Ca2+ entry during the action potential stimulates muscle contraction. During repetitive low frequency stimulation, skeletal muscle undergoes staircase potentiation (SP), a progressive increase in the peak twitch force induced by each successive stimulus. Multiple mechanisms, including myosin regulatory light chain phosphorylation, likely contribute to SP, a temperature-dependent process. Here, we used the Ca2+-sensitive fluorescence indicators acetoxymethyl (AM)-furaptra and AM-fura-2 to examine the intracellular Ca2+ transient (ICT) and the baseline Ca2+ level at the onset of each ICT during SP at 30 and 37°C in mouse lumbrical muscle. The stimulation protocol, 8 Hz for 8 s, resulted in a 27 ± 3% increase in twitch force at 37°C and a 7 ± 2% decrease in twitch force at 30°C (P < 0.05). Regardless of temperature, the peak rate of force production (+df/dt) was higher in all twitches relative to the first twitch (P < 0.05). Consistent with the differential effects of stimulation on twitch force at the two temperatures, raw ICT amplitude decreased during repetitive stimulation at 30°C (P < 0.05) but not at 37°C. Cytosolic Ca2+ accumulated during SP such that baseline Ca2+ at the onset of ICTs occurring late in the train was higher (P < 0.05) than that of those occurring early in the train. ICT duration increased progressively at both temperatures. This effect was not entirely proportional to the changes in twitch duration, as twitch duration characteristically decreased before increasing late in the protocol. This is the first study identifying a changing ICT as an important, and temperature-sensitive, modulator of muscle force during repetitive stimulation. Moreover, we extend previous observations by demonstrating that contraction-induced increases in baseline Ca2+ coincide with greater +df/dt but not necessarily with higher twitch force.
Excitation–contraction coupling is the process by which depolarization of the muscle membrane is converted into mechanical forces by striated muscle cells. The voltage-gated release of Ca2+ from the terminal cisternae of the SR produces transient elevations in cytosolic Ca2+ levels that regulate several processes including muscle force. Ca2+ binding to troponin C relieves stearic hindrances to cross-bridge formation, i.e., formation of the strongly bound actomyosin complex on the thin filament, and thus allows force production. Cytosolic Ca2+ concentration is returned to basal levels by the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), an ATP-dependent pump that translocates Ca2+ across the SR membrane. Although a single action potential produces only a “twitch,” repeated action potentials yielding more sustained elevations in Ca2+ produce a “tetanus” with much greater force-time integral output of striated muscle contraction.
The twitch force produced by a muscle is extremely history dependent and is thus highly labile. As an example, repetitive stimulation of fast-twitch skeletal muscle at low stimulation frequencies (<10 Hz) produces a stepwise or progressive increase in twitch force to a new peak (Isaacson, 1969), known as staircase potentiation (SP). The magnitude of SP is dependent on several factors including temperature, being reduced as muscle cools (Walker, 1951; Close and Hoh, 1968; Hanson, 1974; Krarup, 1981; Moore et al., 1990; Vandenboom et al., 2013). Based on the characteristics of the action potential measured during brief but repetitive stimulation of rat intercostal skeletal muscle (in vitro) at 22 and 37°C (Hanson, 1974), the temperature dependence for SP likely originates downstream of membrane excitability and voltage-gated Ca2+ release channel open probability per se (Vandenboom et al., 2013).
The primary intracellular mechanism for SP may be posttranslational modifications of the myosin motor molecule concomitant with repetitive stimulation of fast-twitch skeletal muscle. As an example, repetitive stimulation of rodent fast-twitch skeletal muscle has been demonstrated to increase the phosphate content of the myosin regulatory light chain (RLC) subunits (Klug et al., 1982; Manning and Stull, 1982), a reaction that increases the Ca2+ sensitivity, but not maximal force, of permeabilized skeletal fibers (Persechini et al., 1985; Metzger et al., 1989). These studies thus provide a mechanistic explanation for the ability of repetitive stimulation to enhance twitch without influencing tetanic force (Sweeney and Stull, 1990). A small but growing body of evidence, however, suggests the existence of multiple or complementary mechanisms for SP. For example, extensor digitorum longus (EDL) muscles devoid of skeletal myosin light chain kinase, the enzyme responsible for phosphorylating the RLC, exhibit SP in the absence of RLC phosphate incorporation (Zhi et al., 2005; Gittings et al., 2011). Moreover, denervated rat gastrocnemius muscles displaying reduced levels of RLC phosphorylation retain SP (Rassier et al., 1999; MacIntosh et al., 2008). Although an alternative mechanism has not definitively been identified, our recent work has shown that posttetanic potentiation (PTP) of the mouse lumbrical, a muscle without detectable RLC phosphorylation, is associated with elevations in cytosolic Ca2+ immediately before the onset of an intracellular Ca2+ transient (ICT), such that baseline Ca2+ is higher at the onset of potentiated contractions than unpotentiated contractions (Smith et al., 2013). However, it remains to be determined if changes in the ICT and/or baseline Ca2+ at the onset of the ICT contribute to SP in the mouse lumbrical.
The primary purpose of this investigation was to compare and contrast the changes in force with the changes in the cytosolic Ca2+ during SP at two different temperatures (i.e., 30 and 37°C) to determine if stimulation-induced changes in either the ICTs or the baseline Ca2+ level at the onset of each ICT could contribute to the SP response. To this end, we measured the changes in the isometric twitch force in mouse lumbrical muscles after loading with Ca2+-sensitive fluorescence indicators, either acetoxymethyl (AM)-furaptra or AM-fura-2, which were, respectively, used to monitor changes in the ICT and baseline Ca2+. We hypothesized that there would be temperature-dependent declines in the amplitude and area of the Ca2+-time integral of the ICT, which may help account for the reduced potentiation seen at low versus high temperatures. We hypothesized that just as in PTP (Smith et al., 2013), baseline Ca2+ would be elevated at the onset of twitches exhibiting SP, regardless of temperature.
MATERIALS AND METHODS
All methods used in this study were approved by the University of Waterloo Animal Care Committee, and all experiments were performed in the laboratory of A.R. Tupling at the University of Waterloo. Detailed descriptions of the methods used in this study were reported recently (Smith et al., 2013). In brief, after sacrifice by cervical dislocation, the lumbrical muscles from the hind feet of 3–6-mo-old male C57BL/6 mice were excised in Tyrode’s dissecting solution (mM: 136.5 NaCl, 5.0 KCl, 11.9 NaHCO3, 1.8 CaCl2, 0.40 NaH2PO4, 0.10 EDTA, and 0.50 MgCl2, pH 7.5; on ice). Muscles were then suspended at optimum length for twitch-force production (Lo) between a high speed length controller (model 322C; Aurora Scientific Inc.) and a force transducer (model 400A; Aurora Scientific Inc.) in a bath containing circulating oxygenated (95% O2, 5% CO2) Tyrode’s experimental solution (mM: 121.0 NaCl, 5.0 KCl, 24.0 NaHCO3, 1.8 CaCl2, 0.40 NaH2PO4, 5.50 glucose, 0.10 EDTA, and 0.50 MgCl2, pH 7.3) at either 30 or 37°C. Stimulation was applied through flanking platinum plate field stimulus electrodes using a stimulator (model 701C; Aurora Scientific Inc.) at supramaximal voltage. Lumbrical muscles were loaded with the low affinity ratiometric Ca2+ indicator AM-furaptra (Molecular Probes) to detect transient changes in the intracellular Ca2+ concentration. Separate muscles were loaded with the high affinity, ratiometric Ca2+ indicator AM-fura-2 to monitor changes in baseline Ca2+. Monochromator-controlled light (Photon Technology International) at either 380 or 344 nm provided the excitation signal to the muscle-bound indicators, and the resulting 510-nm fluorescence emission signals were collected through an inverted microscope photometer system (D-104; Photon Technology International) affixed with a photomultiplier tube (model 814; Photon Technology International). Analogue force and fluorescence signals were digitized and collected at 10,000 Hz and stored for later analysis.
A schematic illustration of the stimulation protocols used in this study is shown in Fig. 1 (A and C). AM-furaptra–loaded lumbricals were stimulated once per 30 s to produce a steady isometric twitch-force response. Fluorescence emission signals from the last six twitches were used to determine the characteristics of the unpotentiated ICT. This pacing procedure was then halted, and an 8-Hz stimulation protocol lasting for 8 s was initiated with continuous fluorescence emission monitoring. This protocol was used as it allowed force to return to resting values between consecutive twitches at both 30 and 37°C. The muscle was then subjected to the pacing protocol for 5 min to monitor dissipation of the potentiation response. The excitation wavelength was adjusted and the stimulation protocol was repeated 10 times per muscle, alternating between 380- and 344-nm excitation to produce five fluorescence ratio signals, which were averaged together during data analysis. Data were further condensed by averaging the five unpotentiated twitches into a single record, whereas the 8-Hz stimulation data were condensed into four windows where all twitches within 2-s blocks were averaged together, i.e., 16 twitches per window. Muscles generally tolerated this protocol well, as tetanic force did not change over the course of the protocol. Twitch force, however, declined between the first and 10th trials. There was a trend toward greater reduction of twitch force at 37°C than at 30°C (30°C: 16 ± 3% vs. 37°C: 23 ± 5%; P = 0.14), in contrast to the greater reduction in ICT amplitude seen at 30°C compared with 37°C. This suggests that even if a systematic decline in Ca2+ release occurs, it should not affect our conclusions. Furthermore, the early decline in ICT amplitude seen during SP, the lack of a difference in ICT characteristics during PTP (Smith et al., 2013), and the return of twitch force to prestimulation levels within 30 s of recovery all suggest fast recovery of ICT amplitude after cessation of repetitive stimulation. At 37°C, potentiation systematically increased between the first and 10th trials, and at 30°C, force depression systematically decreased (final force was relatively higher in later trials, although it never achieved potentiation). Both ICT signals and force data were averaged across the 10 repetitions. Thus, all data reflect the average response of the 10 trials for each muscle, which should control for any systematic decline in twitch force (and Ca2+ release or sensitivity).
To gain greater insight into the changes in the ICT during the first 2 s of SP, a second analysis of the furaptra recordings was performed using finer groupings, specifically the combination of twitches 1–6, 6–11, and 11–16 in the SP protocol. Asynchronous measurement of emission signals is a salient feature of dual-excitation mono-emission ratioable indicators, which leaves room for signal contamination by motion artifact. For example, small changes in the contraction between consecutive trials may result in varying amounts of indicator in the field of view; thus, it cannot be said that our measurements are entirely free of motion artifact. However, extensive testing has verified the effectiveness of our protocols to cancel the motion during sinusoidal length changes (Smith, 2014; see also Morgan et al., 1997). Additionally, we have carefully checked the data for any large mismatches in the emission signals, and in the hundreds of repetitions, we found only one instance of mismatch with significant consequence on the resultant ratio, which was subsequently removed from the data. Thus, we are confident that the signals presented are a good representation of the changes in cellular Ca2+.
An additional component of this study was to characterize the expression of the muscle relaxation enzymes (SERCA1a, SERCA2a, and parvalbumin) in whole muscle homogenates of the lumbrical, soleus, and EDL of the mouse using Western blotting techniques (n = 8). Separation of proteins was performed by electrophoresis using either 7.5% polyacrylamide glycine gels (SERCA1a and SERCA2a) or 13% polyacrylamide tricine gels (parvalbumin) and standard SDS/PAGE protocols (Laemmli, 1970). Protein concentrations of homogenates were determined by the bicinchoninic acid method (Sigma-Aldrich) and verified using Ponceau S (BioShop Canada Inc.). A linear relationship between band density and protein load was established for each mouse using either EDL (SERCA1a and parvalbumin) or soleus (SERCA2a) to form a scale, against which the other muscles were compared. After semi-dry transfer of proteins to polyvinyl difluoride membranes, primary probing was performed using monoclonal anti-SERCA1a A52 (provided by D. MacLennan, University of Toronto, Toronto, Canada), anti-SERCA2a 2A7-A1 (Thermo Fisher Scientific), and PVG 214 goat anti-parvalbumin (Swant). Secondary probing was performed using goat anti–mouse (SERCA1a and SERCA2a; Santa Cruz Biotechnology, Inc.) or donkey anti–goat (parvalbumin; Santa Cruz Biotechnology, Inc.). Detection was performed using a Luminata HRP substrate detection kit (EMD Millipore), and subsequent quantification was based on densitometric analysis obtained using GeneSnap and GeneTools software (GE Healthcare).
All data are reported as mean ± SEM. Relative changes in mean values for various twitch-force and Ca2+ parameters were compared by two-way split-plot ANOVA as appropriate using temperature (between groups) and time (repeated measures), followed by Tukey’s honestly significant difference post-hoc analysis using Statistica 7 software. Differences were considered significant at P < 0.05.
Representative force tracings and corresponding ICT measurements from the 8-s stimulation protocol are shown in Fig. 2, whereas the mean data are presented in Fig. 3. Consistent with previous reports (Krarup, 1981; Zhi et al., 2005; Ryder et al., 2007), the force and kinetic responses were complex as the first 1–2 s were marked by declining force and accelerating twitch-time course at both temperatures. The results from this initial period are reported in greater detail below. Beyond ∼2 s, twitch force, the time to peak tension (TPT), and time to 50% relaxation (1/2RT) progressively increased at 37°C, whereas at 30°C, force remained relatively stable at a depressed level, TPT declined slowly, and the 1/2RT progressively increased. The changes in the peak rate of force production (+df/dt) exhibited a similar pattern to the changes in twitch force at 37°C. Interestingly, +df/dt was consistently elevated during the stimulation protocol at 30°C, but this change did not track with the changes in peak twitch force at this temperature. The peak rate of relaxation (−df/dt) initially increased at both temperatures, before reaching a plateau and finally declining in the final seconds of the protocol (Fig. 3 E). Overall, at 37°C, 8 s of 8-Hz stimulation resulted in a twitch with 26.8 ± 3.2% higher force, 4.4 ± 1.9% lower TPT, 11.4 ± 2.5% higher 1/2RT, 43.6 ± 1.6% faster +df/dt, and 11.1 ± 2.5% faster −df/dt than an unpotentiated twitch (all P < 0.05; see representative tracings in Fig. 2 A and raw force values in Table 1). In comparison, at 30°C, 8 s of 8-Hz stimulation resulted in a final twitch with 6.8 ± 2.0% lower force (P < 0.05), 19.0 ± 3.7% lower TPT (P < 0.05), 8.8 ± 3.9% lower 1/2RT (P < 0.05), 18.3 ± 3.6% faster +df/dt (P < 0.05), and 1.6 ± 3.2% slower −df/dt (not significant) than an unpotentiated twitch. Interestingly, 30 s after the stimulation protocol, the differences in the kinetic characteristics of the twitches at 30 and 37°C became quite similar, with both temperatures exhibiting potentiated force (3.4 ± 1.1 and 7.4 ± 2.4% relative to unpotentiated levels at 30 and 37°C, respectively), whereas −df/dt was faster and 1/2RT was reduced relative to unpotentiated twitches (all P < 0.05; not depicted).
Fluorescence ratio signals from lumbricals loaded with AM-furaptra were averaged together in 2-s windows during the 8-Hz stimulation protocol and across six unpotentiated twitches occurring at 30-s intervals before initiating the 8-Hz stimulation. Relative changes in the peak amplitude of the resultant ratio signals are presented in Fig. 3 F. The peak amplitude significantly declined (P < 0.05) by the end of the protocol at 30°C, whereas peak amplitude was maintained at 37°C. The raw furaptra ratio records were next adjusted to eliminate the differences in the baseline fluorescence ratio at the onset of each stimulus. The resultant records were averaged together as above. Analysis of these records revealed that regardless of temperature, peak change in amplitude was lower during the 8-Hz stimulation protocol relative to the unpotentiated ICT (Fig. 3 G). The decline in peak amplitude was reflected by a decline in the Ca2+ time integral during the first 2 s of staircase (P < 0.05); however, the Ca2+-time integral was no longer significantly different than initial unpotentiated values by beyond 2–4 s, and was significantly greater than 0–2 s at 6–8 s (Fig. 3 H). As the time-dependent increase in the Ca2+-time integral was absent if the integration time was reduced from 20 to 10 ms (not depicted), this particular effect can be attributed to increases in the decay time of the ICT. Consistent with this attribution were the progressive elevations in 1/2RT and full width at half-maximum (Fig. 3, I and J). Although these were all temperature-independent effects, the magnitudes of the initial decreases in the peak ratios (30°C 3.13 × 10−3 ± 0.44 × 10−3 vs. 37°C 1.68 × 10−3 ± 0.44 × 10−3 ratio units; P < 0.05) and Ca2+-time integrals (30°C 1.74 × 10−2 ± 0.45 × 10−2 vs. 37°C 0.87 × 10−2 ± 0.42 × 10−2 ratio units × ms; P = 0.09) were higher at 30°C than at 37°C. These particular comparisons must be made cautiously, as although the Ca2+ sensitivity of furaptra is largely temperature independent, its relatively high temperature sensitivity to Mg2+ is a potential confounding factor (Konishi et al., 1991). However, when the integration time is reduced to 10 ms poststimulus, the declines in Ca2+-time integral are only significant at 30°C (P < 0.05 vs. P = 0.19 at 37°C), thus supporting the temperature dependence of this effect. These results collectively indicate that the changes in force and force kinetics seen late in the protocol correspond well to changes seen in the ICT.
Analysis of the furaptra ICT records using finer groupings of the first 2 s of the SP protocol, specifically of twitches 1–6, 6–11, and 11–16 (Fig. 4), revealed that relative to unpotentiated levels, the Ca2+-time integral is reduced throughout the 8-Hz stimulation protocol, whereas peak amplitude of the ICT tended to decrease relative to unpotentiated values throughout the protocol, but only reached statistically significant reductions by twitches 6–11 at 30°C and by twitches 11–16 at 37°C.
Lumbricals loaded with the high affinity Ca2+-sensitive indicator AM-fura-2 demonstrated increases in baseline Ca2+, as summarized in Fig. 5. Importantly, because temperature can affect the fura-2 fluorescence ratio independently of changes in Ca2+ (Oliver et al., 2000) and no calibration attempt was made in this study, attempting to directly compare [Ca2+]i between temperatures is inappropriate; however, relative changes over time are perfectly valid. To facilitate this comparison, the change in fluorescence ratio for each temperature was normalized on a scale of 0–1, with the pre-SP fura-2 ratios at twitch 1 serving as zero (i.e., the unpotentiated baseline cytosolic Ca2+ levels) and the peak fluorescence ratio during twitch 1 serving as 1.0. The resulting data demonstrate that the twitch-to-twitch increases in baseline Ca2+ were largest in the first three to four contractions and achieved a plateau by ∼1.5 s. The time course of the increase in baseline Ca2+ was not influenced by temperature. Fura-2 recordings were only performed for 2 s because of photo-bleaching concerns, as AM-fura-2 loading was much less effective than AM-furaptra loading.
Western blotting results
An additional part of this study was the characterization of the proteins responsible for muscle relaxation in the lumbrical relative to the EDL and soleus muscles, as illustrated in Fig. 6. In general, the expression profile of Ca2+-sequestering proteins of lumbricals lies between the extremes set by the slow-relaxing soleus and fast-relaxing EDL.
The primary purpose of this study was to characterize the ICT during SP to determine if the ICT might contribute to the potentiation response seen at physiological temperatures. Although the mouse lumbrical is a relatively unique model with which to study potentiation phenomena, our results reproduce many classic features of SP at 20–37.5°C shown by Krarup (1981) in rat EDL muscle. As a result, our results seem to be generally applicable to both species. As the first study to characterize the ICT during SP in either species, our findings provide novel insights into potential force-modulating mechanisms, which have implications for potentiation of vertebrate striated muscle.
Comparison of force and Ca2+ records
Previous work has shown that the mouse lumbrical, despite its fast fiber–type composition, displays no detectable RLC phosphorylation either at rest or after stimulation (Smith et al., 2013). Despite this shortcoming, the lumbrical exhibits PTP at 37°C (Smith et al., 2013). In the present study, we have demonstrated that the lumbrical also exhibits SP at 37°C, but not at 30°C (Fig. 3). Additionally, at both temperatures, there are enhancements in the rate of force production during 8-Hz stimulation, indicating a general increase in the apparent Ca2+ sensitivity of force production, which does not necessarily translate to elevated peak twitch force (Fig. 3). This may be an effect of elevations in baseline Ca2+ that were seen at both temperatures (Fig. 5). We have demonstrated previously a parallel between PTP and elevated Ca2+ levels at the onset of stimulation, which may enhance twitch force by enhancing the Ca2+ occupancy of troponin C at rest or by reducing competition for troponin C Ca2+ binding through saturation of other cytosolic Ca2+-binding proteins such as parvalbumin (Smith et al., 2013), which we show to be present in significant quantities in the lumbrical (Fig. 6). It is also conceivable that elevations in baseline Ca2+ could indirectly influence the ICT to enhance or alter subsequent twitch-force development. Redox signaling may also play a role in enhancing Ca2+ sensitivity of the contractile proteins. For example, S-glutathionylation of the fast isoform of troponin I can enhance the Ca2+ sensitivity of force production (Dutka et al., 2011; Mollica et al., 2012). Whatever the mechanism, it must be both quickly inducible and reversible to account for the potentiation and increases in Ca2+ sensitivity seen in this study.
At 37°C, the peak amplitude of the raw furaptra ratios remained unchanged throughout the protocol (Fig. 3 F) despite diminutions in the amplitude of the baseline-adjusted ICT (Fig. 3 G). The maintenance of the amplitude of the raw furaptra ratio is presumably caused by the elevations in baseline Ca2+ (Fig. 5). In contrast, both the raw and baseline-adjusted furaptra ratios were depressed from unpotentiated levels at 30°C throughout the 8-Hz stimulation protocol. This may have contributed to the reduced twitch force seen throughout the protocol at this lower temperature. A comparison of the twitch relaxation times and the ICT decay times presents a curious feature of this investigation. Although twitch relaxation parameters were initially hastened by the 8-Hz stimulation, there were no corresponding increases in the decay time of the ICT. In contrast, both ICT and twitch decay times slow beyond ∼2 s of 8-Hz stimulation (Fig. 3). The lack of a general consistency in the pattern of changes in twitch and ICT decay times is a surprising finding, as accelerated relaxation during repeated submaximal contractions has been ascribed to faster cytosolic Ca2+ removal (Vøllestad et al., 1997; Tupling, 2009). Our present finding suggests that this may not be the case. Finally, evidence from intact frog skeletal fibers has shown that it is the duration, and not the amplitude, of the ICT that is most important for isometric twitch-force development (Sun et al., 1996); thus, the slowing of the ICT may have been a force-enhancing factor in SP.
It is clear that the ICT had some influence on the force response, but the extent to which this extends is unknown because of myriad confounding factors including the effects of metabolites and metabolic byproducts, reactive oxygen and nitrogen species, parvalbumin saturation levels, elevated baseline Ca2+, and posttranslational modifications of key proteins. Specific investigation of these aspects and their role in potentiation as modulators of force, the contractile proteins, and cytosolic Ca2+ levels is beyond the scope of this investigation. Considerable effort will be required to discern the specific effects of each of these factors on the force response during SP. This area of research will undoubtedly yield fascinating results in the future.
Changes in Ca2+ in relation to other studies
Many of the features of the changes in cytosolic Ca2+ are consistent with the features of many known physiological phenomena. For example, Hollingworth and Baylor (2013) report that a significant parvalbumin concentration in fast-twitch muscle fibers delays the return of cytosolic Ca2+ concentration to resting levels. As the lumbrical contains significant parvalbumin, this may contribute to the elevations in baseline Ca2+ seen in this and in our previous investigation on PTP (Smith et al., 2013). Additionally, the declines in baseline-adjusted ICT amplitude seen during SP are consistent with the general properties of Ca2+-dependent inactivation of Ca2+ release from the SR seen during closely spaced contractions (Hollingworth et al., 1996; Posterino and Lamb, 2003; Caputo et al., 2004; Barclay, 2012). As the degree of Ca2+ inactivation of Ca2+ release is generally greater at lower temperatures, the greater reductions in ICT amplitude and area at 30°C than at 37°C are also generally consistent with this property. Finally, the presence of parvalbumin in the lumbrical poses a degree of complexity to the mechanism underlying the prolongation of the ICT we observed in this study. As the Ca2+-binding properties of parvalbumin account for 50–73% of the rate of ICT decay during relaxation of fast-twitch rodent and frog muscle (Hou et al., 1993; Hollingworth et al., 1996; Carroll et al., 1997), an elevation in baseline Ca2+ could indirectly slow the rate of decay of the ICT by saturating the Ca2+-buffering capacity of parvalbumin (Hollingworth et al., 1996). However, this mechanism is presently difficult to reconcile in the absence of slowed ICT decay during PTP where baseline Ca2+ is similarly elevated (Smith et al., 2013). An equally probable cause of ICT slowing is the accumulation of metabolic byproducts such as ADP and/or Pi, which slow the Ca2+-pumping activities of SERCA, and would accordingly be expected to prolong ICT decay times (Stienen et al., 1993; Macdonald and Stephenson, 2001). Although our data cannot confirm the validity of any of these proposed mechanisms, it provides a theoretical framework upon which to base further testing.
Both Mg2+ and Ca2+ increase the fluorescence ratio of furaptra; therefore, these results most likely contain contamination from the Mg2+ signal at rest, and may contain a progressively increasing Mg2+ artifact during the repetitive stimulation protocol (Konishi et al., 1991; Hollingworth et al., 1996). However, because of the low intensity of this protocol, the progressive component of this Mg2+ contamination is unlikely to be of significant consequence to the conclusions made in this study. Although it was assumed that the locus of the furaptra was the cytosol, AM dyes are known to compartmentalize forming localized noncytosolic contributions to the universal fluorescence, which may have a confounding effect on the results (see Morgan et al., 1997). Finally, although the ability of furaptra to precisely track the ICT has not been definitively demonstrated for 37°C (Hollingworth et al., 1996), the parallel responses at 30 and 37°C lend ostensible validity to the 37°C data.
The force response to repetitive low frequency stimulation is the result of the balance between mechanisms of force enhancement and force diminishment. Although the force-enhancing effects of myosin RLC phosphorylation and its effects on the Ca2+ sensitivity of cross-bridge formation are well documented (Stull et al., 2011), this study has highlighted both parallels and discrepancies between changes in the ICT characteristics and the force response during SP. Not the least of these is our finding that just as in PTP, elevations in baseline Ca2+ occur during SP, an effect that is likely to contribute to the potentiation response as a means of increasing the Ca2+ sensitivity of force production. Although much work remains to definitively establish any causal links between changes in Ca2+ and force responses, the findings in this study provide a basis for further investigations into both Ca2+-based and Ca2+-independent mechanisms and how they specifically influence the characteristics of the force response during repeated stimulation.
The authors would like to acknowledge the kind gift of monoclonal anti-SERCA1a A52 antibody from Dr. David MacLennan (University of Toronto).
Financial support for this study was received from the Natural Sciences and Engineering Research Council of Canada (grant 311922-05 to A. R. Tupling and grant RGPIN-2014-05122 to R. Vandenboom).
The authors declare no competing financial interests.
Richard L. Moss served as editor.
- Abbreviations used in this paper:
- time to 50% relaxation
- peak rate of force production
- peak rate of relaxation
- extensor digitorum longus
- intracellular Ca2+ transient
- posttetanic potentiation
- regulatory light chain
- sarco-endoplasmic reticulum Ca2+-ATPase
- staircase potentiation
- time to peak tension
- Submitted: 10 July 2014
- Accepted: 22 October 2014
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