IL-1α Reversibly Inhibits Skeletal Muscle Ryanodine Receptor. A Novel Mechanism for Critical Illness Myopathy?

Abstract

Critical illness myopathies in patients with sepsis or sustained mechanical ventilation prolong intensive care treatment and threaten both patients and health budgets; no specific therapy is available. Underlying pathophysiological mechanisms are still patchy. We characterized IL-1α action on muscle performance in “skinned” muscle fibers using force transducers and confocal Ca²⁺ fluorescence microscopy for force/Ca²⁺ transients and Ca²⁺ sparks. Association of IL-1α with sarcoplasmic reticulum (SR) release channel, ryanodine receptor (RyR) 1, was investigated with coimmunoprecipitation and confocal immunofluorescence colocalization. Membrane integrity was studied in single, intact fibers challenged with IL-1α. IL-1α reversibly stabilized Mg²⁺ inhibition of Ca²⁺ release. Low Mg²⁺–induced force and Ca²⁺ transients were reversibly abolished by IL-1α. At normal Mg²⁺, IL-1α reversibly increased caffeine-induced force and Ca²⁺ transients. IL-1α reduced SR Ca²⁺ leak via RyR1, as judged by (1) increased SR Ca²⁺ retention, (2) increased IL-1α force transients being reproduced by 25 μM tetracaine, and (3) reduced Ca²⁺ spark frequencies by IL-1α or tetracaine. Coimmunoprecipitation confirmed RyR1/IL-1 association. RyR1/IL-1 immunofluorescence patterns perfectly colocalized. Long-term, 8-hour IL-1α challenge of intact muscle fibers compromised membrane integrity in approximately 50% of fibers, and confirmed intracellular IL-1α deposition. IL-1α exerts a novel, specific, and reversible interaction mechanism with the skeletal muscle RyR1 macromolecular release complex without the need to act via its membrane IL-1 receptor, as IL-1R membrane expression levels were not detectable in Western blots or immunostaining of single fibers. We present a potential explanation of how the inflammatory mediator, IL-1α, may contribute to muscle weakness in critical illness.

Clinical Relevance

Muscle weakness of the diaphragm and limb muscles is one of the most abundant complications in critically ill patients, which vastly prolongs treatment, intensive care unit stays, and health budgets. No specific cure is at hand, as the pathophysiology of the muscle weakness is still cryptic. This study describes a novel mechanism for inflammatory cytokine IL-1 action directed at the skeletal muscle ryanodine receptor to interfere with muscle activation and Ca²⁺ homeostasis. This IL-1 “off-receptor-site” target might be responsible to explain muscle dysfunction during inflammatory and critical illness myopathies. It also represents a unique, novel signaling property of this inflammatory mediator.

Skeletal muscle is a very common target in inflammatory diseases. Increased proteolysis and muscle wasting is seen in tumor cachexia (1), chronic inflammation (2, 3), and critical illness (4, 5). Many patients in intensive care units (ICUs) develop sepsis and multiorgan failure. In many developed countries, sepsis ranks among conditions with the highest mortality (6). Most critically ill patients in the ICU develop neuromuscular dysfunctions (e.g., critical illness myopathies [CIMs]) that present with flaccid paralysis and impose vast threats to patients and health budgets. CIM is not restricted to sepsis, but can also be triggered by steroids or neuromuscular blockers (7, 8). Neuromuscular dysfunctions, such as seen in CIMs or critical illness polyneuropathies, are considered a form of organ failure secondary to critical illness, whether sepsis related or not (9).

Skeletal muscle is highly specialized and tuned for fast activation and force development. Action potentials spread along the sarcolemma and penetrate deep into the transverse tubules to activate dihydropyridine receptor (DHPR) voltage sensors. In skeletal muscle, DHPRs are conformationally coupled to sarcoplasmic ryanodine receptor (RyR) 1 Ca²⁺ release channels (10). Upon activation of DHPR, its II–III loop acutely lowers Mg²⁺ affinity of the inhibitory Mg²⁺ binding site on the RyR1 to enable sarcoplasmic reticulum (SR) Ca²⁺ release (11). Elevated myoplasmic Ca²⁺ activates cross-bridge cycling, force production, and motility. Apart from this “physiological activation” involving “Mg²⁺ disinhibition,” the RyR1 contains a multitude of other regulatory binding sites (12). For example, methylxanthines (e.g., caffeine) increase Ca²⁺ affinity for the Ca²⁺ activation site, and induce strong SR Ca²⁺ release (13).

Cellular mechanisms of muscle failure in critical illness conditions are still poorly understood. Skeletal muscles mostly affected are limb muscles and the diaphragm, which translates to the symptoms seen in affected patients (i.e., acute quadriplegia or weaning failure [14]). Experimental approaches that cover the sepsis aspect of CIM range from LPS- (15) to cecal-ligation and puncture-induced sepsis (16), as well as acute septic serum challenge in vitro models (17), whereas other trigger factors apart from inflammation have been employed using steroid-denervation animal models (18). Although decreased membrane excitability (15, 18), decreased contractility and increased fatigability (16), or metabolic failure (19) have been noted across all different conditioning models, findings for altered Ca²⁺ homeostasis (20) have been only descriptive, and events that coin the mechanism of CIM have not been unraveled. During systemic inflammation, global and local cytokines are up-regulated (21). In mixed ICU patients, pro- and anti-inflammatory cytokines were vastly up-regulated in the blood, regardless of whether sepsis was present or not (22). In limb skeletal muscle biopsies from critically ill patients, vast immune cell infiltration and proinflammatory cytokine expression was detected (e.g., TNF-α and IL-1 [23]). This tempted us to investigate the role of selected cytokines on skeletal muscle dysfunction in a defined in vitro setting involving the extensor digitorum longus (EDL) muscle as a widely used fast-twitch–type limb muscle in muscle research. We chose IL-1 (IL-1α isoform; hereafter referred to as IL-1) as one prominent proinflammatory cytokine in sepsis and critical illness. Our results show a very unexpected and novel mechanism of IL-1–RyR1 interaction that may explain some aspects of muscle failure in conditions of CIM associated with high cytokine burden. This is the first demonstration of a direct cytokine action on a release channel that is not mediated via the cytokine receptor.

Materials and Methods

Force Transducer Recordings

Recordings were performed in small, C57Bl6 mouse EDL fiber bundles after chemical skinning (10 μg/ml saponin; 90 s) to gain diffusional access and control over the myoplasmic environment. Before global SR Ca²⁺ releases with either caffeine (30 mM) or introducing low-Mg²⁺ (10 μM) solution, SR was loaded in roughly 250 nM Ca²⁺–containing internal solution. Force transients were recorded and amplitudes related to maximum force responses induced by high activating solution (−log (Ca²⁺) [pCa], ∼ 4.2). pCa-force recordings were obtained in solutions with different pCas. IL-1 was added at 10, 25, or 100 ng/L before the releases for at least 1 minute (see Materials and Methods in the online supplement).

Confocal Ca²⁺ Recordings

Rhod-2 (10 μM) global Ca²⁺ transients were elicited in mechanically skinned single EDL fibers with caffeine or low Mg²⁺ with or without prior IL-1 incubation (25 ng/L). A complete sequence contained a control, an IL-1, and a final washout run. Ca²⁺ transients were analyzed from whole-fiber regions of interest of at least three individual single fibers. “Ca²⁺ sparks” were recorded in saponin-skinned single EDL fibers after staining with 10 μM Fluo-4. Confocal XYT series (100 frames at 0.9 frames per second) were taken under control conditions and 5 minutes after application of either IL-1 or tetracaine. Images were denoised and sparks detected using a wavelet-based technique and expressed as specific frequency. Further details are available in the Materials and Methods in the online supplement.

Immunofluorescence Recordings

Single saponin-skinned EDL fibers were incubated with 100 ng/L IL-1 for 1 hour to allow IL-1 to distribute inside the myoplasm. After fixation and applying an immunolabeling protocol for IL-1 and labeling RyR with Ry-BODIPY, sequential confocal 488 nm and 633 nm excitation fluorescence images were obtained (see the Materials and Methods in the online supplement).

IL-1 Incubation Cell Culture and Biochemistry Experiments

See the Materials and Methods in the online supplement for full details.

Results

IL-1 Reduces SR Ca²⁺ Leak by RyR1 Binding

In initial cytokine screens, IL-1 (10–100 ng/L) robustly and reversibly increased amplitudes of caffeine-induced force transients without affecting maximum force level at pCa 4.2 (Figure 1A). The increase in force was significant at 25 ng/L, a concentration corresponding to plasma levels in septic shock (21). TNF-α failed to show such an effect (data not shown), and was not further pursued in this study. Ca²⁺ sensitivity of the contractile apparatus at any tested IL-1 concentration was unaffected (Figure 1B). Because this suggests that IL-1 increases SR Ca²⁺ release, we directly visualized myoplasmic Ca²⁺ in mechanically skinned single fibers (with the sarcolemma manually removed) by confocal microscopy as an independent validation and observed a reversible increase in Ca²⁺ fluorescence amplitude by IL-1 (Figure 1C). Because all fibers used were skinned, the IL-1–induced effect can be regarded as instantaneous, and does not involve IL-1 receptor (IL-1R) signaling, due to diffusional access to the myoplasm and full control of the intracellular milieu (i.e., all myoplasmic second messenger molecules removed by the artificial intracellular solution [> 1:1,000,000 dilution]). A more direct proof is shown in Figure E1 in the online supplement, where Western blots probing for IL-1R1 receptor subtype in small fiber bundles (Figure E1A), as well as immunostains in either chemically or mechanically skinned single fibers (Figure E1B), did not show any significant receptor expression levels in the muscle sarcolemma. We then tested whether the increased Ca²⁺ release observed by acute IL-1 incubation originated from a reduction of SR Ca²⁺ leak by IL-1 using a defined SR Ca²⁺ leak approach (24). Each cycle consists of 5-second–5-minute–5-second leak periods (the last one to check for reversibility) under control conditions after preincubation with IL-1 and washout (control) (Figure 2A). The relative decrease in force after prolonged leakage was substantially smaller with IL-1 than under control conditions, and was completely reversible (Ca²⁺ retention index; Figure 2C, paired experiments). During such long-lasting experiments (∼ 80 min), maximum force levels remained stable under Ca²⁺ saturation conditions (pCa, 4.2). Even after tens of release-reloading cycles, caffeine-induced force was still larger with IL-1 in relation to maximum force than without IL-1 (Figure 2B). To elucidate the mechanism by which IL-1 interfered with SR Ca²⁺ leakage, we compared the action of IL-1 on caffeine-induced force transients with that of tetracaine, a known inhibitor of RyR1. In some experiments, tetracaine (25 μM) replaced IL-1. Again, a robust and reversible increase in force was seen after IL-1 preincubation (25 ng/L; Figure 2D). After another control release, the next cycle included 25 μM tetracaine. This was completely reversible as well. Analysis from three such complete cycles revealed an almost identical increase in relative force (related to the previous control) by either IL-1 or tetracaine (Figure 2E). This strongly suggests that IL-1 inhibits SR Ca²⁺ release by a robust and reversible RyR1 block.

Figure 1.

IL-1 directly increases caffeine-induced Ca²⁺ release in skeletal muscle. (A) Force transients in a skinned extensor digitorum longus (EDL) fiber bundle after incubation with different IL-1 concentrations. Force levels are consistently and reversibly increased by IL-1 (significant at 25 ng/L) and restored after washout. Maximum force (high-activating solution [HA]: −log[Ca²⁺] [pCa], 4.2) is unaltered (A), as is Ca²⁺ sensitivity of the contractile apparatus (B; pCa₅₀, pCa for 50% force activation); n_H, Hill coefficient. n.s., not significant. (C) Direct confocal observation of caffeine-induced sarcoplasmic reticulum (SR) Ca²⁺ release in mechanically skinned single fibers confirms force transducer results. The red cloud reflects the global release of Ca²⁺ into the myoplasm and the surrounding solution. Sequences in (A) and (C) are from a single preparation (horizontal lines in [C], control levels). Similar results as in (C) were obtained in three preparations. n = 5 in (A) (right panel), n = 2–8 in (B). *P < 0.05, ANOVA. LR, low relaxing solution. Dashed lines in (A) correspond to initial control peak force level (left panel) and projected linear run-down (right panel). Dashed and solid lines in (C) correspond to the amplitude of the control transients of fiber (a) and (b), respectively. caff., caffeine; F/F(0), fluorescence normalized to time point 0; rel. Force, relative force.

Figure 2.

IL-1 increases SR Ca²⁺ by reducing SR Ca²⁺ leak through ryanodine receptor (RyR) 1. (A) Force recordings from one skinned EDL small fiber bundle to assess relative releasable SR Ca²⁺ content after leaking Ca²⁺ from the SR by defined incubation times. Extended leakage was completely reversible. Decrease in peak amplitude is reduced by IL-1. Note that the increase in force by IL-1 under identical conditions is robustly observed (e.g., last transient of control and first transient of IL-1, both 5-s leakage). (B) In long-lasting experiments (∼80 min), relative increase of force amplitude after the final run was still very prominent and approached the maximum force at pCa 4.2. (C) Paired evaluation from four preparations confirms significant reduction in SR Ca²⁺ leak (i.e., larger SR Ca²⁺ retention index by IL-1). (D) Cycle of force transients aimed at identifying the source of IL-1 action at the SR. Two initial control releases after either 2- or 4-minute LR. Peak force amplitudes were compared between 4-minute LR control conditions (100%) and after either IL-1 (25 ng/L) or tetracaine preincubation. (E) The increase in force amplitude by IL-1 was mimicked by 25 μM tetracaine, indicating IL-1 binding to RyR1 to reduce SR Ca²⁺ leak. a.u., arbitray units; RS, release solution (LR + 30 mM caff.).

As an independent test for this hypothesis, we recorded elementary Ca²⁺ release events (ECREs) as an indicator of RyR1 activity. With the SR loaded as in force recordings, a large number of ECREs was observed (Figure 3A). IL-1 reduced ECRE frequencies from approximately 15 × 10⁻³/100 μm²/s to approximately 4 × 10⁻³/100 μm²/s (unpaired analysis, P < 0.01; Figure 3B). In paired XYT recordings, relative spark frequency was decreased by roughly 75% (P < 0.01). This was mimicked by approximately 100 μM tetracaine (n = 5), in good agreement with the force data. Larger tetracaine concentrations reduced ECRE frequencies even further.

Figure 3.

IL-1 partially blocks RyR1 and reduces Ca²⁺ spark frequencies in single skinned skeletal muscle fibers. (A) Images from XYT confocal Fluo-4 Ca²⁺ spark recordings of two single skinned EDL muscle fiber segments under control conditions and approximately 5 minutes after either application of 25 ng/L IL-1 or 300 μM tetracaine. Both drugs markedly reduced Ca²⁺ spark occurrence. Automated denoising and event detection allowed precise counts of Ca²⁺ sparks. Arrows represent detected sparks. Scale bar, 10 μm. (B) Ca²⁺ spark frequency was reduced approximately 75% by IL-1, and this corresponded to the effect exerted by approximately 100 μM tetracaine. ECRE, elementary Ca²⁺ release event.

We next evaluated biochemical evidence for an association of IL-1 to RyR1. Intrinsic IL-1 was already detected in skeletal muscle homogenates (gastrocnemius, Figure 4A, EDL Figure 4D). Pulldown of IL-1 with subsequent immunoblotting for RyR1 showed substantial coimmunoprecipitation (Figure 4B). An immunoblot showing that the target protein (IL-1) was immunoprecipitated is shown in Figure 4C (control). The reverse procedure (immunoprecipitate [IP]:RyR, immunoblot:IL-1) confirmed this association (Figure 4D). To verify that the IL-1 band in the immunoblot of the IP:RyR assay indeed identified IL-1, another control probing for IL-1 in a blot running an IP:IL-1 on the recombinant IL-1α as input was additionally performed (Figure 4D). In contrast to the RyR1–IL-1 association, there was no association of IL-1 to sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) (Figure 4D). We next performed confocal immunofluorescence experiments in single skinned EDL fibers after incubation with IL-1 to allow for RyR1 interaction. Both partners showed substantial overlap (Figures 4E and 4F). Striation pattern profiles and periodicity were virtually the same for IL-1 and RyR (Figures 4F and 4G). Spatial signal shifts between IL-1 and RyR1 signals for all maxima and minima were negligible (Figure 4H). These results show that the in vitro association translates to colocalization of IL-1 and RyR1 in situ within the resolution limits of the confocal microscope.

Figure 4.

IL-1 coimmunoprecipitates and colocalizes with RyR1 in skeletal muscle. (A) Western blot demonstrating expression of IL-1 in gastrocnemius muscle; lysate samples (as indicated) were obtained from two independent control animals. (B) Coimmunoprecipitation of IL-1 and RyR in skeletal muscle: gastrocnemius lysate was incubated with anti–IL-1 antibody or IgG (control), precipitated using protein A beads and resolved on SDS-PAGE. Proteins were transferred to nitrocellulose membrane and probed with the antibodies against (B) RyR (immunoprecipitate [IP]:IL-1, immunoblot [IB]:RyR) or (C) IL-1 (IP:IL-1, IB:IL-1); RyR was detected as a band migrating at greater than 500 kD, but no signal was detected when lysates were incubated with the IgG control (IP:IgG, IB:RyR, IB:IL-1). (D) Co-IP of IL-1 and RyR in EDL skeletal muscle lysates using the reverse procedure as in (B) confirmed association of both proteins (IP:RyR, IB:IL-1). Unlike RyR, IL-1 did not coimmunoprecipitate with sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA; IP:SERCA, IB:IL-1). (E) Confocal immunofluorescence microscopy shows IL-1 and RyR colocalization in situ in permeabilized single EDL fibers after IL-1 incubation. (F) Magnification of the boxed white area in (E), the Fourier transforms of the periodic pattern, as well as the signal intensity overlay. (G) RyR1 and IL-1 periodicity patterns match perfectly. In addition, there is no shift in the maximum or minimum intensity of the spatial IL-1–to–RyR signal within the microscope resolution (H). IgG_HC, IgG heavy chain; rec.IL-1, recombinant IL-1; trans, transillumination image.

IL-1 Blocks Low Mg²⁺–Induced Ca²⁺ Release

We suggest a reversible blocking action of IL-1 on the RyR1, which can still be overcome by the methylxanthine caffeine. However, normal muscle activation usually involves acute lowering of the RyR1 Mg²⁺ binding affinity in response to DHPR activation (11), thus relieving Mg²⁺ inhibition. Therefore, Mg²⁺ disinhibition of RyR1 in several fiber bundles was acutely induced by lowering cytoplasmic Mg²⁺, yielding a short force transient (representative example shown in Figure 5A). After SR reloading and IL-1 preincubation, lowering Mg²⁺ failed to induce a force transient. Subsequent force transient after caffeine application ruled out compromised SR load (Figure 5A). A small force transient after IL-1 washout indicates recovery. As a last test to directly observe IL-1 washout on restoring the RyR1 Mg²⁺ site sensitivity toward low Mg²⁺, in a different fiber bundle, Mg²⁺ was lowered in the presence of IL-1. Again, this prevented SR Ca²⁺ release. Then, only washing out IL-1 resulted in a prominent force transient (Figure 5A). To validate this observation directly for SR Ca²⁺ release, low Mg²⁺–induced Ca²⁺ transients were tracked with the confocal microscope in the absence (control) or presence of IL-1, fully confirming the force recordings (Figure 5B). Because these findings were consistent in several preparations and independent methods, the effect seems to be robust.

Figure 5.

IL-1 reversibly blocks low Mg²⁺–induced SR Ca²⁺ release. (A) Force transient recordings in two single muscle fibers under control caffeine and low Mg²⁺ conditions and after IL-1 preincubation. Low Mg²⁺ release was completely blocked by IL-1, but caffeine still induced a large release. After IL-1 washout and SR reloading with Ca²⁺, low Mg²⁺ release was partially restored. In another preparation, while IL-1 was present, again, low Mg²⁺ did not induce Ca²⁺ release. After removal of IL-1, still in low Mg²⁺, force transient was restored within seconds, while IL-1 diffused away from its binding partner into the bath solution. (B) Confocal Ca²⁺ transients in single, mechanically skinned fibers fully confirm the effect of IL-1 on blocking low Mg²⁺–induced release, which, again, is partially reversible after IL-1 washout. Similar results were observed in three other preparations. abf, file extension of raw data file.

IL-1 Compromises Membrane Integrity

Our data provide functional evidence for a selective RyR1 block by IL-1, most likely via stabilizing RyR1 Mg²⁺ inhibition. IL-1 usually acts through its plasmalemmal IL-1R (mostly the type I isoform), which was not detectable in our preparations (Figure E1). To be able to interact directly with RyR1, there must be a pathway for IL-1 to enter the cell, or IL-1 being overexpressed, or a combination of both. To address this possibility, we incubated and cultured intact single muscle cells in the presence of IL-1 for various durations. Subsequent staining with propidium iodide (PI) reveals compromised membrane integrity by IL-1 at concentrations from 25 ng/L starting after 4 hours (Figure 6A). These experiments show that IL-1 itself induces membrane leaks (by as yet unidentified mechanisms) that might serve as a pathway for this small signaling molecule to penetrate into the cell. Electrical excitability of PI-positive single fibers after 8-hour IL-1 incubation was qualitatively assessed with external field stimulation. About 30% of fibers responded with a twitch. Fibers that did not respond probably were already too depolarized (see Discussion). To actually prove the presence of IL-1 in the muscle cells after prolonged IL-1 challenge, single intact muscle fibers were cocultured with IL-1 overnight (∼ 10 h), washed (to completely remove external IL-1), and probed for IL-1 immunofluorescence. IL-1 immunofluorescence was increased several fold in IL-1– treated fibers (P < 0.01; Figure 6B), thus confirming the intracellular presence of this cytokine.

Figure 6.

Prolonged IL-1 exposure compromises muscle membrane integrity to provide an IL-1 entry pathway. (A) Propidium iodide (PI) staining after 8-hour challenge with IL-1 shows compromised membrane integrity at 25 ng/L. From 25 ng/L, approximately 50% of cells are PI positive after 8 hours (30% of which responded to external field stimulation with a twitch). (B) After overnight IL-1 incubation (25 ng/L), almost no IL-1 fluorescence was seen in control cells, but substantial IL-1 signal was detected in IL-1–challenged cells, proving that IL-1 was located inside the fibers (P < 0.01, ANOVA). Scale bars, 100 μm. ΔF/F₀, relative fluorescence change normalized to initial fluorescence at time point 0; IF, immunofluorescence; Trans, transillumination image.

Discussion

The main findings of the present study define a rather unexpected, novel mechanism for intracellular cytokine action that may explain some of the early muscle weakness and failure related to septic or other inflammatory myopathies, as well as nonseptic CIMs associated with increased cytokine profiles: (1) IL-1 (1α isoform) interacts with RyR1 in situ and in vitro; (2) IL-1 reduces SR Ca²⁺ leak through the RyR1 by stabilizing the Mg²⁺ inhibition; and (3) prolonged IL-1 incubation of intact muscle fibers compromises membrane integrity to provide a potential penetration pathway. That indeed SR Ca²⁺ release was affected by IL-1 was shown by the unaltered Ca²⁺ sensitivity of the contractile apparatus by IL-1 in the force transducer assay, and also directly by confocal Ca²⁺ fluorescence experiments. The effects of IL-1 on SR Ca²⁺ release were almost instantaneous and reversible once IL-1 interacted with RyR1 in the skinned fiber assay. This is not in contrast to the long incubation times needed for intact fibers to exert the action of IL-1 on membrane integrity, as, in the skinned fiber, the sarcolemma had been permeabilized either chemically or mechanically and immediate diffusional access to the myoplasm established (25). Physiological Ca²⁺ release occurs via DHPR–RyR1 interaction to reduce the Mg²⁺ affinity of RyR1 and, thus, disinhibition of Mg²⁺ block (11). Depolarization of the t-system and DHPR activation is a rather strong stimulus to open RyR1 (26). In the skinned fiber, this can be mimicked by acutely lowering myoplasmic Mg²⁺ concentration (25), although this is a somewhat milder stimulus for release (26). In our hands, lowering Mg²⁺ produced robust Ca²⁺ and force transients, which were completely and reversibly abolished in the presence of IL-1. The fact that caffeine could still induce SR Ca²⁺ release is reflected by caffeine (at 30 mM) strongly increasing the Ca²⁺ affinity on the RyR1 activation site (27). An explanation for the increase in caffeine-induced force levels after IL-1 incubations is most likely reflected by a reduced SR Ca²⁺ leak and consequent larger releasable SR Ca²⁺ pools. Apart from RyR1, SR Ca²⁺ leakage is also possible via the SERCA, particularly in heart muscle (28), whereas, in skeletal muscle, SR Ca²⁺ leak seems predominantly via RyR1 (29) or a recently described transient receptor channel, transient receptor potential canonical type 1 pathway (30). The fact that the SR Ca²⁺ retention index (24) increase and the spark frequency decrease upon IL-1 application were well reproduced by tetracaine, together with the finding that IL-1 coimmunoprecipitated with RyR1, but not with SERCA (Figure 4), clearly point toward RyR1 as the main target for this cytokine. Tetracaine has been used in previous studies in muscle to block spark frequencies (i.e., 6-fold reduction at 75–100 μM [31]), or caffeine-induced force transients (32). As pointed out by Pike and colleagues (32), tetracaine action strongly depends on both drug concentrations and pCa. In particular, our combination of incubation and washout times eliminated persistent blocking effects of tetracaine between recordings.

Concentration Effects of IL-1 and Potential Relations to Sepsis and Inflammatory Myopathies

The effects of IL-1 were most prominent at 25 ng/L, a concentration intriguingly reflecting blood levels found in patients with sepsis, whereas 10 ng/L typically correspond to healthy individuals (21, 33). In patients with sepsis who developed myopathy, substantial tissue infiltrates with macrophages and high local IL-1 expression patterns have been detected (21), pointing toward a required precondition for IL to invade individual muscle cells to exert the pathophysiological effects documented in the present study. Indeed, the compromised membrane integrity after long-term IL-1 incubation might be sufficient to provide an uptake pathway for this relatively small molecule by endocytosis, diffusion, or some other, yet to be determined transport mechanism. In diaphragm muscle, experimental sepsis increased the percentage of myofibers with compromised membrane integrity from less than 1% to up to 20%, whereas no effect was observed in soleus muscle (34, 35). Interestingly, in cultured cerebromicrovascular endothelial cells, an IL-1R–dependent transport mechanism of IL-1 across cell membranes has been suggested (36). Whether a similar, IL-1R–dependent mechanism also applies to skeletal muscle would be most interesting to address in future studies involving, for example, IL-1R–deficient mice (37). Both Western blots of small fiber bundles and immunostaining of chemically or mechanically skinned single EDL fibers could not detect any specific IL-1R1 protein expression. In muscle biopsies from patients with idiopathic inflammatory myopathies, Grundtman and colleagues (38) described IL-1R1 and IL-1R2 expression in muscle histology sections at the level of the sarcolemma and the nuclei. However, because no single fibers were employed, signal contributions from immune cell infiltrates in the interstitial space cannot be excluded. Because the mechanically skinned fiber allowed the comparison of IL-1R1 signals both from the unskinned (still sarcolemma containing) and the skinned side of the “cuff,” we can confidently say that IL-1R1 expression levels, if present, are below the detection limit of the Western blot and immunostaining performed, which confirms previous results (39). Finally, it is also possible that immune activation of the skeletal muscle inflammasome with secretion of IL-1 (40) may additionally contribute to the internal deposition of IL-1, as detected by our long-term overnight challenge of intact single fibers.

To the best of our knowledge, this is the very first study systematically investigating the action of the cytokine, IL-1α, on skeletal muscle Ca²⁺ homeostasis. Studies involving isolated cytokines or cytokine cocktails are important, yet very scarce for skeletal muscle. Probably best studied is the proinflammatory cytokine, TNF-α, incubation of which with intact diaphragm and limb muscle (flexor digitorum brevis [FDB]) fibers for 4 hours produced substantial decrement in tetanic force without altering underlying Ca²⁺ transients. Thus, it was concluded that TNF-α somehow reduced myofilament sensitivity toward Ca²⁺ (41). In C₂C₁₂ skeletal myotubes incubated with TNF-α for a 48-hour period, however, the opposite effect had been observed (i.e., TNF-α markedly depressed electrically evoked Ca²⁺ transients [42]). It is very likely that some of the multiple suggested slow signaling mechanisms involved in cytokine-mediated muscle weakness (reactive oxygen species, nitric oxide production, peroxidation, ATP depletion [5]) respond differentially to different prolonged exposure time versus concentration products. However, in the case of IL-1α in our study, it is highly unlikely that second messenger–related pathways contributed significantly to the observed effects in our skinned fiber preparation, where the myoplasmic environment is in equilibrium with a defined indefinite bath volume controlling the fiber cytosol, and no longer containing signaling molecules in any significant amounts (calculated dilution >1:10⁶). Therefore, the documented IL-1 effects are likely to be direct and are almost completely reversible. In the heart, IL-1 has been used to elucidate mechanisms of cardiac failure in models of septic cardiomyopathy. A very recent study showed that a 3-hour treatment of rat cardiomyocytes significantly reduced Ca²⁺ transients, fractional shortening, and SR Ca²⁺ content, but only when cells were cocultured with TNF-α (43). Either cytokine on its own only induced minor changes. However, IL-1 concentrations were approximately 100-fold higher compared with those used in our studies. Under these conditions, the authors deduced increased SR Ca²⁺ leak from increased spark frequencies in the presence of cytokines, in contrast to our findings in skeletal muscle and at more clinically relevant concentrations.

A New Model Explaining Critical Illness and Inflammatory Myopathies?

We suggest a new model that may explain some aspects of early muscle failure and weakness in critically ill patients, in particular in conditions involving a strong inflammatory response. Systemic inflammation is associated with leukocytosis and local tissue infiltration by immune cells (23). Activated macrophages secrete IL-1 (mostly IL-1β, but also the IL-1α isoform) into the muscle tissue, accumulating high tissue levels. As critical illness progresses, patient muscle fibers may become exposed to high local concentrations of IL-1 (23). In time, muscle membrane integrity is compromised, providing a passage for IL-1 to enter myocytes. These transient membrane leaks will be repaired by most cells (44), but also allow entry of cations and subsequent membrane depolarization that additionally interfere with muscle performance (45). This also explains why not all single fibers were still electrically excitable after extended IL-1 challenge. In addition, activated inflammasome also increases IL-1 expression within muscle cells (40). All this would enable IL-1 to associate with RyR1 within the cell, by-passing the usual IL-1R–mediated signaling cascade (46), or using it for IL-1R–mediated IL-1 entry (36) if surface receptors were present at large enough densities. Given sufficient repair mechanisms of the membrane after localized membrane damage (44), a general Ca²⁺ overload of muscle fibers and subsequent cell death seems unlikely. IL-1 bound to the RyR1 macromolecular complex then stabilizes Mg²⁺ inhibition of SR Ca²⁺ release, representing a reversible block of the release channel. Although reduced SR Ca²⁺ leak via RyR1 stabilizes or may even increase SR Ca²⁺ content, RyR1 inhibition remains during the course of cytokine burden. Our acute IL-1 assay was almost completely reversible in terms of the SR effects. However, this may be more puzzling in the critically ill patient. Prolonged sepsis, for example, induced vast ultrastructural changes to muscle architecture by selective proteolysis of myofilaments (47, 48). Some of these effects can be triggered by isolated cytokines (47). Although proteolysis is already stimulated within hours after mechanical ventilation (49) or sepsis (48), and can be just as rapidly reversible, it is probably the altered contractile architecture as a consequence of maintained proteolysis in ongoing critical illness that leads to sarcomere disruptions (50) and even “ghost sarcomeres” with selective myosin loss (48). The latter might be reversible only at a much longer time scale, and may be responsible for structurally related contractile dysfunction. Indeed, patients with myosin loss myopathy in CIMs usually take months to recover (51).

Past clinical studies employing anti–IL-1 therapy did not show any benefit for patients with sepsis, despite most promising results in rodents (52). However, all those studies explored mortality as the endpoint to evaluate treatment efficiency rather than muscle function. Thus, whether anti–IL-1 treatment would be able to improve muscle function or prevent/ameliorate CIM would be an interesting hypothesis for future studies.