Altered excitation-contraction coupling with skeletal muscle specific FKBP12 deficiency

The FASEB Journal express article 10.1096/fj.04-1587fje. Published online August 2, 2004.

Altered excitation-contraction coupling with skeletal muscle specific FKBP12 deficiency Wei Tang,* Christopher P. Ingalls,¶ William J. Durham,* Jessica Snider,* Michael B. Reid,† Gangyi Wu,* Martin M. Matzuk, ‡,§,║ and Susan L. Hamilton*

*Departments of Molecular Physiology and Biophysics, †Medicine, ‡Pathology, §Molecular and Cellular Biology and ║Molecular and Human Genetics Baylor College of Medicine, Houston, Texas 77030; and ¶Department of Kinesiology and Health, Georgia State University, Atlanta, Georgia 30303

Corresponding author: Susan L. Hamilton, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail: [email protected] ABSTRACT The immunophilin FKBP12 binds the skeletal muscle Ca2+ release channel or ryanodine receptor (RyR1), but the functional consequences of this interaction are not known. In this study, we have generated skeletal muscle specific FKBP12-deficient mice to investigate the role of FKBP12 in skeletal muscle. Primary myotubes from these mice show no obvious change in either Ca2+ stores or resting Ca2+ levels but display decreased voltage-gated intracellular Ca2+ release and increased L-type Ca2+ currents. Consistent with the decreased voltage-gated Ca2+ release, maximal tetanic force production is decreased and the force frequency curves are shifted to the right in extensor digitorum longus (EDL) muscles of the mutant mice. In contrast, there is no decrease in maximal tetanic force production in the mutant diaphragm or soleus muscle. The force frequency curve is shifted to the left in the FKBP12-deficient diaphragm muscle compared with controls. No changes in myosin heavy chain (MHC) phenotype are observed in EDL or soleus muscle of the FKBP12-deficient mice, but diaphragm muscle displays an increased ratio of slow to fast MHC isoforms. Also, calcineurin levels are increased in the diaphragm of the mutant mice but not in the soleus or EDL. In summary, FKBP12 deficiency alters both orthograde and retrograde coupling between the L-type Ca2+ channel and RyR1 and the consequences of these changes depend on muscle type and activity. In highly used muscles such as the diaphragm, adaptation to the loss of FKBP12 occurs, possibly due to the increased Ca2+ influx.

Key words: ryanodine receptor ● cre/loxP mouse models

he physical interaction between the sarcoplasmic reticulum Ca2+ release channel (RyR1) and voltage-gated L-type channels (DHPRs) in the transverse tubule is thought to be essential for skeletal muscle excitation-contraction (E-C) coupling (1, 2), the process

whereby electrical signals at the muscle membrane lead to muscle contraction. This interaction involves both an orthograde signal whereby the DHPR signals RyR1 to open and a retrograde signal whereby RyR1 changes DHPR organization in the membrane and increases the Ca2+ current through DHPR (3). FKBP12 is a 12 kDa binding protein that binds to and regulates the

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activity of intracellular Ca2+ release channels including RyRs (4–6). Each RyR1 is a homotetramer comprised of four 565 kDa polypeptides and four FKBP12 proteins.

In vitro studies have demonstrated that the association of FKBP12 with RyR1 stabilizes a closed state of the channel (4) and facilitates coupled gating between neighboring RyR channels (7). In addition to binding to RyR1, FKBP12 is a member of the immunophilin family that binds the immunosuppressive drugs FK506 and rapamycin (8, 9). Depletion of FKBP12 with rapamycin or FK506 increases the time that the channel spends in the open state but causes it to open primarily to subconductance states (4, 10). Similar alterations in RyR1 single channel properties are found in RyR1 expressed in insect cells and RyR1 isolated from FKBP12 null mouse muscle (4, 6). Lamb and Stephenson (11) reported that treatment of skeletal muscle with FK506 causes a rapid loss of depolarization-induced contraction without altering the SR Ca2+ store contents. Avila et al. (12) demonstrated that myotubes expressing either wild-type RyR1 or a mutated form of RyR1 that disrupts FKBP12 binding have similar resting Ca2+ levels, SR Ca2+ content, and L-type currents. However, maximal voltage gated sarcoplasmic reticulum (SR) Ca2+ release was reduced by ~50%, suggesting a direct role of FKBP12 in modulating the gain of skeletal muscle E-C coupling. Reiken et al. (13) showed that RyR1 from skeletal muscle of a mouse with heart failure was depleted of FKBP12, hyperphosphorylated, and had increased channel activity. Soleus muscles from these animals had prolonged tetanic half-contraction times and early fatigue. These studies suggest that FKBP12 may modulate skeletal muscle function by regulating Ca2+ homeostasis.

The majority of FKBP12 null mice die between embryonic day 14.5 and birth due to severe cardiac defects (6). These mice have apparently normal skeletal muscle development through embryogenesis, although both skeletal RyR1 and cardiac RyR2 have altered single-channel properties consistent with previous pharmacological studies. The early lethality, however, precludes an assessment of the role of FKBP12 in adult skeletal muscle function. Therefore, our first goal was to generate a skeletal muscle-restricted FKBP12-deficient mouse strain using Cre-loxP-mediated gene recombination (14, 15). The second goal of the study was to assess the effects of FKBP12 depletion on skeletal muscle function.

We demonstrate that the loss of FKBP12 in myotubes alters E-C coupling, as evidenced by reduced voltage-gated SR Ca2+ release but increased L-type channel currents. Contractile properties were differentially affected according to muscle fiber type and chronic activity level. The EDL, a fast type muscle with relatively low contractile activity levels and low oxidative capacity, exhibited maladaptive responses (lower maximal tetanic force and a rightward shift in the force frequency relationship) consistent with the findings in myotubes. In contrast, the diaphragm, a chronically active fast type muscle composed of more oxidative fibers than EDL, displayed adaptive response to FKBP12 deficiency. The slow-type soleus, consisting of primarily oxidative fibers, was not significantly affected by FKBP12 deficiency.

When complexed with the immunosuppressive drug FK506, FKBP12 binds and inhibits calcineurin, an enzyme that dephosphorylates the nuclear factor of activated T cells (NFAT). Dephosphorylation of NFAT activates its translocation to the nucleus, a step thought to be important for skeletal muscle adaptation (16). Therefore, the third goal of this study was to assess the effect of FKBP12 deficiency on calcineurin mRNA and protein levels, as well as myosin heavy chain (MHC) isoform distributions, in diaphragm muscle. We found that the

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absence of FKBP12 increases calcineurin mRNA and protein levels and produces a shift to slower MHC isoforms, consistent with the altered contractile properties in FKBP12-deficient diaphragm muscle.

MATERIALS AND METHODS

Creation of the Fkbp12 (lox) mice

Linearized Fkbp12 loxP targeting vector was electroporated into the AB2.1 ES cell line containing the original Fkbp12 null allele as described previously (6). The Fkbp12 null, floxed, and wild-type alleles were distinguished by Southern blot analysis using probes as indicated in Fig. 1. PCR analysis was performed to genotype the Cre transgene (forward primer: 5′-GCCACCAGCCAGCTATCAAC-3′ and reverse primer: 5′-GCTAATCGCCATCTTCCAGC-3′). For Southern blot analysis, genomic DNAs were digested using BamHI (for 5′ probe) or SalI plus EcoRV (for 3′ probe).

Immunoprecipitations, Western blot analyses, and immunoassays

Muscle homogenates (100 µg) were electrophoresed on SDS polyacrylamide gels and then transferred to PVDF membranes. Western blot analyses were performed using a polyclonal anti-FKBP12 antibody and an anti-calcineurin A polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a monoclonal anti-DHPR 1 subunit antibody, a monoclonal anti-actin antibody (Santa Cruz), and a monoclonal anti-SERCA1 antibody (ABR, Golden, CO). Horseradish peroxidase linked goat anti-rabbit or anti-mouse antibodies (Pierce, Rockford, IL) were used as secondary antibodies. Chemiluminescent assays were performed using SuperSignal West Pico Chemiluminescent substrate (Pierce). For immunoprecipitation, muscle homogenates (1 mg) were resuspended in 0.5 ml of IP buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% deoxycholate, and protease inhibitors) and incubated with monoclonal anti-RyR1 antibody (34C, ABR) overnight at 4°C.

Equilibrium [3H]ryanodine and [3H]PN200-110 binding

Crude SR membranes were prepared as described previously (17). Membranes (25 µg/assay) were incubated with 5 nM [3H]ryanodine (Amersham Bioscience, Piscataway, NJ) or 20 nM [3H]PN200-110 (Amersham Bioscience), and binding studies were performed as described previously (17).

Muscle contractile studies

Diaphragm muscle bundles were cut and EDL or soleus muscles were dissected free using an in vitro preparation as described previously (18, 19). Briefly, diaphragm, EDL, and soleus muscles were mounted in a chamber containing a Krebs-Ringer buffer (pH 7.4). After 10 min of equilibration at 35-37°C, physiological muscle optimal length (Lo) was set with a series of twitch contractions. For diaphragm muscle, peak isometric force as a function of stimulation frequency (15, 30, 50, 80, 120, 160, 250, and 300 Hz) was measured with 3 min between stimulations. To assess caffeine sensitivity, diaphragm strips were exposed to increasing concentrations of caffeine (0.5, 1.0, 1.5, 2.0, 4.0, 8.0, and 16.0 mM). For EDL and soleus muscle, peak isometric


force as a function of stimulation frequency (10-300 Hz for EDL and 5-250 Hz for soleus) was measured with 3 min between stimulations. Caffeine sensitivity of the EDL and soleus was assessed by measuring basal and peak forces of twitch contractions during 5 min exposure to Krebs-Ringer solution containing 1, 2, 4, 8, 10, 16, and 50 mM caffeine. Upon completion of procedures, Lo and weight were measured. Muscle cross-sectional area and stress were calculated as described previously (19, 20).

Skeletal muscle hematoxylin & eosin staining

Skeletal muscle (diaphragm, EDL, or soleus) cryosections (10 µm) were stained with hematoxylin and eosin. Muscle fibers containing internal nuclei or peripheral nuclei were counted. For each group, 3-4 sections from an individual mouse and an average of 400 muscle fibers from each section were analyzed.

Muscle MHC isoform analyses

Diaphragm, EDL, and soleus muscles were homogenized in buffer containing 250 mM sucrose, 100 mM KCl, 5 mM EDTA, and 10 mM Tris-HCl, pH 6.8. The homogenates were used for the preparation of washed myofibrils (21). The washed myofibrils (1 µg) were boiled in sample buffer and electrophoresed on an 8.0% SDS polyacrylamide gel for 24 h at 4°C to separate MHC isoforms. Gels were then stained with Bio-SafeTM Coomassie (Bio-Rad, Hercules, CA).

Quantitative RT-PCR

RNAs were extracted from diaphragm muscles with Trizol (Sigma), according to the protocol of the manufacturer. Reverse transcription was performed on 2 µg of total RNA with the RETROscript Kit (Ambion, Austin, TX) using random decamers. Real-time PCR was performed with SYBR Green PCR master mix kit (Applied Biosystems, Warrington, UK) and specific primers. The thermal cycle is as follows: 95°C 15 s, 60°C 1 min for 40 cycles. The primers are as follows: calcineurin Aα (5′-AAGGAGGGCAGGCTGGAA-3′, 5′–GAGAATCGAAGCACCCTCTGTT-3′); calcineurin Aβ (5′-TGCTGCACTTCTAAACCAACAATT-3′, 5′-TCCAGTGTGTGTATTTCTGGTGAA-3′); calcineurin Aγ (5′-CATGCACACTTTCGACTGTCTTC-3′, 5′-CATGTACACAGAGAAACTGCTGGTT-3′); and the glyceraldehyde-3-phosphate dehydrogenase (Gapdh) primers (5′-CATGGCCTTCCGTGTTCCTA-3′, 5′-GCGGCACGTCAGATCCA-3′) were used for normalization. Data were analyzed with comparative CT method according to the manual from the ABI PRISM 7700 sequence detection system (Applied Biosystems) to achieve the relative fold increase.

Measurement of whole cell Ca2+ currents and Ca2+ transients in primary myotubes

Mouse primary skeletal muscle myotubes were prepared as described previously (22). Ca2+ currents (ICa) were recorded from cultured single myotubes at room temperature (20–23°C) using an EPC-9 amplifier (HEKA, Lambrecht, Germany; ref 23). The recording chamber was perfused with Tyrode's solution (24). The pipettes had a resistance of 2–4 mOhm when filled with pipette solution (containing: 130 mM cesium gluconate, 10 mM tetraethylammonium chloride, 10 mM HEPES, 10 mM EGTA, 3 mM MgATP, 0.3 mM Na3GTP). Membrane currents were filtered at 3 kHz, digitized at 5-10 kHz, recorded, and analyzed with PULSE and PULSEFIT software (HEKA). A prepulse protocol (300-700 ms to -30 mV followed by 50 ms to


-50 mV) before each test pulse was used to inactivate T-type Ca2+ channels and Na+ channels. Inward L-currents were elicited by 50-250 ms test pulses of variable amplitude (-40, -30, -20, -10, 0, 10, 20, 30, 40, 50, and 60 mV) from a holding potential of –60 mV. The interval between test pulses was 15 s.

Simultaneous intracellular calcium ([Ca2+]i) imaging was conducted using a Nikon 20x Fluor objective (N. A. 0.75), a Videoscope ICCD camera, a Sutter filter wheel-shutter assembly, and software from Universal Imaging (25). The myotubes were loaded with fura-2 through the patch pipette. The imaging sample rate was ~5 Hz, which may underestimate actual amplitudes of the fast Ca2+ transients. Therefore, the absolute value of intracellular [Ca2+] was not calculated and all the Ca2+ changes were expressed as ratio changes of the background-corrected fluorescence changes at 340 nm divided by the background-corrected fluorescence changes at 380 nm, ∆340 nm/380 nm. The whole cell Ca2+ current density, voltage-dependent Ca2+ activation, and Ca2+ transients as a function of membrane potential were fit by the Boltzmann distribution and statistically compared as described by Garcia and Beam (24).

To measure resting Ca2+and electrical- and caffeine-induced Ca2+ transients in intact myotubes, myotubes were incubated for 1 h with 2.5 µM fura-2/AM in normal Tyrode’s solution at room temperature. Action potential (AP)-induced Ca2+ transients were triggered by field electrical stimulation with a 1 ms pulse via a pair of platinum electrodes connected to a high constant current isolator (WPI). A U-shaped fast perfusion tube controlled by an electrical valve was used for fast application of caffeine. Cells were exposed to each caffeine concentration for 3 min.

RESULTS

Creation of skeletal muscle restricted FKBP12-deficient mice (SFK12KO)

To create a spatially-regulated deficiency in a protein by the Cre-loxP system, two mouse lines are needed. One line requires an allele with the region to-be-deleted flanked by loxP site at the 5′ and 3′ ends, a so-called "floxed" allele. The other transgenic mouse line expresses a Cre transgene driven by a tissue-restricted promoter. After crossing these two transgenic mouse lines, the gene of interest will be deleted in Cre-expressing cells. Fkbp12 (lox) mice were generated using a targeting construct (Fig. 1A) that introduced a neomycin cassette flanked by two loxP sites upstream of exon 3 of Fkbp12 gene and one additional loxP site downstream of exon 3. The mutants are phenotypically normal and the three expected genotypes from Fkbp12 loxP heterozygous matings are distributed according to a Mendelian frequency.

To create skeletal muscle-restricted FKBP12-deficient mice, we used muscle creatine kinase (Mck)-Cre transgenic mice (26), and these Mck-Cre mice were crossed with the Fkbp12 loxP mutants. Cre expression in these mice is regulated by the Mck promoter. Mck is highly restricted to skeletal muscle and heart and its expression begins at embryonic day 17 in rats and maintains a high level in muscle throughout life. Mck-Cre transgenic mice were first crossed with Fkbp12 heterozygous null (Fkbp12tm1zuk, herein called Fkbp12- mice) to obtain Mck-Cre, Fkbp12+/− mice. These Mck-Cre+, Fkbp12+/− mice were then intercrossed with Fkbp12 Flox/Flox mutants to generate the skeletal muscle-specific Fkbp12KO (Fig. 1B, mice #218 and 219) with the genotype Mck-Cre+, Fkbp12Flox/-. The skeletal muscle-specific Fkbp12 knockout (herein called SFK12KO) mice display no apparent altered phenotype, and ambulation is normal. The four expected


genotypes in the progeny are equally distributed. There were no differences in weight at age 6- to 8-wk old between control and knockout groups when we carried out our experiments. However, SFK12KO male mice start to weigh less (26.3±1.2 g; n=8) than wild type (31.9±0.6 g; n=18) at the age of 3 months, while female mice have similar weights at this age.

To determine the effectiveness of the Cre-recombinase for reducing FKBP12 levels, we first assayed the FKBP12 protein levels in skeletal muscle homogenates using Western blot analyses. FKBP12 protein was markedly decreased (~80-90%) in both skeletal and cardiac muscle homogenates (data not shown) of SFK12KOs. In SFK12KO diaphragm, EDL, and soleus muscle homogenates, FKBP12 protein was decreased 91 ± 4, 92 ± 3, and 94 ± 3% (n=3), respectively, compared with wild type. The residual FKBP12 could come from nonmuscle cells or arise from the mosaic expression of Cre in the muscle. To determine the FKBP12 association with RyR1, we performed immunoprecipitations using skeletal muscle (including diaphragm) homogenates with monoclonal RyR1 antibodies. No FKBP12 signal was detected in SFK12KO immunoprecipitates by Western blot analysis (Fig. 1C), suggesting markedly reduced FKBP12 association with RyR1.

The FKBP12 decrease in cardiac muscle is due to the expression of Cre recombinase in heart (26). In contrast to the ubiquitous knockout where FKBP12 is deficient at all embryonic stages, the reduction in FKBP12 in these mice occurs at a late stage and does not produce any apparent cardiac abnormalities in mice that are <8-wk-old. We are currently examining older mice to determine if decreased FKBP12 levels alter cardiac function in older mice.

FKBP12 depletion had no effect on the expression levels of RyR1, L-type Ca2+ channel (DHPR), and SERCA1 proteins as determined by Western blot analyses (Fig. 1C and D). In addition, [3H]PN200-110 and [3H]ryanodine, which specifically bind to the L-type Ca2+ channel and RyR1, respectively, were used to assess the relative amounts of E-C coupling proteins in skeletal muscle crude membranes. There was no alteration in absolute levels or in the ratio of PN200-110 binding to ryanodine binding (Fig. 1E) in SFK12KO mice.

Changes of muscle contractile properties in SFK12KO

To assess the effects of FKBP12 deficiency on muscle function, the contractile properties of EDL, soleus, and diaphragm muscle fiber bundles of 6- to 8-week-old SFK12 KO mice were compared with muscles from age and sex-matched wild-type mice. In the diaphragm, the force-frequency relationship was shifted leftward in the SFK12KO mice (Fig. 2A). Tetanic force was greater than control (P<0.01) at stimulation frequencies between 15 and 50 Hz, within the physiological range (10-100Hz) of activation frequencies for this muscle. There were no differences between groups in fiber bundle cross-section, twitch force, and maximal force in the diaphragm (Fig. 2A). In contrast, isometric tetanic force (mN·mm-2) in the EDL muscle from SFK12KOs was less (19-32%) than control at stimulation frequencies between 60 and 300 Hz (Fig. 2B). Isometric tetanic force in the soleus muscle from SFK12KOs was indistinguishable from controls at tested stimulation frequencies (Fig. 2C).

Malignant hyperthermia (MH) is an autosomal inherited myopathy associated with mutations in RyR1 (27, 28). Some of the MH mutations are within or close to the putative FKBP12 binding sites, and, therefore, the MH mutations could alter muscle function by altering FKBP12 binding.


Conversely, a deficiency in FKBP12 might produce MH like symptoms. Susceptibility to MH is diagnosed by in vitro contracture tests using caffeine or halothane (29). We used in vitro caffeine contracture tests to determine if the SFK12KO mice are susceptible to MH-like responses. The EDL, soleus, and diaphragm fiber bundles were exposed to increasing concentrations (0-16 mM) of caffeine. We did not observe any difference in caffeine-induced contractures between control and SFK12KO groups (data not shown).

Intracellular Ca2+ transients and L-type current recordings

Alterations in muscle contractile properties in SFK12KO mice could be due to alterations in E-C coupling. To determine whether E-C coupling is altered in SFK12KO skeletal muscle, L-type currents and Ca2+ transients were simultaneously measured in primary myotubes using whole cell patch clamp techniques and ratiometric Ca2+ imaging. A high affinity ratiometric Ca2+ indicator (fura-2) with a high concentration of an extrinsic Ca2+ buffer (10 mM EGTA) was used to monitor the voltage-activated Ca2+ transients (30). As shown in Fig. 3A and C, enhanced maximal L-type currents were detected in SFK12KO myotubes. The increase in L-type current is not due to changes in voltage-dependent activation (data not shown). Consistent with the findings of Avila et. al. (12) using RyR1 mutants that did not bind FKBP12, SFK12KO myotubes exhibited a reduced maximal voltage-gated Ca2+ release (Fig. 3B and D). The decay of the Ca2+ transients (Fig. 3B), which can be adequately fit with a single exponential equation, was not significantly different between the SFK12KO and the wild-type group (t1/2=0.025±0.0025 s, n=10 in wild type vs. 0.024±0.0024, n=11 in SFK12KO). Our data suggest that the loss of FKBP12 from muscle appears to alter both orthograde and retrograde signaling between the L-type channel and RyR1.

To evaluate further the consequences of FKBP12 loss to Ca2+ homeostasis and E-C coupling, we measured both the resting Ca2+ levels and the caffeine-evoked Ca2+ release in fura-2/AM loaded myotubes. We found no significant differences in resting Ca2+ levels in wild type (F340/380: 0.396±0.013, n=19) vs. in SFK12KO (0.394±0.0157, n=15). Nor was there any difference in frequency of the signal. There was, however, a difference in the caffeine sensitivity. The effects of different concentrations of caffeine on intracellular Ca2+ release and the electrically evoked Ca2+ transients are shown (Fig. 3E and F). The peak amplitude of the caffeine-induced Ca2+ release was quantitatively compared between the SFK12 KO and wild-type groups. The SFK12 KO myotubes were more sensitive to caffeine induced Ca2+ release with EC50 of 3.2 ± 0.2 mM vs. 5.1 ± 0.3 mM (P<0.05) in wild-type myotubes (Fig. 3F). Maximal responses to caffeine (20 mM) were similar between two groups, indicating that depletion of intracellular Ca2+ stores did not occur.

Muscle MHC phenotype alterations and up-regulation of calcineurin levels in SFK12KO mice

Muscle has a great capacity to undergo remodeling in response to both physical activity and pathological stimuli. Changes in Ca2+ homeostasis arising from the FKBP12 deficiency could, therefore, lead to adaptive changes in muscle. To examine this, we used SDS polyacrylamide gels to separate MHC isoforms. As shown (Fig. 4A, inset), diaphragm, composed of both slow and fast twitch fibers, contains all four MHC isoforms (type I, type IIa, type IIb, and type IIx). The EDL muscle, a primarily fast twitch muscle, contains the type IIb as the predominant


isoform and the soleus muscle, a slow twitch muscle, contains the type IIa and type I isoforms. The FKBP12-deficient diaphragms showed an increase in the ratio of type I to type II fibers (0.20±0.007) compared with the wild-type diaphragm (0.16±0.003, n=3, P<0.05; Fig. 4A). In contrast, there was no change in MHC components in SFK12KO EDL and soleus muscles compared with the control group (Fig. 4A). A skeletal muscle fiber is a multinucleated cell with most nuclei at the periphery of the fiber. Internal nuclei are associated with muscle remodeling and adaptation in response to muscle damage or contractile activity. Consistent with adaptive changes in diaphragm, there was an increase in the percentage of fibers containing internal nuclei in SFK12KO 2.12 ± 0.52% (n=4) vs. 0.77 ± 0.11% (n=4) in wild-type mice (Fig. 4D). There was no significant difference in fibers containing internal nuclei in EDL and soleus between control and knockout groups (data not shown).

Fiber type determination has been suggested to involve the phosphatase calcineurin (16). We determined the relative levels of calcineurin mRNA using real-time PCR and calcineurin protein using Western blot analysis. Calcineurin is composed of two subunits, a 59 kDa catalytic subunit (calcineurin A) and a 19 kDa regulatory subunit (calcineurin B). The mRNAs for all three isoforms of calcineurin A (α, β, and γ) were significantly elevated in the diaphragm muscle of SFK12KOs (Fig. 4B). Using an antibody that recognizes all three isoforms of calcineurin A, we also showed a significant increase (P<0.05) in calcineurin protein levels in diaphragm muscle homogenates from the SFK12KOs (Fig. 4C). There is no difference in calcineurin protein level in EDL and soleus muscles between groups (data not shown). Therefore, reduction of FKBP12 in diaphragm muscle leads to up-regulation of calcineurin mRNA and protein levels. The increased calcineurin levels could contribute to the alteration in the ratio of the fast to slow muscle fiber types in diaphragm.

DISCUSSION

FKBP12 modulates the activity of the skeletal muscle Ca2+ release channel via a tight association with this channel. Several possible mechanisms for modulation of RyR1 include the following: 1) stabilization of both a closed state of the channel and cooperative interactions between subunits within the tetramer (4), 2) coupled gating between neighboring channels (7), and 3) protection of RyR1 from hyper-phosphorylation (13). In myotubes a mutation in the FKBP12 binding site on RyR1 that destroys its ability to bind FKBP12 results in decreased maximal voltage gated SR Ca2+ release, suggesting a direct role of FKBP12 in modulating the gain of skeletal muscle E-C coupling (12). To investigate the role of FKBP12 modulation in skeletal muscle function, we created a mouse line with a skeletal muscle specific deficiency in FKBP12 (SFK12KO). Myotubes from these SFK12KO mice did not exhibit changes in either resting Ca2+ levels or SR Ca2+ content, similar to the findings with myotubes containing the mutation that destroys FKBP12 binding (12). These findings are inconsistent with the hypothesis that the absence of FKBP12 increases Ca2+ leak from the SR. Our data suggest that either FKBP12 depleted RyR1 is not leaky in its muscle environment or other cellular events compensate and/or mask “Ca2+ leak” from FKBP12-depleted RyR1. However, similar to the findings of Avila et al. (12) using the RyR1 mutant that cannot bind FKBP12, FKBP12 depletion reduced voltage-dependent Ca2+ release. FKBP12, therefore, appears to contribute to E-C coupling gain. Consistent with this, the FKBP12 deficiency in EDL muscle significantly decreased voltage-activated force producing capacity.


A major difference between our findings and those of Avila et al. (12) was that our FKBP12-deficient myotubes also showed an increase in voltage dependent Ca2+ currents without an increase in L-type channel protein expression. These findings suggest that FKBP12 also plays a role in retrograde coupling. An effect on retrograde coupling was not detected with the mutant RyR1 that could not bind FKBP12 (12). However, such an effect could have been obscured by the overexpression of the mutated RyR1 in those experiments.

Our second finding is that Ca2+ transients in the FKBP12-deficient myotubes show an increased sensitivity to caffeine compared with the myotubes from the wild-type mice. Myotubes are an attractive experimental system in which to study calcium regulation because they allow study of calcium-mediated events with minimal interference from the effects of muscle contraction (e.g., high energy phosphate utilization, alterations in redox state, activation of mechanical signaling pathways). We were, however, unable to detect a change in caffeine sensitivity of [3H]ryanodine binding to isolated membranes from FKBP12-deficient mice compared with membranes from wild-type mice (data not shown). These findings suggest that caffeine sensitivity may be modulated by other factors in intact myotubes that are not present in isolated membranes. An increase in sensitivity to caffeine has been detected in RyR1s that have mutations associated with malignant hyperthermia (27, 28). Also, most patients with central core disease (CCD) have been found to be MH susceptible. However, we did not detect MH responses in in vitro caffeine contracture tests with EDL, soleus, and diaphragm muscles from FKBP12-deficient mice. The lack of caffeine sensitivity suggests that mechanical and/or adaptive factors (e.g., altered SERCA activity or calcium buffering) present in intact muscle may compensate or obscure the primary effect of FKBP12 deficiency that we observed in FKBP12-deficient myotubes. We did not detect cores in the SFK12KO muscle with NADH tetrazolium reductase staining (data not shown). We observed a significant increase in the number of internal nuclei in SFK12KO diaphragm fiber, but not in EDL or soleus. Internal nuclei are typically associated with muscle repair and remodeling (31).

The diaphragm muscle in SFK12KO exhibits a leftward shift in the force-frequency curve. The changes in the diaphragm muscle could arise from either primary or adaptive changes in the muscle. Slow twitch muscles (type I) are known to show a leftward shift in the force-frequency relationship compared with fast twitch fibers. These differences may be due to the lower nitric oxide synthase levels (32) and the greater Ca2+ sensitivity of the contractile machinery (troponin complex) in slow twitch fibers (33). Consistent with a fiber type shift, we found an increase in the ratio of type I to type II MHCs. Therefore, some of the functional differences in diaphragm muscle of FKBP12-deficient mice compared with wild-type mice are likely to arise from adaptive changes. In contrast, neither EDL nor soleus muscle exhibited a leftward shift in the force-frequency relationship or a change in MHC isoforms. Instead, EDL muscle in SFK12KO showed reduced tetanic force, consistent with decreased voltage gated Ca2+ release. Therefore, the altered contractile properties in the EDL muscle most likely stems from the FKBP12 deficiency itself or from a lack of adaptation of the muscle to FKBP12 deficiency. We did not detect any difference between the contractile properties of SFK12KO and wild-type soleus. We interpret this finding to indicate that either FKBP12 has a different role in modulating E-C coupling in slow twitch muscle, or the soleus muscle has a higher capacity to compensate for the reduced Ca2+ signal compared with the EDL muscle.


One possible consequence of increased Ca2+ influx as well as the altered voltage dependent Ca2+ release is a change in signaling pathways. A number of studies have demonstrated that Ca2+ influx through L-type calcium channels is required for the activation of a number of transcription factors such as CREB and MEF-2 (34). Consistent with this, we detected an increase in phospho-CREB levels in SFK12KO diaphragm (unpublished observation). This may indirectly lead to the increased calcineurin mRNA and protein levels in the diaphragm of FKBP12-deficient animals. The increased expression of calcineurin and possibly the loss of the negative regulation of calcineurin by FKBP12 (35) could result in a dysregulated calcineurin/NFAT pathway, thereby promoting fiber type alterations (16).

Although FKBP12 deficiency markedly altered the intrinsic contractile properties of the EDL muscle, a lower recruitment demand of the EDL compared with the diaphragm muscle may explain the lack of remodeling of this fast twitch hindlimb muscle. Future studies using this muscle specific FKBP12-deficient mouse model may yield further insight into the complex mechanisms underlying E-C coupling, Ca2+ homeostasis, and skeletal muscle function as well as the signaling pathways underlying skeletal muscle remodeling.

ACKNOWLEDGMENTS This work is supported by grants from the Muscular Dystrophy Association (to S. L. Hamilton) and the National Institutes of Health AR-41802 and AR-44864 (to S. L. Hamilton); HD32067 (to M. M. Matzuk); and HL-45721 (to M. B. Reid).We thank L. Chen and K. Dong for technical assistance, W. Shou for help in designing the original targeting vector, and C. R. Kohn for the gift of the Mck-Cre transgenic mice.

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Received April 16, 2003; accepted June 10, 2004.


Fig. 1


Figure 1. Generation of skeletal muscle restricted FKBP12-deficient mice. A) Identification of recombinant loxP allele. Production of recombinant Fkbp12 allele with loxP sites flanking exon 3. B) Genotype analysis of SFK12KO and littermates. Left panel) Southern blot analysis of Fkbp12 alleles. Right panel) PCR analysis of the Mck-Cre transgene. C) Coimmunoprecipitation of FKBP12 protein with RyR1 using anti-RyR1 antibody in skeletal muscle homogenates. D) Western blotting analysis of DHPR and SERCA 1 proteins in skeletal muscle homogenates. E) PN200-110 (PN) binding and [3H]ryanodine (RY) binding to skeletal muscle membranes. C and KO are samples from age and sex-matched wild-type and SFK12KO mice, respectively (n=3).


Fig. 2

Figure 2. Contractile properties of SFK12KO and age and sex-matched wild-type mice. Closed squares are age- and sex-matched wild-type mice, and open squares are SFK12KO mice. A) Force-frequency relationship of diaphragm strips (n=5). B) Force-frequency relationship of EDL muscle (n=8-10). C) Force-frequency relationship of soleus muscle (n=7-8). *Statistical difference between SFK12KO mice and controls (P<0.05).


Fig. 3

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Figure 3. Voltage dependent L-type Ca2+ current and SR Ca2+ release and electrical and caffeine induced Ca2+ transients. Closed squares and C are myotubes from wild-type mice, and open squares and KO for SFK12KO mice. A–D) Simultaneous measurement of voltage-dependent L-type Ca2+ current and SR Ca2+ release. A) Voltage-dependent L-type Ca2+ current trace. B) Voltage-dependent intracellular Ca2+ transient traces. C) Average peak I-V curves. Lcurrents were normalized to cell capacitance (pA/pF). D) Average voltage-dependent intracellular Ca2+ transients. E–F) Myotubes were under continuous 0.1 Hz electric stimuli and different concentrations of caffeine were applied additionally. E) Ca2+ transient traces under different concentrations of caffeine (0-10 mM). Baseline shift was marked in bottom trace. F) Plot of normalized ratio 340/380 in response to different concentrations of caffeine. Values were normalized to their respective maximal response at 10 mM caffeine. *Statistical difference between SFK12KO mice and controls (P<0.05).


Fig. 4

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Figure 4. MHC phenotype alteration associated with up-regulation of calcineurin mRNA and protein levels in SFK12KO mice. C and KO are samples (n=3) from wild-type and knockout mice, respectively. A) Myosin heavy chain isoform separation in diaphragm, EDL, and soleus muscles. Diaphragm, EDL, and soleus muscle myofibrils (1 µg) were electrophoresed on a 8.0% SDS polyacrylamide gel to separate myosin heavy chain isoforms. Type IIa, type IIb, type IIx, and type I are 4 MHC isoforms and the migration rate in the gel is I > IIb > IIx > IIa. B) Upregulation of calcineurin Aα, Aβ, and Aγ mRNA levels in knockout mouse diaphragms. C) Summary of Western blot analysis data of calcineurin A normalized to actin level in diaphragm homogenates; inset is Western blot. D) H&E staining of diaphragm strips. Arrowhead indicates a muscle fiber containing an internal nucleus.


Altered excitation-contraction coupling with skeletal muscle specific FKBP12 deficiency

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