Ca2+ sparks are initiated by Ca2+ entry in embryonic mouse skeletal muscle and decrease in frequency postnatally

Lois G. Chun, Christopher W. Ward, and Martin F. Schneider

Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Submitted 21 February 2003 ; accepted in final form 22 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
"Spontaneous" Ca2+ sparks and ryanodine receptor type 3 (RyR3) expression are readily detected in embryonic mammalian skeletal muscle but not in adult mammalian muscle, which rarely exhibits Ca2+ sparks and expresses predominantly RyR1. We have used confocal fluorescence imaging and systematic sampling of enzymatically dissociated single striated muscle fibers containing the Ca2+ indicator dye fluo 4 to show that the frequency of spontaneous Ca2+ sparks decreases dramatically from embryonic day 18 (E18) to postnatal day 14 (P14) in mouse diaphragm and from P1 to P14 in mouse extensor digitorum longus fibers. In contrast, the relative levels of RyR3 to RyR1 protein remained constant in diaphragm muscles from E18 to P14, indicating that changes in relative levels of RyR isoform expression did not cause the decline in Ca2+ spark frequency. E18 diaphragm fibers were used to investigate possible mechanisms underlying spark initiation in embryonic fibers. Spark frequency increased or decreased, respectively, when E18 diaphragm fibers were exposed to 8 or 0 mM Ca2+ in the extracellular Ringer solution, with no change in either the average resting fiber fluo 4 fluorescence or the average properties of the sparks. Either CoCl2 (5 mM) or nifedipine (30 µM) markedly decreased spark frequency in E18 diaphragm fibers. These results indicate that Ca2+ sparks may be triggered by locally elevated [Ca2+] due to Ca2+ influx via dihydropyridine receptor L-type Ca2+ channels in embryonic mammalian skeletal muscle.

calcium; ryanodine receptor; dihydropyridine receptor; muscle development


SPONTANEOUS CA2+ SPARKS are discrete, localized elevations of Ca2+ that can be detected using a Ca2+ indicator dye and laser-scanning confocal microscope. These spatially and temporally isolated fluorescence transients are believed to be due to openings of one or a small functional group of ryanodine receptor (RyR) Ca2+ release channels. Ca2+ sparks have been observed in a variety of tissues, including embryonic mammalian skeletal muscle (10) and cultured myotubes (29). However, Ca2+ sparks rarely occur in intact adult mammalian skeletal muscle (10, 29). Taken together, these findings may suggest that spontaneous Ca2+ sparks in mammalian skeletal muscle play a role in the process of muscle development.

Differences in the expression profile as well as the structural arrangement of excitation-contraction (EC) coupling proteins between embryonic and adult mammalian skeletal muscle may contribute to the rare detection of Ca2+ sparks in the adult muscle. One protein that is differentially expressed during development of mammalian skeletal muscle is the RyR. In adult skeletal muscle the type 1 isoform (RyR1) is predominantly expressed (21, 31), whereas in the embryonic tissue RyR3 is coexpressed with RyR1 at high levels. The expression level of RyR3 declines to very low levels in adult muscle (16) during the postnatal period. Whereas RyR1 is characterized by its ability to functionally couple to dihydropyridine receptor (DHPR), giving skeletal muscle a voltage-dependent sarcoplasmic reticulum (SR) Ca2+ release mechanism independent of Ca2+ flux across the sarcolemma (28), RyR3 does not couple to the DHPR, instead being activated by voltage-independent mechanisms (14, 27), presumably by Ca2+-induced Ca2+ release (CICR). Given the developmental expression of RyR3, it is tempting to speculate that the presence of RyR3 contributes to the relative abundance of Ca2+ sparks in embryonic but not in adult mammalian skeletal muscle.

The occurrence of Ca2+ sparks in embryonic mammalian skeletal muscle may also be attributed to an altered EC coupling mechanism in the embryonic muscle. In adult skeletal muscle, EC coupling occurs independently of external Ca2+ (1), whereas the twitch tension in neonatal mouse and rat EDL muscles was found to decrease with lowered extracellular Ca2+ concentration ([Ca2+]o) (11, 25). Other investigators have observed a Ca2+ current-dependent component of contraction in developing cultured myotubes (8). Embryonic and neonatal mammalian skeletal muscles thus appear to posses a cardiac type of EC coupling in which influx of extracellular Ca2+ initiates RyR Ca2+ release by CICR due to a local elevation of Ca2+ in the vicinity of the RyR. This raises the possibility that extracellular Ca2+ might also modulate the production of Ca2+ sparks in embryonic fibers.

The presence of Ca2+ sparks in embryonic but not intact adult mammalian skeletal muscle appears to be a complex issue. To examine the production of Ca2+ sparks during muscle development, we have performed a detailed and objective examination of the frequency of occurrence and spatial-temporal properties of skeletal muscle Ca2+ release events over the course of muscle development (i.e., late embryonic through postnatal) in mouse diaphragm and extensor digitorum longus (EDL) skeletal muscle. We have shown that the frequency of occurrence of spontaneous Ca2+ sparks declines dramatically in isolated mouse skeletal muscle fibers after only 1 wk postbirth. The decreasing frequency is not associated with a decline in the relative expression of RyR3 compared with RyR1; in fact, RyR3 shows little decline through postnatal day 14. Thus differential expression of RyR isoforms does not appear to play a central role in Ca2+ spark production in developing mammalian skeletal muscle or in the decline of spark occurrence during early postnatal development.

We also explored possible mechanisms by which RyRs are activated to produce Ca2+ sparks in embryonic skeletal muscle. We have shown that the frequency of occurrence of Ca2+ sparks in embryonic diaphragm is influenced by the [Ca2+]o and specifically by Ca2+ influx via L-type Ca2+ channels. A possible interpretation of our findings is that Ca2+ sparks in embryonic muscle are triggered by local elevation of [Ca2+] due to Ca2+ influx via L-type Ca2+ channels. A decrease in Ca2+ influx via L-type channels during early postnatal development, possibly due to formation of molecular coupling between the DHPRs and RyR1 (30) or to some other developmental change resulting in decreased opening of DHPRs in resting fibers, might account for the observed decrease in spontaneous event occurrence after birth.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Muscle Fiber Preparation

Embryonic and postnatal CD-1 mice (Charles River) at gestation day 18 (E18) and postnatal days 1, 7, and 14 (P1, P7, and P14) were killed by CO2 asphyxiation followed by rapid decapitation. Diaphragm and EDL muscles were dissected and enzymatically dissociated with 2 mg/ml collagenase (type 1; Sigma) dissolved in medium (MEM with Earle's salts; GIBCO) containing 10% fetal bovine serum (Biofluids) and 100 µM gentamicin (Sigma) at 37°C for 2.5–3.5 h. Fiber bundles were then transferred to serum-supplemented medium without collagenase, teased apart, and separated by gentle trituration. Intact single fibers were plated on extracellular matrix (Sigma)-coated coverslips attached across a 15-mm hole in the bottom of 35-mm petri dishes (MatTek) in serum-supplemented medium. The isolated diaphragm and EDL fibers were incubated overnight in a 5% CO2 incubator at 37° in serum-supplemented medium to allow retention of only viable fibers.

Ca2+ Imaging

Isolated developing diaphragm and EDL fibers were loaded by exposure to 10 µM fluo 4-AM (Ca2+ indicator dye; Molecular Probes) for 30 min and prepared in Ringer solution (in mM: 135 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES-Na2). Fibers were then bathed in Ringer containing no dye (30 min) to allow dye deesterification. Visual screening was used to prescreen fibers for apparent stress. Only fibers possessing a regular striation pattern and no visible signs of stress (e.g., elevated cytosolic Ca2+, membrane blebs/sprouting) were imaged for this investigation. Local Ca2+ events in diaphragm and EDL fibers were monitored on an inverted microscope (Olympus IX-70 with a x60, 1.3 NA water-immersion objective) coupled to a Bio-Rad MRC-600 laser scanning confocal imaging system, focused near the lower surface of the fiber slightly above the coverslip. An argon ion laser was used to excite fluorescent molecules at 488 nm. Fibers were then imaged in either full-frame (XY) or line-scan (Xt) mode. In XY mode, each 138.2 x 92.2-µm image was acquired in 1 s (2 ms per line). The fiber was oriented roughly parallel to the X scan direction. A series of 30 full-frame images was collected at each of four randomly selected locations on each fiber. In line-scan mode, a 138.2-µm line was placed longitudinally along the fiber and repeatedly scanned at the same location 512 times at 2 ms per line, generating an image of distance (X) vs. time (t). Five sequential line-scan images were captured at each spatial location. When possible, line-scan images were acquired at locations that demonstrated Ca2+ activity during full-frame imaging. In P7 EDL, P14 EDL, and P14 diaphragm fibers, line-scan images were acquired without regard to active sites; rather, the entire diameter of the fiber was scanned at increments of 10 pixels across the fiber.

Effect of Ca2+ Entry on Spark Frequency

Isolated embryonic diaphragm fibers were loaded and imaged as described for the developmental studies, with the following exceptions: each E18 diaphragm fiber was imaged at three separate locations, with 30 or 50 sequential full-frame images (X vs. Y) (768 x 512 pixels; 1 pixel = 0.18 µm) obtained at each of three locations on the fiber while bathed in control Ringer solution. The external solution bathing the fiber was then replaced, and the fiber was imaged again after 10 min in 1.8 mM Ca2+ Ringer (control with physiological concentration of Ca2+), 8 mM Ca2+ Ringer, or 0 Ca2+ Ringer containing 1 mM EGTA (Sigma) at the same fiber locations as imaged in Ringer before the external solution was changed. In other experiments E18 diaphragm fibers were first imaged at three locations in Ringer solution and then imaged again at the same locations of the fiber as imaged in Ringer after 30 min in normal Ringer or Ringer solution containing 5 mM CoCl2, 30 µM nifedipine (Sigma), or 5 µM NiCl2 (Sigma).

Ca2+ Event Identification and Analysis

XY images. Ca2+ sparks were identified as spatially localized regions of elevated fluorescence using an automatic detection method (6) modified for identification of Ca2+ release events in XY confocal images (33). First, an average fiber fluorescence image was obtained by summing a sequence of 30 images pixel by pixel and calculating the mean fluorescence at each pixel. The region of the image corresponding to the fiber was then manually defined as an area of interest, and the standard deviation (SD) of each fiber pixel was calculated over the sequence of 30 images. Potential local Ca2+ release events were identified as contiguous pixels exhibiting fluorescence >=1.5 standard deviations above the mean fiber fluorescence. Mean fiber fluorescence (F) was then recalculated in each image pixel by pixel over the 30-image sequence excluding any potential event areas. The normalized change in fluorescence ({Delta}F/F) was calculated pixel by pixel after three-point smoothing of the F images. Regions of potential local Ca2+ events were identified in {Delta}F/F images as contiguous regions of pixels having fluorescence values >=2 SD above the mean fluorescence and were selected by the criterion that at least 1 pixel must exceed 3 SD above the mean. Ca2+ release events were characterized in the {Delta}F/F image by the measured parameters peak amplitude (peak {Delta}F/F) and full area at half-maximal fluorescence (FAHM; µm2) and by the derived parameters equivalent diameter (EDHM) and equivalent volume integral at half-maximal fluorescence (VIHM). The equivalent diameter of Ca2+ events was calculated from a circle created by fitting pixels from the area at 50% of the peak amplitude as EDHM = 2(FAHM/{pi})1/2 (µm). Similarly, the equivalent volume integral was derived from the area at 50% of the peak amplitude, with the assumption that the events have a spherical geometry, as a product of the derived sphere and the mean {Delta}F/F at half-maximal fluorescence (mean {Delta}F/F): VIHM = ({pi}/6)(EDHM)3(mean {Delta}F/F).

Line-scan images. Ca2+ sparks from line-scan images were also identified as spatially localized regions of elevated {Delta}F/F fluorescence by using an automatic spark detection algorithm (6) with slight modifications as previously described (32, 33). Ca2+ sparks were characterized by calculating peak amplitude ({Delta}F/F), spatial width (fit to the Gaussian curve) at 50% of the peak amplitude (FWHM, µm), duration at 50% of the peak amplitude (FDHM, ms), and the rise time (RT) between 10–90% of the peak amplitude (ms). Only events adhering to the following criteria were selected: amplitude {Delta}F/F values >0.2, FWHM <=9 µm, and FDHM <=30 ms. Long-duration events without a poorly defined rising or falling phase of the transient were categorized by examination of the time course records. The time courses were measured as the mean {Delta}F/F of five adjacent spatial pixels centered at the peak of the event. Image processing and data analysis for both XY and line-scan images were performed with custom image analysis routines written in IDL 5.0 (Boulder, CO).

Statistical Analysis

Statistical significance among variables was determined using the Kruskal-Wallis nonparametric test followed by the Dunn's multiple comparison test (P < 0.05).

Assay of SR Store Content

E18 diaphragm fibers were loaded with fura 2-AM for 20 min and then allowed to equilibrate in normal Ringer containing 20 µM N-benzyl-p-toluene sulfonamide (BTS; Sigma S949760) for 20 min. All solutions thereafter contained 20 µm BTS to inhibit contraction (7). Fiber fluorescence emission ratios R = F380/F358 for excitation at 380 or 358 nm were monitored to assay relative cytoplasmic [Ca2+]. Fibers were briefly exposed to 20 mM caffeine in Ringer solution containing 1.8 mM Ca2+ (control) and then washed with caffeine-free Ringer after the peak of the Ca2+ transient. When the fiber had recovered completely from the caffeine-induced Ca2+ transient, the fibers were then incubated in 0 Ca2+ or 8 mM Ca2+ Ringer for 10 min and then reexposed to 20 mM caffeine in 1.8 mM Ca2+ Ringer. The peak amplitudes of the caffeine-induced {Delta}R transients were measured and normalized to the preceding control caffeine response in the same fiber, and the mean of the normalized responses was calculated.

Crude Membrane Preparation and Western Blot Analysis

Adult frog limb muscles and mouse diaphragm muscles from E18, P1, P7, P14, and adult (>60 days) mice were rapidly dissected and flash frozen in 2-methylbutane (cooled on dry ice) and stored at –80°C. In each of three litters of embryos, the tissue from the first two animals was used to prevent degradative proteolysis of the tissue after asphyxiation. Mouse diaphragm samples at each time point were pooled because of the small amount of protein in each sample in E18 and P1 time points. As a control we used frog hindlimb muscle, which is known to express RyR{alpha} and RyR{beta} (RyR1/RyR3 homologues) in equal amounts. Frozen tissue (60–80 mg) was ground in a mortar containing liquid nitrogen and then homogenized in 500 µl of ice-cold homogenization buffer [20 mM HEPES, pH 7.4, 250 mM sucrose, 0.2% sodium azide, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 10 µM pepstatin, 10 µM leupeptin, 10 µM aprotinin, 10 µM E-64, and 10 µM antipain] with a Dounce homogenizer. Crude membrane proteins were extracted by adding 500 µl of extraction buffer (5 mM NaPO3, pH 7.4, 75 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 0.2 mM PMSF, 10 µM pepstatin, 10 µM leupeptin, 10 µM aprotinin, 10 µM E-64, and 10 µM antipain) and then centrifuging at 10,000 g for 15 min (18, 35). The supernatant, which contained the membrane proteins, was stored at –80°C. Protein concentrations were determined using the Bio-Rad DC protein assay. Membrane proteins were separated on 4% SDS-PAGE gels as described by Laemmli (20). Proteins were then transferred to a polyvinylidene difluoride membrane (Novex) in transfer buffer containing 12 mM Tris-base, 92 mM glycine, 0.02% SDS, and 20% methanol at 25 V at 4°C overnight. The membrane was blocked for 3 h in blocking solution [5% Carnation nonfat dry milk in 150 mM NaCl, 10 mM Tris · HCl, pH 7.4, 0.1% Tween 20 (TBST)] at room temperature and then incubated in monoclonal anti-RyR antibody (Affinity Bioreagents) diluted 1:5,000 in blocking solution for 1 h at room temperature. Antigen detection was performed using a peroxidase-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories) diluted 1:10,000 in blocking solution for 1 h at room temperature, followed by a chemiluminescent substrate-detection system (Pierce SuperSignal West Pico chemiluminescent substrate no. 34080).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Release Events in XY Images

Figure 1A presents 30 sequential {Delta}F/F images of a randomly selected region of an E18 diaphragm fiber loaded with fluo 4. These images exemplify the low Ca2+ event occurrence typically observed in embryonic skeletal muscle fibers. Surface plots (Fig. 1B) of the fiber displayed in Fig. 1A show accepted events (dark arrows) selected by the objective technique utilized in identification and selection of Ca2+ release events. Light arrows point to areas of increased fluorescence that do not adhere to the selection criteria and were not selected as events.



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Fig. 1. Embryonic day 18 (E18) diaphragm fibers have a low frequency of Ca2+ event occurrence. A: 30 sequential full-frame {Delta}F/F (normalized change in fluorescence) images of E18 diaphragm fibers loaded with fluo 4-AM display Ca2+ release events. Selected events are denoted by white arrows, whereas events not meeting predetermined criterion values are denoted by shaded arrows (see MATERIALS AND METHODS). Pixels having negative values of {Delta}F/F are set to zero. B: surface plots of images displaying accepted events (images 6, 10, 19, and 27). Black arrows point to accepted event peaks, whereas the shaded arrow indicates an elevation of Ca2+ that was not defined as an event with the selection criterion (see MATERIALS AND METHODS). Surface plot images were processed with a 7-point smoothing filter for presentation purposes.

 

Our first aim was to objectively quantify the frequency of occurrence of spontaneous Ca2+ events in muscles at various stages of embryonic and postnatal development. Embryonic and postnatal diaphragm and postnatal EDL fibers were loaded with fluo 4, and 30 sequential images were taken on three to four randomly selected regions on each fiber. Ca2+ event frequency was calculated as the mean number of events per image and normalized to the fiber area, which increases during development as a result of splitting of myofibrils and addition of new myofibrils (15). The fiber area of interest increased by 28.6% from E18 to P14 in diaphragm fibers and by 12.4% from P1 to P14 in EDL fibers (data not shown). The mean frequencies were measured in E18, P1, P7, and P14 diaphragm fibers and in P1, P7, and P14 EDL fibers. E18 EDL fibers were not observed because of the difficulty in isolating healthy fibers in which a regular sarcomere pattern was present. Both diaphragm and EDL fibers displayed low levels of spontaneous Ca2+ event activity even from the early stages of development observed. On average, only one event was detected for every five XY images monitored in E18 diaphragm fibers (e.g., Fig. 1A), and the event frequency decreased to much lower levels in postnatal diaphragm and EDL fibers. Normalized to the fiber size, the frequency of events in mouse diaphragm fibers declined 30.2% from E18 to P1, 71.0% from E18 to P7, and 95.4% from E18 to P14 (Fig. 2A). The decline in event frequency during postnatal development was even more dramatic in EDL fibers, which showed a 95.1% decline from P1 to P7 (Fig. 2B). At P14 the event frequency remained at a low level significantly greater than zero.



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Fig. 2. Frequency of spontaneous Ca2+ release events decreases as a function of postnatal development in both diaphragm and extensor digitorum longus (EDL) muscle fibers. Frequencies of Ca2+ events in developing mouse diaphragm (A) and EDL fibers (B) were calculated as the mean number of events per XY image and normalized to fiber area (±SE). The number of fibers used to determine the frequencies in E18, postnatal day 1 (P1), P7, and P14 diaphragm were 63, 9, 8, and 13, respectively, and 13, 13, and 10 for P1, P7, and P14 EDL fibers, respectively. Statistical significance differences were determined using the Kruskal-Wallis test followed by Dunn's method (P <= 0.05). a,b,cP < 0.05, groups with the same letter exhibit significant difference from each other.

 

The mean values of spatial and mass parameters for the population of identified sparks in both diaphragm and EDL fibers exhibited generally decreasing trends as the fibers become more developed. In diaphragm fibers, the peak amplitude ({Delta}F/F) decreased signifi-cantly from 1.24 ± 0.02 for E18 diaphragm fibers to 0.65 ± 0.05 for P14 fibers (P < 0.05) (Fig. 3A). In EDL fibers, peak amplitude did not change significantly during postnatal development (Fig. 3B). However, there was a significant decrease in the equivalent diameter, which dropped from 1.56 ± 0.04 to 1.10 ± 0.05 µm (Fig. 3D). In contrast, the decrease in equivalent diameter with postnatal age for the events in diaphragm fibers was not significant (Fig. 3C). The VIHM decreased significantly for both diaphragm and EDL fibers, from 3.79 ± 0.22 to 0.80 ± 0.25 µm3 · {Delta}F/F in E18 to P14 diaphragm fibers (Fig. 3E) and from 2.17 ± 0.20 to 0.66 ± 0.12 µm3 · {Delta}F/F in P1 to P14 EDL fibers (Fig. 3F). Taken together, these results suggest either that in the more developed fibers less Ca2+ was released during a spontaneous release event or that increases in endogenous Ca2+ buffering or some other changes during development cause the spark parameters to decrease.



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Fig. 3. Properties of Ca2+ events identified in XY images of developing diaphragm and EDL fibers. Mean values (±SE) of peak amplitude ({Delta}F/F), equivalent diameter at half-maximal fluorescence (EDHM; µm), and equivalent volume integral at half-maximal fluorescence (VIHM; µm3 · {Delta}F/F) for E18, P1, P7, and P14 diaphragm fibers (A, C, and E) and P1, P7, and P14 EDL fibers (B, D, and F). Statistical significance was determined using the Kruskal-Wallis test followed by Dunn's method (P <= 0.05). a,b,cP < 0.05, groups with the same letter exhibit significant difference from each other. For diaphragm fibers from E18, P1, P7, and P14 mice, n = 983, 114, 56, and 14, respectively, and for P1, P7, and P14 EDL fibers, n = 319, 55, and 56, respectively.

 

To evaluate the effect of the Ca2+ event selection criterion on the developmental change in spark frequency and properties, images were reanalyzed using a less stringent criterion of at least 1 pixel having F >=2.5 SD above the mean. The resulting frequency of selected events increased in relative proportion at all stages of development. The same events identified using the >=3 SD criterion were also identified when the >=2.5 SD criterion was used. Moreover, use of the >=2.5 SD criterion identified many additional events that displayed smaller areas and lower amplitudes that were more difficult to discern from background noise (data not shown). However, a similar relative declining trend in event frequency with development as shown in Fig. 2 was still seen.

Lack of Correlation Between Ca2+ Event Occurrence and RyR3 Expression

Expression of RyR3 has been detected in embryonic mammalian skeletal muscle but occurs at diminished levels in adult mammalian diaphragm and is nonexistent in adult mammalian fast twitch skeletal muscle (16). RyR1 ({alpha}) and RyR3 ({beta}) are known to exhibit different mobilities on SDS-PAGE (23), so to evaluate a possible relationship between the occurrence of Ca2+ events and the expression of RyR3, we performed Western blot analysis on pooled mouse diaphragm samples at E18, P1, P7, P14, and adult (>60 days). As a control we used frog hindlimb muscle, which is known to express RyR{alpha} and RyR{beta} in approximately equal amounts (23) (Fig. 4, A and B, lane 6). Densitometry was used to quantify expression of RyR1 ({alpha}) and RyR3 ({beta}). Because the amounts of total RyR protein may vary compared with the total membrane proteins isolated at each stage of development, we chose to take the ratio of RyR3 to RyR1, which also helped to eliminate discrepancies in gel loading. In agreement with previous reports (16), our results show that RyR3/RyR1 ratios exhibit only a very slight decrease from E18 to P14 (Fig. 4, A and B, lanes 1–4) but a marked reduction in the adult (>60 days) diaphragm (lane 5). Compared with the frequency of Ca2+ event occurrence, which declines very rapidly during postnatal development and is already at very low levels by P14, the RyR3/RyR1 ratio remains relatively high in P14 diaphragm fibers, suggesting that the occurrence of Ca2+ sparks does not correlate to the expression of RyR3.



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Fig. 4. Ryanodine receptor (RyR)3/RyR1 expression ratios demonstrate no correlation to frequency of Ca2+ spark occurrence in mouse diaphragm fibers. A: Western blot displaying the relative amounts of RyR1 or RyR{alpha} (top) and RyR3 or RyR{beta} (bottom) in mouse diaphragm and frog, respectively. Lanes 1–5 were loaded with 10 µg of E18, P1, P7, P14, and adult diaphragm protein, respectively, whereas lane 6 was loaded with 4 µg of frog hindlimb protein. B: graph of the densitometric ratio of the relative amounts of RyR3 vs. RyR1 protein.

 

Ca2+ Release Events in Line-Scan Images

In the series of 30 images in Fig. 1A, three of the four Ca2+ events are shown to have occurred at the same location in this fiber. Within the entire population of fibers examined, the repetitive nature of Ca2+ spark occurrence was examined by evaluating events that recurred within 3 µm of each other in two or more different images in the same image series. With this analysis it was determined that ~29% of all event locations had reoccurring Ca2+ release events. Thus events often occur repetitively at the same locations.

The repetitive occurrence of Ca2+ release events at particular spatial locations was used advantageously in placement of the line location during detection in the line-scan mode of the confocal microscope. Spontaneous Ca2+ release events were identified in line-scan images, and their spatial and temporal properties were characterized using a computer algorithm. Figure 5 presents {Delta}F/F image strips and corresponding fluorescence time courses of Ca2+ sparks recorded in line-scan mode. For those events satisfying our predefined spatial and temporal selection criteria (see MATERIALS AND METHODS), the mean property values were similar for both E18 diaphragm and P1 EDL fibers (Table 1). Because of the low frequency of Ca2+ event occurrence at later developmental stages, many line-scan images were monitored in an attempt to capture enough events for characterization. However, the numbers of events characterized in line-scan mode were limited because of the rare occurrence of events in postnatal skeletal muscle fibers and the challenge of finding and placing a line through an event before it occurs. The numbers of events acquired were too few for comparisons of event properties as a function of developmental stage.



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Fig. 5. {Delta}F/F image strips and fluorescent time courses of representative local Ca2+ release events. A: singly occurring and repetitively occurring Ca2+ sparks events imaged in line-scan mode. B: long-duration (>30 ms) Ca2+ release events were seen infrequently and represented <5% of the total population of events imaged. The first image depicts an apparent fusion of events occurring over time. The last panel depicts a very long-duration Ca2+ release event that extends past this image boundary but was absent in a subsequent image ~1 s later. This event is reminiscent of long-duration Ca2+ sparks seen previously (19, 32). In A and B image strips, pixels having negative values of {Delta}F/F were set to zero for display purposes.

 

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Table 1. Mean spatial properties of Ca2+ events identified in line-scan images of developing diaphragm and EDL fibers

 

In E18 diaphragm fibers as well as P1, P7, and P14 EDL fibers, some events were detected in line-scan images that could not be characterized with our predefined spatial and temporal parameters. Time courses of Ca2+ events that possessed a FDHM >30 ms (4.3% of all events fulfilling the other selection criteria; Fig. 5B) were compared with accepted Ca2+ spark time courses that exhibited property values within the accepted range (Fig. 5A). Temporal traces of longer duration events displayed a variety of fluorescence time courses (Fig. 5B). Some time courses displayed a repetitive firing pattern, whereas others showed a sustained fluorescence, reminiscent of long-duration Ca2+ sparks seen previously (19, 32), often preceded by a graded fluorescence increase and/or followed by a graded decrease in fluorescence.

Extracellular Ca2+ Modulates Ca2+ Event Frequency

Ca2+ entry via L-type Ca2+ channels is reported to be important for EC coupling in embryonic muscle (11, 25). We therefore investigated the possible role of Ca2+ influx in activation of Ca2+ sparks in E18 diaphragm fibers. We first examined the effects of [Ca2+]o on the occurrence of Ca2+ sparks. For these studies, each enzymatically isolated E18 diaphragm fiber was first imaged at three locations in XY mode while bathed in 1.8 mM Ca2+ Ringer solution (control). The same fiber was then imaged again at the same locations after 10 min of additional exposure to control Ringer (1.8 mM Ca2+), 0 Ca2+ Ringer containing 1 mM EGTA, or 8 mM Ca2+ Ringer, and the Ca2+ event frequency was determined while the fiber was bathed in the test solution (0, 1.8, or 8 mM Ca2+ Ringer). The mean event frequency of all fibers in a given solution was normalized to the mean event frequency of the same fibers while bathed in the initial control Ringer solution. The resulting mean relative Ca2+ spark rates of fibers bathed in 0, 1.8, or 8 mM Ca2+ Ringer during the test period were, respectively, 0.61 ± 0.09, 1.11 ± 0.15, and 1.90 ± 0.21 of the rate of events from the same fibers in the initial 1.8 mM Ca2+ Ringer (control) (Fig. 6A). The increase of the mean Ca2+ spark rate in fibers bathed in high concentrations of external Ca2+ and the decrease of event frequency in fibers bathed in 0 Ca2+ clearly demonstrate that Ca2+ spark production is strongly influenced by extracellular Ca2+, possibly due to changes in Ca2+ influx. However, it has been previously shown that Ca2+-free solution causes slight depolarization of neonatal fibers (11), which could also increase spark frequency. Our experiments argue against this possibility because application of a nonspecific Ca2+ channel blocker, CoCl2 (5 mM), also suppressed spark frequency (Fig. 7), an effect independent of fiber depolarization (11). Furthermore, although surface charge effects might partially contribute to the observed effect of 0 mM Ca2+ Ringer solution, changes in surface charge would be opposite with cobalt addition.



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Fig. 6. Extracellular Ca2+ influences event frequency. Dissociated E18 diaphragm fibers were imaged (30 or 50 consecutive XY images) while initially bathed in 1.8 mM Ca2+ Ringer and then imaged again after 10 min in 0 mM Ca2+ Ringer containing 1 mM EGTA, 1.8 mM Ca2+ Ringer (control), or 8 mM Ca2+ Ringer as indicated. A: mean spark rate in the test condition (0, 1.8, or 8 mM Ca2+) was normalized to the mean frequency value in the initial Ringer solution (±SE; n = 27, 20, and 21 fiber sections imaged in 0, 1.8, and 8 mM Ca2+ Ringer, respectively). B: mean fiber fluorescence for each test condition was calculated as the mean ratio F/Fcontrol as a relative measure of the cytosolic Ca2+ (±SE; n = 9, 7, and 8). *P < 0.05, significant difference from control.

 


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Fig. 7. Ca2+ channel blockers decrease event frequency. Isolated embryonic diaphragm fibers were imaged in XY mode while bathed in 1.8 mM Ca2+ Ringer and then imaged again 30 min after the bath solution was changed to normal Ringer or Ringer containing 5 mM CoCl2, 30 µM nifedipine, or 5 µM NiCl2 as indicated. A: Ca2+ spark frequency was calculated as described for Fig. 6A (± SE; n = 36, 22, 38, and 8 fibers imaged in Ringer, CoCl2, nifedipine, and NiCl2, respectively). B: mean fiber fluorescence was calculated as described in Fig. 6B. *P < 0.05, significant difference from control.

 

Possible alterations in SR store Ca2+ content were assayed in a separate group of fibers by using fura 2 (see MATERIALS AND METHODS). Figure 8A presents fura 2 ratio signals from a fiber in response to application of 20 mM caffeine in control (1.8 mM Ca2+) Ringer solution and from the same fiber after 10 min of to exposure to 0 mM Ca2+ Ringer. The similarity of the Ca2+ transients indicates minimal alteration in store content after 10 min in 0 Ca2+ solution. Average peak amplitudes of test caffeine responses after 0 mM (n = 2) or 8 mM (n = 3) Ca2+ Ringer, each normalized to control response in the same fiber, were similar (Fig. 8B). This finding indicates minimal external Ca2+-dependent change in store content. Thus the observed effect of Ca2+ on Ca2+ spark frequency is not likely to be due to changes in store content.



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Fig. 8. Assay of sarcoplasmic reticulum (SR) store Ca2+ content. A: a representative fura 2 fluorescence ratio record (380/358) is shown. After a 40-min incubation in 1.8 mM Ca2+ Ringer, the fiber was challenged with a brief caffeine application (20 mM; ~10 s) in 1.8 mM Ca2+ Ringer and allowed to fully recover. The fiber was then exposed to a test solution of 0 mM Ca2+ Ringer for 10 min, after which a second caffeine challenge (20 mM; ~10 s) was applied in 1.8 mM Ca2+ Ringer. B: store content was evaluated by determining the peak of the fura 2 ratio during a caffeine challenge applied after exposure to a test solution (0 or 8 mM Ca2+ Ringer; 10 min). The response of each fiber was normalized to the peak of the control response (1.8 mM Ca2+ Ringer) in the same fiber. No significant differences between the test conditions (0 and 8 mM Ca2+ Ringer; 0.53 ± 0.10, n = 2, and 0.54 ± 0.17, n = 3, respectively) were found.

 

An influx of extracellular Ca2+ could conceivably increase the frequency of Ca2+ sparks by increasing the cytosolic [Ca2+] in the vicinity of the RyRs, thereby increasing the probability that a RyR Ca2+ release channel would open by a CICR mechanism. Ca2+ influx could initiate the opening of RyR Ca2+ release channels via CICR either by raising the average level of cytosolic Ca2+ or by producing local increases of Ca2+ in the vicinity of the RyRs. To determine whether average cytosolic [Ca2+] was altered when Ca2+ spark frequency was found to vary with [Ca2+]o, relative levels of cytosolic Ca2+ were monitored using the fiber fluorescence within the same fiber images, as used for spark detection (above). Fiber fluorescence was measured in the fluorescence (F) images after exclusion of areas encompassing potential events ({Delta}F/F >= 1.5 SD). The mean fluorescence of each region of each fiber bathed in 0, 1.8, or 8 mM Ca2+ Ringer was normalized to the mean fiber fluorescence of the same region of the same fiber while bathed in the initial normal Ringer. The relative fiber fluorescence exhibited no significant change with regard to [Ca2+]o (Fig. 6B), suggesting that the mean cytosolic [Ca2+] does not play a signifi-cant role in stimulating Ca2+ spark production. However, even though there was no significant change in global cytosolic [Ca2+], it is still possible that [Ca2+] in the microdomain of the triad junction varied with the applied [Ca2+]o without any observed changes in average fiber fluorescence.

Ca2+ Influx from L-type Ca2+ Channels

To determine whether Ca2+ influx via plasmalemma or T-tubule Ca2+ channels was modulating the observed event frequency, we measured the spark rate after blocking specific Ca2+ channels known to contribute to the inward Ca2+ current. The L-type Ca2+ current (Islow) is the primary Ca2+ current measured in adult mammalian skeletal muscle and increases substantially during postnatal development. Thus E18 diaphragm fibers were imaged while in normal Ringer and again after a 30-min exposure to Ringer solution containing 30 µM nifedipine, which is a dihydropyridine that selectively inhibits L-type Ca2+ channels found in the plasmalemma and T-tubules. As a control, embryonic fibers were imaged in Ringer and then imaged again after being bathed in Ringer solution without nifedipine for 30 min. The mean frequency of events in fibers treated with nifedipine dropped to 17.6% compared with the Ringer control after both were normalized to the mean event frequency of those same fibers bathed in Ringer before treatment (Fig. 7A). The significant pronounced decline in Ca2+ spark rate in nifedipine demonstrates that inhibition of the DHPRs drastically reduces the frequency of Ca2+ sparks and indicates that Ca2+ influx via L-type Ca2+ channels can modulate the Ca2+ spark rate in embryonic muscle fibers.

Additionally, embryonic and neonatal mouse skeletal muscles also exhibit a T-type (24) Ca2+ current (Ifast), which disappears by the third week after birth (2). Islow is present in skeletal muscle cells of normal animals but absent from skeletal muscle cells of mdg animals, whereas Ifast is present in developing skeletal muscle cells of both normal and mutant animals, indicating that different channel species give rise to these two currents (3). To determine whether Ca2+ influx via T-type Ca2+ channels has an effect on the Ca2+ spark frequency, we imaged E18 diaphragm fibers in control Ringer solution and then again after a 30-min exposure to 5 µM NiCl2 to block T-type Ca2+ channels (4). Inhibition of T-type Ca2+ channels without affecting L-type Ca2+ channels requires a very low concentration of NiCl2. Berthier et al. (4) reported NiCl2 inhibition of the T-type Ca2+ current with an IC50 of 5.4 µM in embryonic mouse skeletal muscle. Thus in our studies we used 5 µM NiCl2, which should block about half of the T-type Ca2+ channels. Application of 5 µM NiCl2 had no significant affect on the Ca2+ spark frequency (Fig. 7A), suggesting that Ca2+ entering via T-type channels may not play a role or may only play a minor role in contributing to the Ca2+ spark frequency. There were no significant differences in the resting fiber fluorescence (Fig. 7B).

Characterization of Events Influenced by Ca2+ Influx

We also compared the spatial properties of events imaged in XY mode of fibers bathed in Ringer containing varying concentrations of Ca2+ or in fibers treated with Ca2+ channel blockers. The mean properties of peak amplitude ({Delta}F/F), EDHM (µm), and VIHM (µm3 · {Delta}F/F) did not change significantly when [Ca2+]o was altered (Table 2). With Ca2+ channel blockers, significant differences were observed between a limited number of parameter values, but systematic patterns were not observed (Table 2). Histograms, normalized to the total number of events, of property characteristics in each condition showed similar distributions (data not shown).


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Table 2. Mean spherical and mass properties of Ca2+ sparks in XY images

 

Spontaneous Electrical Activity

We also examined whether the Ca2+ sparks detected in the embryonic diaphragm preparation were produced as a result of spontaneous Na+-dependent action potentials. Treatment with tetrodotoxin (TTX), which blocks Na+ channels, was used to prevent Na+-dependent fiber action potentials. To overcome TTX insensitivity observed in embryonic and neonatal mammalian skeletal muscle (17), we exposed embryonic fibers to high concentrations of TTX (10 µM) (12). Three E18 diaphragm fibers were imaged while in Ringer and were then imaged again immediately after the fiber was exposed to Ringer containing TTX. The mean spark frequency did not change significantly (data not shown), demonstrating that Na+-dependent action potentials are not the cause of Ca2+ sparks in embryonic mammalian skeletal muscle. We also saw no evidence for spontaneous Ca2+ waves in the fibers used for these experiments (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this article we present three major findings, two new and one confirmatory. We begin by providing the first objective documentation of the frequency of spontaneous occurrence of Ca2+ sparks in intact murine muscle fibers at various stages of development. We have found that the occurrence of Ca2+ sparks decreases dramatically from late embryonic (E18) through early postnatal (P14) development in mouse diaphragm and from P1 to P14 in mouse EDL muscles. This result might have been anticipated from previous reports of the difficulty in observing Ca2+ sparks in intact adult rodent skeletal muscle fibers (10, 29) compared with the apparent relative ease of detecting such events in embryonic muscle (10). However, our present results now provide an objective quantitative assessment of this decline in occurrence of Ca2+ sparks during early postnatal development of murine skeletal muscle.

We next demonstrated that in mouse diaphragm, the ratio of the protein expression levels of the two muscle RyR isoforms, RyR3 to RyR1, does not change appreciably over the developmental period from E18 to P14. This observation confirms the earlier findings of Flucher et al. (16) regarding RyR isoform expression changes during mouse muscle development and establishes that in the diaphragm, the decline in Ca2+ spark frequency from E18 to P14 cannot be attributed to a relative decline in RyR3 protein expression compared with RyR1.

In a third and unanticipated new observation, we have now found that the frequency of occurrence of Ca2+ sparks in embryonic muscle fibers is markedly influenced by Ca2+ influx via L-type Ca2+ channels in the plasmalemma or transverse tubules. Raising or lowering [Ca2+]o causes the frequency of spontaneous Ca2+ sparks in E18 diaphragm to respectively increase or decrease, and inhibition of plasmalemmal or T-tubule Ca2+ channels by either the nonspecific Ca2+ channel blocker CoCl2 (5 mM) or the specific L-type Ca2+ channel blocker nifedipine (30 µM) causes a marked reduction in spark frequency. Thus Ca influx via DHPR (L-type) Ca2+ channels appears to be involved in triggering the opening of the RyR Ca2+ release channel(s) that underlies the spark. The likely mechanism for such an effect in embryonic muscle would be a local elevation of cytosolic [Ca2+] in the immediate vicinity of an open L-type channel and the resulting activation of a RyR Ca2+ release channel by CICR (Fig. 9A). The effect of Ca2+ influx via L-type channels appears to be quite localized, because there were no detectable changes in global cytosolic fluorescence under conditions that caused clear variations in Ca2+ spark frequency. Changes in SR Ca2+ content were not involved in mediating the observed effects of [Ca2+]o on spark frequency.



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Fig. 9. Proposed mechanism of Ca2+ spark modulation in mouse skeletal muscle. A: in immature skeletal muscle fibers (e.g., E18 diaphragm), dihydropyridine receptors (DHPRs) are not coupled to RyRs. Ca2+ influx through DHPR Ca2+ channels could locally activate Ca2+ release from nearby RyRs via Ca2+-induced Ca2+ release (CICR) in a cardiac-like ECC manner. B: in mature muscle fibers, the DHPRs are coupled to alternating RyR1s. Ca2+ influx via coupled DHPRs may be less likely to activate SR Ca2+ release either because coupling decreases the probability of spontaneous opening of the DHPR Ca2+ channel or because coupling decreases the sensitivity of RyR1s to activation by Ca2+ and coupled DHPRs are now positioned too far from uncoupled RyRs to activate Ca2+ release. C: alternatively, in maturing fibers, DHPRs may not yet be coupled to RyR. In this case, reduced DHPR activity could perhaps result from increased T-tubule membrane polarization during development (34).

 

Development of DHPR-RyR1 Coupling and Cessation of Spontaneous Ca2+ Sparks

Together, our three major findings now suggest the hypothesis that the decline in spark frequency in early postnatal development might be due to either a decline in Ca2+ influx in resting fibers via DHPR L-type channels from E18 to P14 or to a decline in the ability of such Ca2+ influx to initiate opening of RyRs from E18 to P14. Interestingly, the previously studied development of functional and/or structural couplings between the DHPR and RyR1 (26, 27) could provide a molecular mechanism for a developmental decrease in activation of Ca2+ sparks by Ca2+ entry via DHPR L-type channels. Consistent with this hypothesis, it has previously been suggested that sparks in RyR3 knockout myotubes were produced by islands of RyR1 channels, which were not coupled to DHPR voltage sensors (29).

There are several possible mechanisms whereby the development of DHPR-RyR1 coupling could give rise to the observed decrease in the frequency of occurrence of Ca2+ sparks. First, if coupled DHPRs generated much less Ca2+ influx in resting fibers than uncoupled DHPRs (Fig. 9B), the Ca2+ signal for RyR opening by CICR would be decreased by DHPR-RyR1 coupling and fewer sparks would occur.

Alternatively, coupled DHPRs could still carry the same current and exhibit the same opening probabilities and open times in resting fibers as uncoupled DHPRs, but the likelihood that a RyR Ca2+ release channel would be activated by Ca2+ from a nearby DHPR could still be decreased as a result of DHPRRyR1 coupling (not shown in Fig. 9). Decreased RyR activation in response to a given DHPR current after coupling of DHPRs to RyR1 could occur by either or both of two possible mechanisms. In the first mechanism, coupling to a DHPR could directly decrease the Ca2+ sensitivity for CICR activation of a coupled RyR1. In this case, a given Ca2+ current through a coupled DHPR could be unlikely to open its coupled RyR1, whereas the same current through an uncoupled DHPR would be more likely to activate a nearby but uncoupled RyR1 or RyR3. As a second possibility, RyR1s (coupled or uncoupled) could be less susceptible to activation by CICR than RyR3s (5, 9, 14, 22, 32). In this case, the structural reorganization induced by coupling, where RyR1s but not RyR3s are now coupled and thus closest to the DHPR, would restrict RyR3s from closest proximity to coupled DHPRs (13). The RyR3s would then be further from the activating trigger Ca2+ signal and, consequently, would experience lower local [Ca2+] elevation and thus be less likely to open in response to a given Ca2+ influx through coupled than through uncoupled DHPRs.

It is also possible that changes in Ca2+ influx via DHPRs could occur independently of changes in DHPR/RyR coupling (Fig. 9C). For example, Ward and Wareham (34) reported membrane hyperpolarization during postnatal development of mouse EDL fibers (from –42 mV at P4 to –54 mV at P8 and –67 mV at P16), which could give rise to less frequent opening of DHPR Ca2+ channels, resulting in decreased frequency of Ca2+ spark occurrence. These and other hypothetical possibilities require experimental investigation in future studies.

In conclusion, the frequency of spontaneous Ca2+ events is greatest in embryonic mouse skeletal muscle and declines significantly over a 2-wk period postbirth. The Ca2+ sparks in embryonic mouse diaphragm fibers can be modulated by Ca2+ influx via L-type Ca2+ channels.


    DISCLOSURES
 
This work was supported by National Institutes of Health (NIH) Grants R01-NS-23346 (to M. F. Schneider) and K01-AR-02177 (to C. W. Ward). L. Chun was supported by predoctoral fellowships from the Interdisciplinary Training Program in Muscle Biology (NIH Grant T32-AR-07592) and the Training Program in Integrative Membrane Biology (NIH Grant T32-GM-08181).


    ACKNOWLEDGMENTS
 
We acknowledge the assistance of Dr. Ian Farrance with the immunoblot methodologies and Dr. Jeanne Digel with providing computer programming in support of the image analysis.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. F. Schneider, 108 N. Greene St., Baltimore, MD 21201 (E-mail: mschneid{at}umaryland.edu).

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. Section 1734 solely to indicate this fact.


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