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Calcium transients in single fibers of low-frequency stimulated fast-twitch muscle of rat

Stefanie Carroll, Pierluigi Nicotera, and Dirk Pette

Faculty of Biology, University of Konstanz, D-78457 Constance, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Ca2+ transients were investigated in single fibers isolated from rat extensor digitorum longus muscles exposed to chronic low-frequency stimulation for different time periods up to 10 days. Approximately 2.5-fold increases in resting Ca2+ concentration ([Ca2+]) were observed 2 h after stimulation onset and persisted throughout the stimulation period. The elevated [Ca2+] levels were in the range characteristic of slow-twitch fibers from soleus muscle. In addition, we noticed a transitory elevation of the integral [Ca2+] per pulse with a maximum (~5-fold) after 1 day. Steep decreases in rate constant of [Ca2+] decay could be explained by an immediate impairment of Ca2+ uptake and, with longer stimulation periods, by an additional loss of cytosolic Ca2+ binding capacity resulting from a decay in parvalbumin content. A partial recovery of the rate constant of [Ca2+] decay in 10-day stimulated muscle could be explained by an increasing mitochondrial contribution to Ca2+ sequestration.

calcium buffering; free calcium; mitochondria; parvalbumin; sarcoplasmic reticulum calcium-adenosinetriphosphatase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHRONIC LOW-FREQUENCY STIMULATION (CLFS) induces in fast-twitch muscle a wide variety of molecular and functional changes that ultimately turn the muscle slow (for reviews see Refs. 25 and 26). As a result of qualitative and quantitative alterations in gene expression, fast protein isoforms are exchanged in all major functional elements of the muscle fiber with their slow counterparts. Proteins involved in Ca2+ release and Ca2+ sequestration appear to be affected during relatively early stages of this transformation process. Preceding the fast-to-slow exchange of the SERCA isoforms, there is a decrease in the catalytic activity of the sarcoplasmic reticulum Ca2+-ATPase a few hours after stimulation onset (8, 16). This partial inactivation of the enzyme is probably due to protein oxidation and nitration of tyrosyl groups (20). In parallel with the decrease in the capacity of Ca2+ uptake by the sarcoplasmic reticulum, there is a reduction in the cytosolic Ca2+ binding that results from a decay in parvalbumin expression and content (17). In addition, significant reductions in the contents of major proteins involved in excitation-contraction coupling have also been demonstrated (16). Ryanodine receptor, dihydropyridine receptor, and triadin exhibit steep reductions in muscles stimulated longer than 6 days, reaching levels comparable to their contents in slow-twitch soleus muscle.

These early perturbations in Ca2+ handling proteins indicate that CLFS might indirectly or directly affect calcium concentration ([Ca2+]) homeostasis in the muscle fiber. Indeed, earlier studies using Ca2+-sensitive electrodes for in vivo measurements in fibers of rabbit extensor digitorum longus muscle undergoing fast-to-slow conversion demonstrated that intracellular Ca2+ content was transiently elevated (32). Fivefold elevated peak levels of intracellular [Ca2+] were observed by 2 wk but recovered to near normal values with ongoing stimulation. A study on low-frequency stimulated extensor digitorum longus (EDL) and tibialis anterior (TA) muscles of rat revealed, in addition to increases in Na+ and decreases in K+, progressive increases in total [Ca2+] with 2.5- to 3.8-fold elevations after 24 h, respectively (10).

Although these studies provided first evidence that CLFS induces perturbations in the ionic milieu of the muscle fiber, these observations did not allow to relate alterations in intracellular free [Ca2+] to the effects of low-frequency stimulation on gene expression in the transforming muscle. However, the development of highly sensitive Ca2+ indicators and the development of high-resolution Ca2+ detection setups in the past decade has provided powerful tools for such a study. In other cell systems, high-affinity Ca2+ indicators have been used to show that small nanomolar changes in intracellular free Ca2+ activate specific transcription factors involved in cell growth, death, and differentiation (1, 7). For example, Alevizopoulos and co-workers (1), using fura 2, detected a transitory 125 nM increase in calcium influx initiated by addition of transforming growth factor-beta that could be linked to induction of the transcription factor CTF-1. Also using fura 2, Dolmetsch and co-workers (7) determined that transcription factors such as NF-kappa B and JNK1/ATF-2 are activated by large transitory increases in free calcium, whereas other transcription factors such as NFATc/p are sensitive to small sustained elevations in free calcium. Applying this technology, we set out in the present study to investigate [Ca2+] transients in single fibers of rat EDL muscle exposed to CLFS for different periods of time. We were interested in early effects of CLFS and, therefore, confined our study to time points between 2 h and 10 days after the onset of stimulation. Because rat muscle undergoes fast-to-slow transformation less readily than rabbit muscle, we chose to apply CLFS to the hypothyroid rat. This protocol has previously been shown to enhance the transformation process (19). Calcium transients calculated from indo-1 fluorescence signals were analyzed for changes in peak [Ca2+], resting [Ca2+], integral [Ca2+], and rate of [Ca2+] decay. In addition to changes in [Ca2+], we monitored changes in other factors involved in Ca2+ handling, such as parvalbumin content, activity of the sarcoplasmic reticulum Ca2+-ATPase, and mitochondrial content. The degree of the induced fast-to-slow transformation was determined by analysis of the myosin heavy chain (MHC) isoform complement. Our results show significant changes in Ca2+ dynamics during the first hours and 10 days of CLFS, thus indicating that alterations in Ca2+ homeostasis may be related to fiber type transition.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Animals, hypothyroidism, and CLFS. The experiments were performed on adult (4-5 mo) male Wistar rats (Thomae, Biberach, Germany). Hypothyroidism was produced by 7 wk of feeding an iodine-poor diet (Altromin C1042; Altromin, Lage, Germany) and by the addition of 0.1% propylthiouracil to the drinking water (27). Electrodes were implanted laterally to the peroneal nerve of the left hindlimb. After 1 wk of recovery, CLFS (10 Hz, 24 h/day) was started (28). The following time points (4-5 rats per time point) were investigated: 2 h, 12 h, 1 day, 4 days, and 10 days. After disconnection from the stimulator, the animals were killed by cervical fracture and EDL and TA muscles were quickly dissected from both hindlimbs. The muscles of the right hindlimb served as controls. For Ca2+ measurements, the EDL muscles were placed on Styrofoam-lined culture dishes containing Ringer solution (2.5 mM KCl, 1.0 mM MgSO4, 145 mM NaCl, 10 mM HEPES, 10 mM glucose, and 2.5 mM CaCl2; pH 7.4). The TA muscles were frozen in liquid N2, stored at -80°C, and later used for monitoring changes in Ca2+-ATPase activity, parvalbumin, and citrate synthase activity. Similar analyses were performed on EDL muscles collected in a second experimental series using the same time periods of CLFS. For comparison, normal soleus muscles were included in the measurements.

Measurement of [Ca2+] transients. The methods for enzymatic dissociation and agarose suspension were essentially the same as previously described (3). EDL and soleus muscles were trimmed of connective tissue, then spread out and pinned to the Styrofoam-lined culture dish to allow maximum exposure of the muscle to the collagenase medium, containing 0.2% collagenase (Sigma, type I) and 1% gentamycin in Ringer solution. EDL and soleus muscles were incubated 4-5 h at 37-40°C. Following collagenase digestion, EDL and soleus muscles were washed 2-3 times with Ringer solution and separated with forceps into smaller bundles that were triturated until single fibers were liberated. Prior to agarose suspension, fibers were loaded with 5 µM Ca2+ indicator indo-1-AM (cell-permeant form, Molecular Probes) for 40 min at 37°C, and pipetted onto a coverslip serving as the bottom of a small culture dish placed on a Leica TCS 4D confocal microscope. Indo-1 was excited using an ultraviolet laser source set at less than 10% maximum output to minimize photo bleaching (bleaching was calculated to account for less than 5% of the total signal per second). Fibers were stimulated by trains of 2-20 pulses (3 ms in duration, separated by a 7-ms rest interval) from a programmable voltage source via platinum wires. The fluorescence signals were collected at 405 and 490 nm by photomultiplier tubes using a fast line scan mode (13 ms/point). The ratio of the indo-1 emission signals (F405/F490) was used to calculate the [Ca2+] transients as previously described for fura 2 (3). Kinetic corrections for the Ca2+-indo-1 reaction were determined according to Ref. 33. Maximal Ca2+ binding to indo-1 (Rmax) was determined from long pulse trains (10-20 pulses) that clearly saturated the indicator with calcium. Minimum Ca2+ binding (Rmin) was estimated by subtracting 15% of the mean resting ratio value. The decay of the [Ca2+] transients was determined by single exponential plus a constant function fit as described previously (4). Rate constants determined from soleus fibers were consistent with previously reported values and were in agreement with the assumption that indo-1 does not significantly buffer Ca2+ in the fiber myoplasm (4). To insure that any change in the rate constant values was not due to fiber rundown, a "bracket" pulse was applied. A bracket pulse was a pulse train of 2 pulses or more that followed the last pulse train of the experiment. All fibers included in this analysis exhibited a bracket ratio record that was at least 94% the amplitude of the first ratio record. Measurements on fibers from the control EDL muscle of the right hindlimb were pooled and considered as zero time point in the low-frequency stimulation time course.

Ca2+-ATPase activity. Muscle homogenates were used for measuring Ca2+-ATPase activity spectrophotometrically in a coupled enzyme assay (29). Frozen EDL and TA muscles were pulverized under liquid N2 in a steel mortar. An aliquot of muscle powder was diluted 1:14 in homogenization buffer (20 mM Tris · HCl, 300 mM sucrose, 0.2 mM phenylmethylsulfonyl fluoride) and homogenized on ice with the Kinematica Polytron three times for 30 s with speed 9000 (14). The activity measurements were performed using a thermostated cuvette holder at 37°C. The assay medium (1.0 ml) contained 100 mM KCl, 5 mM MgCl2, 5 mM NaN3, 2 mM EGTA, 2.8 mM phosphoenolpyruvate, 30 U/ml pyruvate kinase, 45 U/ml lactate dehydrogenase, 0.4 mM NADH, 25 mM HEPES, and 1 µg of the Ca2+ ionophore A23187 to prevent intravesicular Ca2+ accumulation. The assay was initiated by adding ATP (6 mM final concentration), and the measurement was performed as previously described (14).

Quantification of parvalbumin. Parvalbumin was detected and quantified in whole muscle homogenates. Samples were prepared by adding 2 µl of the homogenate used for the Ca2+-ATPase assay to a total 16 µl of sample buffer (87.5 mM Tris · HCl, 2.0 mM dithiothreitol, 15% glycerol, 2% SDS, and 0.1% bromophenol blue, pH 6.8), heated to 65°C for 10 min, and separated on a 15% SDS polyacrylamide minigel with a 5% SDS-polacrylamide stacking gel (21). Standards for actin and parvalbumin were treated in the same manner. Gels were run at 25-40 mA. After electrophoresis, proteins were electrotransferred to a nitrocellulose membrane for 12-16 h at a current of 15 V on ice. The nitrocellulose membrane was fixed and blocked with 5% low-fat milk Tris · HCl-buffered saline with 0.1% Tween-20 (18). Monoclonal antibodies for actin and parvalbumin (Sigma) were used at dilutions 1:20,000 and 1:1,000, respectively. After 2 h at room temperature, secondary antibody (goat anti-mouse IgG) reaction was detected by chemiluminescence using Hyperfilm (Amersham). Densitometric evaluation of the films was performed, and total amount of parvalbumin and actin were calculated from comparison of standard curves. The total amount of parvalbumin detected in each sample was referred to the amount of protein loaded in each lane. Protein concentration of the homogenates was determined by the method of Bradford (2).

Myosin heavy chain analysis. Crude myosin was extracted from frozen EDL and TA muscle powder and analyzed electrophoretically for MHC isoform complement (13). Gels were silver-stained and evaluated densitometrically.

Citrate synthase activity. Citrate synthase activity was determined according to Ref. 31 and used as a marker of mitochondrial content.

Statistical analysis. All data are given as means ± SE. Analysis for statistical significance was done with Student's t-test for unpaired data. Differences were considered as significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in [Ca2+] transients induced by low-frequency stimulation. Figure 1 shows [Ca2+] transients in single EDL fibers resulting from 2 and 10 pulse trains for four different time periods of CLFS: 0, 2 h, 1 day, and 10 days. The most obvious differences were observed in the 1-day time point, where the [Ca2+] transient for both pulse trains was significantly prolonged and larger than the 0, 2-h, or 10-day time points. An even clearer picture of the [Ca2+] changes emerges in Fig. 2 when the peak Delta [Ca2+] (Fig. 2A), resting [Ca2+] (Fig. 2B), integral [Ca2+] (Fig. 2C), and the rate constants of the [Ca2+] transient decay (Fig. 2D) are shown as a function of CLFS duration. Although there were no significant differences in the peak [Ca2+] (determined from the peak level attained for a 2-pulse train duration), throughout the low-frequency stimulation period (Fig. 2A), we observed pronounced changes in the other characteristics of Ca2+ transport. Resting [Ca2+] (determined from the baseline level before the first pulse train) started to rise after 2 h of CLFS, reaching values that were 2.4- to 3.0-fold elevated after 12 h and 1 day of CLFS (Fig. 2B). This elevation was significant and maintained after 10 days of stimulation. The integral of the [Ca2+] transient (Fig. 2C) reached a maximum (~5-fold elevated) after 1 day but recovered to near control levels after 4 days of CLFS. Finally, the rate constants of the [Ca2+] transient decay (Fig. 2D), as determined from single exponential fits (see METHODS) for the 2-pulse (Fig. 2, solid symbols) and 10-pulse (Fig. 2, open symbols) train durations, decreased after 12 h. Whereas the 2-pulse rate constant remained reduced throughout the rest of the stimulation period, the 10-pulse rate constant slowly recovered, reaching near control values after 10 days of CLFS.


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Fig. 1.   Ca2+ transients in low-frequency stimulated extensor digitorum longus (EDL) fibers. Ca2+ transients calculated from fluorescence signals recorded from EDL fibers stimulated for 0 (A), 2-h (B), 1-day (C), and 10-day (D) time periods are shown for a 2-pulse (thin line) and a 10-pulse train duration (thick line).



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Fig. 2.   Peak Delta [Ca2+], resting [Ca2+], integral [Ca2+] per pulse, and rate constants of [Ca2+] decay in single fibers from EDL muscles exposed to chronic low-frequency stimulation (CLFS) for different time periods. Peak Delta [Ca2+] (A) was not significantly altered throughout the time course of CLFS, but there were significant differences in resting [Ca2+] (B), integral [Ca2+] per pulse (C), and rate constants of [Ca2+] decay (D). Significant increases in resting [Ca2+] (see RESULTS for calculation) were observed as early as 12 h of CLFS, and were maintained after 10 days of stimulation. Integral [Ca2+] per pulse (see RESULTS for explanation) reached a significant peak level after 1 day of CLFS but recovered to control levels after 4 days of stimulation. Rate constant of [Ca2+] decay for the short 2-pulse train (solid symbols) was significantly reduced for the 12-h and 1-day CLFS time periods and remained reduced after 10 days of stimulation. In contrast, rate constant of [Ca2+] decay for the longer 10-pulse train duration dropped initially after 12 h of CLFS, but returned to control levels thereafter. Half-filled symbol for the soleus data (D) represents the rate constant value for both the 2-pulse train and the 10-pulse train. The n values for 0, 2-h, 12-h, 1-day, 4-day, and 10-day CLFS time periods for A-D were 5, 3, 7, 4, 4, and 6 ,respectively; n value for soleus data was 3. *Significance compared with control values (P < 0.05).

It should be noted that the end values for the resting [Ca2+], integral [Ca2+], and rate constants of [Ca2+] decay were consistent with the data collected from soleus fibers. The resting [Ca2+] in the soleus fibers tended to be twofold higher than the EDL controls and was not significantly different from the 10-day low-frequency stimulated EDL fibers. Likewise, the rate constants of [Ca2+] decay for the short 2-pulse train was significantly lower compared with the value determined for control EDL, but there was no significant difference between the rate constant values determined from the [Ca2+] transients of the 10-day low-frequency stimulated EDL fibers and the rate constant values determined from the [Ca2+] transients of soleus fibers.

Changes in the relative contributions of parvalbumin and Ca2+-ATPase to the decay of the [Ca2+] transient. The effect of CLFS on the rate constants of [Ca2+] transient decay was further explored by analyzing the relationship between the rate constant values and the duration of the pulse train for each time point during CLFS (Fig. 3). Control (time point 0) fibers exhibited a pronounced reduction in the rate constant values with increasing pulse train durations, which is characteristic of fast-twitch fibers with high concentrations of parvalbumin. As more and more Ca2+ is released into the myoplasm, parvalbumin becomes increasingly saturated until it can no longer contribute to the rate of [Ca2+] decay. However as CLFS continued, this relationship between rate constants of [Ca2+] decay and pulse train duration changed. There was still a reduction in the value of the rate constant with longer pulse train durations, but the initial value for the short 2-pulse train was lower, and, therefore, the decline in the rate constant values was less dramatic. After 10 days of CLFS, the relationship between rate constant and pulse train duration seemed to resemble the relationship observed for soleus fibers and thus was more indicative of a slower fiber type with intermediate levels of parvalbumin.


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Fig. 3.   Time-dependent changes of the relationship between rate constants of [Ca2+] decay and pulse train duration. Control fibers (top) exhibited a high rate constant of [Ca2+] decay that decreased dramatically with increasing pulse duration. As the duration of CLFS continued to 2 h, 12 h, and 1 day (top), the relationship between the rate constant of [Ca2+] decay and the pulse duration changed. Initial rate constant became progressively lower, and, therefore, the saturation effect with increasing pulse duration was less. Relationship between the rate constants and the pulse train duration for the 4-day and 10-day stimulated fibers (bottom) resembled the relationship exhibited by soleus fibers (bottom). Control, 2-h, 12-h, 1-day, 4-day, and 10-day data were fit by a single exponential function. Soleus data were fit by a constant function.

The relationship between the rate constant values of [Ca2+] decay was fit by a single exponential as previously described (4). This analysis was conducted to determine the relative contributions of the saturable component (YS), i.e., the contribution of parvalbumin, and the nonsaturable component (YNS), which reflects the contribution of the Ca2+-ATPase. The results were plotted as a function the duration of CLFS in Fig. 4. Up to 1 day of CLFS, the saturable parvalbumin component appeared to be the major contributor to [Ca2+] transient decay. It accounted for about 80% of the rate constant of [Ca2+] decay, whereas the nonsaturable Ca2+-ATPase component only amounted to approximately 20%. However, between 1 and 4 days of CLFS, there was a shift in the relative contributions of the two components. Figure 4 shows that the contribution of the saturable component to [Ca2+] transient decay was reduced to approximately 40% and the contribution of the nonsaturable component had increased to 60%.


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Fig. 4.   Relative contributions of Ca2+-ATPase and parvalbumin throughout the time course of CLFS. Contribution of YNS and YS, representing the contribution of Ca2+-ATPase and parvalbumin, respectively (see RESULTS for explanation) changed throughout the time course of CLFS. From 0 to 1 day of stimulation, the YS component (open symbols) contributed to 80% of the rate constant, whereas the YNS component (solid symbols) accounted for only 20%. After 4 days of CLFS, there was a shift in contributions. Between 4 and 10 days of CLFS, the YS component contributed approximately 40% to the decay of Ca2+, and the YNS contributed 60%.

Changes in parvalbumin content, Ca2+-ATPase activity, and mitochondrial content. Presumably, the 2-pulse train data are indicative of the maximal Ca2+ binding capacity of parvalbumin and its full contribution to the [Ca2+] transient decay. During such short train durations, Ca2+ release is small and not expected to saturate parvalbumin. Therefore, the change in the rate constant determined under this condition should be consistent with the changes in the integral [Ca2+] demonstrated in Fig. 2C. As Ca2+ levels increase by 1 day of CLFS, the rate constant of [Ca2+] decay would be expected to decrease for the 2-pulse train, due to an increased saturation of parvalbumin. With ongoing CLFS, the rise in integral [Ca2+] falls and this should lead to a recovery of the rate constant of [Ca2+] decay, because the amount of Ca2+ bound to parvalbumin would then be less. However, the change in the rate constant of [Ca2+] decay with the 2-pulse train duration was not generally consistent with this prediction. Although, as shown in Fig. 5A, there was the expected initial drop in the rate constant around 1 day, this reduction did not recover after 10 days of CLFS. To explain this apparent inconsistency, the parvalbumin content was determined in both EDL and TA muscles for all time points of CLFS. According to the results shown in Fig. 5B, parvalbumin was reduced in the EDL homogenates by approximately 34% after 10 days of CLFS. The TA homogenates also exhibited decreased parvalbumin levels, but the reductions were smaller (~10%, data not shown). Using the reduction in parvalbumin content for "correcting" the rate constants of [Ca2+] decay for the 2-pulse train duration, the 10-day value reached 85% of the control, as predicted.


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Fig. 5.   Change in parvalbumin content in low-frequency stimulated EDL fibers. Change in rate constant of [Ca2+] decay for the 2-pulse train, depicted as % of the control value (A), was not consistent with the assumption that this value is indicative of parvalbumin's full Ca2+ binding capacity (see RESULTS for further explanation). A 34% reduction in the parvalbumin content of the 10-day stimulated muscle (B) was considered and used to correct the 2-pulse train rate constant value (open symbol). Correction of the rate constant was conducted by adding the change in parvalbumin content to the 10-day value. The n values in B for the 0, 2-h, 12-h, 1-day, 4-day, and 10-day CLFS time points were 5, 5, 3, 5, 4, and 5, respectively.

We assumed that the longer pulse duration should release sufficient calcium to fully saturate parvalbumin. The removal of Ca2 would, therefore, primarily be effected by the Ca2+-ATPase, such that the change in the 10-pulse train duration rate constant should correspond to changes in the Ca2+-ATPase activity. As shown in Fig. 6B, Ca2+-ATPase activity was 30% reduced in EDL homogenates 12 h after the onset of CLFS and gradually decreased to 45% of the control value in 10-day stimulated muscles. Ca2+-ATPase activity was also reduced in TA homogenates. It dropped by 20% after 1 day and was approximately 30% reduced in 10-day stimulated TA muscles (data not shown).


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Fig. 6.   Change in Ca2+-ATPase activity in low-frequency stimulated EDL fibers. Change in the rate constant of [Ca2+] decay for the 10-pulse train duration, shown as % of the control (A), was not consistent with the assumption that this value was solely indicative of the Ca2+-ATPase activity. Measured Ca2+-ATPase activity (B, solid symbols) dropped after 2 h of CLFS and continued to decrease from 1 to 10 days of stimulation. An increase in citrate synthase activity, used as a mitochondrial marker, was subtracted from the 10-pulse train value (open symbol). The n values in B for the 0, 2-h , 12-h, 1-day, 4-day and 10-day CLFS time points were 5, 5, 3, 5, 3, and 3, respectively.

A comparison of the changes in the 10-pulse rate constant of [Ca2+] decay (Fig. 6A) with the changes in Ca2+-ATPase activity (Fig. 6B) revealed inconsistencies. Thus, although the rate constant of [Ca2+] decay displayed an initial drop, it had recovered to control levels after 10 days. To reconcile this inconsistency, we decided to explore the possible mitochondrial contribution to Ca2+ sequestration. Such a role has previously been reported for slow-twitch fibers rich in mitochondria (11). Changes in mitochondrial content were investigated by measuring the activity of citrate synthase as a mitochondrial marker enzyme. Citrate synthase activity amounted to 11.3 ± 0.9 U/g muscle in control EDL and 14.8 ± 0.9 U/g muscle in 10-day stimulated EDL. Using this 30% increase for "correction" of the 10-pulse train value in the 10-day stimulated EDL muscle (Fig. 6A), the rate constant of [Ca2+] decay fit better the change in Ca2+-ATPase activity.

Changes in myosin heavy chain isoforms. Whole muscle analyses for changes in myosin heavy chain complement are shown in Fig. 7. Changes significant with regard to a decrease in MHCIIb and an increase in MHCIIa and MHCI were observed after 10 days of CLFS. Thus, the measurements on changes in Ca2+ dynamics fell into the time period during which a significant change occurred in phenotype expression.


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Fig. 7.   Stimulation-induced changes in the relative concentrations of myosin heavy chain (MHC) isoforms in 10-day stimulated EDL muscle. MHC isoforms in control (n = 3) and 10-day stimulated (n = 3) muscles were electrophoretically separated, and their relative concentrations were evaluated by integrating densitometry. *Significance compared with control values (P < 0.05).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CLFS induces in fast-twitch muscles fiber type transitions in the direction from fast to slow. The present data show that these transitions are preceded by marked alterations in intrafiber Ca2+ dynamics. Pronounced impairment of Ca2+ uptake by microsomal fractions from rabbit muscle exposed to CLFS for only 2 days has led us 20 years ago to suggest that CLFS-induced changes in gene expression might be related to elevations in cytosolic free [Ca2+] (15). It has now become possible to test this hypothesis by applying Ca2+ imaging methods for the study of time-dependent effects of CLFS on Ca2+ dynamics and cytosolic free [Ca2+] in single fibers. The rat was chosen as an experimental animal in the present study because, contrary to the rabbit (23), the induced fast-to-slow conversion occurs without signs of fiber injury or degeneration (27). This was a prerequisite for the study of Ca2+ transients as fiber injury could easily produce erroneous findings or misinterpretation. Fiber injury may have contributed to the very high increases in myoplasmic [Ca2+] reported in a previous study in which measurements with Ca2+-sensitive microelectrodes were performed on single fibers from low-frequency stimulated rabbit muscles (32).

The present findings clearly show that CLFS has a pronounced effect on the Ca2+ dynamics of rat muscle fibers. It is obvious that some of the observed changes in Ca2+ dynamics are transitory, whereas others are persistent. Among the transitory changes during short pulse trains are marked decreases in peak Delta [Ca2+] and elevation in integral [Ca2+] during the first hours after the onset of CLFS. Both changes are fully reversible. The reduced peak Delta [Ca2+] after only 2 h may be an artifact and result from a transient edema of the muscle fiber. A transient edema of muscle fibers has been shown to occur in rabbit muscle during the early phases of CLFS (9, 30). Considering this possibility, the transitory increase in integral [Ca2+] would even be greater, especially between 2 and 12 h when this rise may be dampened by an edema. Similarly, the reduction in parvalbumin content observed after 2 h of CLFS might be an effect of the edema.

In contrast to the transitory changes in peak Delta [Ca2+] and integral [Ca2+], the alterations in resting [Ca2+] are maintained throughout the investigated 10-day period of CLFS. The increase in resting [Ca2+] that is significant after 12 h attains a 2.5-fold elevation after 1 day and persists in this range after 10 days. It is noteworthy that the elevation in resting [Ca2+] reaches a level characteristic of slow-twitch fibers in soleus muscle (4). The increased resting [Ca2+] levels most likely result from the observed decrease in Ca2+ uptake potential due to the reduced Ca2+-ATPase activity and, probably also, to the loss of Ca2+ binding capacity by reduced parvalbumin levels. The reduction in parvalbumin after 10 days of CLFS is in agreement with similar findings from a previous study on rat muscles (17). As for the Ca2+-ATPase, loss of activity during the early phase of CLFS has been observed in several studies on rat (S. Matsunaga and D. Pette, unpublished results) and rabbit (8, 16, 22). This partial inactivation of the enzyme that precedes the SERCA1-to-SERCA2 transition, most likely results from an attack of free radicals (20, 24). However, an impaired function of the Ca2+-ATPase, which would not be noticed by activity measurement under in vitro conditions, results from the abrupt drop of the ATP phosphorylation potential in low-frequency stimulated muscle. As previously shown by studies on whole muscle and single fibers, CLFS leads to a steep and persistent decay of this magnitude a few minutes after the onset of stimulation (6, 12). Because the function of the Ca2+ pump of the sarcoplasmic reticulum depends on a high ATP phosphorylation potential (34), the decrease in Ca2+-ATPase activity would be even greater in vivo than assessed by the in vitro assay. Therefore, decreases in Ca2+-ATPase activity and parvalbumin content seem both to be reflected by the changes in the rate constant of [Ca2+] decay.

The effect of the parvalbumin reduction is mirrored by the significant drop in the rate constant of [Ca2+] decay for the 2-pulse train duration and by the pronounced decrease in the saturation effect by the slowing of the rate constants with increasing pulse train duration. A closer look at the relationship between the rate constants of [Ca2+] decay with increasing pulse train duration reveals that as the saturation effect becomes less, the level at which saturation finally occurs is lower at the 12-h, 1-day, and 4-day CLFS time periods. This reflects the action of the Ca2+-ATPase alone, before increases in mitochondria occur during the first 4 days of stimulation, or the combined efforts both of Ca2+-ATPase and mitochondria after 10 days of CLFS. Analysis of the nonsaturable and saturable components, representing the contribution of the Ca2+-ATPase and parvalbumin, respectively, show that up to 1 day of CLFS the parvalbumin component is the major contributor to the removal of Ca2+. This is consistent with the contribution of the parvalbumin component previously determined in fibers assumed to be type IIB with high parvalbumin content (4). However, as CLFS continues, there is a shift in the contributions of parvalbumin and Ca2+-ATPase. Both contribute approximately equally to the removal of Ca2+ after 4 days of CLFS. This is consistent with their relative contributions observed in fibers assumed to be type IIA with intermediate or low levels of parvalbumin (4). Indeed, this is in agreement with the changes in myosin heavy chain complement.

In summary, measurements on single fibers from rat EDL muscle exposed to CLFS demonstrate pronounced (~2.5-fold) elevations in intracellular free [Ca2+] reaching the level normally found in slow-twitch fibers from soleus muscle. These changes occur soon (2 h) after stimulation onset and persist up to 10 days, the longest time point investigated. Obviously, the increase in free [Ca2+] results from an impaired function of the sarcoplasmic reticulum Ca2+-ATPase and is complemented, with longer stimulation periods, by a decrease in parvalbumin content. Interestingly, the increases in [Ca2+] observed in this study are within the range of [Ca2+] elevations detected in other cell systems, in which changes in transcription occur (1, 7). These findings substantiate our original hypothesis that increases in free intracellular [Ca2+] are causally related to altered gene expression in low-frequency stimulated muscle (15). In fact, a direct relationship to elevated [Ca2+] levels has recently been suggested by a calcineurin-dependent transcriptional pathway involved in the control of muscle fiber phenotype expression (5).


    ACKNOWLEDGEMENTS

We are grateful to Elmi Leisner and I. Traub for excellent technical assistance in the animal experiments and the measurement of citrate synthase and MHC electrophoresis.


    FOOTNOTES

This study was supported by the Deutsche Forschungsgemeinschaft (PE 62-19-3). S. Carroll thanks the Alexander von Humboldt Foundation for a stipend.

Addresses for reprint requests and other correspondence: S. Carroll, National Institutes of Health, Building 6, Room 408, 6 Center Dr. MSC 2755, Bethesda, MD 20892-2755; and D. Pette, Faculty of Biology, University of Konstanz, D-78457 Konstanz, Germany.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 29 March 1999; accepted in final form 22 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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