large increases in unstimulated force during fatigue in KATP-channel-deficient muscles. A second objective was to determine if verapamil reduces unstimulated [Ca(2+)]i in KATP-channel-deficient fibers. We measured changes in myoplasmic [Ca(2+)] ([Ca(2+)]i) using two KATP-channel-deficient models: (1) a pharmacological approach exposing fibers to glibenclamide, a channel blocker, and (2) a genetic approach using fibers from null mice for the Kir6.2 gene. Fatigue was elicited with one tetanic contraction every sec for 3 min. For all conditions, large differences in fatigue kinetics were observed from fibers which had greater tetanic [Ca(2+)]i at the end than at the beginning of fatigue to fibers which eventually completely failed to release Ca(2+) upon stimulation. Compared to control conditions, KATP-channel-deficient fibers had a greater proportion of fiber with large decreases in tetanic [Ca(2+)]i, fade and complete failure to release Ca(2+) upon stimulation. There was, however, a group of KATP-channel-deficient fibers that had similar fatigue kinetics to those of the most fatigue-resistant control fibers. For the first time, differences in fatigue kinetics were observed between Kir6.2(-/-) and glibenclamide-exposed muscle fibers. Verapamil significantly reduced unstimulated and tetanic [Ca(2+)]i. It is concluded that not all fibers are dependent on the myoprotection of KATP channels and that the decrease in unstimulated force by verapamil reported in a previous studies in glibenclamide-exposed fibers is due to a reduction in Ca(2+) load by reducing Ca(2+) influx through CaV1.1 channels between and during contractions.