Selvin D, Hesse E, Renaud JM. Properties of single FDB fibers following a collagenase digestion for studying contractility, fatigue, and pCa-sarcomere shortening relationship. Am J Physiol Regul Integr Comp Physiol 308: R467-R479, 2015. First published January 7, 2015 doi:10.1152/ajpregu.00144.2014.-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 (ϳ80%) 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 2ϩ ] 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. calcium; culture medium; flexor digitorum brevis; membrane potential; sarcomere shortening
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 [Ca2+]i in KATP-channel-deficient fibers. We measured changes in myoplasmic [Ca2+] ([Ca2+]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 [Ca2+]i at the end than at the beginning of fatigue to fibers which eventually completely failed to release Ca2+ upon stimulation. Compared to control conditions, KATP-channel-deficient fibers had a greater proportion of fiber with large decreases in tetanic [Ca2+]i, fade and complete failure to release Ca2+ 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 [Ca2+]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 Ca2+ load by reducing Ca2+ influx through CaV1.1 channels between and during contractions.
Most studies on muscle fatigue use whole muscle. The disadvantages of such preparations are the development of an anoxic core and the impossibility of studying differences between fiber types. While some studies have used mechanically dissected single muscle fibers, the preparation is time consuming and difficult to use for electrophysiological measurements. Another method is to isolate single fibers in large numbers following a collagenase treatment to allow for dispersal of fibers by trituration. Although variations on the method to obtain such single fiber preparations exist, there is no quantitative data indicating quality of preparation in terms of contractility. The objective of this study was to determine the best protocol for fiber isolation from mouse FDB fibers. Fibers isolated following collagenase digestion in MEM culture medium with 10% FBS had better morphology and contractility (i.e., number of contracting fibers and threshold values) than those in which DMEM was used as a culture medium. Also, in the absence of FBS in culture medium, all fibers supercontracted during trituration regardless of the culture medium used. The addition of 0.2% FBS in the physiological solution bathing fibers during experiments also improved morphology, contractility and stability.
During fatigue, KATP channels are crucial in reducing action potential amplitude to eventually lower Ca2+ release by sarcoplasmic reticulum, which helps preserve ATP during a metabolic stress by decreasing Ca2+ ATPase and myosin ATPase activity. In the absence of KATP channel activity, skeletal muscle muscles suffer major contractile dysfunctions, which lead to faster fatigue rate because of fiber damage. In our studies at 37°C, we have observed a tremendous variability in the fatigue kinetics among mouse single FDB muscle fibers. Under control conditions, such variability is expected as fatigue resistance is in the order of type I>IIA>IIX>IIB (note IIB fibers are not present in FDB). However, we also observed a large variability in fatigue kinetics when KATP channels are completely blocked with 10 µM glibenclamide. Our results suggest that the importance of the KATP channel for myoprotection during fatigue is in the order of type IIX > IIA > I types, an order similar to the difference in KATP channel content between fiber types; i.e., the KATP channels are the most important in glycolytic fibers and the least important in oxidative fibers. Grant Funding Source: NSERC
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