Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle^,

Background: Osmotic stress triggers RyR-mediated Ca²⁺ sparks that are spatially confined at the periphery of muscle fibers.

Results: Pharmacological intervention of IP₃ production and genetic ablation of IP₃R suppressed RyR-mediated Ca²⁺ sparks.

Conclusion: Activation of the type 1 IP₃R is necessary to induce Ca²⁺ sparks.

Significance: This work highlights a potential interaction between IP₃R and RyR in mediating Ca²⁺ signaling in adult skeletal muscle fibers.

Abstract

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca²⁺ signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca²⁺ sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca²⁺ sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca²⁺ sparks involves interaction between activation of IP₃R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP₃ production or inhibition of IP₃R channel activity abolishes stress-induced Ca²⁺ sparks in skeletal muscle. Although genetic ablation of the type 2 IP₃R does not appear to affect Ca²⁺ sparks in skeletal muscle, specific silencing of the type 1 IP₃R leads to ablation of stress-induced Ca²⁺ sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP₃R1 and RyR1 contributes to Ca²⁺ spark activation in skeletal muscle.

Introduction

Ca²⁺ sparks are localized Ca²⁺ release events originating from the opening of clustered ryanodine receptors (RyR)⁴ on the sarcoplasmic reticulum in muscle cells (1–3). In cardiac muscle, these spontaneous Ca²⁺ release events underlie the rhythmic contractile activity of the heart. In skeletal muscle, opening of the type 1 ryanodine receptor (RyR1) is strictly controlled by the voltage sensor located on the sarcolemmal membrane, and thus spontaneous Ca²⁺ sparks are rarely observed during resting conditions. We previously showed that application of transient osmotic stress led to robust Ca²⁺ sparks that are predominantly distributed at the periphery of the fibers from young, normal skeletal muscles (4). These Ca²⁺ spark events involve Ca²⁺ release from RyR1 as knock-out of RyR3 does not prevent the activation of Ca²⁺ sparks (5). Immunostaining and electron microscopy studies established that RyR1 is distributed throughout the junctional sarcoplasmic reticulum membrane in association with the transverse tubular invagination of the sarcolemmal membrane (6); thus, activation of these spatially confined Ca²⁺ sparks must reflect localized activation of RyR1 channels near the periphery of the muscle fiber. At present, it is not known whether physical perturbation of the junctional membrane structure and/or production of a local second messenger are essential for the initiation of stress-induced Ca²⁺ sparks in skeletal muscle.

It is known that application of osmotic stress can affect stretch-activated phospholipases on the plasma membrane and lead to production of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and inositol (1,4,5)-trisphosphate (IP₃), which could impact intracellular Ca²⁺ signaling (7, 8). Several studies in smooth muscle and cardiomyocytes demonstrated that activation of IP₃ receptors (IP₃R) can contribute to RyR-mediated Ca²⁺ signaling in these cells and shape the spatial distribution and kinetic properties of Ca²⁺ sparks in smooth and cardiac muscle cells (9–14). Although some studies have shown that the IP₃R can affect intracellular Ca²⁺ signaling in skeletal muscle (15–17) and potentially contribute to excitation-transcription coupling (18), the influence of IP₃R on RyR-mediated Ca²⁺ spark signaling in skeletal muscle has not been clearly resolved.

In this study, we tested the hypothesis that cross-talk between IP₃R and RyR underlies activation of stress-induced Ca²⁺ sparks in mammalian skeletal muscle. We found that inhibition of IP₃R by pharmacological agents suppresses osmotic stress-induced Ca²⁺ sparks and that UV photo-release of caged IP₃ could increase Ca²⁺ spark activity in intact skeletal muscle fibers. Selective activation of RyR1 near the periphery of the sarcolemma through coupling with IP₃R appears to contribute to the onset of localized Ca²⁺ sparks as RNAi silencing of IP₃R1 abolishes stress-induced skeletal muscle Ca²⁺ sparks. Our data provide direct evidence that IP₃R1 modulates RyR1-mediated Ca²⁺ spark signaling in skeletal muscle.

EXPERIMENTAL PROCEDURES

Isolation of Flexor Digitorum Brevis (FDB) Muscle Fibers

The method was described in previous work (4, 19). Briefly, FDB muscles were surgically removed from mice in zero Ca²⁺ Tyrode's buffer (in mm) 140 NaCl, 5 KCl, 10 HEPES, 2 MgCl₂ (pH 7.2) and incubated in Tyrode's buffer containing 2 mg/ml type I collagenase (Sigma C-5138) for 90 min at 37 °C. After two washes in isotonic Tyrode's buffer containing 2.5 mm Ca²⁺ (osmolality = 290 mosm), muscle fibers were gently dissociated by several passages through a series of micropipette tips of gradually decreasing diameter. Individual FDB muscle fibers were plated onto ΔTC3 glass-bottomed Petri dishes (Fisher Scientific) in Tyrode's buffer and used for experimentation within 6 h.

Confocal Ca²⁺ Imaging and Spark Analysis

Imaging of intracellular Ca²⁺ levels used previously described methods (4, 19). Individually isolated muscle fibers were loaded with Fluo4-AM (10 μm) for 60 min at room temperature. Measurements of Ca²⁺ sparks were performed using a Bio-Rad Radiance-2100 confocal microscope equipped with an argon laser (488 nm) and a ×40, 1.3 NA oil immersion objective. For Ca²⁺ spark measurements, fibers were perfused with hypotonic solution (osmolality = 170 mosm) containing (in mm) 70 NaCl, 5 KCl, 10 Hepes, 2.5 CaCl₂, 2 MgCl₂ (pH 7.2), for 100 s to induce swelling before perfusion was switched back to the initial isotonic Tyrode's buffer (osmolality = 290 mosm). The osmolality of all solutions was measured using an Advanced model 3300 micro-osmometer (Advanced Instruments). Line scan images of 512 pixels in length were acquired at a sampling rate of 2 ms/line, and serial x-y images of muscle fibers were acquired at 3.08 s/frame.

Patch Clamp Electrophysiology

This approach was adapted from the methods developed by Jacquemond and colleagues (20, 21) and adapted in our recent publication (19).

Photo-release of IP₃

ciIP₃/PM (caged membrane-permeant derivative of IP₃) was dissolved in DMSO to yield a final concentration of 10 μm. FDB fibers were loaded with 10 μm ciIP₃/PM for 45 min and an additional 30 min with 10 μm Fluo-4 AM. Photo-uncaging was performed according to Zhang et al. (12). The caged IP₃ was released into the fibers by photolysis of the compound using a UV lamp following the protocol previously described (12). Only one photolysis was induced in each fiber.

shRNA Design

Multiple short hairpin RNA (shRNA) probes targeting common sequences on the mRNA for IP₃R1 and IP₃R2 were screened for their efficacy in knocking down IP₃R1 and IP₃R2 protein expression using transfection in C2C12 cells. We found one shRNA IP₃R probe, 5′-GATCGACTACAGGAAGAACCAGGAGTACTTCAAGAGAGTACTCCTGGTTCTTCCTGTAGTCTTTTTT-3′, that was effective in knocking down the expression of IP₃R1 and IP₃R2. shRNA IP₃R1 for specific knockdown of IP₃R1 expression was according to the published sequence (22). Bold underlined nucleotides represent the conserved sequence on the target mRNAs. shRNA control targets sequence in the luciferase cDNA as described previously (19). These shRNA probes were annealed to the pU6-mRFP expression vector (19).

Electroporation of Plasmid DNA into Adult Muscle

Plasmid delivery followed previously published methods (19). For all experimental results reported here, mice were sacrificed at 14 days after electroporation, and FDB muscles were surgically removed. IP₃R2^−/− mice and age-matched wild-type control mice (11) were kindly provided by Dr. Ju Chen.

Western Blot Preparation

RFP expression was used as an indicator to measure the transfection efficiency of shRNA into the FDB muscle. Untransfected muscle bundles were trimmed out from the RFP-positive muscle for preparation of tissue lysates. The lysates were separated on a 6–10% SDS-PAGE gel. Antibodies used for immunoblots were as follows: rabbit polyclonal antibodies against IP₃R1 and IP₃R2 (gifts from Drs. J. Maxwell and G. Mignery (Loyola University) and J. Chen (University of California, San Diego (UCSD)); polyclonal anti-CSQ1 (from Dr. E. K. Kim (Gwangju Institute of Science and Technology)); mouse monoclonal antibodies against RyR1 (34c-ABR from Affinity BioReagents), actinin (from Sigma), and sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) (from Affinity BioReagents); and goat polyclonal antibody of phospholipase Cδ (PLCδ) (from Dr. L. Runnels (University of Medicine and Dentistry of New Jersey (UMDNJ)). HRP-conjugated RFP antibody was from Abcam. Densitometry analysis was performed using ImageJ software.

Resting and Store Ca²⁺ Measurement

Individual RFP-positive FDB fibers (as indicator for transfection of shRNA) were loaded with 10 μm fura-2 AM. The ratio of fura-2 fluorescence at excitation wavelength of 340 and 380 nm was measured using a PTI spectrofluorometer (Photon Technology International) to assess the resting [Ca²⁺]_i level. Zero Ca²⁺ Tyrode's solution was perfused onto the fiber before adding either caffeine/ryanodine or ionomycin (Sigma) to induce Ca²⁺ store release.

Chemicals

U73122 and U73343 (Cayman Chemicals), xestospongin C (Enzo Life Sciences), anthracene 9-carboxylic acid (Sigma), nifedipine (Sigma, MP Biomedicals), and ciIP₃/PM (siChem) were dissolved in DMSO and diluted in experimental buffer to yield final concentration of 0.1% DMSO.

Statistical Analysis

Results are presented as mean ± S.E. as tested for statistical significance by Student's t test, *, p < 0.01.

RESULTS

Interference with IP₃R Signaling Affects Stress-induced Ca²⁺ Sparks in Skeletal Muscle

We showed in our previous publication (4) that a characteristic feature of osmotic stress-induced Ca²⁺ sparks in intact skeletal muscle is their spatial distribution at the periphery of the muscle fiber directly underneath the sarcolemma (Fig. 1a). These osmotic stress-induced Ca²⁺ sparks are mediated by activation of RyR channel because preincubation of the FDB muscle fibers with 10 μm ryanodine could completely abolish the appearance of Ca²⁺ sparks (4). To test whether changes in sarcolemmal membrane potential could influence the onset of Ca²⁺ sparks in skeletal muscle, we used electrophysiological means to clamp the membrane potential at −70 mV following the method developed by Jacquemond and colleagues (20, 21). As shown in Fig. 1b, an apparently normal Ca²⁺ spark response was observed when the muscle fibers were clamped at −70 mV during application of osmotic stress. This suggests that changes in sarcolemmal membrane potential are not required in activation of osmotic stress-induced Ca²⁺ sparks in muscle fibers. Additional studies show that changes in Cl⁻ flux associated osmotic stress did not appear to contribute to the onset of Ca²⁺ sparks (supplemental Fig. S1a).

FIGURE 1.

Interference of IP₃R function affects osmotic stress-induced Ca²⁺ sparks in skeletal muscle. A binary plot of Fluo-4 fluorescence in skeletal muscle fibers, illustrating the peripheral distribution of Ca²⁺ sparks triggered by osmotic stress is shown (for details, see Refs. 4, 5, and 19). a, spatially confined Ca²⁺ sparks near the sarcolemmal region were observed in FDB muscle fiber following transient exposure to a hypo-osmotic stress (170 mosm) with a normal Tyrode's solution containing 2.5 mm Ca²⁺ (290 mosm) and 0.1% DMSO (n = 20 fibers). b, clamping of FDB fiber at −70 mV did not affect osmotic stress-induced Ca²⁺ sparks in a normal Tyrode's solution supplemented with 0.1 mm anthracene 9-carboxylic acid (n = 10 fibers). c, FDB fibers treated with U73343 (as a negative control) showed abundant Ca²⁺ sparks similar to untreated controls (n = 15 fibers). d, application of PLC inhibitor (5 μm U73122) prevented onset of osmotic stress-induced Ca²⁺ sparks (n = 12 fibers). e, application of 10 μm xestospongin C, an IP₃R antagonist, prevented appearance of Ca²⁺ sparks (n = 15 fibers).

Exposure of cells to osmotic stress has been shown to increase PI(4,5)P₂ production (8) and to activate PLC that can hydrolyze membrane-delimited PI(4,5)P₂ to IP₃ and diacylglycerol (7). Given the well known contribution of this signaling cascade to Ca²⁺ spark activation in various tissues (9–14), we used pharmacological inhibitors of PLC to test whether IP₃R was involved in the generation of osmotic stress-induced Ca²⁺ sparks. Preincubation of FDB fibers with U73122 (5 μm), a pharmacological inhibitor of PLC, led to complete suppression of the osmotic stress-induced Ca²⁺ spark activity (Fig. 1d). As a control, FDB fibers treated with U73343 (an inactive chemical analog for U73122) displayed normal onset of osmotic stress Ca²⁺ sparks (Fig. 1c). Moreover, pretreatment of FDB fibers with xestospongin C (10 μm) (Fig. 1e) that specifically inhibited IP₃R channel activation (18) greatly suppressed the Ca²⁺ spark activity following osmotic stress. Quantification analysis showed an average of 0.5 ± 0.1 Ca²⁺ spark events/min when compared with 4 ± 0.2 Ca²⁺ spark events/min in DMSO-treated fibers (Fig. 1a). Kinetic analysis of the amplitude (ΔF/F₀) and full duration at half-maximum (FDHM) of the remaining Ca²⁺ sparks showed similar Ca²⁺ spark amplitude (0.85 ± 0.02; 0.92 ± 0.03) but reduced FDHM (67 ± 21.0ms; 102.2 ± 7.6 ms) in xestospongin C-treated fibers (Fig. 1e) when compared with DMSO-treated fibers (Fig. 1a). These findings provide initial evidence that activation of IP₃R might be required for RyR-mediated Ca²⁺ sparks in skeletal muscle.

Facilitation of Ca²⁺ Spark Activity through Uncaging of IP₃

To test whether local activation of IP₃R could produce a Ca²⁺ spark response in intact skeletal muscle fibers, we used UV flash photo-uncaging to produce local elevation of IP₃ by photo-uncaging of a caged IP₃ analog, ciIP₃/PM (12). A pulse of UV flash photo-uncaging of IP₃ was applied at 20 s after the start of a 60-s confocal line scan of the fibers. Application of UV photo-uncaging in the DMSO-treated fibers did not affect the resting cytosolic Ca²⁺ level (Fig. 2a, top panel). Uncaging of IP₃ in a resting muscle fiber bathed in an isotonic solution also did not produce any immediate Ca²⁺ release (Fig. 2a, middle panel).

FIGURE 2.

Uncaging of IP₃ elicits additional Ca²⁺ sparks following osmotic stress in skeletal muscle. a, exposure of FDB fibers with UV laser did not induce changes in cytosolic Ca²⁺ at resting condition (top panel, n = 10 fibers). Fibers loaded with ciIP₃/PM did not show localized Ca²⁺ sparks following UV flash photo-uncaging at resting condition (middle panel, n = 30 fibers). Fibers pretreated with osmotic stress showed significant elevation of Ca²⁺ sparks following photo-uncaging of ciIP₃/PM. (bottom panel, n = 35 fibers); the line scan image was conducted at 7 min after exposure to osmotic stress. b, quantification of Ca²⁺ spark frequency before and after photo-uncaging of IP₃ (calculated from the experiments shown in the bottom panel of a (n = 20 fibers). c, statistical analysis of the amplitude of Ca²⁺ sparks before and after photo-uncaging of IP₃. These analyses were conducted on the first 20 s following photo-uncaging. Results are presented as mean ± S.E.

Other muscle fibers were challenged with osmotic stress, and IP₃ uncaging was performed at 7 min after osmotic stress, a point when Ca²⁺ spark activity has greatly subsided. In greater than 50% of the fibers tested, we observed a significant increase in Ca²⁺ spark events within 20 s of the uncaging of IP₃ in fibers that had previously been exposed to osmotic stress (Fig. 2a, lower panel, and 2b). The average amplitude of Ca²⁺ sparks was similar before and after uncaging of IP₃ (Fig. 2c). In both resting (Fig. 2a, middle panel) and osmotic stress- (Fig. 2a, bottom panel) treated fibers, we observed diffuse Ca²⁺ release along with gradual elevation of cytosolic Ca²⁺ at later stages of the experiment (beyond 20 s after UV flash). This Ca²⁺ release could result from diffusion of IP₃ to other IP₃R targets (18, 23) rather than direct photo-damage to the muscle fiber as application of UV flash to the DMSO only-treated fibers did not result in any distinct Ca²⁺ release (Fig. 2a). Considering the immediate effect of IP₃R in activating RyR-associated Ca²⁺ sparks in smooth muscle cells (12), we therefore focused our analysis on the production of Ca²⁺ sparks immediately following uncaging of IP₃ by analyzing the characteristics of Ca²⁺ sparks appearing within 20 s of photo-uncaging of IP₃. In this experiment, line scans were conducted at the periphery of the fiber as we normally observed the Ca²⁺ sparks in this region. Additional line scan recording at the middle of fiber upon photo-uncaging of IP₃ did not reveal any increase in Ca²⁺ release events over base line (not shown). These findings provide direct evidence that IP₃ can act as a diffusible second messenger to facilitate activation of Ca²⁺ sparks in skeletal muscle.

Our results are consistent with early studies from Pozzan and colleagues (16), who demonstrated that injection of IP₃ in skinned muscle preparation could elicit intracellular Ca²⁺ release from skeletal muscle. Many subsequent studies from other investigators have revealed a role for IP₃R in facilitating the RyR-mediated Ca²⁺ release in smooth muscle and cardiac muscle. Recent studies from Jaimovich and colleagues (18) demonstrated a role for IP₃R in modulation of excitation-transcription coupling in skeletal muscle; however, controversy exists in this topic as Blaauw et al. (24) did not find a significant role for IP₃ in skeletal muscle Ca²⁺ signaling. The observation that uncaging of IP₃ alone could not produce Ca²⁺ spark events in muscle fibers without pre-exposure to osmotic stress suggests the possibility that other factors such as physical uncoupling of voltage sensor from RyR during osmotic stress could be required for induction of the robust Ca²⁺ spark events (4, 25).

Knockdown of IP₃R Ablates Stress-induced Ca²⁺ Sparks in Skeletal Muscle

Toward understanding the role of IP₃Rs in regulation of Ca²⁺ spark signaling in skeletal muscle, we examined the distribution of IP₃R isoforms in skeletal muscle. Western blots showed that both IP₃R1 and IP₃R2 were present in the skeletal muscle and that IP₃R3 was absent (Fig. 3a). Immunolocalization revealed staining of IP₃R1 and IP₃R2 in the perinuclear and subsarcolemmal region where Ca²⁺ sparks appear (supplemental Fig. S2). To directly test whether IP₃R is required for induction of Ca²⁺ sparks in skeletal muscle, we used shRNA to simultaneously knock down both IP₃R1 and IP₃R2 in the FDB muscle using a probe targeting conserved sequences in the two mRNAs (Fig. 3b). As a control, we used an shRNA sequence targeting the luciferase cDNA (19). Plasmid DNA was delivered to FDB muscles in mice using electroporation-mediated transfection (19), and after 14 days, FDB muscles were removed. Western blotting showed that both IP₃R1 and IP₃R2 expression was significantly reduced in these muscles, whereas the expression of other Ca²⁺ regulatory proteins was not affected (Fig. 3c). The shRNA sequence was cloned into an RFP expression vector, allowing for identification of FDB muscle fibers with targeted knockdown of IP₃R1 and IP₃R2 (Fig. 3d). Muscle fibers with knockdown of both IP₃R1 and IP₃R2 (based on the presence of RFP fluorescence) were challenged with osmotic stress. Although robust Ca²⁺ sparks were observed in the shRNA control fibers (Fig. 4a, top panel), shRNA IP₃R fibers displayed very few, if any, Ca²⁺ sparks following osmotic stress (Fig. 4a, bottom panel). Quantification of Ca²⁺ spark frequency showed that these events were extremely rare in the shRNA IP₃R when compared with the shRNA control fibers (Fig. 4c). This result was in accordance with our data shown in Fig. 1 where pharmacological interference of IP₃R signaling had a significant impact on the activation of Ca²⁺ sparks in skeletal muscle. Kinetic analysis of the remaining Ca²⁺ sparks showed that knockdown of IP₃R1 and IP₃R2 did not affect the Ca²⁺ spark amplitude (ΔF/F₀) between shRNA control (0.96 ± 0.04, n = 20) and shRNA IP₃R (0.91 ± 0.04, n = 30), whereas FDHM changed from 88.3 ± 5.5 ms in the shRNA control to 44.9 ± 21.0 ms in the shRNA IP₃R fibers. The reduction in frequency of Ca²⁺ sparks following knockdown of IP₃R was not the result of changes in the resting intracellular Ca²⁺ levels or loading of the internal Ca²⁺ stores. fura-2 ratiometric measurements showed that the resting [Ca²⁺]_i levels were similar between shRNA IP₃R (0.47 ± 0.01, n = 15) and shRNA control (0.43 ± 0.02, n = 15) (Fig. 4c). Ionomycin and caffeine plus ryanodine-induced release of intracellular Ca²⁺ stores showed no significant difference between shRNA control and shRNA IP₃R fibers (Fig. 4c).

FIGURE 3.

shRNA-mediated knockdown of IP₃R1 and IP₃R2 in skeletal muscle. a, Western blots were performed using lysates from gastrocnemius muscle, lung, and brain tissues derived from WT mice (n = 6 mice). 10-fold of muscle lysates when compared with brain and lung lysates were loaded. IP₃R1 and IP₃R2, but not IP₃R3, were present in the skeletal muscle. b, shRNA IP₃R was designed to target a common sequence on IP₃R1 and IP₃R2. Knocking down both IP₃Rs did not alter the expression of other Ca²⁺ regulatory proteins. c, densitometry analysis showed that shRNA IP₃R resulted in significant knockdown of IP₃R1 and IP₃R2 in FDB muscle (n = 8 mice). A. U., arbitrary units. d, FDB fibers with knockdown of IP₃Rs could be identified by the appearance of red fluorescence.

FIGURE 4.

Silencing of IP₃R abolishes osmotic stress-induced Ca²⁺ sparks in skeletal muscle. a, robust peripheral Ca²⁺ sparks following osmotic stress was observed in the shRNA control fibers (n = 20 fibers), but not in the shRNA IP₃R fibers (bottom panel) (n = 30 fibers). b, quantification analysis showed reduced Ca²⁺ spark activity after hypotonic perfusion in the shRNA IP₃R fibers (green square) when compared with the shRNA control fibers (black square). c, representative fura-2 fluorescence in FDB fibers following treatment with 5 μm ionomycin to release the total intracellular [Ca²⁺]_i store (left panel, n = 15 fibers) or following the addition of 20 mm caffeine + 5 μm ryanodine to release the sarcoplasmic reticulum Ca²⁺ store (middle panel, n = 15 fibers). Fiber was previously perfused with zero Ca²⁺ Tyrode's solution before application of either caffeine/ryanodine or ionomycin to induce store Ca²⁺release. Data are presented as mean ± S.E.

Activation of IP₃R1, but Not IP₃R2, Is Involved in RyR1-mediated Ca²⁺ Spark in Skeletal Muscle

Previous studies have shown that different IP₃R isoforms exhibit different functional properties in Ca²⁺ signaling (22, 26, 27). We next asked whether a specific IP₃R isoform is required for RyR-mediated Ca²⁺ sparks. Genetic ablation of IP₃R2 produced a viable mouse model (11), allowing for examination of stress-induced Ca²⁺ sparks in adult skeletal muscle. When examined under our experimental conditions, we found that the IP₃R2^−/− skeletal muscle fibers displayed robust peripheral Ca²⁺ sparks upon osmotic stress stimulation (Fig. 5b), which was similar to the results in wild-type (WT) control or fibers transfected with the shRNA control (Fig. 5a).

FIGURE 5.

IP₃R1 but not IP₃R2 contributes to regulation of RyR-mediated Ca²⁺ spark activity. FDB muscle derived from the IP₃R2^−/− mice displayed robust peripheral Ca²⁺ sparks following osmotic stress (b, n = 18 fibers), similar to those observed in age-matched WT muscle (a, n = 18 fibers). c, knockdown of IP₃R1 alone suppressed Ca²⁺ sparks in FDB muscle following osmotic stress (n = 22 WT/shRNA IR₃P1 fibers). d, compromised Ca²⁺ spark response was also observed in the IP₃R2^−/− muscle fiber following electroporation of shRNA IP₃R1 (n = 16 fibers). e, Western blots showed the specificity of shRNA IP₃R1 in knocking down IP₃R1, but not IP₃R2 (n = 4 mice). f, time-dependent changes in Ca²⁺ spark activity in FDB fibers following hypo-osmotic stress were quantified. Knocking down of IP₃R1 from WT muscle led to near complete suppression of Ca²⁺ spark activity (red) when compared with robust Ca²⁺ spark events in WT muscle that decline with time (black). Muscle fibers derived from IP₃R2^−/− mice showed normal Ca²⁺ spark response (blue), and knockdown of IP₃R1 led to complete ablation of Ca²⁺ events (aqua).

We then tested whether IP₃R1 was required for the onset of Ca²⁺ sparks in skeletal muscle. We used electroporation to deliver an shRNA probe that specifically targets knockdown of IP₃R1 (shRNA IP₃R1) (22), without affecting the expression of IP₃R2 (Fig. 5e). We found that knocking down IP₃R1 alone in FDB muscle derived from WT mice could completely suppress Ca²⁺ sparks following osmotic stress stimulation (Fig. 5c). Similar results were observed with electroporation of shRNA IP₃R1 into the IP₃R2^−/− FDB muscle, where Ca²⁺ spark response was nearly completely ablated (Fig. 5, d and f). No significant differences were observed in the average Ca²⁺ spark amplitude (ΔF/F₀) of WT (0.97 ± 0.05, n = 18), IP₃R2^−/− (0.93 ± 0.04, n = 18), shRNA IP₃R1 (0.89 ± 0.06, n = 22), and shRNA IP₃R1 in IP₃R2^−/− background (0.85 ± 0.08, n = 16). Although the average FDHM was similar between WT (105 ± 10.7 ms) and IP₃R2^−/− (100 ± 11.3 ms), it was significantly reduced in the shRNA IP₃R1 in WT (45.7 ± 10.9 ms) or in IP₃R2^−/− fibers (42.5 ± 15.4 ms). Overall, our findings indicate that activation of IP₃R1, and not IP₃R2, was required to trigger RyR-mediated Ca²⁺ sparks in skeletal muscle.

DISCUSSION

We present evidence for a role for IP₃R in regulation of RyR-mediated Ca²⁺ sparks in skeletal muscle. The Ca²⁺ spark response in skeletal muscle was absent with pharmacological intervention that inhibited activation of the IP₃R channels. It was also absent in muscle fibers following knockdown of IP₃R1; thus, activation of IP₃R1 is required for the onset of osmotic stress-induced Ca²⁺ sparks in mammalian skeletal muscle. Based on our findings, we propose that initial Ca²⁺ release through IP₃R1 channel coupled with transient membrane deformation activates neighboring RyR1, leading to the production of peripherally localized Ca²⁺ sparks in intact skeletal muscle fibers.

Although transient elevation of IP₃ through photo-uncaging could amplify RyR-mediated Ca²⁺ release, the frequency of Ca²⁺ sparks triggered by IP₃ was much less when compared with the osmotic stress-induced Ca²⁺ sparks. Moreover, significant elevation of Ca²⁺ sparks associated with photo-uncaging of IP₃ was only observed in muscle fibers following exposure to osmotic stress. Thus, stress-induced Ca²⁺ sparks in mammalian skeletal muscle would likely require two cellular events, one involving production of a local IP₃ second messenger and the other involving structural changes at the triad junction that would lead to uncoupling of RyR1 channel inhibition by the voltage sensor on the sarcolemmal membrane (4, 25). Our study added additional evidence that osmotic stress-induced Ca²⁺ sparks in adult skeletal muscle does not involve changes in ion flux across the sarcolemmal membrane or changes in membrane potential.

A distinct feature of the stress-induced Ca²⁺ sparks in skeletal muscle is their confinement to the periphery of the muscle fiber. We show that the IP₃R channels are preferentially localized near the subsarcolemmal region of the muscle fiber, which could potentially facilitate local activation of the RyR1 channel through Ca²⁺ as an intermediate messenger. The asymmetrical distribution of phospholipids on the sarcolemmal membrane (28, 29) could result in localized activation of PLC and IP₃ production, which could represent a peripherally confined signal for activation of the RyR1 channel in skeletal muscle. Although we showed that IP₃R2 is not required for the osmotic stress-induced Ca²⁺ sparks in skeletal muscle, a specific role for IP₃R1 in the activation of Ca²⁺ sparks is established through our shRNA silencing approaches. Although the different Ca²⁺ signaling properties of the different IP₃R isoforms (26, 27) could contribute to the specificity of IP₃R1 in regulating RyR1-mediated Ca²⁺ sparks, it is not clear why IP₃R2 is not required for Ca²⁺ spark activation. Future studies are necessary to test whether a direct interaction between the different isoforms of IP₃R and RyR plays a role in their functional cross-talk in modulating the spatial and temporal aspects of Ca²⁺ signaling in skeletal muscle or whether other intermediate messengers that favor the IP₃R1 activation pathway could participate in modulation of the stress-induced changes in Ca²⁺ signaling in skeletal muscle.

The role for IP₃ in Ca²⁺ signaling in skeletal muscle was discovered over 25 years ago (15, 16); however, cross-talk between IP₃R and RyR in modulation of Ca²⁺ signaling has mostly been studied in cardiac and smooth muscles (9–13). Altered Ca²⁺ release mediated by cross-talk between IP₃R and RyR has been linked to hypertrophic cardiomyopathy and arrhythmia (9, 11, 13, 14, 30–33), indicating the role of such cross-talk in physiology and pathophysiology. Peripherally confined Ca²⁺ sparks in smooth muscle have been linked to activation of Ca²⁺-dependent K⁺ channel, which is important for vasodilation (2, 34). Numerous studies in other cell types have shown the physiological relevance of localized Ca²⁺ signals in vesicle fusion (28) and cytoskeletal reorganization (35). Our findings of IP₃R cross-talk with RyR1 in skeletal muscle suggest that this may be a mechanism governing normal Ca²⁺ regulation in skeletal muscle, and these findings also open a new line of investigation into potential pathophysiologic mechanism during muscle diseases. Several studies showed that the pathological consequence of altered Ca²⁺ signals observed in the dystrophic skeletal muscle fibers was associated with abnormal IP₃R distribution (36, 37). Knockdown of IP₃R1 could minimize cell death in dystrophin-deficient muscle fibers (38). Our previous studies showed that Ca²⁺ spark in the dystrophic (mdx) muscle fibers was irreversible and no longer peripherally confined (4). Hence, aberrant Ca²⁺ sparks observed in the dystrophic skeletal muscle could be associated with altered IP₃R1 expression, localization, or activation.