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The ROLE OF CYTOSOLIC CALCIUM IN POTENTIATION OF MOUSE LUMBRICAL MUSCLE.

出版	University of Waterloo, 2014
URL	http://books.google.com.hk/books?id=SAGezgEACAAJ&hl=&source=gbs_api

註釋Following contractile activity, fast twitch skeletal muscle exhibits increases in submaximal force known as potentiation. Although there is no consensus on the purpose of potentiation, it is known to enhance power during rapid dynamic contractions and counteract the early stages of peripheral fatigue. Potentiation is primarily attributed to phosphorylation of the myosin regulatory light chain (RLC) through a calcium-mediated process which results in increased calcium-sensitivity of crossbridge formation. However, there is a growing body of evidence showing that potentiation can be achieved in the absence of RLC phosphorylation, albeit to a lesser degree. A secondary characteristic of the potentiated contraction is an acceleration of relaxation properties, which could be teleologically beneficial to enhance the cycling rate of rapid motions (e.g. running). However, accelerated relaxation is inconsistent with elevations in calcium-sensitivity as this would tend to slow the time course and slow relaxation. Therefore there are multiple mechanisms involved in potentiation, some of which enhance crossbridge formation, and some of which enhance crossbridge detachment. A possible explanation for these events involves contraction-induced changes in the intracellular cytosolic calcium signal that triggers muscle contraction. For example, elevations in submaximal force could be achieved by increasing the amplitude of the calcium signal while enhanced relaxation speed could be achieved by a shorter duration of the calcium signal. Thus the main objective of this thesis was to investigate the contribution of changes in cytosolic Ca2+ to force potentiation. To achieve this objective, intact lumbrical muscles were extracted from the hind feet of C57BL/6 mice for use as the experimental model. The first study in this thesis examined cytosolic calcium signals during posttetanic potentiation using high (AM-fura-2 and AM-indo-1) and low (AM-furaptra) affinity calcium-sensitive fluorescent indicators to monitor resting and peak calcium respectively, both before and after a potentiating stimulation protocol of 2.5 s of 20 Hz stimulation at 37oC. This protocol resulted in an immediate 17±3% increase in twitch force (n=10; P0.05), though this potentiation dissipated quickly, lasting only 30 s. Resting cytosolic Casup2+/sup was also increased following the potentiating stimulus as indicated by increases of 11.1 ± 1.3% and 8.1 ± 1.3% in the fura-2 and indo-1 fluorescence ratios respectively. Like the force potentiation, these increases were short lived, lasting 20-30 s. No changes were detected in either the amplitude or kinetics of the Casup2+/sup transients following the potentiating stimulus. Western blotting analysis of the myosin heavy chain isoforms which determine the contractile phenotype of lumbrical muscle revealed predominance of fast type IIX fibres, while immunohistochemical analysis of proteins important for relaxation, namely parvalbumin, sarco-endoplasmic reticulum Casup2+/sup ATPase (SERCA) 1a and SERCA2a, revealed that the expression of these proteins in lumbrical moderated those found in the soleus (slow) and EDL (fast) archetypes. Surprisingly, despite the fast phenotype of the lumbrical, it exhibited low expression of the skeletal muscle isoform of myosin light chain kinase, the enzyme responsible for phosphorylating the myosin RLC, and high expression of myosin targeting phosphatase subunit 2, the enzyme responsible for dephosphorylating the myosin RLC. These data were corroborated by a complete lack of myosin RLC phosphorylation in either the rested or potentiated states. It was thus concluded that elevations in resting cytosolic calcium concentration, in the absence of changes in the intracellular calcium transient and RLC phosphorylation, can potentiate twitch force. The next objective of this thesis was to determine if there are changes in the cytosolic calcium transient during staircase potentiation, defined as a stepwise increase in twitch force during low frequency stimulation (10 Hz). Staircase potentiation has been repeatedly demonstrated to exhibit more robust potentiation than posttetanic potentiation in the absence of RLC phosphorylation. It was hypothesized that while the calcium transient is not altered during posttetanic potentiation, it may be an important potentiating factor in staircase due to the lower rest intervals between successive contractions. The effects of temperature on the intracellular calcium transient during staircase potentiation were also examined as part of this investigation. Here, lumbricals were loaded with AM- furaptra and then subjected to stimulation at 8 Hz for 8.0 s to induce staircase potentiation at either 30 or 37supo/supC. This stimulation protocol resulted in a 26.8 ± 3.2 % increase in twitch force at 37supo/supC (P0.05) and a 6.8 ± 1.9 % decrease in twitch force at 30supo/supC (P0.05) at the 8 s mark. Both the peak amplitude and the calcium-time integral of the calcium transient decreased during the first 2.0 s of the protocol (P0.05), however these decreases were greater at 30supo/supC than 37supo/supC (P0.05 amplitude; P=0.09 area). While peak amplitude remained low throughout the duration of the protocol, the calcium-time integral began to increase after the 2 s time point (P0.05), a change reflective of the progressive increases in the 50% decay time and full width at half maximum of the calcium transient (P0.05). Regression analysis of raw furaptra fluorescence ratios revealed a progressive decline in the peak amplitude of the calcium transients throughout the protocol which was not present at 37supo/supC. The increases in the duration of the calcium transient were mirrored by increases in the half relaxation time of the twitch contractions at both 30 and 37supo/supC, which had initially been reduced by ~20 and 9 % at 30 and 37