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Roles of the calcineurin and CaMK signaling pathways in fast-to-slow fiber type transformation of cultured adult mouse skeletal muscle fibers

¹ Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine Baltimore, Maryland
² Department of Biology, Morgan State University, Baltimore, Maryland

	ABSTRACT

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Two Ca²⁺-dependent signaling pathways, mediated by the Ca²⁺-activated phosphatase calcineurin and by the Ca²⁺-activated kinase Ca²⁺/calmodulin-dependent kinase (CaMK), are both believed to function in fast-to-slow skeletal muscle fiber type transformation, but questions about the relative importance of the two pathways still remain. Here, the differential gene expression during fast-to-slow fiber type transformation was studied using cultured adult flexor digitorum brevis (FDB) fibers and a custom minimicroarray system containing 21 fiber type-specific marker genes. After 3 days of culture, unstimulated fibers showed a generally slower gene expression profile; 3 days of electric field stimulation of cultured FDB fibers with a slow fiber-type pattern transformed the fibers to an even slower gene expression profile. Unstimulated FDB fibers overexpressing constitutively active calcineurin featured a slower gene expression profile, except four genes, indicating that transformation occurred, but was incomplete with activation of the calcineurin pathway alone. In both unstimulated FDB fibers and slow-type electrically stimulated FDB fibers, blocking of CaMK pathway with KN93 generated a faster gene expression profile compared with the negative control KN92, indicating that CaMK pathway functions during the transformation induced by both unstimulated culturing and slow fiber-type electrical stimulation. Moreover, neither the calcineurin nor the CaMK pathway alone could maximally activate the transformation, and coordination of the two pathways is required to accomplish a complete fast-to-slow fiber type transformation.

plasticity; electrical stimulation; calcium-dependent signaling pathways; Ca²⁺/calmodulin-dependent kinase

	INTRODUCTION

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ADULT SKELETAL MUSCLE IS COMPOSED of functionally diverse fiber types that differ in contractile properties, myofibrillar protein isoforms, metabolic enzyme profile, amount of mitochondria, and ability to utilize oxidative or glycolytic metabolism (35, 41). According to the myosin heavy chain (MHC) isoforms expressed in the fibers, skeletal muscle fibers are generally categorized as slow-twitch oxidative type I, fast-twitch oxidative type IIa, fast-twitch glycolytic type IIx/d, and fast-twitch glycolytic type IIb (35, 41). There are also fiber subtypes that are hybrid fibers expressing more than one MHC isoform simultaneously (35).

It is well established that transformation of adult skeletal muscle fibers from one phenotype to another can be accomplished by denervation, cross-innervation, or direct electrical stimulation (34, 37, 50). In fast skeletal muscle of live animals, denervation transforms the expression of fiber type-specific protein isoforms toward that of slow-type muscle (36, 50). In addition to the effect of denervation alone, direct electrical stimulation of the denervated muscle with patterns mimicking action potential patterns of slow muscle fibers augments fast-to-slow fiber type transformation (36, 50). These changes in composition of fiber type-specific protein isoforms usually take from 2 to 8 wk to occur (21, 36, 50). On the gene transcriptional (mRNA) level and following a faster time course, it has also been shown that both denervation and slow twitch-type electrical stimulation change gene expression of fast skeletal muscle in both animals (8, 23, 40), in cultured whole muscle (4), and in cultured adult muscle fibers (29).

Despite many observations on fast-to-slow fiber type transformation due to cross-innervation, denervation, or slow-type electrical stimulation, the molecular basis of fiber type-specific gene expression and fast-to-slow fiber type transformation in skeletal muscle is still not clear. A variety of Ca²⁺-dependent signal transduction pathways, including the calcineurin pathway and the Ca²⁺/calmodulin-dependent kinase (CaMK) pathway, have been implicated in upregulating slow type-specific genes and mediating fast-to-slow fiber type transformation (11, 13, 15, 25, 32). Our laboratory has shown that both these pathways are activated in fast muscle fibers by slow-type patterns of action potentials, presumably by elevated intracellular free calcium concentration ([Ca²⁺]_i). Both pathways may thus contribute to the upregulation of slow gene expression (30). However, there has been controversy concerning studies in this area, and it is still unclear whether these pathways are independent or coordinated, whether there is specificity in each of the two pathways, and whether additional pathways may be involved.

Based on real-time PCR studies of gene transcription in transgenic mice overexpressing constitutively active calcineurin (CaN*), calcineurin activation was suggested to be sufficient to induce the slow fiber gene regulatory program in vivo (53). However, other studies showed that calcineurin only upregulates a subpopulation of slow genes (43). On the other hand, it has been shown that calcineurin does not downregulate all fast genes and that some fast genes are actually upregulated by calcineurin in skeletal muscle (2, 3, 43). On the basis of these and other observations, it now appears that calcineurin alone is not sufficient to complete fast-to-slow fiber type transformation and does not fully dominate the induction and maintenance of slow or fast fiber type in adult skeletal muscle. The role of calcineurin will be further investigated here.

There is also evidence supporting a role of CaMK in fast-to-slow fiber type transformation. Two isoforms of CaMK proteins, CaMKII and CaMKIV, have been examined. CaMKII activation was shown to cause phosphorylation of serum response factor, which is related to the hypertrophic response (19) and to promote glucose transport, which is correlated with oxidative capacity (51). Overexpression of constitutively active CaMKIV in a transgenic mouse strain promotes fast-to-slow fiber type transformation with increased abundance of type I slow fibers and upregulation of mitochondrial enzymes and muscle oxidative capacity (52). However, the role of CaMKIV in skeletal muscle has been questioned, because CaMKIV mRNA was not identified in rat muscle (12). CaMKIV was thus concluded to be not required for the maintenance of slow-twitch muscle phenotype, endurance training-induced mitochondrial biogenesis, and IIb-to-IIa fiber type switching in mouse skeletal muscle (1). With these inconsistent observations concerning CaMKII or CaMKIV, the role of endogenous CaMK proteins in fiber type-specific gene expression and fast-to-slow fiber type transformation of skeletal muscle remains questionable.

We have developed an in vitro system for maintaining adult rodent fast-twitch FDB (flexor digitorum brevis) skeletal muscle fibers in culture with or without stimulation and have used this system to study in vitro modification of fiber type gene expression in mature muscle fibers (29). In vitro cultured FDB fibers provide a convenient system for studying skeletal muscle plasticity. The cultured FDB fibers are not innervated by motor neurons and do not exhibit spontaneous action potentials, so that such unstimulated cultures may be in status equivalent to denervated skeletal muscle.

Here our main purpose is to examine the possible roles of the calcineurin and CaMK signaling pathways in fast-to-slow fiber type transformation by studying the differential gene expression profiles in fast-to-slow fiber type transformation of in vitro cultured primary adult skeletal muscle fibers. Specifically, in cultured FDB fibers, we wish to determine: 1) whether the calcineurin pathway alone can determine the transformation, 2) whether CaMK pathway functions in fiber type-specific gene expression and fast-to-slow fiber type transformation, and 3) whether the two pathways are coordinated in determining fast-to-slow fiber type transformation? We are also interested in determining whether unstimulated culturing or electrical stimulation with slow type-fiber pattern can result in coordinated fast-to-slow fiber type transformation of expression of multiple genes in cultured adult fast skeletal muscle fibers.

For these studies, a custom "mini" microarray system containing 21 marker genes plus one negative control gene was set up (Table 1) to identify the differential gene expression profiles in cultured adult FDB fibers subjected to various experimental manipulation. Many of the 21 genes were selected with reference to the paper of Campbell et al.(10), in which a standard Affymetrix microarray system was applied to globally screen for differential gene expression profiles between red/slow (soleus) and white/fast (quadriceps) mouse skeletal muscles. Twelve of our marker genes are in their list as being most different between fast and slow muscles. Another eight marker genes are well-established slow or fast fiber-specific genes, including slow or fast isoforms of MHC, myosin light chain (MLC), troponin C, troponin I, and phospholamban. -Actin, the skeletal muscle actin that is expressed in both slow and fast twitch skeletal muscle, was also included in the list.

	MATERIALS AND METHODS

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FDB Fiber Culturing
Single FDB fibers were enzymatically dissociated from FDB muscle of 4- to 5-wk-old CD-1 mice and cultured in MEM containing 10% fetal bovine serum and 50 µg/ml gentamicin sulfate as previously described (27), but now the plastic bottom of plastic petri dishes (35 mm diameter) was used for fiber plating. Fibers were maintained in an incubator at 5% CO₂, 37°C. All our cultured FDB fibers, with or without stimulation, were harvested for mRNA isolation 3 days after the initiation of the treatment, when possible variations in gene transcriptional profiles were detectable by the microarray system. After 3 days of culture, our fibers remained healthy and responsive under all manipulations that we examined.

Slow Fiber-type Pattern of Electrical Stimulation
The slow twitch-type pattern of electrical stimulation that was applied to cultured FDB fibers consisted of a 5-s 10-Hz train delivered once every 50 s (28). The pulse voltage was set at 20 V and alternated in polarity for each pulse application. Almost all the fibers in the plastic dish were observed to vigorously contract during the 5-s train of stimulation, which confirms the success of electrical stimulation No contraction of fibers was observed between the 10-Hz trains. Electrical stimulation was initiated about 20 h after fiber plating, and the fibers were cultured for 3 days afterward with or without electrical stimulation. The electrical stimulation was applied via stainless steel wire electrodes glued into the petri dishes.

Chemical Application and Protein Overexpression in FDB Fibers
The functional effects of the CaMKII pathway were studied by comparing the effect of the CaMK inhibitor KN93 (3 µM, Calbiochem) or KN62 (5 µM, Sigma), with KN92 (3 µM, Sigma) used as negative control. The role of the calcineurin pathways was studied by overexpressing Ca²⁺-independent, CaN*, in which the autoinhibitory motif, CaM binding motif, and CnB binding motif were removed (31). For these studies, nuclear factor of activated T cells c1-green fluorescent fusion protein (NFATc1-GFP) was expressed in FDB fibers either with or without coexpressed calcineurin, to serve as a marker of calcineurin activity. Overexpression of the proteins was accomplished by infection of FDB fibers with recombinant adenoviruses carrying the coding sequence of CaN* or NFATc1-GFP. Chemical treatment and viral infection were conducted 20 h after fiber plating, as previously described (28), and the fibers were cultured for 3 days afterward. NFATc1-GFP, either in cytoplasm or nucleus, was visible by confocal microscopy around 36 h after virus infection.

CaMKII Autophosphorylation Studies
The efficiency of CaMKII inhibition by various chemical treatments was tested using FDB fibers cultured in coverslip-bottomed culture dishes with two stainless steel wire electrodes glued to the bottom of the dish. Fibers were stimulated inside the cell culture incubator at 37°C with 10-Hz trains for 72 h, during which they were exposed to either KN93 or KN92. The cultures were fixed with paraformaldehyde and immunostained with a phospho-specific antibody that recognizes CaMKII only when it is autophosphorylated at Thr-286 () or Thr-287 (ß, , and ) (Promega).

MEF2 Activity Monitoring
A recombinant adenovirus containing a myocyte enhancer factor (MEF) 2-luciferase reporter cassette, composed of six concatomerized MEF2 sites from the MCK muscle-specific enhancer upstream of a minimal TATA box-containing promoter (generously provided by Dr. J. D. Molkentin) (48) was used to infect FDB cultures. After 24 h of infection, the cultures were subject to 10-Hz train electrical stimulation for 48 h, either with no addition or in the presence of CaMK inhibitor KN93 or of the control compound KN92. The cultures were then lysed in passive lysis buffer (Promega), and luciferase activity was determined with the luciferase assay kit (Promega).

Setup of the Microarray System
A total of 22 genes was selected for the microarray, including a negative control (a human gene), -actin, 11 anticipated slow muscle-specific genes, and 9 anticipated fast muscle-specific genes (Table 1). These genes were categorized as contractile structure, contractile regulatory, energy metabolism, ion transport, extracellular matrix, signal transduction, transcription factor, and calcium signaling.

In our microarray system, oligonucleotides (60mers) representing 21 mouse genes and one human gene (negative control) were selected according to their sequences in the National Center for Biotechnology Information GenBank (Designed by TeleChem/ArrayIt.com). The oligonucleotide sequences are presented in the supplemental materials.¹ The DNA microarray was custom spotted in the Biopolymer/Genomics Core Facility, University of Maryland-Baltimore, with a Cartesian Microarray Printer. Each oligonucleotide has six replicates in one array, spotted in two sets of three adjacent spots.

Procedure of Microarray Experiment
Experimental design.
The dye-swap design (46), reference design (46), and loop design (24) were each used in our experiments. In the dye-swap design, two mRNA samples were directly compared with balanced dye switch (46). In the reference design, the reference sample was the amplified RNA from the combined equal amounts of both FDB mRNA and soleus mRNA. This combined mRNA from both fast and slow muscle was used to provide generally balanced mRNA abundance of all the genes and would thus be a good reference for both fast and slow fiber-type genes (42). In the loop design, each mRNA sample was compared with two other mRNA samples with different dye orientation (24).

mRNA isolation, amplification, and probe labeling.
For every individual sample, >100 FDB fibers cultured in one 35-mm plastic dish were harvested by taking each single fiber off the bottom of the culture dish with a 200-µl pipette. This harvesting procedure minimizes the contamination by nonmuscle-fiber cell types (i.e., fibroblast) in the culture. mRNA was isolated with TRIzol reagent (Invitrogen). We harvested 0.5 to 1 µg of purified total RNA from each group of 100 FDB fibers. The extraction of mRNA from cultured FDB fibers is limited, and mRNA from 100 fibers was found to be insufficient for reliable dye-coupled cDNA probes for our microarray system (>2 µg of mRNA needed). Therefore, we amplified the original mRNA and generated amino-ally-UTP incorporated anti-sense RNA (cRNA). mRNA amplification and probe labeling were conducted with Amino Ally MessageAMP II aRNA kit (Ambion), to obtain Cy3-coupled or Cy5-coupled cRNA probes. The cRNA probes were then fragmented with the RNA Fragmentation Reagent (Ambion), to avoid secondary structure formation.

cDNA probes of unamplified mRNA were prepared using protocols from Brown's lab (7) and The Institute for Genomic Research (TIGR) (22a), with some modification. Total RNA from FDB or soleus was reverse transcribed with reverse transcriptase SuperScript II (Life Technologies), to get amino-ally-dUTP (Sigma) incorporated cDNA, and then coupled with Cy3 or Cy5 dye (Amersham Pharmacia) to get dye-coupled cDNA probes.

All dye-coupling reactions were carried out in the presence of 16.67% 1 M sodium bicarbonate (Invitrogen/Molecular Probes) and 50% DMSO (Sigma).

Array hybridization.
Array hybridization was conducted in the presence of 50% formamide (Invitrogen). Although the RNA:DNA duplex has a higher melting temperature than DNA:DNA duplex, cRNA probes degraded faster than cDNA probes. In our experiments, the incubation temperature was 37°C rather than 42°C (7, 22a) to avoid degradation of cRNA probes in 14 h of hybridization, which produced better results.

Slide scanning and data acquisition.
Slides were scanned with a GSI Lumonics ScanArray 5000 chip scanner. The laser system was specified for dye Cy3 and Cy5, with Cy3 and Cy5 signals being scanned at wavelengths of 532 and 635 nm, respectively. ImaGene5.5 (BioDiscovery) was used for image analysis.

Data normalization and filtering.
TIGR-Midas was used for checking data quality and data normalization/filtering. The "total intensity normalization" method was selected for data normalization (39). "Slice analysis" in TIGR-Midas was used to classify and filter data to eliminate bad spots.

Data analysis and interpretation.
Data analysis and interpretation were conducted with TIGR MultiExperiment Viewer (TMEV). Methods of hierarchical clustering (18), SAM (Significant Analysis for Microarray) (47), and t-test were utilized in data analysis (16, 33). "SAM-one-class" was used in the control experiments to validate reliability of our microarray system and amplification method. "t-Test one-class" was used in the formal experiments to pick up genes that were differentially expressed between different treatments (P value based on permutation and P value <0.05 was accepted as statistically significant). Statistical analysis of microarray data is presented as means ± SE, of at least three biological replicates for each formal experiment.

The array data are available at GEO accession number GSE6729.

	RESULTS

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Validation of our Microarray System and Amplification Method
Before the formal experiments on the effects of various treatments on gene expression in FDB fibers, a number of control experiments were carried out to validate: 1) the reliability of the microarray system, 2) the reliability of the mRNA amplification method, and 3) whether our system can successfully identify differential mRNA expression profiles between fast and slow muscle.

For validation purposes 1) and 2), self-hybridization experiments were conducted with commercial total RNA of mouse skeletal muscle (Stratagene), using both unamplified RNA and amplified RNA. Total RNA of mouse skeletal muscle was reverse transcribed to get Cy3- and Cy5-cDNA probes or amplified to get Cy3 and Cy5-cRNA probes and then two self-hybridization experiments were conducted individually with cDNA probes or cRNA probes. In both of the self-hybridization experiments, the signal intensities of almost all spots in the Cy3 channel and the Cy5 channel were classified to be not significantly changed according to the TIGR-Midas slice analysis method (95% confidence), and amplified RNA resulted in fewer bad spots than unamplified RNA (Fig. 1).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Signal intensity comparison between 2 channels for all spots in 2 self-hybridization experiments. Plots of Log (channel A intensity) vs. log(channel B intensity) are shown. Green-colored spots are not significantly different comparing Cy3 and Cy5, and red-colored spots are significantly different comparing Cy3 and Cy5 (classified with "slice analysis," at a confidence level >95%). A: self-hybridization with cDNA probes from un-amplified mouse muscle total RNA; 10 bad spots were identified. B: self-hybridization with cRNA probes from amplified mouse muscle total RNA; 3 bad spots are identified.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Comparison of differential gene expression profiles of soleus (slow) and flexor digitorum brevis (FDB, fast) using both unamplified RNA and amplified RNA. The comparison contains 2 independent experiments. Results of the 2 experiments were comparable for both unamplified RNA and amplified RNA, and genes are similarly at high or low relative abundance in unamplified RNA group and amplified RNA group. The fidelity and enhanced sensitivity of differential transcription profiles following linear amplification were confirmed. In both diagrams (left and right), from top to bottom, the first 9 genes are human CaMB (negative control), -actin, and nominally slow genes (indicated with green line); the next 9 genes are nominally fast genes (indicated with red line). Left: "color gradient view" of 4 arrays (average of 6 spots for each gene per array) in The Institute for Genomic Research's MultiExperiment Viewer (TMEV) (columns represent arrays, rows represent genes). From left to right: the first 2 arrays were conducted with unamplified RNA, and the next 2 arrays were conducted with amplified RNA; array 1, 3 are from experiment 1, and array 2, 4 are from experiment 2. The differential Log2 (ratio) of 22 genes are shown in colored boxes, with green-black-red scheme (green is <0, black is = 0, and red is >0) indicating slower-to-faster profile. Gene IDs are also shown. Right: statistical analysis of the 2 experiments is depicted with horizontal bar chart, with both unamplified mRNA (black-colored bars) and amplified RNA (gray-colored bars). Here and in all subsequent bar charts, the differential Log2 (ratio) of 22 genes is shown as value bar with SE. Log2(ratio) >0 is upregulation, and Log2(ratio) <0 is downregulation. Hu-CaMB, human calmodulin; MHC, myosin heavy chain; TnI s, troponin I slow; LDH, lactate dehydrogenase; NaK, Na+/K+ ATPase; MGP, matrix Gla protein; MLC, myosin light chain; CaN, calcineurin; Par, parvalbumin; CaM, calmodulin.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Day 3 unstimulated cultured FDB fibers compared with day 0 FDB control. We conducted 3 experiments; 3 days of unstimulated culturing resulted in an incomplete fast-to-slow transformation in cultured FDB fibers compared with day 0 control FDB fibers. Here and in all the subsequent bar charts, stars mark the genes that are significantly different.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Slow-type electrically stimulated FDB fibers compared with unstimulated control FDB fibers. We conducted 5 experiments; 3 days of slow-type electrical stimulation resulted in a general fast-to-slow fiber type transformation in cultured FDB fibers.

View larger version (75K): [in this window] [in a new window]   Fig. 5. Comparison of day 3 FDB fibers coexpressing nuclear factor of activated T cells c1-green fluorescent protein (NFATc1-GFP) plus constitutively active calcineurin (CaN*) and FDB fibers expressing only NFATc1-GFP.Nuclear localization of NFATc1-GFP indicates calcineurin activity. CaN* overexpression successfully activated NFAT-GFP and drove NFATc1-GFP cytoplasm-to-nucleus translocation. In both A and B,the picture to the left is NFATc1-GFP, and the picture to the right is NFAT-GFP plus CaN*. A: FDB fibers observed in fluorescence imaging. Successful protein overexpression and positive calcineurin activity are shown. B: FDB fibers observed in transmitted imaging (top) and in fluorescent light (bottom). High efficiency of adenoviral infection and protein overexpression are shown.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Calcineurin and NFATc1-GFP overexpressing FDB fibers compared with the control overexpressing only NFATc1-GFP. We conducted 3 experiments. Calcineurin activation induced an incomplete fast-to-slow transformation in 3-day cultured FDB fibers.

View larger version (12K): [in this window] [in a new window]   Fig. 7. KN93-treated FDB fibers (inactive CaMK) compared with KN92-treated FDB fibers (active CaMK) in unstimulated cultures. We conducted 5 experiments. KN93 blockage of CaMK in resting FDB fibers attenuated the denervation-induced fast-to-slow transformation. Unstimulated culture induced fast-to-slow fiber type transformation is thus less effective when CaMK pathways are inhibited.

View larger version (13K): [in this window] [in a new window]   Fig. 8. KN93-treated FDB fibers compared with KN92-treated fibers, both subjected to slow-type electrical stimulation (ES). We conducted 3 experiments. KN93 blocking of CaMK attenuated the fast-to-slow fiber type transformation induced by slow-type ES. Inhibition of CaMK pathways attenuates fast-to-slow fiber type transformation induced by slow fiber-type ES.

View larger version (11K): [in this window] [in a new window]   Fig. 9. CaMK II inhibitors KN-93 blocked the activation of CaMK II and MEF2. A: mean nuclear pixel fluorescence (normalized to control) in resting fibers and fibers repetitively stimulated for 72 h using 10-Hz trains in the presence or absence of KN-62, KN-93, or KN-92 and labeled with antibody for autophosphorylated CaMKII. Only nuclei stimulated in the absence of KN-93 and stained for autophosphorylated CaMKII exhibited a significant increase in nuclear staining after stimulation. From left to right, the data were averages of mean nuclear fluorescence from 13, 15, 12, or 12 nuclei from 9, 10, 10, or 10 fibers, respectively. Error bars represent ± 1 SE. *P < 0.01 vs. control without stimulation. B: MEF2 promoter-driven luciferase reporter activity was increased in fibers infected with adenovirus encoding MEF2-luciferase reporter and stimulated with 10-Hz trains for 48 h. KN-93 (3 µM) blocked the increase in luciferase activity in fibers stimulated with 10-Hz trains. Results represent triplicate measurements from each of 2 independent experiments. Error bars represent ± 1 SE. *P < 0.05 vs. control without stimulation.

View larger version (13K): [in this window] [in a new window]   Fig. 10. Comparison of FDB fibers treated with KN92 plus slow-fiber type ES with FDB fibers treated with KN92 only. We conducted 3 experiments. KN92 treatment did not affect ES induced fast-to-slow transformation. FDB fibers treated with slow-type ES plus KN92 displayed a slower gene expression profile compared with fibers treated with KN92 alone.

View larger version (18K): [in this window] [in a new window]   Fig. 11. Comparison of FDB fibers treated with KN93 or KN62 plus slow-fiber type ES with FDB fibers treated with KN93 or KN62 alone. We conducted 3 experiments, for either KN93 or KN62. KN93/KN62 inhibition of CaMK did not totally inhibit ES induced fast-to-slow transformation. The fibers treated with ES show a slower gene expression profile. A: FDB fibers treated with KN93. B: FDB fibers treated with KN62.

	DISCUSSION

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View larger version (12K): [in this window] [in a new window]   Fig. 12. Summary of transcriptional regulation of each of the 17 marker genes in the 7 types of experiments. Log2 values of the ratios are shown. A: nominally slow marker genes and actin. B: nominally fast marker genes. The 7 types of experiments are: 1) day 3 unstimulated FDB fibers compared with day 0 FDB control (Fig. 3), 2) slow-type electrically stimulated FDB fibers compared with unstimulated control FDB fibers (Fig. 4), 3) calcineurin and NFATc1-GFP overexpressing FDB fibers compared with the control overexpressing only NFATc1-GFP (Fig. 6), 4) KN92-treated FDB fibers (active CaMK) compared with KN93-treated FDB fibers (inactive CaMK), in unstimulated cultures (inverted ratio of Fig. 7), 5) KN92-treated FDB fibers compared with KN93-treated fibers subjected to slow fiber-type ES (inverted ratio of Fig. 8), 6) FDB fibers treated with KN93 plus slow-fiber type ES compared with FDB fibers treated with KN93 without stimulation (Fig. 11A), 7) FDB fibers treated with KN62 plus slow-fiber type ES compared with FDB fibers treated with KN62 without simulation (Fig. 11B).

View larger version (46K): [in this window] [in a new window]   Fig. 13. Log2(ratio) of the marker genes in the 7 types of experiments. Gene upregulation is indicated in red, and gene downregulation is indicated in green. Significant genes are represented with larger font, bold, and marked with asterisks. Note that values for experiments 4 and 5 are expressed as the negative, i.e., log2 (reverse ratio) of the values presented in Figs. 7 and 8.

Fast-to-Slow Fiber Type Transformation in Unstimulated Cultures is Mediated by the CaMK Pathways
The fast-to-slow fiber type transformation that occurred in unstimulated cultured FDB fibers raises the question of which signaling pathway is involved. The responsible signaling pathway may not be a typical Ca²⁺-dependent signaling pathway that requires elevated [Ca²⁺]_i. It must respond to the myoplasmic [Ca²⁺]_i of 100 nM in our cultured resting FDB fibers (49). Calcineurin has been shown to respond to the slow fiber-type pattern of electrical stimulation and thus to require elevated [Ca²⁺]_i (6, 13). In contrast, other studies show that [Ca²⁺]_i as low as 100 nM can activate CaMKII and induce phosphorylation of RyR complexes (9, 14, 17). Furthermore, autophosphorylation of CaMKII in the presence of Ca²⁺-CaM can cause it to remain active independent of Ca²⁺-CaM for a long period (5, 22). A direct observation of calcium modulation by CaMKII in resting muscle fibers has also been demonstrated. By blocking CaMKII in mouse single FDB fibers, Tavi et al. (44) showed that CaMKII activation increases the action potential-mediated SR Ca²⁺ release under resting conditions. Our microarray results show that KN93 inhibition of CaMK pathways results in a faster gene expression profile in unstimulated cultured fibers, suggesting that CaMK pathways upregulate slow gene expression and downregulate fast gene expression, thereby mediating fast-to-slow fiber type transformation in resting cells. Our results provide a further indication of the role of CaMK in mediating calcium signaling in resting cells and constitute the first direct observation that CaMK can regulate gene expression in resting cells.

Fast-to-Slow Fiber Type Transformation Occurs in Cultured FDB Fibers Subjected to Slow Fiber-Type Electrical Stimulation
Consistent with the well-established role of slow fiber-type electrical stimulation in determining fast-to-slow fiber type transformation in animals (8, 23, 40), we observed that slow fiber-type electrical stimulation can also induce fast-to-slow fiber type transformation in muscle fibers cultured in vitro. This effect enhances the transformation already induced by unstimulated culture itself, indicating that either already activated CaMK pathways are further activated by slow-type electrical stimulation and thus elevated [Ca²⁺]_i, or calcineurin pathways are activated, or both CaMK and calcineurin (and/or other as yet unidentified pathways) are functioning simultaneously. In the present studies we utilized 10-Hz train stimulations for 24 h per day, which may mimic the situation in the diaphragm but not in other skeletal muscles. The effect of stimulation with a normal daily pattern of other slow twitch muscles is a possible topic for investigation in future studies.

The in vitro culture of FDB fibers is free of in vivo environmental interferences and is now shown here to be an effective system for studying fiber type transformation by slow fiber-type electrical stimulation. Therefore, this cultured FDB fiber system can serve as a model for studying molecular mechanisms underlying skeletal muscle plasticity, which has previously been carried out primarily in living animals. This system was further used here to study the roles of the calcineurin and CaMK pathways in fast-to-slow fiber type transformation induced by slow fiber-type pattern of electrical simulation.

Both Calcineurin and CaMK Pathways Mediate the Fast-to-Slow Fiber-type Transformation Induced by Slow Fiber-type Electrical Stimulation
Our results show that, in cultured FDB fibers, besides upregulating slow gene expression in unstimulated fibers, CaMK pathways can also mediate fast-to-slow fiber type transformation induced by slow fiber-type electrical stimulation. In FDB fibers with CaMK pathways inhibited by either KN93 or KN62, a clearly detectable extent of fast-to-slow fiber type transformation still occurs in fibers stimulated with the slow fiber-type electrical stimulation, indicating that there must be other Ca²⁺-dependent signaling pathways, including the calcineurin pathways, mediating this transformation.

Thus, both the CaMK and calcineurin pathways may function in mediating the fast-to-slow fiber type transformation induced by slow fiber-type electrical stimulation in cultured FDB fibers. However, it is possible that these inhibitors may also partially suppress other HDAC kinases that have not yet been identified.

Calcineurin and CaMK Pathways Have Individual Specificities, and Both Contribute to Fast-to-Slow Fiber Type Transformation
Activated calcineurin resulted in an expected upregulation of most slow genes and downregulation of most fast genes, with the exception of four genes (anticipated fast genes MHC IIa, MHC IIb, and calmodulin 2; anticipated slow gene troponin C-slow). In contrast, the gene expression profile resulting from CaMK inhibition shows essentially uniform changes of most anticipated fast and slow genes (except calmodulin 2). Thus, for CaMK, there seems to be some difference in strength or type of regulation compared with calcineurin in the control of "atypical" calcineurin-regulated genes (MHC IIa, MHC IIb, and troponin C-slow).

MHC IIa is in fact not strictly a fast gene and was previously shown to be preferentially activated by both calcineurin and CaMK pathways (3!). However, our data show that gene expression of MHC IIa was obviously upregulated only in the calcineurin overexpression experiment and was downregulated by a pathway sensitive to KN93. The previously reported positive effects of CaMK on MHC IIa gene expression were detected in a CaMK blocking experiment with KN62 (3), in which myocytes treated with KN62 were directly compared with normal myocytes, but this comparison could be questioned since no control was carried out to rule out the unspecific chemical side effects of KN62, as done here using the negative control KN92.

In unstimulated cultures of FDB fibers, the fast-to-slow fiber type transformation possibly mediated by CaMK pathways is not complete, with the genes MHC I and LDH-2 being differentially regulated in the opposite direction as anticipated, perhaps due to the relatively short period of FDB culturing (3 days).

Fibers treated with the slow fiber-type pattern of electrical stimulation plus KN93 show slower gene expression profile than fibers treated with KN93 alone, which indicates that CaMK-independent pathways (calcineurin alone or calcineurin plus other pathways) are sufficient to mediate at least partial fast-to-slow fiber type transformation. However, fibers treated with KN93 plus slow-fiber-type electrical stimulation are still faster than fibers treated with control compound KN92 plus slow-fiber-type electrical stimulation, which may indicate that CaMK pathways could also be partially responsible for fast-to-slow fiber type transformation and that CaMK-independent pathways are not sufficient to mediate the maximal transformation induced by electrical stimulation. Here we conclude that the calcineurin and CaMK pathways each contribute partially to establishing the maximal fast-to-slow transformation induced by slow-type electrical stimulation.

Some Other Signaling Pathways may be Involved in the Fast-to-Slow Transformation Induced by Slow Fiber-Type Electrical Stimulation
Our results show that both CaMK and calcineurin pathways can contribute to fast-to-slow fiber type transformation. But there is still a question of whether calcineurin pathways alone can accomplish the observed fast-to-slow fiber type transformation that occurs during slow fiber-type electrical stimulation when CaMK pathways are inhibited by KN93 or KN62. Calcineurin activation is Ca²⁺ dependent and an absolute consequence of slow-fiber-type electrical stimulation. Overexpression of CaN*, which is Ca²⁺ independent, shows the effect of activated calcineurin pathways only but would not affect any other Ca²⁺-dependent signaling pathways responding to the slow fiber-type pattern of electrical stimulation.

Our results show that the effect of CaN* overexpression is quite different from that of the CaMK pathways, as revealed by KN93 or KN62 in terms of fiber type gene expression. An incomplete fast-to-slow fiber type transformation was induced by activation of calcineurin pathways alone, with marker genes MHC IIa, MHC IIb, and troponin C-slow being in the opposite direction as anticipated. In contrast, the contribution of CaMK pathways as revealed by KN93 or KN62 inhibition during slow fiber-type stimulation indicated that all genes were uniformly regulated in the anticipated slow or fast direction. This indicates the possible involvement of some other signaling pathways in regulating fiber type transformation.

Isolated Fiber Culture Model
The isolated fiber culture model provides a unique opportunity for well-controlled experimental manipulations, such as molecular biological and pharmacological interventions as well as electrical stimulation by precisely controlled patterns, to adult muscle fibers. However, this model also has some inherent limitations such as lack of normal physiological innervation and absence of fiber interaction with a normally structured in vivo extracellular matrix. The extent to which these limitations might modify our results remains to be determined.

Conclusion
In conclusion, the present results indicate that our custom microarray system is capable of tracking changes in fiber type-specific gene expression induced by a variety of experimental manipulations. We have found that both unstimulated culture, which may mimic fiber denervation, and stimulation with slow fiber-type patterns promote fast-to-slow transitions of muscle gene expression. Furthermore, both calcineurin and CaMK-dependent signaling pathways contribute to these changes in fiber type-specific gene expression.

	GRANTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work is supported by National Institute of Neurological Disorders and Stroke Grant R01-NS-33578.

	ACKNOWLEDGMENTS

 
We thank Dr. Collin Stine, for help with the design and implementation of the custom microarray system used here, Yuansha Chen and Jing Yin for arrays spotting and advice concerning microarray experimental protocols, and Dr. Hegang Chen for advice on experimental designs and data analysis.

	FOOTNOTES

 
Address for reprint requests and other correspondence: Martin F. Schneider, Dept. of Biochemistry and Molecular Biology, Univ. of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201 (e-mail: mschneid{at}umaryland.edu).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

¹ The online version of this article contains supplemental material.

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