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α-Methylprednisolone promotes skeletal myogenesis in dystrophin-deficient and control mouse cultures
Neuroscience Letters, 155 (1993) 171-174
171
© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93l$ 06.00
NSL 09561
-Methylprednisolone promotes skeletal myogenesis in dystrophindeficient and control mouse cultures Laurent Metzinger a, Anne-Catherine Passaquin a, Jean-Marie Warter b and Philippe Poindron a "Equipe de Biologic de la cellule musculaire, Dkpartement d'Immunologie, Immunopharmacologie et Pathologic, Universitk Louis" Pasteur. lllkirch (France) and bEquipe de Neurologie clinique, Service de Neurologie 11, Hospices Civils, Strasbourg (France) (Received 1 February 1993; Accepted 10 March 1993)
Key words. Prednisolone; Muscle regeneration; Duchenne's muscular dystrophy: Glucocorticoid; Skeletal muscle culture We have examined the influence of the glucocorticoid ~-methylprednisolone (PDN) on the morphological differentiation of skeletal muscle cells derived from dystrophin-deficient C57BL/10 mdx, congenic C57BL/10 and allogenic Balb/c newborn mice. We show that PDN enhances myogenic cell numbers in dystrophin-deficient cultures as well as in matched controls. A parallel increase in the fusion rate of myoblasts into myotubes occurs while the size of myotubes, as determined by nuclei per myotube, is slightly increased. This promoting effect of PDN on myogenesis could be related to the enhanced muscular function observed in PDN-treated Duchenne's muscular dystrophy-affected boys.
In addition to their anti-inflammatory effect on muscle necrosis [10], glucocorticoids have been shown to enhance myoblast proliferation rate [5]. Taking this property into account, Mendell et al. [11] have carried out a clinical trial of the glucocorticoid ~-methylprednisolone (PDN) in Duchenne's muscular dystrophy (DMD), an X-linked myopathy characterized by chronic wasting of the skeletal musculature [8]. The defect of the disease is localized on a very large gene located on chromosome X that encodes a protein called 'dystrophin' [7]. An improvement of strength and function in treated patients was observed after 1 month and was demonstrated to last at least 3 yr [4]. We and others have, therefore, studied the effect of PDN on cultured skeletal muscle cells (SMC) and found that PDN enhances myogenesis in different in vitro systems [3,9,17]. The process of transformation of myoblasts into myotubes can be considered in two parts, not necessarily interdependent in vitro, morphological and biochemical differentiation. We have previously shown that PDN increases the expression of acetylcholine receptor and dystrophin-related protein in SMC derived from an animal model of DMD, the C57BL/10 mdx mouse [13]. Like DMD-affected boys, these mice lack dystrophin expression [14]. Here, we have estimated the degree of myogenesis by different morphoCorrespondence: P. Poindron, D6partement d'Immunologie, Immunopharmacologic et Pathologic, Universit6 Louis Pasteur, BP 24, 67401 Illkirch C6dex, France. Fax: (33)(16)88660190.
logical criteria: myoblast numbers, fusion rate (as evaluated by the fusion index evolution) and extent (myotube and total myonuclei numbers) of cell fusion and qualitative estimation of myotube size (nuclei per myotube). C57BL/10 mdx mice were a gift from Dr Gu6net, Institut Pasteur (Paris, France). Balb/c mice were purchased from IFFA-CREDO (L'Arbresle, France) and C57BL/ 10 mice from CSEAL-CNRS (Orl6ans, France). Primary cultures of mouse SMC were prepared as previously described [13]. At the beginning of myoblast fusion (i.e., 3rd day of culture), SMC were treated with PDN (Upjohn, Kalamazoo, MI) at a final concentration of 10-5 M. 20 pl of a 10-3 M freshly prepared stock solution were added to the treated cultures whereas 20 pl of vehicle alone (phosphate buffered saline) were added to the control cultures. Cultures were established in triplicate and experiments were carried out at least two times, one representative experiment being illustrated in the figures. Statistical differences between control and treated cultures were evaluated by a one-tailed Student's t test using the Statview 512+ Software (Apple, Cupertino, CA). Cells were stained with May-Griinwald-Giemsa dye during the time course ofmyogenesis from the 2nd to 8th day in culture. The relative number of myoblasts and myotubes was estimated by counting the cells present in one microscopic field at a 100x magnification. The nuclei, so-called 'myonuclei', were counted in both myoblasts and myotubes in the same fields. Five randomly
172
TABLE I PDN E N H A N C E S MYOBLASTS A N D TOTAL MYON UCLEI N U M B E R
Five randomly chosen fields were counted per dish and three dishes were examined for each condition. Total myonuclei/field represents the average of the sum of all nuclei found in myoblasts and myotubes in one microscopic field Strains
Balb/c
C57BL/10
mdx
Day
Myoblasts/field
Total myonuclei/field
Control
PDN
Control
PDN
2 4 6
30.7 + 3,8 31.5 + 6,5 23.1 + 4,6
34.0 + 5.2 N's 25.7 + 4.1Ns
30.7 + 3.8 44.7 + 13.8 58.7 + 16.5
59.9 + 11.6"** 84.9 + 26.4***
8
19.1 + 3.2
25.9 + 3.6***
45.9 + 13.1
78.9 + 16.2"**
Control
PDN
Control
PDN
43.9 15.9 19.7 15.1
20.9 + 4.8** 26 _+ 6.2** 16.9 + 4.8 N's
ND 9.5 + 8.5 14.8 + 9,3 15.0 _+ 14,8
21.7 + 12.3"** 35.8 _+ 26.5*** 28.0 + 22.0*
Control
PDN
Control
PDN
19.6 _+ 3.7 16.3 _+ 3.7 13.3+_2.7 13.5 _+ 2.8
21.7 + 4.9*** 15.6+3.3" 14.6 _+ 2.6 Ns
19.6 + 3.7 24.7 + 5.6 34.5+ 11.2 41.4 _+ 7.5
34.9 + 7.7*** 70,6 + 19.6"** 64.1 + 20.5***
2 4 6 8
2 4 6 8
+ + + +
3.7 2.9 4.2 2.7
*Significant effect of treatment (P < 0.05). **Idem (P < 0.01). ***Idem (P < 0.001). N.S. No significant effect.
chosen fields were counted per dish and three dishes were examined for each condition. No differences in the yield of viable myoblasts per litter and in morphological aspect of cultures were found between dystrophic (C57BL/10 mdx) and normal (congenic C57BL/10 and allogenic Balb/c) mice (data not shown). Similar results were observed in SMC cultures from the three strains of mouse despite the fact that C57BL/10 mdx satellite cells are known to proliferate more rapidly than control cells [1]. However, our experiments have been carried out on cultures derived from 1-2-day-old newborn which could be too young to be affected by these phenotypic changes. In both control and PDN-treated cells, the number of myoblasts per field tends to decrease with time (Table I). In all cases, the number of myoblasts per field was higher in PDNtreated than in untreated cultures. The acceleration of myoblast proliferation under PDN influence has been reported by others in primary cultures of SMC from rat [5] and human [16]. The latter authors have suggested that P D N decreased myoblast death during cell replication in normal and D M D cultures [6,16]. The effect of P D N is particularly salient in regard to myotube densities which is increased two- to three-fold after PDN treatment of normal and dystrophin-deficient SMC (Fig. 1).
TABLE II E F F E C T OF PDN ON N U C L E I N U M B E R PER M Y O T U B E
Five randomly chosen fields were counted per dish and three dishes were examined for each condition. Nuclei/myotube is the average of nuclei per myotube Strains
Balb/c
C57BL/10
mdx
Day
4 6 8
4 6 8
4 6 8
Nuclei/myotube Control
PDN
3.79 + 1.69 4.94 + 0.77 5.81 + 0.95
3,95 + 0.35 ~s 4,81 + 0.81Ns 5.20 + 0.49 Ns
Control
PDN
2.31 _+ 2.05 3.37 + 1.58 4.10 +_ 1.77
3.66 _+ 0.98** 4.14 + 0.86* 3.86 +_ 1.24 Ns
Control
PDN
4.48 _+ 1.23 4.17 + 0.85 4.65 + 0.91
4.19 + 0.76 N's" 4.88 + 0.61"* 4.69 _+ 0.58 Ns
*Significant effect of treatment (P < 0.05). **Idem (P < 0.01:). ***Idem (P < 0.001). N.S. No significant effect.
173
1
BALB/C
20 7 BALB/C !
0.8
16 ~
I
]
12 @
0.6 0.4
8
0.2
.
0
0
.
.
.
.
.
~
I
----
4 Days in culture
I
4 Days in culture
8
llS
C57BL/10
20
8
C57BL/10
16
0.8 O.6
o
o.4
8
0.2
4
0 0
q
0 4 Days in culture
8
1
20
4 Days in culture
mdx
md,x
***
8
**
0.8
16 .~
0.6
=
0.4
4
"~ 12 @
0.2
0 .
0
.
.
.
F
J
-
l
4 Days in culture
8
Fig. 1. P D N increases myotube numbers in Balb/c-, C57BL/10- and mdx-derived S M C cultures. PDN-treated ( i ) and control (~) cultures were stained at various times during myogenesis (see text for methodological details). Results are expressed as n u m b e r of myotubes/ field + S.D. (n = 15). Significant statistical differences as compared with matched control cultures are indicated on figure as follows: ***P < 0.001; **P < 0.01; *P < 0.05.
4 Days in culture
8
Fig. 2. P D N enhances fusion indexes in Balb/c-, C57BL/10- and mdxderived SMC cultures. PDN-treated ( i ) and control ( ) cultures were stained at various times during myogenesis (see text for methodological details). Fusion index is expressed as the ratio: [total number of nuclei in myotubes/field]/[total n u m b e r of nuclei in myotubes and myoblasts/ field] and reflects therefore the percentage of myoblasts that fused into myotubes. Significant statistical differences as compared with matched control cultures are indicated in this figure as follows: ***P < 0.001; **P < 0.01; *P < 0.05: ns, non-significant.
174 This effect was detectable 24 h after P D N addition and remained constant t h r o u g h o u t in vitro myogenesis. We previously reported similar results in rat S M C [3]. In h u m a n , Sklar and B r o w n [15] reported that the inhibitory effect o f P D N on apoptosis is p r o b a b l y responsible for the increase in m y o t u b e densities o f normal SMC. The p r o m o t i n g effect o f P D N on in vitro myogenesis is also well a r g u m e n t e d by the two- to three-fold increase in the total n u m b e r o f myonuclei (Table I). O u r results are in accordance with the increased expression o f myogenic markers such as acetylcholine receptor [2,3,9], creatine kinase [3,5] and dystrophin and dystrophin-related protein [13,15]. The n u m b e r o f nuclei per m y o t u b e can provide an estimation o f the size o f m y o t u b e s . A time course o f this parameter's value in P D N - t r e a t e d and control cells is given in Table II. M i n o r significant differences were sometimes observed after P D N treatment o f C57BL/10 m d x and C57BL/10 S M C , in contrast to Sklar and B r o w n ' s results [15] in S M C f r o m h u m a n donors. A t each time, fusion indexes were higher in treated than in untreated cultures (Fig. 2). Therefore, P D N can accelerate and enhance the fusion o f myoblasts into myotubes. In summary, statistical analysis o f morphological parameters o f m y o g e n i c differentiation allowed us to demonstrate that P D N is a potent myogenic enhancer o f S M C f r o m n e w b o r n C57BL/10 m d x and control mice. P D N is also effective in S M C derived from healthy h u m a n whereas its effect is age-dependent in D M D boys [6]. G r o w i n g interest has been shown to novel therapeutic strategies in D M D such as m y o b l a s t transfer [12]. One o f the difficulties o f this m e t h o d is the in vitro development o f a large n u m b e r o f myoblasts prior to injection. We hypothesize that P D N treatment o f myoblasts from healthy d o n o r s before or after injection to the patients might enhance satellite cell replication and thus favor D M D muscle regeneration and biochemical (i.e., dystrophin expression) recovery.
This w o r k was supported by the Association Frangaise contre les M y o p a t h i e s and by the Institut National de la Sant6 et de la Recherche M6dicale, C o n t r a t 90.02.09.
1 Anderson, J.E., Bressler, B.H. and Ovalle, W.K., Functional regeneration in the hindlimb skeletal muscle of the mdx mouse, J. Muscle Res. Cell Motil., 9 (1988) 499-515.
2 Askanas. V., Cave, S., Martinuzzi, A. and Engel, W.K., Glucocorti-. coids increase number of acetylcholine receptors (AChRs) and AChRs aggregates in human muscle cultured in serum-lree hormonally/chemically defined medium, Neurology, 36 (1986) 241. 3 Braun, S., Tranchant, C., Vilquin, J-T., Labouret, P., Warier, J.-M. and Poindron, P., Stimulating effect of prednisolone on acetylcholine receptor expression and myogenesis in primary cultures of newborn rat muscle cells, J. Neurol. Sci., 92 (1989) 119 131. 4 Fenichel, G.M., Florence, J.M., Pestronk, A., Mendell, J.R., Moxley, R.T., Griggs, R.C., Brooke, M.H., Miller, J.E, Robison, J., King, W., Signore, L., Pandya, S., Schierbrecker J. and Wilson, B., Long-term benefit from prednisone in Duchenne muscular dystrophy, Neurology, 41 (1991) 1874 1877. 5 Guerriero, V. and Florini. J.R., Dexamethasone effects on myoblast proliferation and differentiation, Endocrinology, 106 (1980) 1198-1202. 6 Hardiman, O., Brown, R.H., Beggs, A.H., Specht, L. and Sklar, R.M., Differential glucocorticoid effects on the fusion of Duchenne/ Becker and control muscle cultures, Neurology, 42 (1992) 1085. 1091. 7 Hoffman, E.E, Brown, R.H. and Kunkel, L.M., Dystrophin: the protein product of the Duchenne muscular dystrophy locus, Cell, 51 (1987) 919- 928. 8 lannacconne, S.T., Current status of Duchenne muscular dystrophy, Pediatr. Neurol., 39 (1992) 879-894. 9 Kaplan, I., Blakely, B.T., Pavlath, U.K., Travis, M. and Blau, H.M., Steroids induce acetylcholine receptors on cultured human muscle: implications for myasthenia gravis, Proc. Natl. Acad. Sci. USA, 87 (1990) 8100-8104. 10 Kissel, J.T., Burrow, K.L., Rammohan, K.W., Mendell, J.R. and the CIDD Study group, Mononuclear cell analysis of muscle biopsies in prednisone-treated and untreated Duchenne muscular dystrophy, Neurology, 41 (1991) 667-672. 11 Mendell, J.R., Moxley, R.T., Griggs, R.C., Brooke, M.H., Miller, J.E. Mandel, S., Robinson, J., Arfken, C. and Gilber, B., Randomized, double-blind six-month trial of prednisone in Duchenne's muscular dystrophy, New Engl. J. Med., 320 (1989) 1592--1597. 12 Partridge, T.A., Muscle transfection made easy, Nature (London), 352 (1991) 757 758. 13 Passaquin, A.C., Metzinger, L., L6ger, J.J., Warter, J.-M. and Poindron, E, Prednisolone enhances myogenesis and dystrophin-related protein in mdx cultures, J. Neurosci. Res., in press. 14 Sicinski, E, Geng, Y., Ryder-Cook, A.S., Barnard, E.A., Darlison, M.G. and Barnard, P.J., The molecular basis of muscular dystrophy in the mdx mouse, Science, 244 (1989) 1578 1580. 15 Sklar, R.M. and Brown, R.H., Methylprednisolone increases dystrophin levels by inhibiting myotube death during myogenesis of normal human muscle in vitro. J. Neurol. Sci., 10t (1991) 73-81. 16 Sklar, R.M., Hudson, A. and Brown, R.H., Glucocorticoids increase myoblast proliferation rates by inhibiting death of cycling cells, In Vitro Cell. Dev. Biol., 27A (1991) 433-434. 17 Vilquin, J-T., Braun, S., Labouret, E, Zuber, G, Tranchant, C., Poindron, E and Warter, J.-M., Specific effect of corticoids on acetylcholine receptor expression in rat skeletal muscle celt cultures, J. Neurosci. Res., 31 (1992) 285-293.
171
© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93l$ 06.00
NSL 09561
-Methylprednisolone promotes skeletal myogenesis in dystrophindeficient and control mouse cultures Laurent Metzinger a, Anne-Catherine Passaquin a, Jean-Marie Warter b and Philippe Poindron a "Equipe de Biologic de la cellule musculaire, Dkpartement d'Immunologie, Immunopharmacologie et Pathologic, Universitk Louis" Pasteur. lllkirch (France) and bEquipe de Neurologie clinique, Service de Neurologie 11, Hospices Civils, Strasbourg (France) (Received 1 February 1993; Accepted 10 March 1993)
Key words. Prednisolone; Muscle regeneration; Duchenne's muscular dystrophy: Glucocorticoid; Skeletal muscle culture We have examined the influence of the glucocorticoid ~-methylprednisolone (PDN) on the morphological differentiation of skeletal muscle cells derived from dystrophin-deficient C57BL/10 mdx, congenic C57BL/10 and allogenic Balb/c newborn mice. We show that PDN enhances myogenic cell numbers in dystrophin-deficient cultures as well as in matched controls. A parallel increase in the fusion rate of myoblasts into myotubes occurs while the size of myotubes, as determined by nuclei per myotube, is slightly increased. This promoting effect of PDN on myogenesis could be related to the enhanced muscular function observed in PDN-treated Duchenne's muscular dystrophy-affected boys.
In addition to their anti-inflammatory effect on muscle necrosis [10], glucocorticoids have been shown to enhance myoblast proliferation rate [5]. Taking this property into account, Mendell et al. [11] have carried out a clinical trial of the glucocorticoid ~-methylprednisolone (PDN) in Duchenne's muscular dystrophy (DMD), an X-linked myopathy characterized by chronic wasting of the skeletal musculature [8]. The defect of the disease is localized on a very large gene located on chromosome X that encodes a protein called 'dystrophin' [7]. An improvement of strength and function in treated patients was observed after 1 month and was demonstrated to last at least 3 yr [4]. We and others have, therefore, studied the effect of PDN on cultured skeletal muscle cells (SMC) and found that PDN enhances myogenesis in different in vitro systems [3,9,17]. The process of transformation of myoblasts into myotubes can be considered in two parts, not necessarily interdependent in vitro, morphological and biochemical differentiation. We have previously shown that PDN increases the expression of acetylcholine receptor and dystrophin-related protein in SMC derived from an animal model of DMD, the C57BL/10 mdx mouse [13]. Like DMD-affected boys, these mice lack dystrophin expression [14]. Here, we have estimated the degree of myogenesis by different morphoCorrespondence: P. Poindron, D6partement d'Immunologie, Immunopharmacologic et Pathologic, Universit6 Louis Pasteur, BP 24, 67401 Illkirch C6dex, France. Fax: (33)(16)88660190.
logical criteria: myoblast numbers, fusion rate (as evaluated by the fusion index evolution) and extent (myotube and total myonuclei numbers) of cell fusion and qualitative estimation of myotube size (nuclei per myotube). C57BL/10 mdx mice were a gift from Dr Gu6net, Institut Pasteur (Paris, France). Balb/c mice were purchased from IFFA-CREDO (L'Arbresle, France) and C57BL/ 10 mice from CSEAL-CNRS (Orl6ans, France). Primary cultures of mouse SMC were prepared as previously described [13]. At the beginning of myoblast fusion (i.e., 3rd day of culture), SMC were treated with PDN (Upjohn, Kalamazoo, MI) at a final concentration of 10-5 M. 20 pl of a 10-3 M freshly prepared stock solution were added to the treated cultures whereas 20 pl of vehicle alone (phosphate buffered saline) were added to the control cultures. Cultures were established in triplicate and experiments were carried out at least two times, one representative experiment being illustrated in the figures. Statistical differences between control and treated cultures were evaluated by a one-tailed Student's t test using the Statview 512+ Software (Apple, Cupertino, CA). Cells were stained with May-Griinwald-Giemsa dye during the time course ofmyogenesis from the 2nd to 8th day in culture. The relative number of myoblasts and myotubes was estimated by counting the cells present in one microscopic field at a 100x magnification. The nuclei, so-called 'myonuclei', were counted in both myoblasts and myotubes in the same fields. Five randomly
172
TABLE I PDN E N H A N C E S MYOBLASTS A N D TOTAL MYON UCLEI N U M B E R
Five randomly chosen fields were counted per dish and three dishes were examined for each condition. Total myonuclei/field represents the average of the sum of all nuclei found in myoblasts and myotubes in one microscopic field Strains
Balb/c
C57BL/10
mdx
Day
Myoblasts/field
Total myonuclei/field
Control
PDN
Control
PDN
2 4 6
30.7 + 3,8 31.5 + 6,5 23.1 + 4,6
34.0 + 5.2 N's 25.7 + 4.1Ns
30.7 + 3.8 44.7 + 13.8 58.7 + 16.5
59.9 + 11.6"** 84.9 + 26.4***
8
19.1 + 3.2
25.9 + 3.6***
45.9 + 13.1
78.9 + 16.2"**
Control
PDN
Control
PDN
43.9 15.9 19.7 15.1
20.9 + 4.8** 26 _+ 6.2** 16.9 + 4.8 N's
ND 9.5 + 8.5 14.8 + 9,3 15.0 _+ 14,8
21.7 + 12.3"** 35.8 _+ 26.5*** 28.0 + 22.0*
Control
PDN
Control
PDN
19.6 _+ 3.7 16.3 _+ 3.7 13.3+_2.7 13.5 _+ 2.8
21.7 + 4.9*** 15.6+3.3" 14.6 _+ 2.6 Ns
19.6 + 3.7 24.7 + 5.6 34.5+ 11.2 41.4 _+ 7.5
34.9 + 7.7*** 70,6 + 19.6"** 64.1 + 20.5***
2 4 6 8
2 4 6 8
+ + + +
3.7 2.9 4.2 2.7
*Significant effect of treatment (P < 0.05). **Idem (P < 0.01). ***Idem (P < 0.001). N.S. No significant effect.
chosen fields were counted per dish and three dishes were examined for each condition. No differences in the yield of viable myoblasts per litter and in morphological aspect of cultures were found between dystrophic (C57BL/10 mdx) and normal (congenic C57BL/10 and allogenic Balb/c) mice (data not shown). Similar results were observed in SMC cultures from the three strains of mouse despite the fact that C57BL/10 mdx satellite cells are known to proliferate more rapidly than control cells [1]. However, our experiments have been carried out on cultures derived from 1-2-day-old newborn which could be too young to be affected by these phenotypic changes. In both control and PDN-treated cells, the number of myoblasts per field tends to decrease with time (Table I). In all cases, the number of myoblasts per field was higher in PDNtreated than in untreated cultures. The acceleration of myoblast proliferation under PDN influence has been reported by others in primary cultures of SMC from rat [5] and human [16]. The latter authors have suggested that P D N decreased myoblast death during cell replication in normal and D M D cultures [6,16]. The effect of P D N is particularly salient in regard to myotube densities which is increased two- to three-fold after PDN treatment of normal and dystrophin-deficient SMC (Fig. 1).
TABLE II E F F E C T OF PDN ON N U C L E I N U M B E R PER M Y O T U B E
Five randomly chosen fields were counted per dish and three dishes were examined for each condition. Nuclei/myotube is the average of nuclei per myotube Strains
Balb/c
C57BL/10
mdx
Day
4 6 8
4 6 8
4 6 8
Nuclei/myotube Control
PDN
3.79 + 1.69 4.94 + 0.77 5.81 + 0.95
3,95 + 0.35 ~s 4,81 + 0.81Ns 5.20 + 0.49 Ns
Control
PDN
2.31 _+ 2.05 3.37 + 1.58 4.10 +_ 1.77
3.66 _+ 0.98** 4.14 + 0.86* 3.86 +_ 1.24 Ns
Control
PDN
4.48 _+ 1.23 4.17 + 0.85 4.65 + 0.91
4.19 + 0.76 N's" 4.88 + 0.61"* 4.69 _+ 0.58 Ns
*Significant effect of treatment (P < 0.05). **Idem (P < 0.01:). ***Idem (P < 0.001). N.S. No significant effect.
173
1
BALB/C
20 7 BALB/C !
0.8
16 ~
I
]
12 @
0.6 0.4
8
0.2
.
0
0
.
.
.
.
.
~
I
----
4 Days in culture
I
4 Days in culture
8
llS
C57BL/10
20
8
C57BL/10
16
0.8 O.6
o
o.4
8
0.2
4
0 0
q
0 4 Days in culture
8
1
20
4 Days in culture
mdx
md,x
***
8
**
0.8
16 .~
0.6
=
0.4
4
"~ 12 @
0.2
0 .
0
.
.
.
F
J
-
l
4 Days in culture
8
Fig. 1. P D N increases myotube numbers in Balb/c-, C57BL/10- and mdx-derived S M C cultures. PDN-treated ( i ) and control (~) cultures were stained at various times during myogenesis (see text for methodological details). Results are expressed as n u m b e r of myotubes/ field + S.D. (n = 15). Significant statistical differences as compared with matched control cultures are indicated on figure as follows: ***P < 0.001; **P < 0.01; *P < 0.05.
4 Days in culture
8
Fig. 2. P D N enhances fusion indexes in Balb/c-, C57BL/10- and mdxderived SMC cultures. PDN-treated ( i ) and control ( ) cultures were stained at various times during myogenesis (see text for methodological details). Fusion index is expressed as the ratio: [total number of nuclei in myotubes/field]/[total n u m b e r of nuclei in myotubes and myoblasts/ field] and reflects therefore the percentage of myoblasts that fused into myotubes. Significant statistical differences as compared with matched control cultures are indicated in this figure as follows: ***P < 0.001; **P < 0.01; *P < 0.05: ns, non-significant.
174 This effect was detectable 24 h after P D N addition and remained constant t h r o u g h o u t in vitro myogenesis. We previously reported similar results in rat S M C [3]. In h u m a n , Sklar and B r o w n [15] reported that the inhibitory effect o f P D N on apoptosis is p r o b a b l y responsible for the increase in m y o t u b e densities o f normal SMC. The p r o m o t i n g effect o f P D N on in vitro myogenesis is also well a r g u m e n t e d by the two- to three-fold increase in the total n u m b e r o f myonuclei (Table I). O u r results are in accordance with the increased expression o f myogenic markers such as acetylcholine receptor [2,3,9], creatine kinase [3,5] and dystrophin and dystrophin-related protein [13,15]. The n u m b e r o f nuclei per m y o t u b e can provide an estimation o f the size o f m y o t u b e s . A time course o f this parameter's value in P D N - t r e a t e d and control cells is given in Table II. M i n o r significant differences were sometimes observed after P D N treatment o f C57BL/10 m d x and C57BL/10 S M C , in contrast to Sklar and B r o w n ' s results [15] in S M C f r o m h u m a n donors. A t each time, fusion indexes were higher in treated than in untreated cultures (Fig. 2). Therefore, P D N can accelerate and enhance the fusion o f myoblasts into myotubes. In summary, statistical analysis o f morphological parameters o f m y o g e n i c differentiation allowed us to demonstrate that P D N is a potent myogenic enhancer o f S M C f r o m n e w b o r n C57BL/10 m d x and control mice. P D N is also effective in S M C derived from healthy h u m a n whereas its effect is age-dependent in D M D boys [6]. G r o w i n g interest has been shown to novel therapeutic strategies in D M D such as m y o b l a s t transfer [12]. One o f the difficulties o f this m e t h o d is the in vitro development o f a large n u m b e r o f myoblasts prior to injection. We hypothesize that P D N treatment o f myoblasts from healthy d o n o r s before or after injection to the patients might enhance satellite cell replication and thus favor D M D muscle regeneration and biochemical (i.e., dystrophin expression) recovery.
This w o r k was supported by the Association Frangaise contre les M y o p a t h i e s and by the Institut National de la Sant6 et de la Recherche M6dicale, C o n t r a t 90.02.09.
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