Changes in myosin heavy chain (MHC) mRNAs were studied in rabbit fasttwitch muscles during continuous electrical stimulation at 10 Hz for periods up to 3 weeks, and during the first 12 days of the recovery process that followed cessation of 6 weeks' stimulation. Two cDNA probes were used to detect MHC mRNAs specific to fast- and slow-twitch skeletal muscle in RNase protection assays and Northern- and slot-blot analyses. The isolation and base sequence of one of these probes, corresponding to the MHC gene expressed in soleus (slow-twitch), is described. At an early stage of the response to stimulation, fast MHC mRNA was replaced by slow MHC mRNA. During recovery, this process occurred in reverse but took longer. The time course of recovery was slightly faster in tibialis anterior than in extensor digitorum longus. The changes in mRNAs during both stimulation and recovery reflected changes in the corresponding muscle proteins. Key words: muscle stimulation recovery myosin mRNA MUSCLE 84 NERVE 15:694-700 1992

RECIPROCAL CHANGES IN MYOSIN ISOFORM mRNAs OF RABBIT SKELETAL MUSCLE IN RESPONSE TO THE INITIATION AND CESSATION OF CHRONIC ELECTRICAL STIMULATION CAROL BROWNSON, BSc, PhD, PAULINE LITTLE, JONATHAN C. JARVIS, BSc, PhD, and STANLEY SALMONS, MSc, PhD

G e n e expression in adult skeletal muscle can respond to changing demands. A model system wellsuited to studying this response is one in which the contractile activity of a muscle is modified by electrical stimulation. There is extensive literature on transitions in the levels and types of protein associated with contractile, metabolic, and transport functions in a fast-twitch muscle subjected to continuous low-frequency electrical stimulation (see refs. 13, 16, and 17). In particular, isoforms of myosin specific to fast-twitch muscle in the rabbit are re ldced by slow-twitch muscle isoforms,*, ," 24 and there are changes in the corresponding myosin mRNAs, determined using

8:-

From the Department of Biological Sciences, City of London Polytechnic, London (Dr. Brownson and Ms. Little); and the Department of Human Anatomy and Cell Biology and the Muscle Research Centre, University of Liverpool, Liverpool, United Kingdom (Dr Jarvis and Prof Salmons) Acknowledgments The authors thank Miss C Mayne and Miss S Danzer Claren for their valuable contribution to the experimental work, and the Wellcome Trust and the British Heart Foundation for financial support. Address reprint requests to Dr Brownson, School of Life Sciences. Polytechnic of North London, Holloway Road, London N7 8DB, UK Accepted for publication July 15, 1991 CCC 0148-639x1921060694-07 $04 00 0 1992 John Wiley & Sons, lnc.

694

Reciprocal Changes in Myosin rnRNAs

cDNA probes to fast (Type 2B) and slow (Type 1) myosin heavy chains (MHC).4s5 l h e r e is also a growing body of information about the events that take place after stimulation has been discontinued.'" T h e starting point for such experiments is normally a stage of stimulation-6 weeks in the present case-at which most of the changes in the physiological, ultrastructural, and biochemical properties associated with long-term stimulation are already complete. On their recovery from such stimulation, rabbit muscles show a complete reversion to fast-muscle characteristics, with a time course more prolonged than that of the original transformation. In general, the associated changes in type-specific proteins occur in a reverse sequence to that seen during fast-to-slow transformation. In particular, calcium-activated myosin ATPase activity regains control fast levels in about 6 to 8 weeks.l6 Histochemical demonstration of myofibrillar ATPase activity indicates that the proportion of Type 1 fibers declines to that of the unstimulated contralatera1 muscle by about 4 weeks, and the proportions of Type 2B and Type 2A fibers reach control levels by 12 weeks.' Under broadly similar conditions of stimulation, rat muscles behave somewhat differently,

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and show no appreciable induction of Type 1 myosin. There is, however, a well-marked transition from Type 213 to Type 2A myosin.'5 Corresponding changes in the mRNA have been demonstrated during recovery as well as stimulation. These changes occur rapidly: Type 2A MHC niRNA (the predominant species after stimulation) declines noticeably over 6 days of recovery, with a concomitant reappearance of Type 2B MHC mRNA.' There is good evidence that the more extensive physiological changes induced by stimulation in rabbit muscle, which are associated with the switch to the expression of slow myosin heavy and light chains, are a consequence of pretranslational events. There is much less evidence of changes at the mRNA level during recovery in the rabbit, although the stimulation-induced decrease in aldolase mRNA and increase in myoglobin mRNA have been shown to undergo reversal during recovery." In this study, w e analyzed the time course of changes in MHC mRNA levels during recovery of fast-twitch muscle after continuous stimulation at 10 Hz for 6 weeks, using rabbit-specific cDNA probes to fast- and slow-muscle MHC mRNA. Although MHCs are derived from a multigene family whose members are highly homologous, the 3'-untranslated regions show no significant homology within a species.15 To achieve maximum specificity for fast and slow MHCs in our studies, we isolated and characterized a DNA fragment containing the 3'-untranslated region of a MHC gene from a cDNA library constructed from soleus muscle. MATERIALS AND METHODS

Total RNA was extracted from rabbit skeletal muscles in 8 mol/L urea and 4 mol/L LiCl. The crude R N A recovered by centrifugation was extracted with phenol and precipitated with ethanol. Poly(A)+ RNA was isolated by passage through an oligo-dl' Sepharose column.

Preparation of RNA.

Construction of a Rabbit Soleus Muscle cDNA Library in the Expression Vector Agt 11. A cDNA li-

brary was constructed from poly(A)+ RNA, prepared from rabbit soleus muscle, essentially as described by Huynh et al.' First, strand synthesis of cDNA was carried out with AMV reverse transcriptase and oligo-dT as primer; second, strand synthesis was done by incubation with Escherichia coli DNA polymerase I and RNase H.6 The resultant double-stranded cDNA was ligated into Agt 11

Reciprocal Changes in Myosin rnRNAs

and the D N A packaged into A phage using a commercial in vitro packaging kit (Amersham); the resultant phage library contained approximately 5 x lo4 independent recombinants and 80% of plaques contained insert DNA. Recombinant phage were plated with E . cola Y1088, arid screened by colony hybridization using two probes (pMHC6OO-F, pMHC450-F)" to the Type 2B MHC gene. Whereas pMHC6OO-F contains only coding sequences, pMHC450-F contains both coding and noncoding sequences at the 3' end of the gene and is, therefore, more specific for Type 2B MHC cDNA fragments. In order to eliminate clones which contained Type 2B MHC cDNA, those which hybridized strongly to pMHC6OO-F but not pMHC450-F were selected for further screening. After 4 rounds of screening, 15 positive clones were selected and the cDNA fragments subcloned into pUC 8. A 450-bp H z n f I fragment (pMHC450S), derived from a 1450-bp cDNA fragment (pMHC1450-S), was the probe used in our studies.

Screening the Library.

DNA Sequence Determinations. The cDNA was digested with the restriction enzymes, Hinf I and Ava I I , according to the manufacturer's instructions, and the fragments separated by agarose gel electrophoresis, subcloned into M13mp18 or M13mp19, and sequenced by the dideoxy chain termination method of Sanger."' Sequences were analyzed using Beckman Microgenie. Electrical Stimulation and Recovery of Skeletal Muscle. Fast muscles of adult New Zealarid white

rabbits were chronically stimulated via the common peroneal nerve.' In each case, a self-contained miniature stimulator'8 was implanted subcutaneously on the left flank; stimulation consisted of a continuous train of supramaximal pulses at a frequency of 10 Hz. For recovery experiments, muscles were stimulated for 6 weeks before the stimulator was turned off via a remote optical link. After periods of stimulation of 4 days to 6 weeks and periods of recovery of 4 to 12 days, the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles were removed, flash frozen in liquid N,, and stored at -70" C. Total or poly(A)+ R N A isolated from muscles was analyzed by Northern- and slot-blotting techniques as described previously4 using the two different myosin cDNA probes, pMHC450-S and pMHC450-F." The densities of RNA Analysis.

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695

the bands on the slot-blot autoradiographs were measured with a Joyce Loebl Scanning Densitometer and related to micrograms of total or poly(A)+ RNA. Integrated peak areas per microgram of RNA isolated from stimulated or recovery muscles were expressed as a percentage of the value obtained for control muscles. The integrity of all RNA preparations was checked on urea gels. Neither these gels, nor Northern-blots, showed any evidence of RNA degradation after stimulation, with or without recovery, of TA and EDL muscles. For RNase protection assays, pMHC450-S and pMHC450-F were subcloned into the Sma I and Bum HI sites, respectively, of pGEM ?Z. Plasmids were linearized at the Hind III or Eco RI sites and anti-sense KNA probes were transcribed using T7 and SP6 DNA polymerase for pMHC450-S and pMHC450-F, respectively, in the presence of 20 pCi of [a-"'PIUTP. In one experiment, the probe was labeled with digoxigenin- 1l-uridine-5'triphosphate (DIG-I 1-UTP) using a similar procedure. The labeled probe was incubated with either 10 p,g total RNA or 2 Fg poly(A)+ RNA before RNase digestion.'" RNase-resistant fragments were denatured in formamide, resolved on formaldehyde agarose gels, and transferred to nylon membranes for autoradiography ([a-:12P]UTP)or imrnunochemical staining (DIG-1 1-UTP). RESULTS

Electrophoretic analysis of the heavy and light chains of myosin, and assay of myosin ATPase activity in rabbit fast-twitch muscle, shows that transitions occur during both stimulation and recovery whose end result is a complete switch between the myosin protein isoforms characteristic of Type 2B and Type 1 To ascertain whether these changes in muscle protein composition are due to pretranslational events, we monitored changes in MHC mRNA, using two specific cDNA probes. A probe for fast (Type 2B) MHC mRNA, pMHC450-F was already available to us"; in addition, a new probe for a slow MHC mRNA was selected from a rabbit soleus muscle cDNA library by differential screening with pMHC6OO-F and pMHC450-F" (see METHODS). Preliminary sequencing indicated that a cDNA fragment, pMHC1450-S (1450 bp), from a clone that hybridized strongly to pMHC6OO-F, contained the poly(A)+ tail, and hence, coding and noncoding sequences at the 3'-end of a MHC gene. T o increase the specificity for slow muscle Isolation of Rabbit Slow-Twitch MHC cDNA.

696

Reciprocal Changes

in

Myosin mRNAs

MHC mRNA, we used a 450-bp fragment (pMHC450-S) obtained from a partial Hinf I digest of pMHC1450-S in this study. The nucleotide sequence of pMHC450-S is shown in Figure 1. It contains 306 bp of coding sequence, with extensive hornology to documented sequences at the 3'-end of other MHC cDNAs, and the full 147-bp section of noncoding region, including the potyadenyiation signal (AATAAA) and the poly(A) tail of the mRNA. Comparison of this sequence with that of pMHC450-F, the other probe used in this study, shows that there is only 76.7% homology between the coding sequences, indicating that these fragments correspond to the 3'-end of different myosin genes. Preliminary Northern-blot analyses indicated that the rabbit probe, pMHC 1450-S, hybridized strongly with mRNA isolated from both soleus and heart muscle of this species. This specificity was confirmed with the shorter probe, pMHC450-S (Fig. 28). We showed previously that pMHC1450-S hybridizied with mRNA isolated from rabbit masseter muscle (58% relative to soleus), and that there was limited hybridization with mRNA isolated from TA (19%) and EDL (23%).5 Subsequent results, obtained by slot-blot analysis of mRNA from various muscles with pMHC450-S, show that the shorter probe is more specific for soleus mRNA, with little cross-hybridization to mRNA from either TA or EDL (about 1% and 4% relative to soleus mRNA, respectively). Some cross-hybridization persisted with mRNA isolated from masseter (26% relative to soleus). Further analysis by RNase protection assays (Fig.

Analysis of pMHC450-S.

1

GAGTCGGTGAAGGGCATGAGGAAGAGTGAGCGGCGCATCAAGGAGCTCAC

51

CTACCAGACGGAGGAGGACAGGAAGAACCTGCTGCGGCTACAGGACCTGG

101

TGGACAAGCTGCAGCTGAAGGTCAAGGCCTACAAGCGCCAGGCCGAGGAG

151

GCGGAGGAGCAGGCCAACACCAACCTGTCCAAGTTCCGCAAGGTGCAGCA

201

CGAGCTGGATGAGGCAGAGGAGCGGGCAGACATTGCAGAGTCCCAGGTCA

251

ACAAGCTGCGGGCCAAGAGCCGCGACATCGGCACCAAGAGCTTGAATGAG

30 1

GAATAGCCrGGTGACGCCTTGATCCGCCCAGCCCTGAGGACGACGCCAGT

351

GAAGTCCCTTGTCTGGGAGCTCACATAGCAGCAGCCCTTGGGAAGAAGCA

401

GAATAAATCAGTTTTCCTCGAAGCTG(A)24

FIGURE 1. Nucleotide sequence of clone pMHC450-S, a Hinf I fragment containing coding and noncoding sequences at the 3'end of the cDNA. The end-codon and polyadenylation signal are underlined.

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PMHC 450 - S

pM H C450- F

Pr

S

E

M

S

E

M

pr

E

H

M

S

T

EDL

I B

A

pMHC 4 50 -S

pMHC450-F

Table 1. Relative fast (pMHC450-F) and slow (pMHC450-S, pMHCl450-S) muscle MHC mRNA levels in EDL and TA muscles after stimulation for 9 days.

TA

pMHC450-F (”/)

pMHC450-S

(”/I

pMHCi450-S (”/)

Total P(A+)

7 6

507 288

423

Total

10

24 1

-

-

Note The hgures given are integrated peak areas per microgram of poly(A)+ or total RNA determmed from slot-blot analysis, expressed as a percentage of the value for the same-day controls

C

C St

pr St

4d

pr St

C

9d

21d

mologous to pMHC450-F were almost completely absent from soleus and masseter muscles.

C

C

4d

9d

21d

FIGURE 2. Distribution of myosin heavy chains in soleus (S), EDL (E), heart (H),TA (T), masseter (M), and in 4; 9- and 21day stimulated (St) EDL muscles assessed by RNase protection assays of poly(A)+ RNA (A and C) or total RNA (B) with pMHC450-F and pMHC450-S as probes. In (A) and (C), the and in (B) with DIG-11-UTP. probe is labeled with [cx-~~P]UTP The labeled transcribed probe is also shown (pr).

2A and B) confirmed that (a) mRNA from soleus, cardiac, and masseter muscles contained a sequence that was precisely homologous to the pMHC450-S probe; and (b) mRNA sequences ho-

In preliminary studies, with pMHC 1450-S as probe, we found slow MHC mKNA to be 4 times more abundant in stimulated EDL muscle than in the contralateral control muscle.5 ‘The present study confirms this finding with the more specific pMHC450-S probe. Similar trends were observed with EDL muscle, whether poly(A)+ RNA (2.9fold increase) or total RNA (5-fold increase) was used for the analysis (Table 1). ‘The relative increase in slow MHC mRNA shows some variation because of the low levels of this message in control Myosin mRNA Levels in Stimulated Muscles.

RECOVERY

STIMULATED

rnlusc 1 e

12

42

4

8

MHC450-F

2

L

3

31

42

MHC 4 5 0 -- S

1596

days probe

EDL

MHC450-F

1

6

40

54

MHC4 5 0 - S

3708

2203

309

352

TA

C

days

S t R 42 4

R 0

R 12

FIGURE 3. Relative fast (pMHC450-F) and slow (pMHC450-S) muscle MHC mRNA levels in EDL and TA muscles following stirnulation (St) for 42 days and recovery (R) for 4, 8, and 12 days. The figures given are integrated peak areas per microgram of total RNA determined by slot-blot analysis, expressed as a percentage of the value for the same-day control muscle. Northern-blots show the distribution and integrity of the MHC mRNAs in control EDL (fast) and SOL (slow) (C), stimulated (St), and recovery (R)muscles.

Reciprocal Changes in Myosin mRNAs

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697

ization with any intermediate species that may be present. Both Northern-blot analysis (Fig. 3) and KNase protection assays (Fig. 4) suggested that there was a more rapid return to control levels of fast MHC in T A muscle than in EDL muscle. The concurrent decline in slow MHC mRNA could be monitored with pMHC450-S as a probe. T h e level appeared to be sustained up to 4 days in EDL muscle, after which it declined at an increasing rate to 30% of the initial value at 12 days (Fig. 3). The results of slot- and Northern-blot analyses and RNase protection assays suggested that the decline in slow MHC mRNA of the T A muscle was similar, but had a more rapid time course (Figs. 3 and 4).

tissues. In T A muscle stimulated for 9 days at 10 Hz, slow MHC rriRNA had increased approximately 2.4-fold. RNase protection assays, with poly(A)+ RNA, confirmed that the mKNAs cletected in the slot-blots contained sequences which were homologous t o the two cDNA probes (Fig. 2C). 'These results again demonstrate a dramatic decline of fast MHC mRNA in EDL, muscle in just 4 days.' Expression of slow MHC mRNA was clearly evident after 9 days of stimulation, and was further elevated at 21 days of stimulation. Myosin mRNA Levels in Muscle Recovering From Stimulation. In the experiments on muscle recov-

ery, all muscles were initially stimulated continuously at 10 Hz for 6 weeks, by which time type transformation is known to be close to completion.'& Specific MHC mKNA levels were analyzed in total and poly(A)+ RNA. Slot-blot analysis using pMHC450-F as probe showed that, compared to the rapid disappearance of fast MHC mRNA following electrical stimulation, the reappearance of this rnRNA during muscle recovery was relatively slow (Fig. 3 ) . After 4 days of recovery, fast MHC mRNA in the experiniental EDL muscle had reached only 2% of the control value and, even after 12 days, it had risen to only 42% of this value (Fig. 3). RNase protection assays (Fig. 4) indicated that the actual levels may have been even lower, because analysis of total RNA on slot-blots does not exclude the possibility of some cross-hybrid-

DISCUSSION

Studies of changes in specific myosin mRNA levels, in response to alterations in the patterns of activity of skeletal muscles, depend on the availability of probes that can differentiate unambiguously between the members of this multigene family. In this study, w e isolated and characterized a rabbit slow-muscle MHC cDNA containing coding and noncoding sequences at the 3'-end of the gene, complementing a fast-muscle MHC probe already available." KNase protection assays showed that this slow MHC probe had complete sequence homology with mRNA isolated from both slow skeletal and cardiac muscle. Our results are comple-

M H C 4 50-F

MHC450-S

Pr days

R

C

6

R

C

R

8

C S t 12

EDL

C S t

8

35

C

C S t R R R 42 6 8 1 2 TA

FIGURE 4. RNase protection assays with pMHC450-F and pMHC450-S as probes of poly(A)+ RNA isolated from EDL muscles after 8 and 35 days of stimulation (St); 6, 8, and 12 days of recovery (R); and from their contralateral controls ( C ) ; and total RNA isolated from TA muscles after 42 days of stimulation and 6, 8, and 12 days of recovery. The labeled transcribed probe is also shown (pr).

698

Reciprocal Changes in Myosin rnRNAs

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mentary to those of Sinha et al.," who have shown that a (3-cardiac cDNA (2000 bps of coding sequence) was homologous with mRNA isolated from soleus muscle. Homology between the slow MHC mRNA and P-cardiac mRNA has also been demonstrated in the rat," and oligoprobes to the 3'-untranslated region of the human P-cardiac MHC gene also hybridized with mRNA from slow musc~e.'~ The present study was concerned primarily with changes in the myosin mRNAs during indirect electrical stimulation and recovery of the rabbit fast skeletal muscles T A and EDL. We reported previously that continuous stimulation of these muscles at 10 Hz reduced their fast MHC mKNA content to 34% (TA) and 58% (EDL) of control levels after only 4 days, and to 6% to 8% after 10 days.4 During the recovery of muscle after an extended period of stimulation with the same pattern, the reappearance of fast MHC mKNA followed a slower time course, reaching only 54% and 42% of control levels in 'I'A and EDL muscles, respectively, after 12 days. This gradual increase in fast myosin mKNA is consistent with the reappearance of the protein product in sections processed histochemically for the demonstration of myosin ATPase.' In the earlier study, stimulation of EDL muscle for 10 days produced a 4-fold increase in slow-muscle- specific MHC mKNA.5 T h e more specific probe, pMHC4503, revealed increases in slow MHC mKNA of approximately 16-fold for TA and 37fold for EDL after 42 days of stimulation. Cessation of stimulation did not affect these elevated levels of slow MHC mRNA immediately, but they decreased fairly rapidly after 4 days and, at 12 days, were at 50% in EDL and 10% in TA of their levels after long-term stimulation. Although changes during recovery occurred in a similar sequence in T A and EDL muscles, the time course differed: the rate of recovery in terms of the disappearance of slow muscle mKNA and the appearance of fast muscle mRNA, was somewhat faster in TA than in EDL. Changes in MHC mRNA levels evidently occur more rapidly during stimulation than during the subsequent recovery. In neither process is there any obvious coordination between the switching of fast and slow MHC genes. Earlier studies suggested that an intermediate type of myosin (possibly Type 2A) was expressed during the transitions between Types 1 and ZB, which occur during stimulation and recovery.',2 There is also evidence that a number of isomyosins can coexist within thc

Reciprocal Changes in Myosin rnRNAs

individual fibers of a given muscle.'* Thus, coordinated regulation of switching could occur between one or more transient forms of MHC and the two predominant forms of MHC (Type 2B and Type 1) during stimulation and recovery. Isolation of cDNA probes corresponding to other MHC genes will provide further opportunities to evaluate the extent of any coordination of MHC gene expression in the rabbit. Our results, which confirm and extend earlier observations, show that an alteration in the pattern of activity sustained by fast muscles triggers events that lead to changes in gene products associated with the contractile apparatus. The fact that a switch between two separately encoded isoforms of a major contractile protein is reflected in changes in the corresponding mRNAs indicates that the underlying regulatory events are taking place at a pretranslational level. Based on present evidence, we cannot exclude the possibility that some of these changes are attributable to modulation of the stability and turnover of individual mRNAs. The strong suggestion remains, however, that these findings constitute evidence of a continued potential for control by gene regulatory factors in a highly specialized adult mammalian tissue.

REFERENCES

I . Brown JMC, Henriksson J , Salmons S: Restoration of fast

2.

3.

4.

5.

6.

7.

muscle characteristics following cessation of chronic stimulation: Physiological, histochemical and metabolic changes during slow-to-fast transformation. Proc K Soc L o i d H 1989;235:32 1-346. Brown W, Salmons S, Whalen R: T h e sequential replacement of myosin subunit isoforms during muscle type transformation induced by long term electrical stimulation. J Bzol Chrm 1983;258:14686- 14692. Brown W, Salmons S, Whalen K: Mechanisms uriderlying the asynchronous replacement of' myosin light chain isoforms during stimulation-induced fibre-type transformation of skeletal muscle. FEBS Lett 1985;192:235-238. Brownson C, Isenberg H, Brown W, Salmons S, Edwards Y: Changes in skeletal muscle gene transcription induced by chronic stimulation. M u c k Nmue 1988;11: 1183- 1189. Brownson C:, Salrnons S, Edwards Y: Changes in the concentrations o f selected mRNA transcripts in response to continuous electrical stimulation of skeletal muscle, in Carraro U (ed): Sarcomeric and Non-Sarcomeric Muscles: Basic arid Applied Research Prospects for the 90's. Padova, Unipress, 1988, pp 353-359. Gubler U, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Genr 1983;25:26?269. Hiiynh 'W, Yoitrig KA, Davis KM': (;onstructing ;trrtl screening c l ) N A libraries in hgt 1 0 arid hgtl 1, i n love^D M (ecl): D N A Cloning. Oxford, I K I . Press, 19x5, vol 1 , p p 4I)-78.

X. Kirsc.hl,aurti B J , Srhricitler S , Izumo S, Mahdavi V , Natlal15, f'ecce I): Rapid and rcvei-sible changes i n myosin ( ,in,ird " .

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10.

1I .

12.

13.

heavy cliain expression in response to incrcascd ncuromirsc-ular activity of rat fast-twitch musck. FLUS I A t 1990;268:75-78. Kraus WE, Bernard TS, Williams RS: Interactions between sustained contractile activity and P-adrenergic receptors iri regulation of gene expression in skeletal muscle. AnlJ P l ~ y zof 1!)8‘.);256:

Reciprocal changes in myosin isoform mRNAs of rabbit skeletal muscle in response to the initiation and cessation of chronic electrical stimulation.

Changes in myosin heavy chain (MHC) mRNAs were studied in rabbit fast-twitch muscles during continuous electrical stimulation at 10 Hz for periods up ...
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