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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 7, 2002 as doi:10.1096/fj.02-0060fje. |
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* Department of Molecular Biology,
Anatomy, and
¶ Microbiology, School of Medicine, Kyung Hee University, Seoul 130–701, Korea;
Department of Biotechnology, Dongseo University, Pusan 617–716, Korea; and
Laboratory of Radiation Effect, Korea Cancer Center Hospital, Seoul 139–706, Korea
2Correspondence: Department of Molecular Biology, School of Medicine, Kyung Hee University, Seoul 130–701, Korea. E-mail: sgskim{at}khu.ac.kr
SPECIFIC AIM
Cyclosporin A (CsA) is a drug currently in clinical trials to investigate its potential to reduce immune rejection after allogenic myoblast transplantation (AMT). Success rates to date have not been satisfactory. Hypothesizing that the low efficacy of CsA is due to its toxicity toward proliferating or differentiating myoblasts, we tested whether 1) CsA toxicity is related to oxidative stress and 2) CsA toxicity is caused by the inhibitory action of CsA on the peptidyl-prolyl-cis-trans-isomerase (PPIase) activity of cyclophilin A (CypA).
PRINCIPAL FINDINGS
1. CsA blocks muscle differentiation through ROS generation and inhibition of the PPIase activity of CypA, but not through inhibition of calcineurin activity
Treatment with CsA and SDZ NIM 811, a CsA analog lacking calcineurin inhibitory activity, blocked myoblast differentiation, but treatment with FK506 and ascomycin did not result in any morphological differences compared with the control (Fig. 1
A). Calcineurin activity was completely inhibited by CsA, FK506, and ascomycin. Protein expression levels of myogenin and MRF4, the early and late myogenic markers, were consistent with the morphological data (Fig. 1B
). Stable transfection with CypA/R55A, a PPIase mutant, also completely blocked muscle differentiation (Fig. 2
A). ROS generation was increased after treatment of differentiating myoblasts with CsA and to an even greater extent by CypA/R55A transfection.
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2. CsA generates ROS at least partly by inhibiting the PPIase activity of CypA, resulting in apoptotic cell death
The TUNEL assay and DNA laddering pattern of either H9c2 cells or primary cultured myoblasts treated with CsA showed that the reduction in cell number which accompanied the differentiation block was due to apoptotic cell death. Flow cytometric DNA analysis also showed that CsA, but not FK 506 or ascomycin, increased apoptotic cell death in a dose-dependent manner. Intracellular expression of CypA/wild-type and CypA/W121F, a CsA binding mutant as well as treatment with DFOM, catalase, or Tiron significantly reduced ROS generation and protected cells from apoptosis. However, neither the additional CypA nor the classic antioxidants reversed CsA-induced differentiation block. Apoptosis by CsA was characterized by increased Bax expression and decreased mitochondrial membrane potential. A caspase 3 inhibitor only partially reversed the apoptotic death.
3. Reversibility and acquired resistance of myoblasts preexposed to CsA
To determine whether CsA could induce CypA expression and whether endogenous CypA induction could provide a protective role against CsA toxicity, myoblasts were treated in differentiation medium with CsA. A dose-dependent induction of CypA by the CsA treatment was observed. Cells preexposed to CsA were able to proliferate and differentiate reversibly on subsequent replacement in the corresponding media and in the media containing a higher CsA concentration.
4. Muscle differentiation requires optimal level of ROS
Significant ROS production was observed when myoblasts were switched to the differentiation medium. The time required for the differentiation of CypA/wild-type and CypA/W121F transfectants was 8–9 and 14–15 days, respectively, vs. 4–5 days in the mock transfectant (Fig. 2A
). Accordingly, the expression of CypA/wild-type and CypA/W121F was equal to and twice that of the basal CypA expression, respectively. ROS scavenging activity was proportional to the expression level. Treatment with DFOM, catalase, or Tiron blocked muscle differentiation (Fig. 2B
).
CONCLUSIONS AND SIGNIFICANCE
We show here that myoblasts at early stages of differentiation are more sensitive to CsA toxicity than proliferating myoblasts or fully differentiated myotubes. At a molecular level, CsA induces apoptotic cell death by generating ROS and induces a differentiation block by generating ROS and inhibiting the PPIase activity of CypA. Since the CsA-mediated inhibition of the PPIase activity of CypA contributes significantly to ROS generation, we believe this to be one of main causes of CsA toxicity. Our data contrasts with other reports suggesting that CsA blocks muscle differentiation by inhibiting the transiently increased calcineurin activity during muscle differentiation. Although CsA toxicity is related to ROS generation in hepatic cells or proximal renal tubular cells, there are no reports to our knowledge that ROS are the main causes of CsA-induced myopathies or the cause of the clinically observed lower efficacy of CsA vs. FK506. Therefore, we believe that our data will stimulate further investigation of the molecular mechanisms involved in the development of CsA-induced ROS toxicity in myocytes.
Since surviving CsA-treated myoblasts could reversibly proliferate and differentiate, a fundamental goal of CsA-based therapies should focus on protection from apoptotic death. Given that antioxidants and the presence of increased CypA protein protected myoblasts from CsA-induced apoptosis, it is likely that coadministration of antioxidants with CsA or CypA gene transfer to myoblasts will protect these cells from apoptosis and thus increase the efficacy of CsA in AMT. Future studies will demonstrate whether this is a therapeutic possibility. In contrast to antioxidants and increased CypA protein, preexposure to CsA not only protects myoblasts from CsA-induced apoptosis but also enables myoblasts to differentiate after subsequent exposure to a higher CsA concentration. Since CypA expression is increased by preexposure to CsA, the advantage of acquired resistance over antioxidants or increased CypA may result from induction of other stress proteins as well as CypA. It will be important to investigate which conditions or agents facilitate the development of acquired resistance with minimal side effects, since they would likely greatly improve the efficacy of CsA in AMT. We note as well that the long-term usage of CsA is associated mainly with toxicity to the kidney and liver via mechanisms involving ROS generation. Thus, our results may potentially have application not only in AMT but in other transplantation systems as well. Finally, our finding of a ROS requirement in muscle differentiation is novel and warrants further investigation.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0060fje; to cite this article, use FASEB J. (August 7, 2002) 10.1096/fj.02-0060fje 
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70% confluency in PM.
