Involvement of Cyclic GMP in the Fusion of Chick Embryonic Myoblasts in Culture SUK Woo CHOI, MI YEONG BAEK, AND MAN-SIK KANG**’ Department of Molecular Biology and *Department of Biology, College of Natural Science, Seoul National University, Seoul 151-742, Korea
occurs when the CAMP synthesis is induced 10 h before the onset of fusion in controls, whereas the depression of fusion is a consequence of exposing myoblasts which are undergoing active fusion to an increase in CAMP [6, 71. These findings have two implications. First, the depression of fusion by CAMP is a separate action from the CAMP-mediated stimulation of cell fusion. Second, the depressions of fusion imposed by CAMP are due to the CAMP’S antagonism of a process that is occurring close to the fusion event itself. Alternatively, it is demonstrated that PGE, induces an increased calcium influx which triggers fusion [8, 91 and that myoblast fusion is regulated by PGE, independently of a rise in CAMP [lo]. Furthermore, it is reported that the Ca2+ ionophore A23187 provokes a precocious fusion and the Ca2+ channel blocker D600 inhibits myoblast fusion without affecting recognition and alignment [B]. This contention is supported by the presence of increased Ca2+ influx at the onset of myoblast fusion. More recently, it has been demonstrated that myoblasts in culture generate PGE, and acetylcholine and that both hormone-like factors are capable of causing the necessary Ca2+ influx and of triggering myoblast fusion [ll, 121. The ability of these hormones to initiate fusion is prevented by the Ca2+ channel antagonists, D600 and lantanum. These results suggest that a net Ca2+ influx into fusion competent myoblasts is a requisite step in membrane fusion, even if an exact mechanism of Ca2+ involvement in myoblast fusion is not yet clear. The studies that utilized A23187 have demonstrated that in the absence of other effects that may be produced by hormone-cell interaction an increase in intracellular Ca2+ promotes the rise in cGMP levels in the artery [ 131, pancreas [ 141, and liver [ 151. The increased cellular accumulation in the cGMP promoted by the ionophore in these tissues usually exhibits the same temporal characteristics as those promoted by the agents that exhibit the Ca2+ dependence. As to calcium regulation of myoblast fusion, Przybylski et al. [ 161 suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation. It thus appears that the Ca2+ influx, which may be the consequence of an interaction with the cell membrane of a number of agents, is tightly
We found that a transient rise in cGMP levels, which was closely associated with the Ca” influx, occurred concomitant with the onset of myoblast fusion. The Ca2+ channel blocker D600 decreased both the cell fusion and the normal rise in cGMP levels. In contrast, the Ca2+ ionophore A23187 transiently increased cGMP levels and induced precocious fusion. In addition, the cGMP analog 8-Br-cGMP induced precocious fusion as A23187 did. The guanylate cyclase inhibitor, methylene blue delayed the fusion in a dose-dependent manner without significantly affecting cell alignment, proliferation, or muscle-specific protein expression. Furthermore, methylene blue delayed the normal rise in cGMP levels, and the fusion block imposed by methylene blue was significantly recovered by 8-Br-cGMP. On the basis of our present findings, we suggest that a Ca2+ influx-dependent rise in cGMP levels is an important Step in myoblast fusion. 0 1992 Academic Press, Inc.
INTRODUCTION The differentiation of skeletal muscle in uiuo and in vitro is accompanied by the fusion of myoblasts into multinucleated muscle fibers. The fusion of myoblasts is certainly a multistep process, including a minimum of the following separable components: (1) cell migration, recognition, and alignment, and (2) membrane fusion leading to cytoplasmic continuity [l, 21. At approximately the same time as the onset of cell fusion, the embryonic muscle cell ceases DNA synthesis and cell division and initiates the elaboration of the specialized proteins associated with skeletal muscle contraction. It is generally accepted that the expression of muscle-specific proteins can be uncoupled from the fusion event [3, 41. It is reported that a prostaglandin E, (PGEJ-dependent rise in CAMP occurs 5-6 h prior to myoblast fusion and is necessary for fusion [5,6]. However, an increase in CAMP levels has contradictory effects on myoblast fusion. The stimulation of myoblast fusion by CAMP
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7 g c3 ”
FIG. 1. Time course change of intracellular cGMP level and percentage fusion in control and DGOO-treated cultures. D600 (20 PM) was added 24 h after plating. A, Percentage fusion in control culture; A, percentage fusion in DGOO-treated culture; 0, cGMP level in control culture; 0, cGMP level in DGOO-treated culture. Each point in control culture represents the mean of triplicate determinations and that in DGOO-treated culture represents the mean of duplicate determinations.
linked to a mechanism by which the cellular levels of cGMP can be transiently increased. In the present study, the correlation between the rise in cGMP levels and the Ca2+ influx in some tissues led us to examine whether an increase in the cGMP levels actually occurs in myoblast differentiation and if so, what role is played by it in myoblast fusion.
mM stock solution in 100% dimethyl sulfoxide (DMSO). The D600 stock solution was diluted, with vigorous vortexing, to 2.4 mM with MEM and treated to culture medium. A23187 was prepared as a 20 mM stock solution in 100% DMSO. This stock solution was diluted, with vigorous vortexing, to prepare 10 PM working solution with MEM. The working solution was immediately added to culture medium. 8-Br-cGMP was prepared as 10 mM stock solution in distilled water, was serially diluted with MEM, and then was added to culture medium. Cyclic GMP assay. Cells seeded onto 60-mm dishes were rapidly harvested by adding 0.5 ml of 50 mM sodium acetate buffer, pH 5.2, which was preheated to 90°C. After incubation at 90°C for 1 h, the tubes were subjected to vigorous vortexing for 2 min, followed by incubation at 0-5°C for 30 min. Samples were stored at -70°C and assayed within 3 days. Immediately prior to assay, the samples were thawed and cell debris was removed by centrifugation at 12,800g at 4°C for 10 min. A loo-p1 aliquot was acetylated , and the cGMP content was determined by the radioimmunoassay of Steiner et al. . The cGMP content was normalized to the number of nuclei. Creatine kinase assay. Cells were harvested and sonicated in icecold 50 mM Tris-HCl buffer with 5 mM magnesium chloride, pH 7.5. After centrifugation at 20,OOOg for 30 min, the supernatant was assayed for creatine kinase (CK) using the method of Kuby et al. . receptor (AChR) was Acetylcholine receptor assay. Acetylcholine assayed as described by Lee and Tseng . iz51-labeled oc-bungarotoxin (10 nCi/ml) was added to each dish for 1 h. The cells were rapidly rinsed four times at 3-min intervals with MEM. The cells were harvested with 4% SDS and were counted for radioactivity in a gamma-counter. DNA synthesis. [3H]Thymidine (0.1 &i) was added to 35-mm dish 24 h after plating and incubated for 34 h. The cells were then rinsed three times with ice-cold phosphate-buffered saline and 1 ml of 10% trichloroacetic acid (TCA) was added. After 30 min, the cells were rinsed twice with 10% TCA. The precipitate was dissolved in 0.5 N NaOH and counted in a liquid scintillation counter.
Materials. Eagle’s minimum essential medium (MEM), horse serum, and antibiotics were obtained from GIBCO Laboratories. Tissue culture dishes were from Nunc, and Naiz51 and [3H]thymidine were from Amersham. Methylene blue, cu-isopropyl-a-[(N-methyl-5N - homoveratryl) - 01-aminopropyl] - 3,4,5 - trimethoxyphenylacetoni trite hydrochloride (D600), A23187, 8bromo-cyclic GMP (8BrcGMP), cGMP antibody, and other reagents were from Sigma Chemical Co. Myoblnst cultures. Myoblasts were obtained from breast muscle of 12.day-oldchick embryos . Briefly, after dissection, the tissue was minced with fine scissors, dispersed by mechanical agitation, and filtered twice through 12.fold lens papers. The cells were plated at a density of 1.2 X lo5 tells/35-mm dish or 3 X lo5 tells/60-mm dish in MEM supplemented with 10% horse serum and 2% embryo extracts at pH 7.2-7.4 and cultured in a humidified atmosphere of 5% CO, and 95% air at 37°C. The culture medium was changed 24 h after plating. Cells were fixed with 1% glutaraldeMeasurement of cell fusion. hyde and stained with Giemsa for the estimation of percentage fusion. Cell fusion was estimated by direct microscopic examination at a magnification of 250X. Cells were considered fused only if there were clear cytoplasmic continuity and at least three nuclei were present in each myotube. Drug additions. Methylene blue was prepared as 30 mM stock solution in distilled water. This stock solution was diluted to 40 PMwith MEM and then added to culture medium. D600 was prepared as 240
FIG. 2. Effect of A23187 on the cGMP level. At 47-50 h after plating, cells were washed twice with 2 ml of MEM. Following a l-h equilibration period, medium was aspirated and cultures were preincubated in 3 ml MEM containing 25 mM Hepes and 2 mg/ml bovine serum albumin at 37°C for 15 min. A23187 was then added to the culture medium and the cells were incubated at 37°C. After incubation for the indicated times, the cells were harvested for cGMP radioimmunoassay. 0, cGMP level in control culture; n , V, and A, cGMP levels in 0.25,0.5, and 0.75 PM A23187-treated cultures, respectively. Each point represents the mean of duplicate determinations.
55 h. The initial rise in cGMP levels was closely associated with the onset of myoblast fusion. To explore a possible cause for the cGMP peak, the cGMP levels in the DGOO-treated and control cultures were compared. As shown in Fig. 1, D600 (20 j&f) delayed both myoblast fusion and elevation in cGMP levels. Even in the DGOO-retarded cultures, the rise in cGMP levels was also closely associated with the fusion. To confirm the possibility that the cGMP peak is closely associated with the Ca2+ influx, we measured the cGMP levels in A23187-treated cultures. As shown in Fig. 2, within 30 s after the addition of A23187, a transient increase in cGMP levels was observed at concentration above 0.25 pM. Furthermore, the addition of A23187 at 0.75 pM to the culture 47 h after plating resulted in precocious fusion as compared to control cultures, but the final extent of fusion was similar in both control and A23187-treated cultures (Fig. 3A). To examine the effect of cGMP levels on myoblast fusion, the cGMP analog 8Br-cGMP was used. Addition of 1 r&f 8-Br-cGMP to the cultures 47 h after plating resulted in a precocious fusion, but the degree of fusion finally attained was the same in both 8-BrcGMP-treated and control cultures (Fig. 3B). To extend the significance of the observation obtained with 8-Br-cGMP, we utilized the guanylate cyclase inhibitor methylene blue [22-241. As shown in Fig. 4A, methylene blue delayed myoblast fusion at a concentration range of 0.4-0.6 pM, and the time course change of percentage fusion for control and methylene
Culture FIG. A23187 A23187 A23187mean +
3. Stimulation of myoblast fusion by externally applied (A) or 8-Br-cGMP (B). The cells were exposed to 0.75 nM or 1 nM 8-Br-cGMP 47 h after plating. 0, Control culture; 0, or 8-Br-cGMP-treated culture. Each value represents the SD.
To investigate whether intracellular cGMP levels change in the progress of myoblast fusion, the cGMP radioimmunoassay was employed. As shown in Fig. 1, intracellular cGMP levels, expressed as fmol cGMP/lO’ nuclei, were found to change with culture time and were transiently increased about sevenfold between 51 and
FIG. 4. (A) Dose-dependent inhibition of myoblast fusion by methylene blue. The cells were exposed to methylene blue 24 h after plating and incubated for 34 h. The control level is shown for comparison (A). (B) Time course change of myoblast fusion in control and methylene blue-treated cultures. The cells were incubated in the medium containing 0.6 pM methylene blue at 24 h after plating. 0, Control culture; 0, methylene blue-treated culture. (C) Effect of 8-Br-cGMP on the fusion-retarded myoblasts by methylene blue. Cell fusion was retarded by treating with 0.6 pM methylene blue at 24 h after plating. The cells were then exposed to 8-Br-cGMP at 56 h after plating and incubated for 2 h. CON, control culture; MB, methylene blue-treated culture; a, b, and c, 1, 0.1, and 0.01 nM of 8-Br-cGMP, respectively. Each value represents the mean f SD.
Percentage fusion Nuclei/field [aH]Thymidine incorporation (cpm/35-mm CK (pmol/mg/min) a-Bungarotoxin binding (cpm/mg) Note. Cells were exposed to 0.6 PM methylene
35 f 3.5 8.7 * 1 10325 t 1100
blue at 24 h after plating.
blue-treated cultures was significantly different (Fig. 4B). In addition, methylene blue had no discernible effect on total cell numbers and [3H]thymidine incorporation (Table 1) and was found not to hinder cell alignment to any detectable degree (data not shown). In order to assess the nature of delay in differentiation imposed by methylene blue, the expression of musclespecific proteins was also examined. As shown in Table 1, methylene blue did not prevent the initial increase of CK activities. The mean CK activities at 58 h were similar in both control and methylene blue-treated cultures. Similarly, methylene blue did not significantly interfere with the expression of AChR. The bindings of ‘251-labeled a-bungarotoxin to control and methylene bluetreated cultures were not significantly different, as compared to the significant difference in percentage fusion. To determine if the retarding effect of methylene blue on myoblast fusion is related to its efficacy as a guanyl-
FIG. 6. Effects of methylene blue on the cGMP level and percentage fusion. Methylene blue (0.6 PM) was added 24 h after plating. A, Percentage fusion in control culture; A, percentage fusion in methylene blue-treated culture; 0, cGMP level in control culture; 0, cGMP level in methylene blue-treated culture. Each point represents the mean of duplicate determinations.
58 h 44.5 115 24141 138.6 68523
Each value represents
+- 2.7 f 9.4 f 1442 k 5.9 + 4033
Methylene blue (58 h) 21.1 105 23943 131.4 55169
k 3.1 +- 10.3 t 1254 k 4.5 +- 2658
the mean f SD.
ate cyclase inhibitor, the cGMP levels in the methylene blue-treated and control cultures were examined. As shown in Fig. 5,0.6 &4 methylene blue delayed both the myoblast fusion and the elevation in cGMP levels. Even in the methylene blue-treated cultures, the rise in cGMP levels was also closely associated with the fusion. Then, we examined the effect of 8Br-cGMP on the fusion-blocked myoblasts imposed by methylene blue. As shown in Fig. 4C, 2 h exposure to 8Br-cGMP significantly reversed the fusion block at 1 nM but not below this concentration.
The most striking finding of the present study is that a large but transient increase in intracellular cGMP levels appears concomitant with the onset of myoblast fusion. The invariability of the match in time between the peak of cGMP and myoblast fusion suggests a link between the two events. A role of Ca2+ in myoblast fusion was previously indicated by the inhibitory effect of Ca2+ removal from the culture medium [ 251 and by the finding of David et al.  that the Ca2+ channel blocker D600 inhibited spontaneous fusion, while the Ca2+ ionophore A23187 induced precocious fusion. Furthermore, David et al.  found an increase in calcium influx during both spontaneous myoblast fusion and precocious fusion initiated by either A23187 or PGE, . In our experiments, D600 and A23187 were effective for modulating both cGMP levels and cell fusion at previously indicated concentration ranges where the net 45Ca2+influx was altered . D600 (20 PM) induced the decrease in both cGMP levels and myoblast fusion. In contrast, A23187 transiently increased the cGMP levels in a dose-dependent manner and accelerated myoblast fusion as well. In addition, 8-Br-cGMP induced precocious fusion as A23187 did. These findings suggest that a transient increase in cGMP levels is closely associated with the net Ca2’ influx and also that this rise in cGMP levels is capable of triggering myoblast fusion.
Numerous agents that inhibit myoblast fusion have been identified . Most of these inhibitions are likely to represent a general block of differentiation or cellular function. However, inhibition of prostanoid synthesis [lo], blocking of AChR , and lowering of external Ca2+ , which all seem to affect the fusion process alone, can dissociate the link between myoblast fusion and expression of muscle-specific proteins. In the present study, the inhibitor of guanylate cyclase, methylene blue, was found to inhibit myoblast fusion in a dose-dependent manner, but not the rate of DNA synthesis, CK activity, or AChR binding. In addition, methylene blue was found not to hinder cell alignment to any detectable degree. Thus, the action of methylene blue on myoblast differentiation is likely to be confined to the fusion process, without any discernible effects on alignment, proliferation, or biochemical differentiation. On the other hand, the specific inhibition of myoblast fusion by methylene blue is likely to block the increase in the cGMP levels for two reasons. First, methylene blue (0.6 piV) rendered the normal rise of cGMP levels delayed and partially inhibited. Second, a 2-h exposure of myoblasts to 8-Br-cGMP (1 n&f) was sufficient to achieve a significant recovery of fusion-retarded myoblasts imposed by methylene blue. On the basis of present findings, we suggest that a transient rise in cGMP levels concomitant with the onset of fusion is an important step in chick myoblast fusion and that this rise in cGMP levels is closely associated with Ca2+ influx. In addition, the relation between Ca2+ influx and cGMP levels suggests the possibility that a Ca2+ influx-dependent rise in cGMP levels mediates the effect of Ca2+ influx on myoblast fusion. However, further studies are needed to get an insight into a more precise role of cGMP and to understand the relationship of Ca2+ influx and cGMP levels in the fusion of chick myoblasts. We thank Mr. In Jun Park for his technical assistance. This work was supported by grants from Seoul National University, Daewoo Research Fund, and Korea Science and Engineering Foundation through SRC for Cell Differentiation. Received August 26,199l Revised version received October 22, 1991
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