Journal 01 Neurorhrrrtisrry Vol. 30, pp. 1239-1243 Pergarnon Press Ltd. 1978. Printed in Great Britain 0 International Soarty lor Neurocheinistry Ltd.
0022-3042/78/0601- I239502.00/0
STIMULATION OF NUCLEAR RNA SYNTHESIS IN DENERVATED SKELETAL MUSCLES IRENER. HELD Neuroscience Research Laboratory, Veterans Administration Hospital, Hines, IL 60141 and Loyola University Stritch School of Medicine, Maywood, IL 60153, U.S.A. (Received 22 August 1977. Accepted 29 November 1977)
Abstract-The endogenous activities of the RNA polymerases A and B are higher in nuclei isolated from slow-twitch soleus and fast-twitch extensor digitorum longus muscles which have been denervated for 2-3 days than in nuclei isolated from sham-operated, contralateral muscles. This stimulation is not observed immediately after denervation and declines by the fifth day. The results suggest that some neural influence is exerted upon the contiguous muscle cell at the level of gene transcription.
ALTHOUGHit is recognized that the motor neuron influences the protein composition as well as the maintenance of protein homeostasis in skeletal muscle, the molecular events by which these are mediated are not known. It is hypothesized that substances which are synthesized in the cell body of the motor neuron and carried by axoplasmic transport to the contiguous muscle cell may play a role in the regulation of protein synthesis in the target cell. Furthermore, indirect evidence suggests that such trophic influences of nerve upon muscle may act at the level of gene expression 1976). The rate of gene tran(GUTH, 1971, GUTMANN, scription by the R N A polymerases is, then, one regulatory site which may be influenced when innervation is altered. Recently, we demonstrated that the distinctive ionic requirements and differential sensitivity to the mushroom toxin a-amanitin, which are found for the soluble, purified RNA polymerases (nucleoside triphosphate: R N A nucleotidyltransferase, EC 2.7.7.6), may be employed to distinguish the activities of these enzymes in isolated skeletal muscle nuclei (HELD, 1977). At low ionic strength, for example, it has been established that Mgz+ preferentially stimulates the a-amanitin resistant, nucleolar R N A polymerase A (or I) to transcribe ribosomal-type RNA, while the a-amanitin sensitive, nucleoplasmic R N A polymerase B (or 11) is stimulated to synthesize heterogeneous, nuclear R N A in the presence of M n 2 + at high ionic et al., 1974; TATA& BAKER,1974; strength (CHAMBON BISWASet al., 1975). These kind of assays, conducted in the presence of endogenous influences within the nucleus and the homologous, though highly restricted D N A template, have been used to demonstrate the hormonal stimulation of the R N A polymerases in nu1974). clei from endocrine tissues (SPELSBERG, This report describes a delayed, transitory activation of the nuclear R N A polymerases A and B after
Abbreviation used: EDL. extensor digitorum longus.
denervation of the slow-twitch soleus and the fasttwitch extensor digitorum longus (EDL) muscles of the rat by cutting the sciatic nerve at the level of the mid-thigh. The results are based upon paired experiments with nuclei isolated from soleus and EDL muscles of the contralateral, sham-operated limb and assayed to obtain ‘internal’ control levels of enzyme activity. MATERIALS AND METHODS Animals and muscles. The soleus and extensor digitorum longus (EDL) muscles in the left hind limb of male, albino, Wistar rats, 3-6 months of age, were denervated by cutting the left sciatic nerve while the animals were lightly anesthetized with ether. The length of the nerve stump remaining attached to the soleus was 3C35mm and that attached to the EDL was W45mm. A sham-operation was performed on the right hind limb. A group of 8-12 rats was prepared for each experiment. The number of groups at each of the denervation periods of 3, 25, 49, 72 and 120 h is given in the Tables. The denervated and sham-operated, control muscles were removed within several minutes after decapitation of the rats and then appropriately pooled in ice-cold homogenizing media (0.32 M-sucrose 3 mM-MgCI,, pH 6.8). Isolation of muscle nuclei. In order to reproducibly recover pure nuclei from the soleus and EDL in yields of 16 and 20%, respectively, distinct procedural variations which we have recently reported in detail are required for each type of muscle (HELDet al., 1977). After the muscles are freed of tendon and connective tissue and minced in a cold room, the isolation of nuclei essentially involves: (I) homogenization with a blade-type homogenizer; (2) vacuum filtration of the homogenates through stainless steel screens; (3) collection of a myofibrillar-nuclear pellet by low speed centrifugation; (4) resuspension of this pellet in 3mM-MgCI2-dense sucrose of 54.5% for EDL and 55.5% for soleus-this critical step must be checked with a hand refractometer; and (5) sedimentation of pure nuclei by ultracentrifugation at 52,8OOg,,, for 45min at 5°C in a Beckman type SW 27 rotor. The pelleted nuclei are resuspended in 0.25 M-sucrose-1 mM-MgCi,, pH 6.5, at a concentration of IW250pg DNA per ml and then aliquots are taken immediately for the RNA polymerase assays.
1239
IRENE R . HELD
1240
E
0 DENEWTED (49 HR) EDL 0 CONTROL EDL
9-0
6 .O
3.0
1
I
I
I
I
I
5
10
I5
20
25
30
J.LG
DNA INCUBATED
FIG. 1. Relationship of the level of activity of RNA polymerase A (-Mg2+ assay) and of RNA polymerase B (-----Mn'+ assay) expressed as the pmol of [14C4]UMP incorporated into acid-insoluble material to the concentration of skeletal muscle nuclei in the incubation media. The number of muscle nuclei incubated is based upon the DNA content of resuspensions of nuclei which were isolated from soleus and EDL (extensor digitorum longus) muscles. The muscles were pooled from 32 rats in order to isolate a sufficient quantity of pure nuclei for the two RNA polymerase assays and for the quantification of DNA. Although each point, therefore, represents only a single determination, linearity was replicated in two other similar experiments. Assays were perlormed as described in Material; and Methods with the following: (A) 0 nuclei isolated from 32 soleus muscles denervated by unilateral sciatectomy for 49 h ; 0 nuclei isolated from 32 soleus muscles of the sham-operated, contralateral limb. (B) o nuclei isolated from 32 EDL muscles denervated by unilateral sciatectomy for 49 h ; 0 nuclei isolated from 32 EDL muscles of the sham-operated, contralateral limb.
Increased nuclear RNA synthesis after denervation
1241
R N A polymerase assays. The assays for the Mg2+-acti- TABLE2. EFFECTOF DENERVATION UPON THE ENDOGENOUS vated RNA polymerase A and the Mn2+ (NH4)$304RNA POLYMERASE ACTIVITIES OF EDL MUSCLE NUCLEI activated RNA polymerase B were conducted under the Denervat ion RNA Polymerase activity conditions which we have recently described as optimal A B period (h) for nuclei from the soleus and EDL muscles (HELD,1977). The levels of enzyme activity were determined as the pmol 102 rf: 4 3 (3) 103 f 3 of CJ4C4]UMP incorporated into cold, acid-insolu ble 101 4 104 4 25 (5) material per mg DNA per incubation period. DNA was 125 2* 131 f 3. 49 (6) quantified by the modified diphenylamine reaction (BUR139 & 4* 72 (3) 160 & 5* TON,1956) after extraction from the nuclear resuspensions 120 (3) 89 4 83 & 4 according to a modification of the Schmidt-Thannhauser Means f S.E.M.are expressed as percentages of the procedure (FLECK & MUNRO,1962). specific enzyme activities (pmol [14C4]UMP incorporated into acid-insoluble material/mg nuclear DNA/incubation RESULTS period) of nuclei isolated from EDL (extensor digitorum longus) muscles denervated by sciatectomy relative to the The endogenous activities of RNA polymerase A specific enzyme activities of nuclei from sham-operated and B were assayed with nuclei isolated from soleus EDL muscles of the contralateral limb. The number of and EDL muscles which had been denervated for 3, groups of rats at each denervation period is given in parenThe isolation and assays of nuclei were conducted 25, 49, 72 and 120 h by cutting the sciatic nerve in theses. as paired experiments with separate pools of denervated the mid-thigh of the rat and, simultaneously, with and sham-operated EDL muscles from 8-12 rats in each ‘control’ nuclei isolated from the sham-operated group. The assays for RNA polymerase A (MgZf-activated muscles of the contralateral limb. Whether ‘control’ or ‘denervated’ nuclei are assayed, the pmol of enzyme) and RNA polymerase B (Mn’+ + (NH4)’S04activated enzyme) were conducted as described in Mater[‘4C4]UMP incorporated into acid-insoluble material ials and Methods.
+
*
is linearly dependent upon the number of nuclei (or amount of DNA) added to the incubation media (Fig. I). The difference in the specific enzyme activities of nuclei from control and 49 h denervated soleus (Fig. 1A) and control and 49 h denervated EDL (Fig. 1B) are consistent, therefore, regardless of the amount of DNA incubated within a range of 530pg. The specific activities for RNA polymerase A and B obtained with ‘control’ soleus nuclei are 270 and 1500pmol of [‘4C4]UMP incorporated per mg DNA per incubation period, respectively, based upon the slopes of the lines in Fig. 1A. For ‘control’ EDL nuclei, the specific activity of RNA polymerase A is 240 TABLE1. EFFECTOF DENERVATION UPON T H E ENDOGENOUS RNA POLYMERASE ACTIVITIES OF SOLEUS MUSCLE NUCLEI Denervation Period (h) ~
3 (3) 25 (5) 49 (8) 72 (3) 120 (3)
RNA Polymerase activity A B ~
~
+4
99 107 & 120 & 125 86
loo & 3 111 & 5 140 3* 110 4 93 f 3
+
*
+
4 2* 3* 4
Means f S.E.M. are expressed as percentages of the specific enzyme activities (pmol [14C4]UMP incorporated into acid-insoluble material/mg nuclear DNA/incubation period) of nuclei isolated from soleus muscles denervated by sciatectomy relative to the specific enzyme activities of nuclei from sham-operated soleus muscles of the contralateral limb. The number of groups of rats at each denervation period is given in parentheses. The isolation and assays of nuclei were conducted as paired experiments with separate pools of denervated and sham-operated soleus muscles from 8-12 rats in each group. The assays for RNA polymerase A (Mg’+-activated enzyme) and RNA polymerase B (Mn2+ (NH4)’S04activated enzyme) were conducted as described in Materials and Methods. * P < 0.01.
+
**
* P < 0.01.
and of RNA polymerase B is ll00pmol of [14C4]UMP incorporated per mg DNA per incubation period, based upon the slopes of the lines in Fig. 1B. These values, obtained with nuclei isolated from sham-operated muscles, are comparable to those reported previously when nuclei were isolated from the soleus and EDL muscles of untreated rats. The higher level of activity of RNA polymerase B, but not of RNA polymerase A, observed with nuclei from the soleus compared to EDL nuclei is statistically significant (HELD,1977). The specific activity for RNA polymerase A by the Mgz+ assay with ‘49 h denervated’ soleus nuclei is consistently 140% higher than that of ‘control’ soleus nuclei; and also, the specific activity for RNA polymerase B by the Mn2+ (NHJ2SO4 assay with ‘49 h denervated’ soleus nuclei is consistently 120% higher than that of ‘control’ soleus nuclei (Fig. 1A and Table 1). Consistent increases for RNA polymerase A (131%) and B (125%) were also observed with ‘49 h denervated’ EDL nuclei (Fig. 1B and Table 2). The expression of the polymerase activites in terms of units of DNA makes the valid assumption that the endogenous levels of DNA per diploid nucleus of a given species is stable (DAVIDSON, 1960). Several lines of evidence have shown that the nuclei within the multinucleated syncitia of the myofiber are inactive in DNA synthesis and can be regarded as postmitotic (FISCHMAN, 1972). If denervation induces mitotic activity, possibly in some nuclei of muscle satellite cells (HANZLIKOVA et al., 1975; BLUNT et al., 1975), then increased levels of DNA would be expected and the specific activity of the polymerases would be apparently decreased, not increased as reported here. The nuclear RNA polymerases do not appear
+
IRENER. HELD
1242
stimulated when the soleus and EDL muscles have been denervated for only 3 h (Tables 1 and 2). Even at 25 h, only a slight increase may be seen with soleus nuclei, but no change is evident with EDL nuclei. Both soleus and EDL nuclei, however, have statistically significant increases in the specific activities of the A and B polymerases after a denervation period of 49 h. This persists with soleus nuclei, and appears to increase further with EDL nuclei, at 72 h after the sciatectomy. By the fifth day after denervation (120 h), however, the RNA polymerase activities appear normal or slightly inhibited. DISCUSSION
Both the Mg*+-activated and the M n 2 + + (NH4)1SO,-activated nuclear RNA polymerases A and B, respectively, of soleus and EDL (extensor digitorum longus) muscles are stimulated after these muscles have been denervated for 2-3 days by section of the sciatic nerve in the mid-thigh of the rat. Whether this is a consequence of an activation or an increase in the polymerase enzymes, or an alteration in the availability of the DNA template for transcription is not known. Previously, we established that the levels of activity of the a-amanitin resistant, RNA polymerase A and the m-amanitin sensitive, RNA polymerase B in these skeletal muscle nuclei are being differentiated with the incubation conditions employed (HELD, 1977). Other investigators have shown that these polymerases stimulate the synthesis of different types of nuclear RNAs; i.e. the nucleolar RNA polymerase A transcribes a ribosomal-type RNA, while the nucleoplasmic RNA polymerase B stimulates the synthesis of heterogeneous, nuclear RNA (CHAMBON er al., 1974; TATA& BAKER,1974; BISWASet al., 1975). The present results suggest, therefore, that the synthesis of these types of nuclear RNAs is increased for a brief period very shortly after denervation. It should also be noted that there is a lag period of 3-25 h before a stimulation of the nuclear RNA polymerases is observed in the soleus and of more than 25h in the EDL. Further experiments are needed to determine whether this delay is related to the length of nerve stump remaining attached to the muscle and, presumably, the time course for the depletion of axonally transported neurotrophic factors. The subsequent decline of polymerase activities which was seen after the longer denervation interval of 5 days may be complicated by other endogenous influences of the muscle per se. Of course, it is of considerable interest to understand how the transitory stimulation of nuclear RNA synthesis in denervated muscle could be maintained. The population of nuclei isolated from denervated muscle may contain ‘activated’ satellite cells which are manifested ultrastructurally by increased numbers of ribosomes and polysornes (ONTELL, 1975). It is not clear, however, whether the small proportion of these cells would make a significant contribution to the
average level of enzyme activity per nucleus obtained in our experiments, The number of satellite cells, which normally comprise 4.5-5.8% of the nuclear population of soleus muscle and less than 8% of other skeletal muscles, only increases to about 7.2% after denervation of the soleus for 4 days (HANZLIKOVA et al., 1975; BURLEIGH,1977). The stimulus for this initial regenerative response following denervation is not known. Although different parameters, types of muscles and periods of denervation were employed in the few biochemical studies of ribosomal RNA changes following denervation of mammalian limb muscles, seemingly discrepant findings were obtained. The concentration and total content of RNA, which is predominantly ribosomal RNA, slightly decreases in soleus and EDL muscles within 1 day after denervation, and then increases in the EDL after 7 days (GOLDSPINK,1976). While the in oioo uptake of radiolabeled uridine markedly increases in soleus muscle denervated for five days (MUCHNIK& KOTSIAS,1975), a reduced in vitro uptake of this precursor by nuclei isolated from denervated gastrocnemius muscle has been reported (GUREVICH et al., 1973). It should be recalled that the incorporation of uridine is dependent upon the uridine kinase reaction which is subject to feedback regulation by endogenous levels of UTP. After a denervation period of 3 days, the protein synthesizing capacity of ribosomes isolated from soleus is reduced, while no change occurs with ribosomes from the EDL (PLUSKAL & PENNINGTON, 1976). Implantation of the fast peroneal nerve into denervated soleus, however, increases the translational effectiveness of the soleus ribosomes (BURESOVA et al., 1975). Long periods of denervation (2-3 weeks) of the semitendinosus and gastrocnemius muscles increases the population of free ribosomes relative to messenger-associated ribosomes (GAUTHIER & DUNN,1973; KLEMPERER, 1972). Following a stimulated transcription of nuclear RNAs, an increase in the production of protein, or the synthesis of new species of protein, or both of these, would be anticipated. The present findings, then, seem contradictory to the well known denervation atrophy of limb muscles. Recently, however, a selective stimulation of myosin synthesis was observed shortly after denervation of the limb muscles of the newt (BERESFORD et al., 1976). Other early effects upon muscle membrane properties also indicate that there may be a transitory activation of the protein synthetic machinery associated with denervation. I n particular, the appearance of extra-junctional acetylcholine (ACh) supersensitivity in denervated muscles has been attributed to the synthesis of new ACh receptors (BROCKES& HALL, 1975). Since the development of these ACh receptors can be blocked by puromycin and Actinomycin D, it has been hypothesized that ACh receptor distribution is regulated through a neuronal modulation of genetic expression (FAMBROUGH,1970; FAMBROUGH et al., 1974; GRAMPP et al., 1972). Ultrastructural observa-
Increased nuclear RNA synthesis after denervation tions reveal aggregations of ribosomes apparently preceding the development of ACh sensitivity (GAUTHIER& SCHAEFFER, 1974). It has also been suggested that the presence of phagocytic elements in denervated muscle induces ACh sensitivity (BLUNTet al., 1975). Recently, however, it was reported that the post-denervation increase in muscle acid hydrolase activities is not related t o the presence of phagocytes and can also be abolished by pre-treatment with et al., 1977). It has also Actinomycin D (MASKREY been found that chronic stimulation of denervated soleus muscle not only inhibits fibrillation activity, but also decreases the uptake of radiolabeled uridine compared to nonstimulated, denervated muscle in which uridine uptake is increased (MUCHNIK& KOTSIAS, 1975). Even if muscle fiber activity plays a role in the distribution of ACh receptor density (DRACHMAN & WITZKE, 1972; L ~ M & O ROSENTHAL,1972; COHEN& FISCHBACH,1973), this may still involve some regulation at the level of gene expression (FAMBROUGH et al., 1974). The results reported here demonstrate that the transcriptional machinery is altered in muscle nuclei after denervation, but experiments with other animal preparations are needed to clarify whether these are attributable t o the influence exerted by the motor neuron through neurotrophic factors or through cholinergic transmission.
1243
BURESOVA M., HANZLIKOVA V. & GUTMANN E. (1975). Pjugers Arch. ges. Physiol. 360, 95-108. BURLEIGH I. G. (1977) J. Cell Sci. 23, 269-282. BURTONK. (1956) Biochem. J . 62, 315-323. CHAMBON P., GISSINGER F., KEDINGER C., MANDELJ. L. & MEILHAC M. (1974) in T h e Cell Nucleus (BUSCHH., ed.) Vol. 111, pp. 269-308. Academic Press, New York. COHENS. A. & FISCHBACH G. D. (1973) Science N.Y. 181, 7678. DAVIDSON J. N. (1960) in The Biochemistry of the Nucleic Acids, pp. 135-163. John Wiley, New York. DRACHMAN D. B. & WITZKEF. (1972) Science, N.Y. 176, 514516. FAMBROUGH D. M. (1970) Science, N.Y. 168, 372-378. H. C., POWELL J. A., RASH FAMBROUGH D. M., HARTZELL J. E., & JOSEPHN. (1974) in Synaptic Transmission and Neuronal Interaction (BENNETTM. V. L., ed.) pp. 285-313. Raven Press, New York. FISCHMAN D. A. (1972) in T h e Structure and Function of Muscle (BOURNE G. H., ed.) Vol. I, pp. 75-148. Academic Press, New York. FLECKA. & MUNROH. N. (1962) Biochim. biophys. Acta. 55, 571-583. GAUTHIER G. F. & DUNNR. A. (1973) J. Cell. Sci. 12, 525547. GAUTHIER G. F. & SCHAEFFER S. F. (1974) J . Cell. Sci. 14, 113-137. GOLDSPINK D. F. (1976) Biochem. J . 156, 71-80. GRAMPP E., HARRIS J. B. & THESLEFF S. (1972) J . Physiol. 221, 743-754. GUREVICH V. S., RAZUMOVSKAYA N. 1. & KHALAFOVA N. M. (1973) Proc. Acad. Sci., U.S.S.R. 211, 501-503. Acknowledgements-The author wishes to thank Mr. HOCK GUTHL. (1971)in Contractility of Muscle Cells and Related R. J., ed.) pp. 189-201. PrenticeProcesses (POWLSKY C. YEOH and Mr. REYNALDOT. RODRIGOfor their valuable Hall, Englewood Cliffs, NJ. technical assistance. This investigation was supported by E. (1976) A . Rev. Physiol. 38, 177-216. the Medical Research Service of the Veterans Administra- GUTMANN V., MACKOVA E. V. & HNIKR. (1975) Cell tion and by USPHS Research Grant NS 11755 from the HANZLIKOVA Tiss. Res. 163, 411421. National Institute for Neurological and Communicative HELDI. R. (1977) Exp. Cell Res. 108, 432-435. Disorders and Stroke. HELD,I. R., RODRIGOR. T., Y E ~ HH. C. & TONAKIH. (1977) Exp. Cell Res. 105, 191-197. KLEMPERER H. G. (1972) FEES. Lett. 28, 169172. LOMOT. & ROSENTHAL J. (1972) J. Physiol. 221, 493-513. REFERENCES MASKREY P., PLUSKAL M. G., HARRISJ. B. & PENNINGTON R. J. T. (1977) J. Neurochem. 28, 403-409. BERF~FORD B. J., RATHBONEM. P. & LOGAND. M. (1976) MUCHNIK S. & KOTSIAS B. A. (1975) Life Sci. 16, 543-550. Expl. Neurol. 52, 177-188. BISWASB. B., GANGULY A. & DASA. (1975) in Progress ONTELLM. (1975) Cell Tiss. Res. 160, 345-353. M. G. & PENNINGTON R. J. (1976) Expl. Neurol. in Nucleic Acid Research and Molecular Biology (COHN PLUSKAL 51, 574578. W. E., ed.) Vol. 15, pp. 145-184. Academic Press, New SPEWBERG T. C. (1974) in Acidic Proteins of the Nucleus York. (CAMERON I. L. & JETERJ. R., JR., eds.) pp. 248-296. BLUNTR. J., JONESR. & VRBOVAG. (1975) Pjugers Arch. Academic Press, New York. ges. Physiol. 355, 189-214. BROCKES J. P. & HALLZ. W. (1975) Proc. natn. Acad. Sci., TATAJ. R. & BAKERB. (1974) Exp. Cell Res. 83, 111-125; 125-138. U.S.A. 12, 1368-1372.
0022-3042/78/0601- I239502.00/0
STIMULATION OF NUCLEAR RNA SYNTHESIS IN DENERVATED SKELETAL MUSCLES IRENER. HELD Neuroscience Research Laboratory, Veterans Administration Hospital, Hines, IL 60141 and Loyola University Stritch School of Medicine, Maywood, IL 60153, U.S.A. (Received 22 August 1977. Accepted 29 November 1977)
Abstract-The endogenous activities of the RNA polymerases A and B are higher in nuclei isolated from slow-twitch soleus and fast-twitch extensor digitorum longus muscles which have been denervated for 2-3 days than in nuclei isolated from sham-operated, contralateral muscles. This stimulation is not observed immediately after denervation and declines by the fifth day. The results suggest that some neural influence is exerted upon the contiguous muscle cell at the level of gene transcription.
ALTHOUGHit is recognized that the motor neuron influences the protein composition as well as the maintenance of protein homeostasis in skeletal muscle, the molecular events by which these are mediated are not known. It is hypothesized that substances which are synthesized in the cell body of the motor neuron and carried by axoplasmic transport to the contiguous muscle cell may play a role in the regulation of protein synthesis in the target cell. Furthermore, indirect evidence suggests that such trophic influences of nerve upon muscle may act at the level of gene expression 1976). The rate of gene tran(GUTH, 1971, GUTMANN, scription by the R N A polymerases is, then, one regulatory site which may be influenced when innervation is altered. Recently, we demonstrated that the distinctive ionic requirements and differential sensitivity to the mushroom toxin a-amanitin, which are found for the soluble, purified RNA polymerases (nucleoside triphosphate: R N A nucleotidyltransferase, EC 2.7.7.6), may be employed to distinguish the activities of these enzymes in isolated skeletal muscle nuclei (HELD, 1977). At low ionic strength, for example, it has been established that Mgz+ preferentially stimulates the a-amanitin resistant, nucleolar R N A polymerase A (or I) to transcribe ribosomal-type RNA, while the a-amanitin sensitive, nucleoplasmic R N A polymerase B (or 11) is stimulated to synthesize heterogeneous, nuclear R N A in the presence of M n 2 + at high ionic et al., 1974; TATA& BAKER,1974; strength (CHAMBON BISWASet al., 1975). These kind of assays, conducted in the presence of endogenous influences within the nucleus and the homologous, though highly restricted D N A template, have been used to demonstrate the hormonal stimulation of the R N A polymerases in nu1974). clei from endocrine tissues (SPELSBERG, This report describes a delayed, transitory activation of the nuclear R N A polymerases A and B after
Abbreviation used: EDL. extensor digitorum longus.
denervation of the slow-twitch soleus and the fasttwitch extensor digitorum longus (EDL) muscles of the rat by cutting the sciatic nerve at the level of the mid-thigh. The results are based upon paired experiments with nuclei isolated from soleus and EDL muscles of the contralateral, sham-operated limb and assayed to obtain ‘internal’ control levels of enzyme activity. MATERIALS AND METHODS Animals and muscles. The soleus and extensor digitorum longus (EDL) muscles in the left hind limb of male, albino, Wistar rats, 3-6 months of age, were denervated by cutting the left sciatic nerve while the animals were lightly anesthetized with ether. The length of the nerve stump remaining attached to the soleus was 3C35mm and that attached to the EDL was W45mm. A sham-operation was performed on the right hind limb. A group of 8-12 rats was prepared for each experiment. The number of groups at each of the denervation periods of 3, 25, 49, 72 and 120 h is given in the Tables. The denervated and sham-operated, control muscles were removed within several minutes after decapitation of the rats and then appropriately pooled in ice-cold homogenizing media (0.32 M-sucrose 3 mM-MgCI,, pH 6.8). Isolation of muscle nuclei. In order to reproducibly recover pure nuclei from the soleus and EDL in yields of 16 and 20%, respectively, distinct procedural variations which we have recently reported in detail are required for each type of muscle (HELDet al., 1977). After the muscles are freed of tendon and connective tissue and minced in a cold room, the isolation of nuclei essentially involves: (I) homogenization with a blade-type homogenizer; (2) vacuum filtration of the homogenates through stainless steel screens; (3) collection of a myofibrillar-nuclear pellet by low speed centrifugation; (4) resuspension of this pellet in 3mM-MgCI2-dense sucrose of 54.5% for EDL and 55.5% for soleus-this critical step must be checked with a hand refractometer; and (5) sedimentation of pure nuclei by ultracentrifugation at 52,8OOg,,, for 45min at 5°C in a Beckman type SW 27 rotor. The pelleted nuclei are resuspended in 0.25 M-sucrose-1 mM-MgCi,, pH 6.5, at a concentration of IW250pg DNA per ml and then aliquots are taken immediately for the RNA polymerase assays.
1239
IRENE R . HELD
1240
E
0 DENEWTED (49 HR) EDL 0 CONTROL EDL
9-0
6 .O
3.0
1
I
I
I
I
I
5
10
I5
20
25
30
J.LG
DNA INCUBATED
FIG. 1. Relationship of the level of activity of RNA polymerase A (-Mg2+ assay) and of RNA polymerase B (-----Mn'+ assay) expressed as the pmol of [14C4]UMP incorporated into acid-insoluble material to the concentration of skeletal muscle nuclei in the incubation media. The number of muscle nuclei incubated is based upon the DNA content of resuspensions of nuclei which were isolated from soleus and EDL (extensor digitorum longus) muscles. The muscles were pooled from 32 rats in order to isolate a sufficient quantity of pure nuclei for the two RNA polymerase assays and for the quantification of DNA. Although each point, therefore, represents only a single determination, linearity was replicated in two other similar experiments. Assays were perlormed as described in Material; and Methods with the following: (A) 0 nuclei isolated from 32 soleus muscles denervated by unilateral sciatectomy for 49 h ; 0 nuclei isolated from 32 soleus muscles of the sham-operated, contralateral limb. (B) o nuclei isolated from 32 EDL muscles denervated by unilateral sciatectomy for 49 h ; 0 nuclei isolated from 32 EDL muscles of the sham-operated, contralateral limb.
Increased nuclear RNA synthesis after denervation
1241
R N A polymerase assays. The assays for the Mg2+-acti- TABLE2. EFFECTOF DENERVATION UPON THE ENDOGENOUS vated RNA polymerase A and the Mn2+ (NH4)$304RNA POLYMERASE ACTIVITIES OF EDL MUSCLE NUCLEI activated RNA polymerase B were conducted under the Denervat ion RNA Polymerase activity conditions which we have recently described as optimal A B period (h) for nuclei from the soleus and EDL muscles (HELD,1977). The levels of enzyme activity were determined as the pmol 102 rf: 4 3 (3) 103 f 3 of CJ4C4]UMP incorporated into cold, acid-insolu ble 101 4 104 4 25 (5) material per mg DNA per incubation period. DNA was 125 2* 131 f 3. 49 (6) quantified by the modified diphenylamine reaction (BUR139 & 4* 72 (3) 160 & 5* TON,1956) after extraction from the nuclear resuspensions 120 (3) 89 4 83 & 4 according to a modification of the Schmidt-Thannhauser Means f S.E.M.are expressed as percentages of the procedure (FLECK & MUNRO,1962). specific enzyme activities (pmol [14C4]UMP incorporated into acid-insoluble material/mg nuclear DNA/incubation RESULTS period) of nuclei isolated from EDL (extensor digitorum longus) muscles denervated by sciatectomy relative to the The endogenous activities of RNA polymerase A specific enzyme activities of nuclei from sham-operated and B were assayed with nuclei isolated from soleus EDL muscles of the contralateral limb. The number of and EDL muscles which had been denervated for 3, groups of rats at each denervation period is given in parenThe isolation and assays of nuclei were conducted 25, 49, 72 and 120 h by cutting the sciatic nerve in theses. as paired experiments with separate pools of denervated the mid-thigh of the rat and, simultaneously, with and sham-operated EDL muscles from 8-12 rats in each ‘control’ nuclei isolated from the sham-operated group. The assays for RNA polymerase A (MgZf-activated muscles of the contralateral limb. Whether ‘control’ or ‘denervated’ nuclei are assayed, the pmol of enzyme) and RNA polymerase B (Mn’+ + (NH4)’S04activated enzyme) were conducted as described in Mater[‘4C4]UMP incorporated into acid-insoluble material ials and Methods.
+
*
is linearly dependent upon the number of nuclei (or amount of DNA) added to the incubation media (Fig. I). The difference in the specific enzyme activities of nuclei from control and 49 h denervated soleus (Fig. 1A) and control and 49 h denervated EDL (Fig. 1B) are consistent, therefore, regardless of the amount of DNA incubated within a range of 530pg. The specific activities for RNA polymerase A and B obtained with ‘control’ soleus nuclei are 270 and 1500pmol of [‘4C4]UMP incorporated per mg DNA per incubation period, respectively, based upon the slopes of the lines in Fig. 1A. For ‘control’ EDL nuclei, the specific activity of RNA polymerase A is 240 TABLE1. EFFECTOF DENERVATION UPON T H E ENDOGENOUS RNA POLYMERASE ACTIVITIES OF SOLEUS MUSCLE NUCLEI Denervation Period (h) ~
3 (3) 25 (5) 49 (8) 72 (3) 120 (3)
RNA Polymerase activity A B ~
~
+4
99 107 & 120 & 125 86
loo & 3 111 & 5 140 3* 110 4 93 f 3
+
*
+
4 2* 3* 4
Means f S.E.M. are expressed as percentages of the specific enzyme activities (pmol [14C4]UMP incorporated into acid-insoluble material/mg nuclear DNA/incubation period) of nuclei isolated from soleus muscles denervated by sciatectomy relative to the specific enzyme activities of nuclei from sham-operated soleus muscles of the contralateral limb. The number of groups of rats at each denervation period is given in parentheses. The isolation and assays of nuclei were conducted as paired experiments with separate pools of denervated and sham-operated soleus muscles from 8-12 rats in each group. The assays for RNA polymerase A (Mg’+-activated enzyme) and RNA polymerase B (Mn2+ (NH4)’S04activated enzyme) were conducted as described in Materials and Methods. * P < 0.01.
+
**
* P < 0.01.
and of RNA polymerase B is ll00pmol of [14C4]UMP incorporated per mg DNA per incubation period, based upon the slopes of the lines in Fig. 1B. These values, obtained with nuclei isolated from sham-operated muscles, are comparable to those reported previously when nuclei were isolated from the soleus and EDL muscles of untreated rats. The higher level of activity of RNA polymerase B, but not of RNA polymerase A, observed with nuclei from the soleus compared to EDL nuclei is statistically significant (HELD,1977). The specific activity for RNA polymerase A by the Mgz+ assay with ‘49 h denervated’ soleus nuclei is consistently 140% higher than that of ‘control’ soleus nuclei; and also, the specific activity for RNA polymerase B by the Mn2+ (NHJ2SO4 assay with ‘49 h denervated’ soleus nuclei is consistently 120% higher than that of ‘control’ soleus nuclei (Fig. 1A and Table 1). Consistent increases for RNA polymerase A (131%) and B (125%) were also observed with ‘49 h denervated’ EDL nuclei (Fig. 1B and Table 2). The expression of the polymerase activites in terms of units of DNA makes the valid assumption that the endogenous levels of DNA per diploid nucleus of a given species is stable (DAVIDSON, 1960). Several lines of evidence have shown that the nuclei within the multinucleated syncitia of the myofiber are inactive in DNA synthesis and can be regarded as postmitotic (FISCHMAN, 1972). If denervation induces mitotic activity, possibly in some nuclei of muscle satellite cells (HANZLIKOVA et al., 1975; BLUNT et al., 1975), then increased levels of DNA would be expected and the specific activity of the polymerases would be apparently decreased, not increased as reported here. The nuclear RNA polymerases do not appear
+
IRENER. HELD
1242
stimulated when the soleus and EDL muscles have been denervated for only 3 h (Tables 1 and 2). Even at 25 h, only a slight increase may be seen with soleus nuclei, but no change is evident with EDL nuclei. Both soleus and EDL nuclei, however, have statistically significant increases in the specific activities of the A and B polymerases after a denervation period of 49 h. This persists with soleus nuclei, and appears to increase further with EDL nuclei, at 72 h after the sciatectomy. By the fifth day after denervation (120 h), however, the RNA polymerase activities appear normal or slightly inhibited. DISCUSSION
Both the Mg*+-activated and the M n 2 + + (NH4)1SO,-activated nuclear RNA polymerases A and B, respectively, of soleus and EDL (extensor digitorum longus) muscles are stimulated after these muscles have been denervated for 2-3 days by section of the sciatic nerve in the mid-thigh of the rat. Whether this is a consequence of an activation or an increase in the polymerase enzymes, or an alteration in the availability of the DNA template for transcription is not known. Previously, we established that the levels of activity of the a-amanitin resistant, RNA polymerase A and the m-amanitin sensitive, RNA polymerase B in these skeletal muscle nuclei are being differentiated with the incubation conditions employed (HELD, 1977). Other investigators have shown that these polymerases stimulate the synthesis of different types of nuclear RNAs; i.e. the nucleolar RNA polymerase A transcribes a ribosomal-type RNA, while the nucleoplasmic RNA polymerase B stimulates the synthesis of heterogeneous, nuclear RNA (CHAMBON er al., 1974; TATA& BAKER,1974; BISWASet al., 1975). The present results suggest, therefore, that the synthesis of these types of nuclear RNAs is increased for a brief period very shortly after denervation. It should also be noted that there is a lag period of 3-25 h before a stimulation of the nuclear RNA polymerases is observed in the soleus and of more than 25h in the EDL. Further experiments are needed to determine whether this delay is related to the length of nerve stump remaining attached to the muscle and, presumably, the time course for the depletion of axonally transported neurotrophic factors. The subsequent decline of polymerase activities which was seen after the longer denervation interval of 5 days may be complicated by other endogenous influences of the muscle per se. Of course, it is of considerable interest to understand how the transitory stimulation of nuclear RNA synthesis in denervated muscle could be maintained. The population of nuclei isolated from denervated muscle may contain ‘activated’ satellite cells which are manifested ultrastructurally by increased numbers of ribosomes and polysornes (ONTELL, 1975). It is not clear, however, whether the small proportion of these cells would make a significant contribution to the
average level of enzyme activity per nucleus obtained in our experiments, The number of satellite cells, which normally comprise 4.5-5.8% of the nuclear population of soleus muscle and less than 8% of other skeletal muscles, only increases to about 7.2% after denervation of the soleus for 4 days (HANZLIKOVA et al., 1975; BURLEIGH,1977). The stimulus for this initial regenerative response following denervation is not known. Although different parameters, types of muscles and periods of denervation were employed in the few biochemical studies of ribosomal RNA changes following denervation of mammalian limb muscles, seemingly discrepant findings were obtained. The concentration and total content of RNA, which is predominantly ribosomal RNA, slightly decreases in soleus and EDL muscles within 1 day after denervation, and then increases in the EDL after 7 days (GOLDSPINK,1976). While the in oioo uptake of radiolabeled uridine markedly increases in soleus muscle denervated for five days (MUCHNIK& KOTSIAS,1975), a reduced in vitro uptake of this precursor by nuclei isolated from denervated gastrocnemius muscle has been reported (GUREVICH et al., 1973). It should be recalled that the incorporation of uridine is dependent upon the uridine kinase reaction which is subject to feedback regulation by endogenous levels of UTP. After a denervation period of 3 days, the protein synthesizing capacity of ribosomes isolated from soleus is reduced, while no change occurs with ribosomes from the EDL (PLUSKAL & PENNINGTON, 1976). Implantation of the fast peroneal nerve into denervated soleus, however, increases the translational effectiveness of the soleus ribosomes (BURESOVA et al., 1975). Long periods of denervation (2-3 weeks) of the semitendinosus and gastrocnemius muscles increases the population of free ribosomes relative to messenger-associated ribosomes (GAUTHIER & DUNN,1973; KLEMPERER, 1972). Following a stimulated transcription of nuclear RNAs, an increase in the production of protein, or the synthesis of new species of protein, or both of these, would be anticipated. The present findings, then, seem contradictory to the well known denervation atrophy of limb muscles. Recently, however, a selective stimulation of myosin synthesis was observed shortly after denervation of the limb muscles of the newt (BERESFORD et al., 1976). Other early effects upon muscle membrane properties also indicate that there may be a transitory activation of the protein synthetic machinery associated with denervation. I n particular, the appearance of extra-junctional acetylcholine (ACh) supersensitivity in denervated muscles has been attributed to the synthesis of new ACh receptors (BROCKES& HALL, 1975). Since the development of these ACh receptors can be blocked by puromycin and Actinomycin D, it has been hypothesized that ACh receptor distribution is regulated through a neuronal modulation of genetic expression (FAMBROUGH,1970; FAMBROUGH et al., 1974; GRAMPP et al., 1972). Ultrastructural observa-
Increased nuclear RNA synthesis after denervation tions reveal aggregations of ribosomes apparently preceding the development of ACh sensitivity (GAUTHIER& SCHAEFFER, 1974). It has also been suggested that the presence of phagocytic elements in denervated muscle induces ACh sensitivity (BLUNTet al., 1975). Recently, however, it was reported that the post-denervation increase in muscle acid hydrolase activities is not related t o the presence of phagocytes and can also be abolished by pre-treatment with et al., 1977). It has also Actinomycin D (MASKREY been found that chronic stimulation of denervated soleus muscle not only inhibits fibrillation activity, but also decreases the uptake of radiolabeled uridine compared to nonstimulated, denervated muscle in which uridine uptake is increased (MUCHNIK& KOTSIAS, 1975). Even if muscle fiber activity plays a role in the distribution of ACh receptor density (DRACHMAN & WITZKE, 1972; L ~ M & O ROSENTHAL,1972; COHEN& FISCHBACH,1973), this may still involve some regulation at the level of gene expression (FAMBROUGH et al., 1974). The results reported here demonstrate that the transcriptional machinery is altered in muscle nuclei after denervation, but experiments with other animal preparations are needed to clarify whether these are attributable t o the influence exerted by the motor neuron through neurotrophic factors or through cholinergic transmission.
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BURESOVA M., HANZLIKOVA V. & GUTMANN E. (1975). Pjugers Arch. ges. Physiol. 360, 95-108. BURLEIGH I. G. (1977) J. Cell Sci. 23, 269-282. BURTONK. (1956) Biochem. J . 62, 315-323. CHAMBON P., GISSINGER F., KEDINGER C., MANDELJ. L. & MEILHAC M. (1974) in T h e Cell Nucleus (BUSCHH., ed.) Vol. 111, pp. 269-308. Academic Press, New York. COHENS. A. & FISCHBACH G. D. (1973) Science N.Y. 181, 7678. DAVIDSON J. N. (1960) in The Biochemistry of the Nucleic Acids, pp. 135-163. John Wiley, New York. DRACHMAN D. B. & WITZKEF. (1972) Science, N.Y. 176, 514516. FAMBROUGH D. M. (1970) Science, N.Y. 168, 372-378. H. C., POWELL J. A., RASH FAMBROUGH D. M., HARTZELL J. E., & JOSEPHN. (1974) in Synaptic Transmission and Neuronal Interaction (BENNETTM. V. L., ed.) pp. 285-313. Raven Press, New York. FISCHMAN D. A. (1972) in T h e Structure and Function of Muscle (BOURNE G. H., ed.) Vol. I, pp. 75-148. Academic Press, New York. FLECKA. & MUNROH. N. (1962) Biochim. biophys. Acta. 55, 571-583. GAUTHIER G. F. & DUNNR. A. (1973) J. Cell. Sci. 12, 525547. GAUTHIER G. F. & SCHAEFFER S. F. (1974) J . Cell. Sci. 14, 113-137. GOLDSPINK D. F. (1976) Biochem. J . 156, 71-80. GRAMPP E., HARRIS J. B. & THESLEFF S. (1972) J . Physiol. 221, 743-754. GUREVICH V. S., RAZUMOVSKAYA N. 1. & KHALAFOVA N. M. (1973) Proc. Acad. Sci., U.S.S.R. 211, 501-503. Acknowledgements-The author wishes to thank Mr. HOCK GUTHL. (1971)in Contractility of Muscle Cells and Related R. J., ed.) pp. 189-201. PrenticeProcesses (POWLSKY C. YEOH and Mr. REYNALDOT. RODRIGOfor their valuable Hall, Englewood Cliffs, NJ. technical assistance. This investigation was supported by E. (1976) A . Rev. Physiol. 38, 177-216. the Medical Research Service of the Veterans Administra- GUTMANN V., MACKOVA E. V. & HNIKR. (1975) Cell tion and by USPHS Research Grant NS 11755 from the HANZLIKOVA Tiss. Res. 163, 411421. National Institute for Neurological and Communicative HELDI. R. (1977) Exp. Cell Res. 108, 432-435. Disorders and Stroke. HELD,I. R., RODRIGOR. T., Y E ~ HH. C. & TONAKIH. (1977) Exp. Cell Res. 105, 191-197. KLEMPERER H. G. (1972) FEES. Lett. 28, 169172. LOMOT. & ROSENTHAL J. (1972) J. Physiol. 221, 493-513. REFERENCES MASKREY P., PLUSKAL M. G., HARRISJ. B. & PENNINGTON R. J. T. (1977) J. Neurochem. 28, 403-409. BERF~FORD B. J., RATHBONEM. P. & LOGAND. M. (1976) MUCHNIK S. & KOTSIAS B. A. (1975) Life Sci. 16, 543-550. Expl. Neurol. 52, 177-188. BISWASB. B., GANGULY A. & DASA. (1975) in Progress ONTELLM. (1975) Cell Tiss. Res. 160, 345-353. M. G. & PENNINGTON R. J. (1976) Expl. Neurol. in Nucleic Acid Research and Molecular Biology (COHN PLUSKAL 51, 574578. W. E., ed.) Vol. 15, pp. 145-184. Academic Press, New SPEWBERG T. C. (1974) in Acidic Proteins of the Nucleus York. (CAMERON I. L. & JETERJ. R., JR., eds.) pp. 248-296. BLUNTR. J., JONESR. & VRBOVAG. (1975) Pjugers Arch. Academic Press, New York. ges. Physiol. 355, 189-214. BROCKES J. P. & HALLZ. W. (1975) Proc. natn. Acad. Sci., TATAJ. R. & BAKERB. (1974) Exp. Cell Res. 83, 111-125; 125-138. U.S.A. 12, 1368-1372.
