©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a Nuclear Protein That Interacts with Regulatory Elements in the Human B Creatine Kinase Gene (*)

Ji-Nan Zhang (§) , James E. Wilks , Joseph J. Billadello (¶)

From the (1)Cardiovascular Division, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The B creatine kinase gene is regulated by an array of positive and negative cis-elements in the 5`-flanking DNA that function in both muscle and nonmuscle cells. In CC myogenic cells M and B creatine kinase mRNAs are coordinately up-regulated in the early stages of myogenesis and then undergo distinct regulatory programs. The B creatine kinase gene is down-regulated in the late stages of myogenesis as M creatine kinase becomes the predominant species in mature myotubes. Sequences between -92 and +80 of the B creatine kinase gene confer a regulated pattern of expression to chimeric plasmids that closely resembles the time course of expression of the endogenous B creatine kinase gene in CC cells undergoing differentiation. We show that sequences within the first exon of the B creatine kinase gene are important for the developmental regulation of the gene in CC cells and that these sequences bind a nuclear protein that shows a similar tissue-specific distribution and developmentally regulated expression to that of the endogenous B creatine kinase gene.


INTRODUCTION

Creatine kinase (CK)()catalyzes the reversible phosphorylation of ADP and creatine and is important in the regulation and maintenance of cellular energy metabolism. The M and B creatine kinase genes encode highly homologous M and B subunit proteins that associate in the cytoplasm to form three dimeric cytoplasmic isoenzymes (MM, MB, and BB). B CK is expressed in many tissues, is regulated by steroid hormones(1, 2) , and undergoes developmental regulation in the lens of the eye(3) , in cartilage(4) , and in osteoblastic cells in culture(5) . B CK is a marker for certain histologic types of lung cancer (6) and for brain damage(7, 8) . B-containing isoenzymes (MB and BB) increase in skeletal muscle during chronic exercise training (9) and in the myocardium in response to hypertrophy, acute myocardial ischemia, and heart failure (10, 11, 12) in adaptation to conditions of decreased energy reserve(10) .

Like other multigene families expressed in muscle the CK genes undergo an isoenzyme switch during development(13, 14, 15, 16) . B CK is expressed in immature proliferating myogenic cells (myoblasts). Muscle differentiation is characterized by down-regulation of the B gene, which is switched off during myogenesis, and induction of M CK, which becomes the major isoform present in both cardiac and skeletal muscle (17, 18). With the use of M- and B- specific cDNA probes we showed that coordinate up-regulation of M and B mRNA occurs in the early stages of myogenesis in CC cells (19) and in the developing heart (20) prior to down-regulation of B mRNA during the final stages of differentiation as M mRNA becomes the predominant species. This fetal pattern of expression of the CK genes is recapitulated in heart in response to acute pressure overload(21) . Interestingly, the developmental expression of B CK mRNA is very similar to that of cardiac actin mRNA, which is also up-regulated in the early stages of differentiation of CC cells and down-regulated as it is replaced by skeletal actin mRNA in the late stages of myogenesis(22) . Thus, the pattern of expression of certain fetal isoforms such as B CK and cardiac actin may represent an evolutionarily conserved developmental program in muscle. This program is different from that of other fetal isoforms such as and actin, which are expressed in myoblasts and are down-regulated during all subsequent stages of myogenesis(23) .

We showed that the human B CK gene is regulated by an array of positive and negative cis-elements in the 5`-flanking DNA that function in both muscle and nonmuscle cells and that sequences between -92 and +80 confer expression to chimeric plasmids that resembles that of the endogenous B CK gene in CC cells undergoing differentiation(19) . We now show that sequences within the first exon of the B CK gene are important for the developmental regulation of the gene in CC cells and that these sequences bind a nuclear protein that may play a role in the developmentally regulated expression of the B CK gene.


MATERIALS AND METHODS

Preparation of BCKCAT and BCKneo Reporter Plasmids

The plasmid BCKCAT92 (Fig. 1A) that contains 92 base pairs of 5`-flanking DNA, the first exon (untranslated), and the first 12 base pairs of the first intron of the human B CK gene inserted in the HindIII site of pSVOCAT was described previously(19) . To determine whether sequences within the first exon and first intron are important for expression of BCKCAT92 we prepared BCKCAT92del (+3 to +80), a construct that contains the first 92 base pairs of upstream DNA and the cap site (Fig. 1B). This construct was prepared by synthesizing oligonucleotides representing the sense and antisense strands of the sequence from -92 to +2 designed to reconstitute HindIII-compatible ends after annealing to facilitate subcloning into the HindIII site of pSVOCAT. We also prepared a 3`-deletion series of constructs with identical 5`-ends containing sequences from -92 to +2 and different 3`-ends designed to delete select sequences from the first exon and intron of the B CK gene (Fig. 1, C-F). The constructs were prepared by ligating annealed double-stranded oligonucleotides representing the desired sequences into the HindIII site of pSVOCAT(24) . All plasmids were sequenced in both strands to ensure a single copy of the desired insert was present in the correct orientation(25) .


Figure 1: Identification of a regulatory element within the first exon of the human B CK gene. A, the plasmid BCKCAT92 was described previously (19). The hatchedbox represents sequences contained in the first exon. B-F, 3`-deletion series through the region +3 to +80 prepared as described under ``Materials and Methods'' and drawn to scale. G, the plasmid MCKCAT2620 (not drawn to scale) that contains the human M CK gene enhancer was described previously (30). CC cells were transfected and harvested as myoblasts or myotubes, and extracts were assayed for [C]chloramphenicol conversion. Induction represents CAT activity (mean ± S.D.) present in extracts of cells transfected with chimeric plasmids (26.5% ± 9.4% conversion [C]chloramphenicol for the most active construct) relative to pSVOCAT (3.1% ± 1.8% conversion [C]chloramphenicol). N, total number of dishes of cells transfected with each construct. At least two preparations of each plasmid and different batches of CC cells were used for transfection experiments.



Because the neomycin resistance (neo) gene encodes a very stable transcript (26) we prepared BCKneo reporter plasmids to facilitate detection of transcripts in transiently transfected cells. BCKCAT92 and BCKCAT92del(+3 to +80) were digested completely with BamHI and then partially with HindIII to release the chloramphenicol acetyltransferase (CAT) gene from the plasmid. The neo gene obtained by digesting pSVneo with BamHI and HindIII was ligated to BamHI/HindIII-digested BCKCAT92 and BCKCAT92del (+3 to +80) resulting in the constructs BCKneo92 and BCKneo92del (+3 to +80).

Cell Culture

The murine skeletal muscle cell line CC (ATCC #CRL1772) was maintained in an atmosphere of 8% CO, 92% air in growth medium (Dulbecco's modified Eagle's medium) supplemented with 20% fetal calf serum, penicillin (50 µg/ml), and streptomycin (50 µg/ml)). Differentiation medium was Dulbecco's modified Eagle's medium supplemented with 10% horse serum and antibiotics.

Cell Transfection and Chloramphenicol Acetyltransferase Assays

CC cells were plated 24 h before transfection at a density of 3.5 10 cells/60-mm dish in 3 ml of growth medium. Transfections were performed by the calcium phosphate coprecipitation method(19) . Precipitates contained a total of 20 µg of DNA comprised of 15 µg of test plasmid and 5 µg of pMSVgal as an internal standard to correct for transfection efficiency. After a 4-h incubation with the precipitate, cells were subjected to a 3-min glycerol shock and harvested 24-48 h later. Other dishes of cells were fed with 3 ml of differentiation medium after the transfected cells had become fully confluent and harvested 60 h later as fully differentiated myotubes. Cell extracts were prepared, and assays for -galactosidase and chloramphenicol acetyltransferase were performed as described previously(19) . The amount of extract used for chloramphenicol acetyltransferase assays was based on the results of the -galactosidase assay and contained 25-100 µg of protein. The assays were terminated after 60 min.

Northern Blot Hybridization

CC cells plated as described above were transfected with 15 µg of either BCKneo92 or BCKneo92del(+3 to +80) and 5 µg of pCMVCAT as an internal standard to control for transfection efficiency. The cells were harvested 28 h after transfection, and Poly(A) mRNA was purified directly from transfected cells with the use of a Micro-FastTrack mRNA isolation kit (Invitrogen). Northern blots were prepared with 7 µg of Poly(A) mRNA and Nytran membranes (Schleicher and Schuell) as recommended by the supplier. The probes were a 1321-base pair HindIII/SmaI fragment of the neo gene and a 550-base pair HindIII/NcoI fragment of the CAT gene radiolabeled with [P]dCTP (Amersham Corp.) as described(19) . Autoradiograms prepared with Kodak XAR film and Cronex intensifying screens were analyzed with an LKB Ultroscan XL laser densitometer.

Preparation of Nuclear Extracts

Extracts were prepared from CC cells and tissues by a modification of the method described by Heberlein et al.(27) . The protein concentration was determined by the method of Bradford(28) .

Preparation of Probes for Gel Mobility Shift Assays

A DNA fragment comprising the first exon and first 13 base pairs of the first intron of the B CK gene was used as the template in a polymerase chain reaction with primers designed to include bases +1 to 19, the reverse complement of +64 to +80, and with [P]dCTP at a final concentration of 0.32 µM(29) .

DNA Gel Mobility Shift Assays

Gel mobility shift assays were performed as described previously (30) with 1.0 10 dpm of double-stranded P-labeled probe and 10 µg of nuclear protein extract.

Phosphatase Treatment of Nuclear Extract

Nuclear extract was incubated with 19.2 milliunits of potato acid phosphatase (Sigma) or with buffer (50 mM Tris HCl, pH = 9.3, 1.0 mM MgCl, 0.1 mM ZnCl, and 1.0 mM spermidine) and 2.0 units of calf intestinal alkaline phosphatase (Promega) for 30 min at 37 °C. After phosphatase treatment P-body-labeled probe was added to the extract, and gel mobility shift assays were performed.


RESULTS

Transfection Experiments

We showed that sequences within -92 to +80 of the B CK gene confer a regulated pattern of expression to CAT reporter plasmids in CC cells that resembles the time course of expression of B CK mRNA(19) . The plasmid BCKCAT92 (Fig. 1A) showed peak expression 24-48 h after transfection and was not expressed above background in fully differentiated myotubes. In nonmyogenic cells that express B CK (HeLa cells, Hep G2 cells, and NIH3T3 cells) BCKCAT92 expression was above background 48-108 h after transfection(19) . These results show that the decrease in expression of BCKCAT92 in CC cells is due to differentiation and does not simply reflect the time course of expression of a plasmid in a transient transfection experiment. To determine the importance of exonic and intronic sequences for expression of BCKCAT92 in CC cells we prepared a construct in which the exonic and intronic sequences were deleted (BCKCAT92del (+3 to +80), Fig. 1B). When compared to the expression of BCKCAT92, which was approximately 5-fold above background in myoblasts, deletion of exon I and intron I sequences from BCKCAT92 resulted in a plasmid that was not expressed above background in either myoblasts or myotubes (Fig. 1B). These results were unexpected and showed that sequences from +2 to +80 were critical for expression of BCKCAT92. Accordingly, to determine the sequences from +2 to +80 that regulate expression of BCKCAT92 we prepared a 3`-deletion series (Fig. 1, C-F). Deletion of sequences from +26 to +80 had no effect on expression of the resultant plasmid (Fig. 1, C-D). Although expression of the plasmid BCKCAT92del (+26 to +80) was higher than that of BCKCAT92 (Fig. 1, A and D) the difference was not statistically significant (Student's t test). However, deletion of sequences from +18 to +25 resulted in a construct that was not expressed above background in myoblasts or myotubes (Fig. 1, E and F). In contrast, the plasmid MCKCAT2620 that contains the human M CK gene enhancer (30) was inactive in myoblasts and was expressed 19-fold above background in fully differentiated myotubes (Fig. 1G). These results show that sequences from +18 to +25 are important for expression of BCKCAT chimeric plasmids in CC myoblasts.

Exon I Regulates Expression of Chimeric Plasmids at the Level of mRNA Accumulation

BCKCAT mRNA transcripts derived from the constructs shown in Fig. 1, A-F are different. This suggests expression may vary with translational efficiency of BCKCAT mRNAs. Accordingly, we sought to determine whether the observed difference in CAT activity in cells transfected with these constructs correlated with CAT mRNA levels. We performed Northern blot analysis of mRNA extracted from CC cells transfected with BCKCAT92 and BCKCAT92del(+3 to +80). Because CAT mRNA was not detectable with this technique we prepared constructs in which the CAT gene was replaced with the neo gene, which encodes a more stable transcript. CC cells were transfected with BCKneo92 and BCKneo92del(+3 to +80), and neo mRNA transcripts were analyzed by Northern blot hybridization. The steady-state level of neo mRNA directed by BCKneo92 was 9-fold greater than that of BCKneo92del(+3 to +80) when normalized to CAT mRNA encoded by the internal standard pCMVCAT (Fig. 2). These results show that sequences within B CK exon I modulate expression of chimeric plasmids at the level of mRNA accumulation. Effects at the level of transcription as well as mRNA processing and stability are all formal possibilities.


Figure 2: B CK exon I affects BCKneo mRNA accumulation in transfected cells. RNA extracted from cells transfected with the plasmid BCKneo92del(+3 to +80) (lane1) or BCKneo92 (lane2) and pCMVCAT as an internal standard was analyzed by Northern blot hybridization with P-labeled cDNA probes derived from the CAT gene and the neo gene as described under ``Materials and Methods.''



Gel Mobility Shift Assays

To characterize the trans-acting factors that interact with sequences within the first exon of the B CK gene we prepared nuclear extracts from CC myoblasts and myotubes at select developmental stages and performed gel mobility shift assays. We were particularly interested in factors that are expressed in myoblasts and are down-regulated with differentiation that could be important mediators of B CK gene expression. A representative result from two independent preparations of extracts is shown in Fig. 3. Three nuclear protein-DNA complexes are depicted with arrows. Complex 1 exhibited marked down-regulation with differentiation. Complex 2 was not consistently detected in different preparations of extract tested, and complex 3 was not regulated with differentiation. These results identify a nuclear protein complex that is expressed in myoblasts and is down-regulated with differentiation resembling the expression of B CK in myogenic cells in culture.


Figure 3: Gel mobility shift assays with B CK exon I probe and nuclear extracts from CC cells at select developmental stages. Nuclear extract was prepared from CC myoblasts (lane2), and from CC myotubes after 24 h (lane3), 48 h (lane4), and 120 h (lane5) in differentiation medium for gel mobility shift assays with P-labeled probe (B CK +1 to +80). Uncomplexed probe is shown in lane1.



To determine whether the DNA-protein complexes shown in Fig. 3represented the specific interaction of nuclear proteins with sequences within B CK exon I shown to be important for expression of chimeric plasmids in transfected cells, additional gel mobility shift experiments were performed with competitor DNA (Fig. 4). Unlabeled DNA including sequences from +1 to +25 was added to the gel mobility shift reaction in molar excess to P-labeled exon I probe. The cold DNA competed with the probe for binding to nuclear protein present in complex 1 but not complex 3 (Fig. 4, lanes 1-4). In comparison, unlabeled DNA that included sequences from +1 to +17 did not compete with the probe for binding to complex 1 (Fig. 4, lanes 5-7). These results show that sequences from +18 to +25 that are critical for expression of chimeric plasmids in transfected cells also represent the binding site for complex 1.


Figure 4: Gel mobility shift assays with the B CK probe and competitor DNA. P-labeled probe was incubated with nuclear extract prepared from CC myoblasts. Lane1 represents a reaction without competitor DNA. In lanes2-4 unlabeled DNA containing sequences from +1 to +25 was added to the reaction in 45-, 90-, and 175-fold molar excess as a competitor. In lanes5-7 unlabeled DNA containing sequences from +1 to +17 was added to the reaction in 90-, 185-, and 375-fold molar excess as a competitor.



We used gel mobility shift assays to evaluate the expression of protein 1 in select tissues. We prepared nuclear extracts from brain, a tissue in which B CK is expressed, and from heart and skeletal muscle, tissues in which the CK genes undergo developmental regulation(20, 31) . The results showed that expression of protein 1 was abundant in brain (Fig. 5, laneBR) and expressed at a lower level in heart, a tissue in which the MB isoenzyme of CK is found and in which B CK mRNA is present (Fig. 5, laneH). Mature skeletal muscle, which expresses only trace amounts of B mRNA, did not show detectable amounts of protein 1 (Fig. 5, laneM). These results show that the expression of protein 1 in the tissues evaluated correlated well with that of B CK mRNA. In contrast, band 3 was detected in all tissues. In skeletal muscle and heart, B CK mRNA is down-regulated with development. To determine whether the developmental regulation of protein 1 in heart and skeletal muscle resembled that of B CK mRNA, we prepared nuclear protein extracts from tissues obtained from 1-day-old neonatal mice, a stage at which B CK mRNA expression is abundant relative to that of mature tissue(20) . The results of gel mobility shift assays showed that protein 1 was abundant in both neonatal tissues and was down-regulated with development, correlating well with the developmental regulation of B CK mRNA in these tissues (Fig. 6). In contrast, band 3 was not developmentally regulated.


Figure 5: Expression of band 1 protein in tissues. P-labeled B CK probe was incubated with nuclear extract prepared from adult mouse skeletal muscle (M), heart (H), and brain (BR) and subjected to a standard gel mobility shift assay.




Figure 6: Developmental expression of band 1 protein in tissues. P-labeled B CK probe was incubated with nuclear extract prepared from heart and skeletal muscle from day 1 neonatal (N) and adult (A) mice and evaluated in a standard gel mobility shift assay.



Role of Phosphorylation in Activation of Band 1 Protein

Developmental down-regulation of band 1 protein could be due to either a decrease in synthesis or a post-translational modification of the protein resulting in lower affinity for the target DNA. Because phosphorylation has been shown to modulate the DNA binding activity of many transcription factors(32) , we determined the effect of treatment of extracts with phosphatases on binding of the probe by band 1 protein. Treatment of extract with either enzyme resulted in marked inhibition of formation of complex 1 (Fig. 7). These results suggest that phosphorylation of band 1 protein may be an important post-transcriptional mechanism that mediates affinity for the target DNA.


Figure 7: Effect of phosphatase treatment of nuclear extract on the formation of complex 1. Nuclear extract was prepared from CC myoblasts as described under ``Materials and Methods.'' A, the extract was either untreated (lane1), treated with acid phosphatase (lane2), or incubated with phosphatase buffer without enzyme (lane3) before the addition of P-labeled B CK probe and the gel mobility shift assay. B, the extract was untreated (lane1), treated with acid phosphatase that was boiled for 15 min (lane2), or treated with acid phosphatase (lane3) prior to the addition of P-labeled probe and the gel mobility shift assay. C, the extract was incubated with phosphatase buffer (lane1) or buffer and alkaline phosphatase (lane2) prior to the addition of P-labeled probe and the gel mobility shift assay.



SDS-Polyacrylamide Gel Electrophoresis Analysis of the DNA Binding Protein

To determine the molecular weight of protein 1 we performed a gel mobility shift assay with the B CK probe and nuclear protein extract from CC myoblasts. Band 1 was excised from a wet gel after a brief exposure to Kodak XAR film to facilitate localization of the band in the gel. The protein was separated from the gel with the use of electroelution, analyzed by electrophoresis in a 7.5% SDS-polyacrylamide gel, and detected with the silver reagent (Bio-Rad). A single protein of approximate molecular weight 150 kDa (n = 6 gels) was seen consistently (Fig. 8). This band was not seen when free DNA probe was separated from a band shift gel by electroelution, subjected to SDS-polyacrylamide gel electrophoresis, and stained with silver reagent. Autoradiography of the gel showed that the band represented protein, and not protein complexed to [P]DNA probe.


Figure 8: A single protein is contained in DNA-protein complex 1. A standard gel mobility shift assay was performed with P-B CK probe and nuclear protein extract from CC myoblasts. The position of DNA-protein complex 1 on the wet gel was identified by autoradiography. The band was excised, and the protein was separated from the gel with the use of electroelution. The protein was evaluated by electrophoresis in a 7.5% SDS-polyacrylamide gel that was stained with the silver reagent (Bio-Rad). The protein is depicted with an arrow. The migration of C-protein standards (Amersham Corp.) is shown to the left of the figure.




DISCUSSION

The creatine kinase gene family represents an interesting model of coordinate regulation of isoproteins that can be studied in convenient cell culture systems. A great deal of attention has been given to elucidating the molecular mechanisms that regulate the M CK gene. The regulatory elements that are essential for expression of this gene have been characterized by transfection experiments in cell culture (33-35), direct gene injection into heart and skeletal muscle(36, 37) and in transgenic animals(38) . The M CK gene is regulated by a tissue-specific enhancer that contains two MEF-1 motifs or E-boxes (CANNTG) that bind myogenic factors (MyoD, myogenin, Myf-5, and MRF-4) and an MEF-2 motif that binds a MADS box transcription factor(39) . In contrast, regulatory mechanisms that control genes that are turned off during myogenesis have received less attention. The down-regulation of -actin mRNA during myogenesis is controlled at the level of transcription by conserved sequences in the 3`-nontranslated region of the gene(40) . The cardiac -actin gene is regulated by the interaction of MyoD1, the serum response factor, and Sp1 with promoter elements(41) . The helix-loop-helix protein Id is expressed in CC myoblasts and is down-regulated with differentiation(42) . Id associates specifically with MyoD and attenuates its ability to trans-activate muscle-specific genes such as the M CK gene. There is no evidence that Id or other helix-loop-helix proteins play a direct or indirect role in the regulation of the B CK gene.

The regulation of the B CK gene has been studied in a number of nonmuscle systems. A 61-base pair sequence between -98 and -37 that contains both the CCAAT and TATA sequences is important for efficient in vitro transcription from a minimal B CK promoter (43-46). The results of transfection experiments have identified 5`-upstream sequence elements and sequences within the first (untranslated) exon that are important for expression in HeLa cells (47) and neuroblastoma cells(48) . The B CK gene promoter has strong sequence similarity to the adenovirus E2E gene promoter and like the E2E gene is regulated by the viral activator Ela. This finding may be of significance for the metabolic energy-requiring events that take place after oncogenic activation(49) . In osteoblastic cells transcriptional up-regulation of the gene during differentiation is associated with the formation of a nuclear protein-DNA complex that binds a cis-element between +1 and +228 of the gene(5) . In U937 cells the highly conserved 3`-NTR of B CK mRNA is important for translational regulation of B CK protein(50) .

We present evidence that sequences within the first exon are important for regulation of expression of the B CK gene in transfected myogenic cells in culture. This sequence does not contain recognition sites for any previously described transcription factor (Genetics Computer Group (GCG) transcription factor recognition sites release 6.5). The presence of regulatory sequences within the first exon of a gene is an unusual finding but is not unique to the B CK gene. Important regulatory sequences within the first exon have been described in the human tissue-type plasminogen activator gene(51) , the skeletal troponin I gene(52) , and a human nonmuscle myosin heavy chain gene(53) . Interestingly, none of the exonic regulatory sequences important for expression of other genes in muscle cells share significant identity with the sequence we describe.

Protein phosphorylation is an important control mechanism for regulation of the myogenic developmental program. Both MyoD and myogenin are phosphoproteins(54, 55) . Phosphatase treatment of MyoD attenuates specific binding of MyoD-E12 heterodimers to the M CK gene enhancer(56) . Fibroblast growth factor inhibits myogenesis by inactivating myogenic helix-loop-helix proteins by phosphorylating a site in the DNA binding domain of myogenin(54) . Treatment of CC myoblasts with the protein phosphatase inhibitor okadaic acid inhibits skeletal muscle cell differentiation and MyoD expression and induces expression of the id gene (57). We describe a nuclear protein that binds specifically to sequence elements that are important for expression of B CK chimeric constructs in CC myoblasts. Evidence that the protein we describe is important for regulation of the B CK gene includes specific binding to sequences within the first exon that confer regulated expression to the CAT gene in transfected CC cells, expression in tissues that correlates with that of B CK mRNA and protein, and developmental expression in myogenic cells and in heart and skeletal muscle that resembles that of B CK mRNA. Our results suggest that expression of B CK in cells and tissues may be regulated through a mechanism that alters phosphorylation of this DNA binding protein. Further investigation may provide insight into these mechanisms that mediate the developmental down-regulation of genes during myogenic development.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Cardiology, Nanjing Medical University, Nanjing, China 210029.

Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiovascular Division, Box 8086, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8914; Fax: 314-362-8957; E-mail: billadel@visar.wustl.edu.

The abbreviations used are: CK, creatine kinase; CAT, chloramphenicol acetyltransferase.


ACKNOWLEDGEMENTS

We thank Kimberly Goodwin, Kimberly Hawker, and Nancy Brada for technical assistance, Kelly Hall for secretarial assistance, and Michael Ritchie for contributions to the early stage of this project.


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