THE ROLE OF CYTOSOLIC CALCIUM IN POTENTIATION OF MOUSE LUMBRICAL MUSCLE

Abstract

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 37^oC. This protocol resulted in an immediate 17±3% increase in twitch force (n=10; P<0.05), though this potentiation dissipated quickly, lasting only 30 s. Resting cytosolic Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 37^oC. This stimulation protocol resulted in a 26.8 ± 3.2 % increase in twitch force at 37^oC (P<0.05) and a 6.8 ± 1.9 % decrease in twitch force at 30^oC (P<0.05) 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 (P<0.05), however these decreases were greater at 30^oC than 37^oC (P<0.05 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 (P<0.05), a change reflective of the progressive increases in the 50% decay time and full width at half maximum of the calcium transient (P<0.05). 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 37^oC. 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 37^oC, which had initially been reduced by ~20 and 9 % at 30 and 37^oC during the first 2 s of the protocol. Therefore the degree of staircase potentiation depends, in part, on the magnitude of the decline in the amplitude and the degree of slowing of the cytosolic calcium transient. The declines in calcium transient amplitude noted above occurred simultaneously with increased rates of relaxation and abbreviated contraction times. To determine if there was a causal relationship between the reduced amplitude and the faster contractions, AM-furaptra-loaded lumbrical muscles were stimulated at 8 Hz for 2 s in the presence and absence of caffeine, an agonist of the calcium release channel. Caffeine treatment attenuated the decline of the calcium transient amplitude (P<0.05), and was associated with greater potentiation at 37^oC (P<0.05), and attenuated force loss at 30^oC (P<0.05). Despite the increases in calcium and force, the relaxation times and rates of relaxation exhibited a greater acceleration following caffeine treatment (P<0.05). Therefore the relaxation-enhancing factor during potentiated twitches cannot be attributed to the calcium transient, and must be localized to changes on the myofilament. The case for inorganic phosphate as the effector is made. Similar to the findings of the posttetanic potentiation study, the resting cytosolic calcium concentration was elevated during staircase potentiation, as revealed by fura-2 ratio signals. The largest increase occurring immediately following the first twitch of the protocol. This coincided with the largest increases in force potentiation at both 30 and 37^oC. This finding is in accordance with the initial conclusion that elevations in resting calcium can enhance twitch force and contribute to potentiation, though the mechanism of action is unclear. One possibility is that increases in resting calcium, sub-threshold for force production, can enhance the number of attached but non-force producing crossbridges, thereby accelerating the transition of crossbridges to force-producing states upon calcium-release following stimulation. To test this hypothesis, the resting stiffness, a measure of crossbridge attachment, of lumbrical muscles was examined before and after a potentiating stimulus of 20 Hz 2.5 s. Resting stiffness was assessed using sinusoidal length oscillations, ~0.5 nm per half sarcomere in amplitude and ranging in frequency from 10-200 Hz. Subsequent analysis revealed decreases in the elastic stiffness (P<0.05) that lasted for ~20 s which were greater in magnitude (P<0.05) than increases in viscous stiffness which only lasted for ~5 s. This finding is consistent with the disappearance of short range elastic component (SREC) upon stretch or muscle activation which is commonly attributed to a population of stable, bound crossbridges in resting muscle. Subsequent analysis using imposed length changes to eliminate the SREC prior to contraction had no effect on the amplitude or duration of a subsequent twitch or tetanic contraction, and the changes in elastic and viscous stiffness of resting muscle were identical whether SREC was ablated by a contraction or imposed length change. Therefore it appears that potentiation occurs without an associated increase in bound crossbridges at rest, and may actually occur with fewer bound crossbridges at rest than the unpotentiated state. The lack of effect may be related to the relaxation-enhancing factor discussed above, and be an important feature of skeletal muscle serving to protect against damage via an involuntary eccentric contraction. This thesis describes potentiation as a complex and important biological function which is the sum of factors that serve to enhance and oppose force production.