Measurement of Calcium Release Due to Inositol Trisphosphate Receptors in Skeletal Muscle

Abstract

Calcium transients elicited by IP₃ receptors upon electrical stimulation of skeletal muscle cells (slow calcium signals) are often hard to visualize due to their relatively small amplitude compared to the large transient originated from ryanodine receptors associated to excitation-contraction coupling. The study of slow calcium transients, however, is relevant due to their function in regulation of muscle gene expression and in the process of excitation-transcription coupling. Discussed here are the procedures used to record slow calcium signals from both cultured mouse myotubes and from cultured adult skeletal muscle fibers.

1 Introduction

The notion of inositol 1,4,5 trisphosphate (IP₃) receptors (IP₃Rs) releasing calcium upon stimulation of skeletal muscle was originated from Julio Vergara’s laboratory (1) who suggested that muscle fibers were capable of producing IP₃ in response to electrical stimulation (ES) and proposed a role for IP₃ in the process of muscle excitation-contraction coupling. Several laboratories followed that lead (reviewed in (2)) and reached the conclusion that calcium release by IP₃ was not fast enough to account for the needs of contracting sarcomeres. The evidence gathered nevertheless pointed to the fact that all the molecular machinery needed to synthesize and degrade IP₃ was present in the muscle cells and actually calcium was released by IP₃ in permeabilized fibers (3) and this release depended on membrane potential! A first hint of a new calcium signal in cultured muscle cells was published in 1994 (4) when calcium transients elicited by potassium depolarization of myotubes were shown to have two components, the slower one highly sensitive to nifedipine. The location of type 1 receptor in cultured myotubes was studied (5, 6), together with detailed description of IP₃-dependent slow calcium transients (see also (7–9)) in rat and mouse myotubes with an important component of the signal occurring at the myonuclei (10).

The IP₃-dependent slow calcium signal was associated to signaling pathways leading to transcription factor activation. MAP kinases and CREB phosphoryation as well as increase in mRNA for early genes c-fos, c-jun, and egr-1 was confirmed (6, 11, 12) and a role for IP₃ regulating the activity of transcription factors in skeletal muscle cells was proposed. This role was reinforced when studying expression of specific genes as interleukin 6 (IL6, (13)) and more than a 100-genes studied using microarrays (and confirming some of them using RT-PCR) (14). More recently, a mechanism involving Cav1.1 voltage sensor (12), ATP release from the muscle cell through pannexin 1 channels, P2Y purinergic receptors (15), G protein, PI3K, and PLC (8) has been proposed (see Note 2).

The recent work in adult muscle fibers (16) shows the presence of IP₃-dependent calcium transients in adult muscle fibers, sharing many characteristics of the signals described in cultured myotubes (see Fig. 2, Note 3).

The methods described below show how to detect slow, IP₃-dependent calcium signals in both cultured muscle cells and in cultured adult skeletal muscle fibers.

2 Materials

2.1 Cultures of Myoblasts from Neonatal Skeletal Muscle

• 1. 5–8 neonatal mice (1–3 days old).

• 2. Sterile surgical material.

• 3. Phosphate-buffered saline (PBS), 0.22 μm filtered.

• 4. Collagenase type II (Worthington Biochemical) solution at 1 mg/mL in PBS, 0.22 μm filtered.

• 5. Proliferation medium: F-10 Nutrient mixture medium (Gibco) supplemented with 5 ng/mL human FGF-basic (Preprotech), 20% bovine growth serum (BGS) or 20% fetal bovine serum (FBS, Hyclone), and 1× Penicillin-Streptomycin-Glutamine solution (Gibco).

• 6. Differentiation medium: DMEM-low glucose (Gibco) supplemented with 4% horse serum (HS, Gibco) and 1× Penicillin-Streptomycin-Glutamine solution (Gibco).

• 7. Collagen type I rat tail, high concentration (BD Bioscience) solution at 0.2 mg/mL.

• 8. Nytex filter.

• 9. Petri dishes, 100 mm.

• 10. Trypsin-EDTA solution, 1× (Hyclone).

2.2 Isolation of Adult Muscle Fibers from Mice

• 1. Phosphate-buffered saline (PBS), sterile.

• 2. Dissection chamber filled with Silgar (60 cm Petri dishes are good enough).

• 3. Steel pins.

• 4. Pasteur pipettes cut to obtain pipettes at different diameters (from 0.5 to 3 mm), fire polished. Choose pipettes made of thick glass to avoid closing them during the fire polish.

• 5. Dissection instruments: iridissection scissors, tweezers.

• 6. Mice from 3 to 20 weeks old.

• 7. Collagenase type II (Worthington Biochemical).

• 8. Matrigel (prepared in our lab from published protocols (17)).

• 9. Horse serum (HS, Gibco).

• 10. Dulbecco’s modified Eagle medium (DMEM, Gibco) with 1% penicillin/streptomycin.

• 11. Dulbecco’s modified Eagle medium with 1% penicillin/streptomycin supplemented with 10% HS.

2.3 Calcium Signal Measurement

• 1. Krebs physiological solution: 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, 10 mM HEPES-Tris pH 7.4. Krebs solution without Ca²⁺ has essentially the same composition, but contains 2 mM MgCl₂ and 0.5 mM EGTA.

• 2. Calcium indicator Fluo3-AM: 2–5 μM in Krebs solution. Fluo3-AM solution is made by dissolving 50 μg of lyophilized compound in 20 μL of 20% pluronic acid in dimethyl sulfoxide (DMSO) and then adding Krebs solution to obtain the desired Fluo3 concentration.

• 3. Electrical stimulator (e.g., Grass S48).

• 4. Confocal or epifluorescence microscope with image acquisition capability.

• 5. 25 μM nifedipine.

• 6. 5 μM Xestospongin C or 5 μM Xestospongin B.

3 Methods

3.1 Cultures of Myoblasts from Neonatal Skeletal Muscle

Primary myoblasts can be easily isolated from neonatal mice and grown in proliferation medium. Myoblasts can be differentiated into myotubes with a reduction of serum and can be used for experimental calcium determination on day 3–5 of differentiation (18).

• 1. Sacrifice the neonatal mice by rapid decapitation with surgical scissors and then submerge the body in 70% ethanol for a few seconds in order to kill the skin bacteria and fungus.

• 2. Remove the hind limbs with scissors and remove the skin with surgical tweezers. Then with small tweezers remove the muscle tissue and discard the bones. Cut the muscle into small pieces and enzymatically dissociate by adding collagenase solution (10 mL) for 15 min at 37°C. Then, repetitively aspirate the solution with a sterile syringe (without needle) in order to mechanically dissociate the tissue, and leave the suspension at 37°C for an additional 15 min to complete the tissue disintegration. Add 10 mL of proliferation medium, filter the solution with Nytex paper (in order to remove the undigested material), and centrifuge for 10 min to pellet the cells. Carefully resuspend the pellet in 10 mL of proliferation medium.

• 3. In order to remove fibroblasts, preplate the cell suspension in 100 mm Petri dishes at 37°C, 5% CO₂. After 1 h incubation, remove the cells in suspension (myoblasts) and preplate on PBS washed, collagen-treated 100 mm Petri dishes for another 30 min to remove fibroblasts. Plate the cell suspension in collagen-treated Petri dishes and incubate at 37°C, 5% CO₂. Change the proliferation medium daily until you get a population highly enriched in myoblasts.

• 4. Trypsinize the cells and repeat the preplating on plastic Petri dishes if fibroblasts remain in the cell culture (1 h, 37°C, 5% CO₂). Plate the cell suspension in order to increase the myoblast population for further experiments.

• 5. For long-term storage, myoblasts can be frozen in liquid nitrogen. Trypsinize a 100-mm dish at 70–80% of confluence, pellet the cells, and resuspend in 1 mL of proliferation media supplemented with 10% culture grade DMSO. Freeze the cells using an isopropanol chamber at -80°C and then in liquid nitrogen.

3.2 Myotube Differentiation

• 1. For calcium determinations by fluorescence microscopy, plate the cells on matrigel-coated fluorescence suitable plastic covers (in glass covers the myotubes are easily detached upon spontaneous contraction) with 30% confluence.

• 2. Change the proliferation medium daily until the culture has a confluence of 70–80%.

• 3. In order to differentiate the myoblasts, change the media to differentiation medium and incubate the cells at 37°C, 5% CO₂. Change the medium daily and you will obtain differentiated myotubes at day 2–4.

3.3 Isolation of Adult Muscle Fibers from Mice

• 1. Sacrifice the animals by cervical dislocation.

• 2. Remove skin from hind limb by cutting it from the proximal end of the limb (above the knee) until the ankle. Place the limbs without skin in PBS and fix it with steel pins to a dissection chamber filled with Silgar; add PBS to cover the piece.

  Perform the following steps under a low amplification microscope and under a laminar flow hood.

• 3. Expose the flexor digitorum brevis (FDB) muscle by delicately cutting the skin in the hind paw. Muscle dissection must be done avoiding cutting muscle fibers.

• 4. Place the muscles in a solution of DMEM with 1% penicillin/streptomycin (without serum) and with 450–500 U/mL of collagenase at 37°C for 90 min. Three to four milliliter of solution is enough for 2 FDB muscles. No shaking is needed.

• 5. Prepare plates or coverslips covered with Matrigel 30 min before seeding fibers. 10–15 μL of Matrigel is enough for a 35 mm Petri dish.

• 6. Before collagenase digestion is finished, prepare two 35 mm Petri dishes by coating them with HS or a solution of 5% BSA. Remove the excess and place 2 mL of 10% HS in DMEM. This procedure prevents fibers from sticking to the Petri dish during the dissociation step.

• 7. After collagensase digestion, place muscles in a Petri dish prepared as above and dissociate fibers by passing the muscle through the fire-polished Pasteur pipettes, successively from large to small diameters.

• 8. Seed the isolated fibers on Matrigel-coated cover slips. Place the desired quantity of fibers in a small volume of medium, wait 5–10 min for fibers to sediment, and adhere to the Matrigel and then delicately add DMEM supplemented with 10% HS. Fibers can be used immediately, but are fragile because of collagenase digestion. We usually do experiments 20–30 h after seeding. This protocol is based on a protocol described in (19).

3.4 Calcium Signal Measurement

The choice of a Ca²⁺ probe must take into account the magnitude of the signal to be recorded. For a slow calcium signal, probes with high affinity, like Fluo3 (Kd  =  390 nM) and Fluo4 (Kd  =  350 nM), should be used. They have the sensitivity needed to detect small amounts of Ca²⁺ and display up to 100-fold Ca²⁺-dependent fluorescence enhancement (dynamic range), allowing visualization of low amplitude Ca²⁺ variations. Figure 1 shows calcium signals obtained in mouse myotubes (see Notes 1 and 2 for more details). Figure 2 shows a representative calcium signal evoked by electrical stimuli and the respective kinetic analysis (see Note 3). Nevertheless, they are not adequate for ratiometric measurements or Ca²⁺ concentration quantization.

Fig. 1.

Calcium transient evoked by electrical stimuli in differentiated C57Bl/6 myotubes. Cells were preloaded with 5 μM Fluo-3AM and stimulated at 250 pulses of 0.5 ms of duration at 20 Hz frequency in Krebs buffer at RT. Image acquisition was obtained with an inverted fluorescence microscope equipped with a CCD cooled camera every 0.7 s for 3 min. Representative Fluo-3AM fluorescence images are shown at the indicated times and the quantification of the fluorescence for fast and slow calcium signals is shown at the bottom. (a) Myotube showing mainly EC-coupling-related Ca²⁺ signal (representative of 8% of cells at this stage). (b) Myotube showing fast calcium transient and a prominent slow calcium signal (representative of 68% of cells at this stage). (c) More differentiated myotube (note the larger amount of nuclei and branching) showing a fused signal with a fast and a slow component (representative of 12% of cells at this stage). Bar indicates the electrical stimuli duration.

Fig. 2.

Slow Ca²⁺ signal induced by electrical stimuli in adult skeletal muscle fibers. Isolated muscle fibers from mouse FDB, loaded with Fluo3-AM, were stimulated with a train of 270 pulses (0.3 ms each) at 20 Hz. Images were obtained by confocal microscopy. (a) Images of fiber fluorescence before, during, and at different times after application of electrical stimulus (end of stimulation indicated by time  =  0 s). (b) The graph shows the variations in relative fluorescence values with time in a fiber stimulated as in (a). We observed a first fast Ca²⁺ signal related to contraction when a tetanic train was applied (bar in the graph) followed by a slower signal, observed as a delayed return to basal fluorescence levels after tetanic stimulus. Experiments were done in the presence of standard Krebs solution containing 1 mM calcium. Experimental points could be fitted by a double exponential function (equation and continuous line in (b)) as shown for a typical record in the graph (b). (c) Single twitch can be fitted by a single exponential decay (equation and continuous line in (c)) as shown for a representative record. (d) When muscle fibers are incubated for 30 min with 20 μM Xestospongin B, a specific inhibitor of IP₃R (16), slow calcium signal is completely abolished (filled squares, mean of n  =  4). Empty squares correspond to mean of four signals obtained from control fibers.

• 1. Wash the covers with myotubes (2–4 days after change to differentiation medium) with 37°C prewarmed Krebs buffer solution. Do not carry out this step for adult muscle fibers for risk of detachment. Simply remove the excess medium from coverslips and load it with the Ca²⁺ probe.

• 2. Load the cells with a Krebs solution containing 2–5 μM of Fluo3-AM. Incubate the cells at 37°C for 25 min. In the case of adult muscle fibers, incubate 30 min at room temperature (20–22°C).

• 3. Wash the cells with Krebs solution and place the coverslips in a microscope chamber.

• 4. Apply ES with a couple of platinum electrodes connected through an isolation unit to a stimulator. Different duration and frequencies for trains of 0.3 ms square pulses can be used in adult fibers (0.5 ms for myotubes). During stimulation experiments, maintain the myotubes or fibers in Krebs solution at 21–23°C. Experiments with no extracellular calcium can be performed in the same Krebs solution without calcium and supplemented with 0.5 mM EGTA.

• 5. With the chamber of myotubes or fibers in a confocal or epifluorescence microscope, set the excitation source at 488 nm with the minor laser or lamp emission in order to avoid photobleaching. Collect the emitted light at 526 nm.

• 6. Start acquiring images. Take several images before stimulation to estimate the basal calcium and then stimulate the cells with the electrical field. Acquire for 2 min after ES in case of adult muscle fibers and for 5 min for myotubes. Collect the fluorescence images every 1.0–2.0 s and analyze frame by frame. This acquisition speed is short enough to resolve the slow signal. For rapid image acquisitions, needed to resolve a fast Ca²⁺ signal (related to EC coupling) or a slow signal after a short tetanus, data must be collected by line scan of fibers every 1–2 ms. These data can be analyzed with the software program WinWCP (J Dempster, Strathclyde University).

• 7. Frame-by-frame fluorescence image analysis can be made using the public domain Image J software (NIH, Bethesda). The average cell fluorescence, F, is calculated by setting a region of interest (ROI) for the image series and normalized to its initial or preintervention value F₀ as (F−F₀)/F₀.

• 8. To block the dihydropyridine receptor (DHPR), add 25 μM nifedipine to the physiological medium for 30 min. To specifically block IP₃ receptors, apply either 5 μM Xestospongin C or 5 μM Xestospongin B (see Fig. 2) for 20 or 30 min, respectively, to fiber preparations. Both toxins have been reported to be effective (20), although some variability between batches makes it advisable to test the alternative toxin in case of negative results.

We have not studied in detail IP₃-dependent, slow calcium signals during muscle development and differentiation. It is worth noting though that calcium signals tend to evolve in myotubes from a distinct late calcium transient, to a fused signal comprising the fast and slow calcium transients (Fig. 1). The latter is also the case for adult skeletal muscle fibers (Fig. 2). These observations suggest that the shape of the slow calcium transient may be related to structural characteristics of the skeletal muscle cell, which may include the degree of development of the transverse tubular system.

4 Notes

• 1. As mentioned before, we studied the calcium kinetics in neonatal myotubes after several stimuli. We found that ES induces RyR and IP₃R calcium release to the cytosol in a biphasic manner (8). In order to discriminate these signals, we used Fluo-3AM single-wavelength calcium indicator that has a 390-nM Kd for calcium. In summary, we incubated the cells with the probe for several minutes and then studied the calcium transients evoked by electrical stimuli in live cells by fluorescence microscopy. Calcium increase during the fast signal (RyR-dependent) occurs rapidly, has a sustained plateau during tetanic stimuli, and a fast decay at the end of the stimuli. Slow calcium (IP₃R-dependent) signals are more variable in both the onset and the amplitude of the signal. We illustrate these events in Fig. 1 and show representative calcium measurements obtained in differentiated myotubes from C57 mice. A small proportion (8%) of cells has only the fast calcium component with no evidence of the slow calcium signal (Fig. 1a). The slow calcium signal appears as a second increase after excitation-contraction coupling or fast calcium signal in the majority of cells at this stage (Fig. 1b, 68% of the cells tested). In more differentiated myotubes, judged by size, abundance of nuclei, and branching, slow Ca²⁺ signal is observed as a slow decay of the fast calcium transient similar to that seen in adult myofibers (Fig. 1c, 14% of the cells).

• 2. Primary differentiated myotubes are a suitable model to study muscle physiology, gene expression, and calcium transient evoked by several stimuli or basal calcium modifications (8, 13, 15, 21, 22). We demonstrated that tetanic ES in myotubes produces a biphasic increase in intracellular Ca²⁺. The first increase or fast Ca²⁺ transient is related to excitation-contraction coupling. The second increase or slow Ca²⁺ transient is generated by DHPR/Gβγ protein/PI3K/PLC activation and IP₃ receptor (IP₃R) Ca²⁺ release and the participation of ATP release and ATP signaling by purinergic receptors (8).

• 3. In adult muscle fibers, a slow Ca²⁺ signal is visualized as a delayed return to basal fluorescent levels after the end of the tetanus (16). This posttetanic signal can be fitted to a double exponential decay. This kind of analysis allows for some quantification of the signal, characterization, and comparison in different experimental situations. In this respect, image acquisition speed has a direct influence on time constants decay calculation, imposing a limit for this time constant determination. In order to have an actual value for time constant, line scan records (500–1,000 Hz acquisition speed) are recommended. The slow Ca²⁺ signal doesn’t have an important Ca²⁺ entry component, as no differences are observed in signal when experiments are done in Krebs without Ca²⁺ and 0.5 mM EGTA. The slow Ca²⁺ signal is dependent on number of pulses and frequency of the electrical stimulus. At a fixed frequency, the slow Ca²⁺ signal increases with an increase in the number of pulses applied, becoming difficult to detect when the number of pulses is less than 20. Slow signal has a maximum amplitude at 10–20 Hz, being smaller at lower and higher frequencies (16).