β3-Integrin mediates satellite cell differentiation in regenerating mouse muscle

Abstract

Skeletal muscle satellite cells can sense various forms of environmental cues and initiate coordinated signaling that activates myogenesis. Although this process involves cellular membrane receptor integrins, the role of integrins in myogenesis is not well defined. Here, we report a regulatory role of β3-integrin, which was previously thought not expressed in muscle, in the initiation of satellite cell differentiation. Undetected in normal muscle, β3-integrin expression in mouse hindlimb muscles is induced dramatically from 1 to 3 d after injury by cardiotoxin. The source of β3-integrin expression is found to be activated satellite cells. Proliferating C2C12 myoblasts also express β3-integrin, which is further up-regulated transiently on differentiation. Knockdown of β3-integrin expression attenuates Rac1 activity, impairs myogenic gene expression, and disrupts focal adhesion formation and actin organization, resulting in impaired myoblast migration and myotube formation. Conversely, overexpression of constitutively active Rac1 rescues myotube formation. In addition, a β3-integrin-neutralizing antibody similarly blocked myotube formation. Comparing with wild-type littermates, myogenic gene expression and muscle regeneration in cardiotoxin-injured β3-integrin-null mice are impaired, as indicated by depressed expression of myogenic markers and morphological disparities. Thus, β3-integrin is a mediator of satellite cell differentiation in regenerating muscle.—Liu, H., Niu, A., Chen, S.-E., Li, Y.-P. β3-Integrin mediates satellite cell differentiation in regenerating mouse muscle.

Adult myogenesis involves the differentiation of muscle stem cells known as satellite cells or muscle progenitor cells (MPCs) through coordinated muscle-specific gene expression, cell migration, and fusion in response to such extrinsic cues as disease, injury, or training (1). However, the mechanism through which satellite cells sense and integrate environmental cues for myogenesis and conduct the progressive steps of myogenesis in an orderly manner remains poorly defined (2).

Integrins are a family of transmembrane heterodimeric glycoprotein receptors that physically link extracellular matrix (ECM) to the intracellular cytoskeleton at sites known as focal adhesions. Ligand binding to the extracellular domain of integrins induces integrin clustering and activation (3). In addition to their role in mediating cell adhesion, integrins integrate environmental cues across the plasma membrane and generate numerous intracellular signals (4, 5). Particularly, by mediating signals from the outside environment, integrins help to regulate many basic cell behaviors, such as cell differentiation, cell proliferation, and cell survival (6). Vertebrate skeletal muscle expresses the β1-integrin and its partners α1, α3, α4, α5, α6, α7, α9, and αv (7), and β1-integrin has been shown to mediate myogenesis through regulating myogenic gene expression, migration, and fusion of MPCs (8–10). However, β1-integrin is dispensable to myogenesis (11). We speculate that there are other β-integrins as part of a network of membrane receptors that sense and propagate myogenic cues in MPCs. Unlike β1-integrin, which is constitutively expressed in skeletal muscle, β3-integrin is not detected in normal muscle (12). However, β3-integrin is expressed in human muscle with inflammatory myopathies (13) and in isolated human myoblasts (14), which raises the possibility that β3-integrin is expressed in MPCs that are activated in regenerating muscle. There are two types of β3-integrin heterodimers, αvβ3 and αIIbβ3. The αvβ3 is widely present in various cells, including human myoblasts (14). On the other hand, the expression of αIIbβ3 is restricted largely to platelets and megakaryocytes (15). On the basis of the reported expression pattern of β3-integrin in muscle, we hypothesized that β3-integrin had a biological role in myogenesis and tested the hypothesis in the present study. Here, we show for the first time that β3-integrin expression is transiently induced in activated satellite cells during mouse muscle regeneration and that β3-integrin mediates myogenesis via regulating myogenic gene expression and migration of satellite cells.

MATERIALS AND METHODS

Animal use

Animal experimental protocols were approved in advance by the Animal Welfare Committee at the University of Texas Health Science Center (Houston, TX, USA). Cardiotoxin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in PBS (10 μM) and injected into soleus (100 μl) of male C57/BL6 or β3-integrin^−/− mice and wild-type littermates (Jackson Laboratory, Bar Harbor, ME, USA) at 6 to 8 wk of age to induce necrotic injury. At various time points, solei were excised from euthanized mice for described analyses.

Myogenic cell culture

C2C12 myoblasts (American Type Culture Collection, Manassas, VA, USA) were cultured in growth medium (GM; DMEM supplemented with 10% FBS) at 37°C under 5% CO₂. At 85% confluence, myoblast differentiation was induced by replacing GM with differentiation medium (DM; DMEM supplemented with 2% heat-inactivated horse serum). When indicated, mouse β3-integrin-neutralizing antibody (2C9.G2, BD Biosciences, San Jose, CA, USA) or preimmune IgG (Sigma-Aldrich) was added to DM at the beginning of differentiation.

Western blot analysis

Western blot analysis was performed as described previously (16), using protein lysates prepared from cells or muscle. Protein concentrations of the samples were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Antibodies for p38 MAPK and β3-integrin were from Cell Signaling Technology (Beverly, MA, USA), antibody against p21 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-Rac1 monoclonal antibody was from Cell Biolabs (San Diego, CA, USA), anti-myogenin monoclonal antibody was from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA), antibody for α-tubulin was from Sigma-Aldrich, and antibody for GAPDH was from Millipore (Billerica, MA, USA).

Transfection of siRNA

The on-target smart pool siRNA specific for β3-integrin and control siRNA were purchased from Dharmacon (Denver, CO, USA) and Ambion (Austin, TX, USA), respectively, and were introduced into C2C12 myoblasts by electroporation (5 μg) with the Nucleofector system (Lonza, Walkersville, MD, USA), according to manufacturer's protocol. Differentiation was induced after 48 h. When indicated, at 24 h of siRNA transfection, Rac1 L61 recombinant adenovirus (Cell Biolabs) was transduced into C2C12 myoblasts at 2.1 × 10⁷ viral particles/cm² of area. Differentiation was induced after 24 h.

Immunofluorescence

Cells were seeded on collagen I (Sigma-Aldrich)-coated coverslips for fusion and differentiation analysis in triplicate. At indicated times, cells were fixed with 3.7% formaldehyde for 15 min at room temperature (RT), permeabilized with 0.2% Triton-X 100 at RT for 10 min, and blocked with 1% BSA in PBS at RT for 30 min. Frozen sections of excised soleus were fixed with 4% paraformaldehyde. Immunofluorescence labeling of muscle sections was carried out using the fluorescein M.O.M. Kit (Vector Laboratories, Burlingame, CA, USA) and the following antibodies. Primary antibodies for myogenin (F5D), MHC (MF20), embryonic MHC (F1.652), and Pax7 were obtained from the Development Studies Hybridoma Bank; anti-MyoD (5.8A) was from BD Biosciences; anti-β3-integrin was obtained from Millipore; and anti-phospho-tyrosine was from Cell Signaling Technology. FITC-labeled phalloidin was from Invitrogen. Secondary antibodies (donkey anti-mouse Alexa-488, donkey anti-mouse Alexa-598, donkey anti-rabbit Alexa 488, and donkey anti-rabbit Alexa 598) were purchased from Invitrogen. Immunofluorescence labeling of cultured cells on coverslips was performed using the standard method. Stained samples were covered with antifade mounting medium with or without DAPI (Vector Laboratories). Images were acquired using a microscope (model Axioskop 2; Carl Zeiss, Jena, Germany) equipped with the MetaView software with an ×10 objective, Applied Precision DeltaVision Deconvolution System with an ×40, 0.65–1.35 NA objective (Applied Precision, Inc., Issaquah, WA, USA), or a Nikon A1 confocal microscope with ×60 objective (Nikon, Tokyo, Japan). Adjustment of brightness, contrast, color balance, and final image size was achieved using Adobe Photoshop CS (Adobe Systems, San Jose, CA, USA). ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA) was used to quantify focal adhesion cluster area.

Histology

Paraffin-embedded and hematoxylin-and-eosin-stained soleus sections were prepared by the Breast Center Pathology Core (Baylor College of Medicine, Houston, TX, USA).

Statistical analysis

Data are presented as means ± se and were analyzed with Student's t test or 1-way ANOVA, as appropriate, using SigmaStat software (Systat Software, Inc., San Jose, CA, USA). A value of P < 0.05 was considered to be statistically significant.

RESULTS

β3-Integrin expression is induced in activated satellite cells of regenerating mouse muscle

Utilizing the cardiotoxin injury model, we examined the protein content of β3-integrin in regenerating adult mouse soleus. Although β3-integrin was hardly detectable in uninjured soleus, a dramatic increase of this protein was observed from 1 to 3 d of injury (Fig. 1A). It is noteworthy that the expression of β3-integrin preceded myogenin, a transcription factor required for muscle gene expression and thus a marker of myogenesis (1). To evaluate whether the expression of β3-integrin in regenerating muscle is fiber type specific, β3-integrin levels in tibialis anterior (TA) were also determined. Similar induction of β3-integrin expression was observed in cardiotoxin-injured TA (Supplemental Fig. S1). Thus, β3-integrin expression is induced similarly in both slow and fast-twitch muscles during regeneration. To determine whether satellite cells are the source of β3-integrin expression, immunofluorescence staining of β3-integrin was performed on frozen sections of soleus before and after injury by cardiotoxin. In uninjured soleus (d 0), β3-integrin was not detected in quiescent satellite cells identified by Pax7 staining (Fig. 1B). However, in soleus injured for 3 d, β3-integrin was detected in activated satellite cells that were MyoD positive (Fig. 1C). Thus, β3-integrin is expressed by activated satellite cells. To further evaluate the relationship between β3-integrin expression and myogenesis in MPCs, we examined β3-integrin levels in the mouse myoblast cell line C2C12 that is frequently used in the study of myogenic differentiation. A readily detectable level of β3-integrin was present in proliferating C2C12 myoblasts, which was then transiently up-regulated during the first 24 h of differentiation induced by switching cells to low-serum DM from serum-rich GM (Fig. 1D). Again, the up-regulation of β3-integrin preceded myogenin expression. Thus, β3-integrin expression takes place in proliferating MPCs, and is further up-regulated during the early phase of differentiation. Therefore, β3-integrin may have a role in muscle regeneration, particularly, in myogenic differentiation.

Figure 1.

Expression of β3-integrin is induced in activated satellite cells in regenerating muscle. A) β3-Integrin expression is induced in regenerating mouse soleus. Mouse soleus was injured by direct injection of 100 μl of 10 μM cardiotoxin. At indicated times, soleus was excised from euthanized mice. Muscle homogenates were analyzed for β3-integrin and myogenin expression by Western blot analysis. Sample loading quality was monitored by probing p38 MAPK that was relatively constant in cardiotoxin-injured muscle. B) Quiescent satellite cells do not express β3-integrin. Frozen sections of uninjured soleus were subjected to immunofluorescence staining of β3-integrin (β3-ITG). Costaining of Pax7 was performed to identify satellite cells. Nuclei were stained with DAPI. C) β3-Integrin expression by regenerating soleus takes place in activated satellite cells. Frozen sections of soleus injured by cardiotoxin for 3 d were subjected to immunofluorescence staining of β3-integrin. Costaining of MyoD was performed to identify activated satellite cells. Scale bar = 50 μm. D) β3-Integrin expression by proliferating C2C12 myoblasts is up-regulated during differentiation. C2C12 myoblasts were cultured in GM and then switched to DM. Cells were harvested at indicated times, and cell lysates were analyzed for β3-integrin and myogenin expression by Western blot analysis. Sample loading quality was monitored by probing α-tubulin. Densitometry analysis was performed to quantify detected bands. *P < 0.05; ANOVA.

β3-Integrin is crucial for myogenic gene expression, migration, and fusion of C2C12 myoblasts

To investigate the potential role of β3-integrin in myogenesis, we conducted in vitro studies in C2C12 myoblasts by knocking down β3-integrin expression utilizing β3-integrin-specific siRNA (Fig. 2A). Myoblasts were then incubated in DM at a low cell density that did not allow cells to contact each other for 72 h, which excluded the influence of cell-cell contact on myoblast differentiation. Loss of β3-integrin reduced the number of myoblasts that express myogenic marker myogenin and myosin heavy chain (MHC; Fig. 2B). This observation suggests that β3-integrin mediates myogenic gene expression.

Figure 2.

Myogenic gene expression in C2C12 myoblasts is dependent on β3-integrin. A) Expression of β3-integrin was knocked down by β3-integrin siRNA in C2C12 myoblasts. C2C12 myoblasts were transfected with control or β3-integrin-specific siRNA. After 48 h, β3-integrin in cell lysates was determined by Western blot analysis. B) Knockdown of β3-integrin impairs myogenic gene expression in C2C12 myoblasts. C2C12 myoblasts transfected with control or β3-integrin (β3-ITG) siRNA were plated at low cell density and incubated in DM for 72 h. Immunofluorescence staining of myogenin or MHC was performed to evaluate myogenic gene expression (green). Nuclei were stained with DAPI (blue). Myogenin- or MHC-expressing cells were counted in random fields and presented as a percentage of total cells in a field. Scale bar = 200 μm. *P < 0.05, **P < 0.01; Student's t test.

Considering that β3-integrin is a focal adhesion protein and focal adhesions mediate cell migration that is necessary for cell fusion and myotube formation, C2C12 myoblasts were subjected to immunofluorescence analysis using an antibody against phosphorylated tyrosine to track focal adhesions and FITC-labeled phalloidin to track F-actin. As shown in Fig. 3A, β3-integrin deficiency caused disruption in the formation of focal adhesion and F-actin organization in myoblasts incubated in GM. After switching to DM from GM for 24 h, focal adhesions in control myoblasts were dramatically enhanced. However, focal adhesion formation in β3-integrin-deficient myoblasts was much weaker, as indicated by shrinkage in focal adhesion cluster area (Fig. 3B). To determine whether the disruption in focal adhesion formation and F-actin organization due to β3-integrin deficiency results in impaired myoblast migration, myoblast migration was evaluated using the transwell assay. We observed that nearly 60% lower number of β3-integrin-deficient myoblasts migrated to the lower chamber during 5 h incubation in DM in comparison to control myoblasts (Fig. 3C). In addition, myotube formation was blocked in β3-integrin-deficient myoblasts after 96 h incubation in DM, resulting in a greatly diminished fusion index (Fig. 3D). To verify whether β3-integrin-mediated signaling, rather than a general cytoskeletal disruption due to the loss of β3-integrin, is responsible for the impaired differentiation observed in β3-integrin knockdown myoblasts, we blocked β3-integrin signaling by including a β3-integrin-neutralizing antibody (17) in DM. The antibody treatment blocked myotube formation by normal C2C12 myoblasts in a dose-dependent manner (Fig. 4). These data suggest that β3-integrin-mediated signaling is critical to myogenic gene expression, MPC migration, and fusion.

Figure 3.

Focal adhesions, F-actin organization, myoblast migration, and myotube formation are dependent on β3-integrin. A) Normal focal adhesions and F-actin organization are dependent on β3-integrin. C2C12 myoblasts transfected with control or β3-integrin (β3-ITG)-specific siRNA were cultured in GM or switched to DM for 24 h. Focal adhesion was evaluated by immunofluorescence staining of phosphorylated tyrosine (p-Tyrosine, red) and F-actin (phalloidin, green). B) β3-Integrin deficiency blocks differentiation-induced increase in focal adhesion cluster. Focal adhesion cluster area per cell was measured by using ImageJ software. *P < 0.05; Student's t test. C) Myoblast migration is dependent on β3-integrin. C2C12 myoblasts transfected with control or β3-integrin-specific siRNA were plated on top of the transwells (2.5×10⁵ cells/well). After incubation for 5 h in DM at 37°C, myoblasts that migrated to the lower face of membrane were stained with crystal violet and counted. *P < 0.001; Student's t test. D) Myotube formation is dependent on β3-integrin. C2C12 myoblasts transfected with control or β3-integrin-specific siRNA were induced to differentiate by incubation in DM for 96 h. Immunofluorescence staining was performed with an antibody against MHC (MF-20, green). Nuclei were stained with DAPI (blue). Myotube formation was quantified by using the fusion index analysis, which was derived by counting the number of myotubes having ≥5 nuclei against the total number of nuclei in the field; ≥ 2000 nuclei/field were counted from randomly selected fields. Scale bars = 100 μm (A); 200 μm (D). *P < 0.01 vs. control siRNA-transfected cells; Student's t test.

Figure 4.

β3-Integrin-mediated signaling is critical to myotube formation. A mouse β3-integrin (β3-ITG)-neutralizing antibody or preimmune IgG (control) was included at 1 or 3 μg/ml in DM during differentiation. After 72 h incubation in DM, myotube formation was analyzed by immunofluorescence staining of MHC. Fusion index was calculated by counting the number of myotubes having ≥3 nuclei against the total number of nuclei in the field. *P < 0.05, **P < 0.01 vs. control antibody; ANOVA.

β3-Integrin is required for maintaining Rac1 GTPase activity in differentiating C2C12 myoblasts

In an effort to identify the intracellular signaling events critical to myogenesis that are dependent on β3-integrin, we found that the level of active Rac1 (Rac1-GTP) was diminished in β3-integrin-deficient myoblasts during the first 24 h of differentiation, while total Rac1 level was not altered (Fig. 5A). Because of the important role of Rac1 in mediating myogenic gene expression and cell migration (18), we further assessed whether attenuated Rac1 activity causes the impairment of myogenesis in β3-integrin-deficient myoblasts by ectopically expressing a constitutively active mutant of Rac1, Rac1 L61, in β3-integrin-deficient myoblasts. The expression of Rac1 L61 resulted in a 2.2-fold increase in active Rac1, as compared to GFP expression (Fig. 5B). Expression of Rac1 L61 partially restored myotube formation (Fig. 5C). These data suggest that β3-integrin promotes myogenesis, at least partially, via Rac1.

Figure 5.

Rac1 GTPase mediates β3-integrin effect on myoblast differentiation. A) Rac1 GTPase activity in differentiating myoblasts is dependent on β3-integrin. C2C12 myoblasts were transfected with control or β3-integrin (β3-ITG)-specific siRNA. After incubation in DM for indicated times, cells were harvested. Rac1-GTP (active form of Rac1) was pulled down from cell lysate using PAK1 PBD agarose beads (Cell Biolabs). Pulled-down Rac1-GTP and total Rac1 in cell lysate were analyzed by Western blot analysis. Densitometry data were analyzed by ANOVA. B) Overexpression of constitutively active Rac1 (Rac1 L61) in C2C12 myoblasts. At 24 h after β-3-integrin-specific siRNA transfection, C2C12 myoblasts were transduced with adenovirus encoding Rac1 L61 or GFP. After 24 h, Rac1 L61 expression was verified by Western blot analysis of Rac1-GTP and total Rac1. C) Active Rac1 rescues myotube formation by β3-integrin-deficient myoblasts. C2C12 myoblasts that had been transfected with β3-integrin-specific siRNA and transduced with adenovirus encoding Rac1 L61 or GFP were switched to DM and differentiated for 96 h. Immunofluorescence staining of MHC was performed to assess myotube formation. Fusion index was calculated as described in Fig. 3. Scale bar = 200 μm. *P < 0.05.

β3-Integrin is critical to myogenesis and muscle regeneration induced by cardiotoxin injury

To assess the physiological significance of β3-integrin in myogenesis and muscle regeneration in vivo, we investigated whether deficiency in β3-integrin impairs myogenesis and muscle regeneration by utilizing β3-integrin^−/− mice. The initiation of myogenesis requires MPC expression of the cyclin-dependent kinase (CDK) inhibitor p21 to exit cell cycle, and myogenin to turn on muscle gene expression. On d 3 of cardiotoxin injury when myogenesis takes place, expression of early differentiation markers p21 and myogenin in β3-integrin-null soleus was depressed (Fig. 6A). Similarly, myogenin expression was depressed in β3-integrin-null TA (Supplemental Fig. S2). Thus, β3-integrin appears to be critical to the initiation of myogenesis. In addition, regenerating myofibers expressing embryonic MHC (eMHC) prevailed on d 3 of injury in wild-type soleus, but were almost nonexistent in β3-integrin-null soleus (Fig. 6B), confirming a dependence of satellite cell differentiation on β3-integrin. Finally, on d 5 of injury, regenerated myofibers with centralized nuclei prevailed in wild-type soleus, but in β3-integrin-null soleus, few regenerated myofibers were seen, and an abnormally high level of infiltrated inflammatory cells was present (Fig. 6C), indicating impairment of regeneration. These data suggest that β3-integrin is crucial to myogenesis and muscle regeneration.

Figure 6.

Myogenesis and muscle regeneration are impaired in β3-integrin^−/− mice. A) Initiation of myogenesis is impaired in β3-integrin-null soleus. Soleus was harvested from β3-integrin^−/− (β3-ITG^−/−) mice and wild-type (WT) littermates before (d 0) and after injury by cardiotoxin injection (d 3). Muscle homogenates were analyzed by Western blot. *P < 0.05 vs. WT d 3; Student's t test. B) Myogenic differentiation is impaired in β3-integrin-null soleus. Frozen sections prepared from mouse soleus injured by cardiotoxin for 3 d were analyzed by immunofluorescence staining of eMHC. Nuclei were stained with DAPI. C) Muscle regeneration is impaired in β3-integrin-null soleus. Paraffin-embedded muscle sections prepared from mouse soleus injured by cardiotoxin for 5 d were subjected to hematoxylin and eosin staining. Scale bars = 100 μm.

DISCUSSION

The current study demonstrates a physiological role of β3-integrin in the initiation of myogenesis in adult muscle. During muscle regeneration, the expression of β3-integrin is induced in activated satellite cells transiently when myogenesis takes place. In the event that this expression is perturbed, myogenesis is impaired and muscle regeneration is disrupted. Detailed analyses in cultured myoblasts reveal that at least two independent components of myogenesis, myogenic gene expression and migration of MPCs, are mediated by β3-integrin. Although deficiency in β3-integrin also results in a blockade in myoblast fusion, it is not clear whether it is secondary to the impairment in myoblast migration that precedes myoblast fusion.

Although a previous study reported β3-integrin expression in cultured human myoblasts (14), the possibility exists that the β3-integrin detected was actually from the nonmuscle cells present in the primary human myoblast cultures (12). In addition, it was not known whether β3-integrin was induced before or after the activation of satellite cells. Therefore, the identification of activated satellite cells in regenerating muscle as the source of β3-integrin expression is important. We further show that the transient up-regulation of β3-integrin during the early phase of myogenesis is important for myogenesis, at least partially, due to the dependence of Rac1 activity on β3-integrin. From a technical point of view, as a cell surface protein (also known as CD61), β3-integrin could be used as a marker for activated satellite cells.

The disruption of focal adhesion formation and actin organization in β3-integrin knockdown myoblasts indicates that β3-integrin is an important component of focal adhesion in MPCs. Blockade of myotube formation by β3-integrin-neutralizing antibody further supports that β3-integrin mediates outside-in signaling initiated by extracellular ligands. In this context, we show that β3-integrin is required for the maintenance of Rac1 activity in differentiating myoblasts. Rac1 promotes myogenesis at least partially through the activation of the critical regulator of myogenic gene expression, p38 MAPK (18). We observed that expression of p38-dependent myogenic markers myogenin, p21, and MHC (19) are attenuated in regenerating β3-integrin-null muscle. In addition, Rac1 mediates cell migration due to its critical role in the polymerization of filamentous actin, which results in lamellipodium formation and membrane ruffling at the leading edge of migrating cells (20). The rescue of myotube formation by β3-integrin-deficient myoblasts by a constitutively active Rac1 mutant supports Rac1 as a mediator of the myogenesis-promoting signaling of β3-integrin.

The actions of β3-integrin on myogenesis demonstrated in the present study appear to overlap with some of the actions of β1-integrin on myogenesis (8, 9) and Rac1 activity (21). This redundancy could explain why β1-integrin is dispensable to myogenesis. On the other hand, deficiency in β3-integrin disrupts myogenesis and muscle regeneration while β1-integrin is intact, suggesting that β3-integrin has a unique role in myogenesis. The different expression pattern of β3-integrin from that of β1-integrin suggests that β3-integrin may act specifically for the initiation of myogenesis, whereas β1-integrin may have broader roles in the maintenance of muscle.

Collectively, the current study demonstrates for the first time a physiological role of β3-integrin in the initiation of myogenesis. Because of the complexity of integrin signaling, further study of the detailed mechanism of β3-integrin signaling in MPCs is warranted.