Atypical behavior of NFATc1 in cultured intercostal myofibers
© Robison et al.; licensee BioMed Central Ltd. 2014
Received: 1 July 2013
Accepted: 28 November 2013
Published: 3 January 2014
The NFATc transcription factor family is responsible for coupling cytoplasmic calcium signals to transcription programs in a wide variety of cell types. In skeletal muscle, these transcription factors control the fiber type in response to muscle activity. This excitation-transcription (E-T) coupling permits functional adaptation of muscle according to use. The activity dependence of these transcription programs is sensitive to the firing patterns of the muscle, not merely the period of activity, enabling a nuanced adaptation to various functional tasks.
Isolated skeletal muscle fibers expressing exogenous fluorescent NFATc1 were studied by confocal microscopy under stimulation both with and without pharmacological inhibitors. Western blots of whole muscle lysates were also used.
This study investigates the activity dependent response of NFATc1 skeletal muscle fibers cultured from mice, comparing fibers of respiratory origin to muscles responsible for limb locomotion. Using patterns of stimulation known to strongly activate NFATc1 in the commonly cultured flexor digitorum brevis and soleus muscles, we have observed significant deactivation of NFATc1 in cultured intercostal muscle fibers. This effect is at least partially dependent on the action of JNK and CaMKII in intercostal fibers.
Our findings highlight the role of lineage in the NFAT pathway, showing that the respiratory intercostal muscle fibers decode similar E-T coupling signals into NFAT transcriptional programs in a different manner from the more commonly studied locomotor muscles of the limbs.
KeywordsNFATc1 Skeletal muscle Respiratory muscle Intercostal muscle Excitation-transcription coupling
The functional adaptability of skeletal muscle is an elegant regulatory phenomenon, coupling transcriptional adaptation to a particular function in the role to which the muscle is adapting. The cellular events coupling excitation and contraction with transcriptional activity are known collectively as excitation-transcription (E-T) coupling [1, 2]. This process is perhaps best illustrated by cross-reinnervation [3, 4] studies in which the target muscle begins to take on the properties of the muscle with which it has had its motor neurons exchanged. However, there is also a substantial effect of muscle lineage which limits the degree to which a muscle is capable of adapting to a new activity paradigm. This has been noted in adult muscle [5, 6], but lineage effects can also be observed early in developmental stages .
Regulation of CN is primarily by relief of autoinhibition upon Ca2+ dependent binding to calmodulin (CaM) , although some additional Ca+2 dependence may be conferred by the B subunit . Adjacent to the SRR of NFATc1 are two CN binding domains , responsible for targeting the Ca2+/CaM dependent phosphatase activity to the SRR, permitting the activation of the canonical NFATc1 activation pathway [10, 14, 15, 18].
Muscle activity is also known to promote the activity of several protein kinases, providing additional layers of potential regulation. The c-Jun N-terminal kinases (JNK) and calmodulin dependent kinases (CaMK) may be particularly relevant. JNK, a MAPK, shows strong activation following activity in skeletal muscle [19, 20]. Although JNK, like many MAPKs, is somewhat promiscuous, it is of relevance to the activation of NFATc1 by phosphorylation of sites in the CN binding domains flanking the SRR. This results in inhibition of the interaction of CN with the transcription factor and inactivation of NFATc1 .
CaMKII actually shares an activation mechanism with CN, and it is therefore unsurprising that it is activated by muscle activity . This kinase has an established role in E-T coupling though the histone deacetylases  but also targets CN for phosphorylation at Ser197, thus inhibiting phosphatase activity .
In this study we report reversed NFATc1 signaling in isolated muscle fibers under identical stimulation depending on the muscle of origin. This behavior is at least partially dependent on the activity regulated kinases CaMKII and JNK. We propose that altered expression levels of these kinases and CN result in a change in the inhibition/activation balance of activity-dependent signaling in different muscles, accounting for this atypical behavior. These findings have substantial implications for the use of limb muscles as a general model for skeletal muscle plasticity and function and highlight the underlying diversity of function and adaptability in skeletal muscle.
Six- to eight-week-old female CD1 mice were euthanized and flexor digitorum brevis (FDB), soleus (Sol) and intercostal (ItC) muscles were removed. The procedures for isolating single muscle fibers have been previously described . In brief, ItC and FDB muscles were dissected and enzymatically digested, then mechanically triturated with a polished glass pipette. Resulting fibers were maintained suspended in MEM supplemented with 10% FBS until plating on laminin coated dishes or use in releasable calcium experiments. Isolated fibers were kept in an incubator at 37°C under 5% CO2. Muscles for tissue lysates were lysed in Tissue Protein Extraction Reagent (Thermo Scientific, Wilmington, DE, USA with protease and phosphatase inhibitors and then stored at −80°C until ready for use.
Extracted muscles were ground with a pestle under TPER lysis buffer supplemented with protease inhibitors and kept on ice with periodic agitation for up to three hours until tendons were clean. Insoluble debris was removed by centrifuging samples at 4°C for ten minutes at 5,000 RPM. The supernatant was removed and concentration was estimated by Nanodrop-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA. Approximately 30 μg per lane was denatured at 74°C for ten minutes and loaded onto precast 4 to 12% polyacrylamide gels under reducing conditions. After transfer, membranes were blocked in 5% milk with 0.1% Tween and then cut according to molecular weight so that samples could be normalized to proteins run in the same lane. Membrane sections were incubated overnight in primary antibodies. Primary antibodies were washed out then secondary fluorescent antibodies were applied for one hour and washed out. All antibodies used are commercially available as follows: α-actinin: 11 M4845, SigmaAldrich, St. Louis, MO, USA; JNK: SAB4200176, SigmaAldrich, St. Louis, MO, USA; CaMKII: sc-9035, Santa Cruz Biotechnology, Dallas, TX, USA; CN: 07–1491 Millipore, Billerica, MA, USA; secondary antibodies: a21235 and a21428, Life Technologies Invitrogen, Grand Island, NY, USA. Membranes were imaged on a Typhoon FLA 9500 biomolecular imager (GE Healthcare Life Sciences, Pittsburg, PA, USA). Bands were measured using ImageJ (NIH, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/), following automated background subtraction. JNK/CaMKII/CN bands were normalized to the α-actinin band from the same lane and to the average of the ItC samples.
After initial processing in ImageJ, data was handled in Origin 8 (OriginLab Corporation, Northampton, MA, USA). Significance of NFATc1 translocation was determined by Mann–Whitney U-tests due to significant deviations from normality in the data. Significance of measurements of JNK/CaMKII/CN was carried out by ANOVA, followed by pairwise unpaired t-tests. Significance of calcium measurements on kinase inhibited fibers was determined by paired t-tests. Bonferroni multiple comparison correction was used to adjust the significance threshold within each experiment. Data from Figures 2 and 3 were considered as a single set for the purposes of this correction due to the pooling of data from intercostal fibers with no pharmacological treatment.
All animals used in this study were housed and used in accordance with procedures approved by the University of Maryland Baltimore IACUC under protocol number 0412012.
Our initial observation that NFATc1 showed abnormally high resting activation in cultured ItC fibers  leads us to examine the activity of NFATc1 in these fibers more closely. Fibers derived from both the soleus (Figure 2D-F) and FDB (Figure 2G-I) show increased nuclear import of NFATc1 characteristic of the canonical activation pathway following 90 minutes of repetitive electrical field stimulation. In contrast, the ItC fibers showed dramatic nuclear efflux of NFATc1 in response to the same activity pattern (Figure 2A-C). This effect was observed in the overwhelming majority of ItC nuclei (Figure 2K) and represents a significant difference from both FDB (P = 6.8 x 10-20, < 0.0125) and Sol (P = 2.7 x 10-17, < 0.0125) fibers undergoing similar treatment (Figure 2L).
Inhibition of activity regulated kinases
A probable mechanism for the activity dependent inactivation of NFATc1 in the intercostal muscles is a comparatively high level of activity regulated kinases relative to the canonical pathway of CN, such that the canonical activity of CN is simply overwhelmed. If this is so, inhibition of these kinases should permit the canonical CN pathway to proceed, resulting in the more typical activity induced activation of NFATc1. Using the inhibitors KN62 and SP600125 (inhibitors of CaMKII and JNK, respectively), we find that antagonizing these kinases results in subpopulations (Figure 3A) which show significant activity dependent NFATc1 activation (Figure 3B) in response to both CaMKII (P = 2.7 x 10-7, < 0.0083) and JNK (P = 5.9 x 10-9, < 0.0083) inhibition. Both KN62 and SP600125 treated intercostal fibers maintain the ability to release calcium (Figure 3D) and twitch when subjected to electrical field stimulation (P > 0.0125). Similar treatment of Sol and FDB fibers had the anticipated result of increasing the level of translocation in response to stimulation, although only in the FDB fibers does the difference reach significance (P = 1.7 x 10-5, 6.5 x 10-9, < 0.0083 for KN62 and SP600125 treated FDB fibers respectively).
Kinase/phosphatase expression levels
The role of NFATc1 in skeletal muscle plasticity and E-T coupling is critical . While the core pathway has been thoroughly studied, this has previously been done in a limited variety of skeletal muscles which are routinely used to model skeletal muscles as a whole . This has led us to overlook interesting phenomena in other muscles. The results reported here highlight the need to examine currently understudied muscles.
In this study we demonstrate a previously unreported activity dependent inactivation of NFATc1 in intercostal skeletal muscle fibers. This inactivation is CaMKII and JNK dependent, and may substantially alter the way in which the intercostal muscles adapt to physical activity compared with more commonly studied skeletal muscles.
Our previously reported observation of elevated basal NFATc1 activation in isolated intercostal muscle fibers  spurred us to examine the activation of NFATc1 in this model. To our surprise, we found the canonical activation pattern reversed when we applied stimulation previously shown to strongly activate NFATc1 . The nature of the isolated fiber model lead us to initially hypothesize that the basal levels of the activity-inducible kinases and phosphatases may be different in intercostal fibers compared with more commonly studied muscles. Changes in the relative levels of these proteins might de-emphasize the canonical role of CN and would persist in the absence of paracrine and nervous input for some time (factors which are largely absent in isolated muscle fiber cultures).
Although our data are unable to support a full discussion of the functional significance of this phenomenon without examining the downstream transcripts under the control of NFAT, it does raise interesting implications. If NFATc1 behaves this way in vivo and no other pathways interfere, it would imply that endurance training of these muscles leads the muscle to transform toward the lower endurance fast phenotypes. Although the fiber types present in respiratory muscles are not frequently considered, they have been extensively studied in respiratory diseases such as chronic obstructive pulmonary disease (COPD). One tantalizing result from this work is that, as a result of continuous labored breathing, the intercostals in COPD have been observed to undergo a slow to fast transformation .
Activity inducible kinase inhibition
The kinase activities of CaMKII and JNK are both increased by muscle activity. Work in other tissues has established that both kinases antagonize the activation of NFATc1 [12, 23]. This makes them prime candidates to drive the unusual inactivation observed in the ItC muscle fibers. JNK has been shown in T-cells to phosphorylate NFATc1 directly on the CN targeting motif, preventing CN dependent dephosphorylation . More recent work in cardiomyocytes indicates that CaMKII directly phosphorylates CN, inhibiting its activity . Our results show that the activity dependent inactivation of NFATc1 in intercostal muscle fibers can be at least partially reversed by inhibition of either of these kinases, indicating that they both also play a significant role in this pathway in at least some skeletal muscles.
Kinase/phosphatase expression levels
Our results do not clearly indicate one kinase/phosphatase as the key player behind the atypical behavior of NFATc1 in isolated intercostal muscle fibers. However we do show significant changes in the expression levels of relevant kinases/phosphatases between different types of muscle. While we do not rule out more transient mechanisms, we hypothesize that lineage dependent differences in expression levels of kinases and phosphatases in the NFATc1 activation pathway are the simplest and most plausible mechanism for the activity dependent NFATc1 inactivation reported here.
The mechanisms coupling excitation to transcription in muscle are complex and delicate. Although we examine only a small part of the E-T coupling system here, we show clearly that the dominant mechanisms can and do differ from muscle to muscle. The completely opposite responses to identical activity patterns demonstrate a need for careful model selection in studying E-T coupling and highlight the role played by lineage in muscle type determination.
Calmodulin dependent kinases
Calcium/calmodulin dependent protein kinase II
Casein kinase 1
Chronic obstructive pulmonary disease
Dual-specificity tyrosine-phosphorylation regulated kinase
- E-T coupling:
Flexor digitorum brevis
Glycogen synthetase kinase 3
c-Jun NH2-terminal kinase
Nuclear factor of activated T-cells, calcineurin-dependent 1
- SP repeats:
Serine rich region.
This work was supported by NIH grant R01-AR056477 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases. PR was supported by NIH training grants T32-AR007592 and T32-HL072751.
- Gundersen K: Excitation-transcription coupling in skeletal muscle: the molecular pathways of exercise. Biol Rev 2011, 86: 564-600. 10.1111/j.1469-185X.2010.00161.xPubMed CentralView ArticlePubMedGoogle Scholar
- Hernández-Ochoa EO, Robison P: Excitation-contraction coupling and excitation-transcription coupling in skeletal muscle. Biochem Pharmacol 2012, 1: e117.View ArticleGoogle Scholar
- Sréter FA, Luff AR, Gergely J: Effect of cross-reinnervation on physiological parameters and on properties of myosin and sarcoplasmic reticulum of fast and slow muscles of the rabbit. J Gen Physiol 1975, 66: 811-821. 10.1085/jgp.66.6.811View ArticlePubMedGoogle Scholar
- Bacou F, Rouanet P, Barjot C, Janmot C, Vigneron P, D’albis A: Expression of myosin isoforms in denervated, cross-reinnervated, and electrically stimulated rabbit muscles. Eur J Biochem 1996, 236: 539-547. 10.1111/j.1432-1033.1996.00539.xView ArticlePubMedGoogle Scholar
- Kalhovde JM, Jerkovic R, Sefland I, Cordonnier C, Calabria E, Schiaffino S, Lømo T: “Fast” and “slow” muscle fibres in hindlimb muscles of adult rats regenerate from intrinsically different satellite cells. J Physiol 2005, 562: 847-857. 10.1113/jphysiol.2004.073684PubMed CentralView ArticlePubMedGoogle Scholar
- Gauthier GF, Burke RE, Lowey S, Hobbs AW: Myosin isozymes in normal and cross-reinnervated cat skeletal muscle fibers. J Cell Biol 1983, 97: 756-771. 10.1083/jcb.97.3.756View ArticlePubMedGoogle Scholar
- DiMario JX, Stockdale FE: Both myoblast lineage and innervation determine fiber type and are required for expression of the slow myosin heavy chain 2 gene. Dev Biol 1997, 188: 167-180. 10.1006/dbio.1997.8619View ArticlePubMedGoogle Scholar
- Liu Y, Cseresnyes Z, Randall WR, Schneider MF: Activity-dependent nuclear translocation and intranuclear distribution of NFATc in adult skeletal muscle fibers. J Cell Biol 2001, 155: 27-39. 10.1083/jcb.200103020PubMed CentralView ArticlePubMedGoogle Scholar
- McCullagh KJ, Calabria E, Pallafacchina G, Ciciliot S, Serrano AL, Argentini C, Kalhovde JM, Lømo T, Schiaffino S: NFAT is a nerve activity sensor in skeletal muscle and controls activity-dependent myosin switching. Proc Natl Acad Sci U S A 2004, 101: 10590-10595. 10.1073/pnas.0308035101PubMed CentralView ArticlePubMedGoogle Scholar
- Schiaffino S, Serrano A: Calcineurin signaling and neural control of skeletal muscle fiber type and size. Trends Pharmacol Sci 2002, 23: 569-575. 10.1016/S0165-6147(02)02111-9View ArticlePubMedGoogle Scholar
- Vihma H, Pruunsild P, Timmusk T: Alternative splicing and expression of human and mouse NFAT genes. Genomics 2008, 92: 279-291. 10.1016/j.ygeno.2008.06.011PubMed CentralView ArticlePubMedGoogle Scholar
- Chow C-W, Dong C, Flavell RA, Davis RJ: c-Jun NH2-terminal kinase inhibits targeting of the protein phosphatase calcineurin to NFATc1. Mol Cell Biol 2000, 20: 5227-5234. 10.1128/MCB.20.14.5227-5234.2000PubMed CentralView ArticlePubMedGoogle Scholar
- Liu W, Youn H-D, Zhou XZ, Lu KP, Liu JO: Binding and regulation of the transcription factor NFAT by the peptidyl prolyl cis-trans isomerase Pin1. Febs Lett 2001, 496: 105-108. 10.1016/S0014-5793(01)02411-5View ArticlePubMedGoogle Scholar
- Okamura H, Aramburu J, García-Rodríguez C, Viola JP, Raghavan A, Tahiliani M, Zhang X, Qin J, Hogan PG, Rao A: Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity. Mol Cell 2000, 6: 539-550. 10.1016/S1097-2765(00)00053-8View ArticlePubMedGoogle Scholar
- Müller MR, Rao A: NFAT, immunity and cancer: a transcription factor comes of age. Nat Rev Immunol 2010, 10: 645-656. 10.1038/nri2818View ArticlePubMedGoogle Scholar
- Hashimoto Y, Perrino BA, Soderling TR: Identification of an autoinhibitory domain in calcineurin. J Biol Chem 1990, 265: 1924-1927.PubMedGoogle Scholar
- Rusnak F, Mertz P: Calcineurin: form and function. Physiol Rev 2000, 80: 1483-1521.PubMedGoogle Scholar
- Graef IA, Mermelstein PG, Stankunas K, Neilson JR, Deisseroth K, Tsien RW, Crabtree GR: L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons. Nature 1999, 401: 703-708. 10.1038/44378View ArticlePubMedGoogle Scholar
- Aronson D, Boppart MD, Dufresne SD, Fielding RA, Goodyear LJ: Exercise stimulates c-Jun NH2Kinase activity and c-Jun transcriptional activity in human skeletal muscle. Biochem Biophys Res Commun 1998, 251: 106-110. 10.1006/bbrc.1998.9435View ArticlePubMedGoogle Scholar
- Aronson D: Contractile activity stimulates the c-Jun NH2-terminal kinase pathway in rat skeletal muscle. J Biol Chem 1997, 272: 25636-25640. 10.1074/jbc.272.41.25636View ArticlePubMedGoogle Scholar
- Rose AJ: Exercise increases Ca2 + −calmodulin-dependent protein kinase II activity in human skeletal muscle. J Physiol 2003, 553: 303-309. 10.1113/jphysiol.2003.054171PubMed CentralView ArticlePubMedGoogle Scholar
- Liu Y, Randall WR, Schneider MF: Activity-dependent and -independent nuclear fluxes of HDAC4 mediated by different kinases in adult skeletal muscle. J Cell Biol 2005, 168: 887-897. 10.1083/jcb.200408128PubMed CentralView ArticlePubMedGoogle Scholar
- MacDonnell SM, Weisser-Thomas J, Kubo H, Hanscome M, Liu Q, Jaleel N, Berretta R, Chen X, Brown JH, Sabri A-K, Molkentin JD, Houser SR: CaMKII negatively regulates calcineurin-NFAT signaling in cardiac myocytes. Circ Res 2009, 105: 316-325. 10.1161/CIRCRESAHA.109.194035PubMed CentralView ArticlePubMedGoogle Scholar
- Robison P, Hernández-Ochoa EO, Schneider MF: Adherent primary cultures of mouse intercostal muscle fibers for isolated fiber studies. J Biomed Biotechnol 2011, 2011: 1-11.View ArticleGoogle Scholar
- Bennett BL, Sasaki D, Murray BW, O'Leary EC, Sakata ST, Xu W, Leisten JC, Motiwala A, Pierce S, Satoh Y, Bhagwat SS, Manning AM, Anderson DW: SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 2001, 98: 13681-13686. 10.1073/pnas.251194298PubMed CentralView ArticlePubMedGoogle Scholar
- Gea JG: Myosin gene expression in the respiratory muscles. Eur Respir J 1997, 10: 2404-2410. 10.1183/09031936.97.10102404View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.