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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 2, 2004 as doi:10.1096/fj.04-1587fje. |
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,||,
* Departments of Molecular Physiology and Biophysics,
Medicine,
Pathology,
|| Molecular and Cellular Biology and
Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA; and
Department of Kinesiology and Health, Georgia State University, Atlanta, Georgia, USA
1Correspondence: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: susanh{at}bcm.tmc.edu
SPECIFIC AIMS
FKBP12 is a 12 kDa FK506 binding protein that binds to and regulates the activity of intracellular Ca2+ release channels, including the skeletal muscle Ca2+ release channel or ryanodine receptor 1 (RyR1). The functional significance of the interaction of FKBP12 and RyR1 is not known. The aim of this study was to investigate the effects of FKBP12 depletion on skeletal muscle function and Ca2+ release using a skeletal muscle-restricted, FKBP12-deficient mouse (SFK12KO) model.
PRINCIPAL FINDINGS
1. Changes of muscle contractile properties in SFK12KO mice
Fkbp12 (lox) mice were generated using a targeting construct that introduced a neomycin cassette flanked by two loxP sites upstream of exon 3 of Fkbp12 gene and an additional loxP site downstream of exon 3. Skeletal muscle-restricted FKBP12-deficient mice were generated by crossing muscle creatine kinase (Mck)-Cre transgenic mice with the Fkbp12 loxP mutants. Cre expression in these mice is regulated by Mck promoter. In the 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.
Contractile properties of the EDL, soleus, and diaphragm muscle fiber bundles of 6- to 8-wk-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. 1
A), with tetanic force greater than control (P<0.01) at stimulation frequencies between 15 and 50 Hz, in the physiological range (10–100 Hz) of activation frequencies. There were no differences between groups in fiber bundle cross section, twitch force, and maximal force in the diaphragm (data not shown). 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. 1B
). Isometric tetanic force in the soleus muscle from SFK12KOs was indistinguishable from controls at the stimulation frequencies tested (Fig. 1C
).
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2. Decreased voltage-gated intracellular Ca2+ release and increased L-type Ca2+ currents in SFK12KO myotubes
As shown in Fig. 2
A, enhanced maximal L-type currents were detected in SFK12KO myotubes. SFK12KO myotubes exhibited a reduced maximal voltage-gated Ca2+ release (Fig. 2B
). Decay of the Ca2+ transients was not significantly different in the SFK12KO and wild-type group (t 1/2 0.025±0.0025 s, n=10 in wild-type vs. 0.024±0.0024, n=11 in SFK12KO). We found no significant differences in resting Ca2+ levels for wild-type (F340/380: 0.396±0.013, n=19) vs. SFK12KO (0.394±0.0157, n=15). There was, however, a difference in caffeine sensitivity. The SFK12KO myotubes were more sensitive to caffeine-induced Ca2+ release, with an EC50 of 3.2 ± 0.2 mM vs. 5.1 ± 0.3 mM (P<0.05) in wild-type myotubes. Maximal responses to caffeine (20 mM) were similar in the two groups, indicating that depletion of intracellular Ca2+ stores did not occur.
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3. Muscle myosin heavy chain (MHC) phenotype alterations and up-regulation of calcineurin levels in SFK12KO diaphragm muscle
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). In contrast, there was no change in MHC components in SFK12KO EDL and soleus muscles compared with the control group. Consistent with adaptive changes in the 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. There was no significant difference in fibers containing internal nuclei in EDL and soleus between control and knockout groups.
Fiber type determination has been suggested to involve the phosphatase calcineurin. The mRNAs for all three isoforms of calcineurin A (
, ß, and 
) were significantly elevated in the diaphragm muscle of SFK12KOs. We also showed a significant increase (P<0.05) in calcineurin protein levels in diaphragm muscle homogenates from the SFK12KOs. There was no difference in calcineurin protein level in EDL and soleus muscles between groups.
CONCLUSIONS AND SIGNIFICANCE
In this study, we have generated skeletal muscle-specific FKBP12-deficient mice. Myotubes from these mice showed reduced voltage-dependent Ca2+ release similar to the finding of Avila et al. using the RyR1 mutant, which cannot bind FKBP12. FKBP12, therefore, appears to contribute to E-C coupling gain. A major difference between our findings and those of Avila et al. was that our FKBP12-deficient myotubes 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. No effect on retrograde coupling was detected by Avila et al. with the mutant RyR1, which could not bind FKBP12. However, this could have been obscured by overexpression of the mutated RyR1 in those experiments.
Bilayer studies have suggested that the absence of FKBP12 creates Ca2+ leak, but this does not appear to occur in either FKBP12-deficient myotubes or the myotubes containing the mutation that destroys FKBP12 binding. Nor did we detect any changes in SR Ca2+ content or in resting Ca2+concentrations. These findings suggest either that FKBP12-depleted RyR1 is not leaky within the muscle or, if leaking occurs, other cellular events compensate and/or mask the "Ca2+ leak" from FKBP12-depleted RyR1.
We have demonstrated that different muscles respond to FKBP12 deficiency differently according to muscle type and activity (Fig. 3
). In accordance with findings in primary myotubes, in vitro voltage-induced force production was reduced in FKBP12-deficient EDL, a muscle with low chronic contractile activity in vivo. In contrast, changes in the contractile properties of the FKBP12-deficient diaphragm, a muscle with high chronic contractile activity in vivo, were consistent with an adaptive up-regulation of the slow-twitch phenotype. Accordingly, we found an increase in the ratio of type I to type II myosin heavy chains associated with increased calcineurin levels in the FKBP12-deficient diaphragm. However, no significant changes were observed in contractile properties of FKBP12-deficient soleus, a chronically active slow-twitch muscle. This suggests that either FKBP12 has a different role in modulating E-C coupling in slow-twitch muscle or the soleus muscle has a greater capacity to compensate for the reduced Ca2+ signal than does EDL muscle.
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In conclusion, we propose that FKBP12 plays a role in both orthograde and retrograde coupling between the L-type Ca2+ channel and RyR1. In highly used muscles such as the diaphragm, adaptation to the loss of FKBP12 occurs, possibly due to the increased Ca2+ influx, to compensate for the defects arising from the absence of FKBP12. Future studies using this muscle-specific FKBP12-deficient mouse model may yield further insight into the complex mechanisms involved in E-C coupling, Ca2+ homeostasis, and skeletal muscle function as well as the signaling pathways underlying skeletal muscle remodeling.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1587fje;
This article has been cited by other articles:
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  A. M. Bellinger, S. Reiken, M. Dura, P. W. Murphy, S.-X. Deng, D. W. Landry, D. Nieman, S. E. Lehnart, M. Samaru, A. LaCampagne, et al. Remodeling of ryanodine receptor complex causes "leaky" channels: A molecular mechanism for decreased exercise capacity PNAS, February 12, 2008; 105(6): 2198 - 2202. [Abstract] [Full Text] [PDF]
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