(Received for publication, April 27, 1995; and in revised form, June 1, 1995)
From the
The protein product of the myotonic dystrophy (DM) gene is a putative serine-threonine protein kinase (DM kinase). Previous reports have characterized the DM gene product as various 50-62-kDa proteins. The predicted protein size from DM cDNA sequence is 69 kDa. We therefore expressed a full-length recombinant human DM kinase protein and compared its size and expression to heart, cardiac Purkinje fibers, and skeletal muscle from normal and DM subjects. Recombinantly expressed DM kinase and endogenous DM kinase in human heart, displayed two immunoreactive DM kinase proteins with apparent molecular sizes of 71 and 80 kDa, suggesting that these prior reports are incorrect. In cardiac Purkinje fibers the 71-kDa protein was the major form, and in skeletal muscle the 80-kDa protein was the major form. Immunostaining showed DM kinase localized to neuromuscular junctions in skeletal muscle and intercalated discs in heart and Purkinje fibers. DM subjects showed low abundance of DM kinase in heart and skeletal muscle, suggesting haplotype insufficiency as a potential mechanism for disease expression. These studies describe differential expression of two protein forms of DM kinase, which are localized to specialized cellular structures associated with impulse transmission.
Myotonic dystrophy (DM) ()is an inherited autosomal
dominant disease characterized by systemic disorders such as skeletal
muscle myotonia, weakness, cardiac conduction abnormalities, frontal
baldness, cataracts, testicular atrophy, and dementia(1) . The DM gene locus on chromosome 19 contains an unstable
trinucleotide, [CTG]
, DNA repeat in the
3`-untranslated region of the gene(2, 3, 4) .
The severity of phenotype, age of onset of myotonia and muscle wasting,
and cardiac conduction abnormalities are strongly correlated with an
increase in the number of CTG repeats, yet there is considerable
variation between individuals and
families(2, 3, 4, 5, 6, 7) .
The mRNA from this gene is expressed in the heart, skeletal muscle,
and, to a lesser extent, in brain(2) . It is hypothesized that
increasing CTG repeat length may cause myotonic dystrophy by reducing
the abundance of its own mRNA (8, 9, 10) ,
although this has not been a consistent observation(11) .
Analysis of the DM cDNA sequence suggests that the protein product
of this gene is a 69-kDa serine-threonine protein kinase (DM kinase)
because of its extensive similarity to other protein kinase catalytic
domains(2, 3, 4, 8, 12) .
Several reports have attempted to biochemically characterize the
protein product of this gene in normal and diseased tissues.
Unfortunately, the synthetic peptide and fusion protein immunogens used
for DM kinase antibody production have detected 50-62-kDa
proteins in various
tissues(8, 13, 14, 15) , which are
all smaller than the predicted size of 69 kDa(12) . In none of
these studies has a full-length recombinant DM kinase protein control
been available to prove DM kinase identity and antibody specificity. We
have expressed a full-length recombinant DM kinase protein in COS and
BC3H1 cells and shown that the DM kinase protein is expressed as 71-
and 80-kDa mobility forms, and that these prior reports are incorrect.
Both DM kinase forms are found and differentially expressed in human
muscle tissues. Analysis of the expression and subcellular localization
of the DM kinase in normal human heart and skeletal muscle implicate
highly specialized cellular structures, the intercalated discs and the
neuromuscular junctions, as potential subcellular targets for cell
dysfunction in DM.
COS cells (ATCC) were grown in T75 flasks
with 10 ml of COS cell medium (DMEM medium (Life Technologies, Inc.)
containing 10% calf serum (HyClone), 10 mM Hepes (Life
Technologies, Inc.), and 4 mML-glutamine (Life
Technologies, Inc.)). PCDNA3 DMK (10 µg/flask) was transfected
using the Pharmacia CellPhect transfection system. Briefly, the culture
medium was aspirated and the COS cells were washed once with
phosphate-buffered saline (pH 7.4), 10 µg of PCDNA3 DMK was added
to each flask with 3 ml of DMEM medium (serum-free) and 300 µl of
DEAE. After 3 h of incubation at 37 °C the transfection mixture was
removed and the cells were incubated with 10% MeSO in DMEM
(serum-free) for 60 s at room temperature. The cells were washed twice
in DMEM, 10 ml of COS cell medium was added to each flask and incubated
at 37 °C for 48-72 h. COS cells were harvested by scraping,
followed by centrifugation at 1200
g for 10 min at 4
°C. The pellet was suspended in 20 mM Tris (pH 7.4),
containing 0.1% Triton X-114, 5 mM
-mercaptoethanol (300
µl/flask), and subjected to five freeze/thaw cycles with dry ice
and a 37 °C water bath. The cell lysate was centrifuged at 9000
g for 5 min at 4 °C, and the supernatant
containing recombinant DM kinase was aspirated and used for Western
blotting.
Biochemical characterization of human DM kinase was explored by recombinant protein expression, Western blot analysis and immunocytochemistry of human tissue, and subcellular fractionation of the protein. Western blot analysis of COS cells transfected with the full-length DM kinase cDNA using anti-DM kinase antibody demonstrated specific immunoreactive proteins compared to control untransfected COS cells, which contained no immunoreactive material (Fig. 1). Two major protein bands at 80 and 71 kDa were specifically detected in the transfected COS cells, suggesting that previous studies of 50-62-kDa proteins were probably not DM gene products (Fig. 1). Proteins with the same apparent molecular weight were also detected in transfected BC3H1 cells (data not shown). Human heart contained a doublet with similar staining intensity of both proteins at 80 and 71 kDa (Fig. 1), and no smaller protein forms were observed. The in vitro recombinantly expressed proteins were similar to that detected in human cardiac and skeletal muscle and probably represents two mobility forms of DM kinase. The two protein forms detected in heart had differential expression in other tissues. Human skeletal muscle demonstrated a predominance of the upper 80-kDa immunoreactive protein with only faint staining of the 71-kDa protein. In contrast, human cardiac Purkinje fibers expressed the 71-kDa protein as the major form (Fig. 1). The 71-kDa protein does not appear to be a degradation product of the 80-kDa form, and neither form appears to be glycosylated, since exposing the two forms to boiling, prolonged incubation at 65 °C, 10% nonionic detergents (Triton X, Nonidet P-40), or glycolytic cleavage enzymes (endoglycosidase H, N-glycosidase F, Boehringer Mannheim) did not alter their molecular size (data not shown).
Figure 1: DM kinase in human skeletal muscle, heart, and cardiac Purkinje fibers. A, recombinantly expressed DM kinase in transfected COS cells (COS DMK) showed two major immunoreactive proteins at 80 and 71 kDa. Untransfected control COS cells (COS CON) showed no detectable DM kinase staining. Human heart (Ht) demonstrated staining of both 80- and 71-kDa proteins with similar intensity. In skeletal muscle (Skm), the 80-kDa protein was the major form. In cardiac Purkinje fibers (Purk.), the 71-kDa protein was the major form. The positions of molecular size markers are indicated at the left. B, Coomassie Blue stain of myosin abundance for the lanes of the gel immunoblotted in A. COS cells did not express myosin. Total protein measurements of each lane were as follows: 3 µg of protein in both COS cells (COSCON and COSDMK), 12 µg of protein in skeletal muscle (Skm) and heart (Ht), 8 µg of protein in Purkinje fibers (Purk.).
To determine whether DM kinase
displays a specific subcellular structure for its localization,
immunostaining of DM kinase in human tissues was performed. Cardiac
myocytes and Purkinje fibers demonstrated specific subcellular
localization to the intercalated discs (Fig. 2, A and C). There were no obvious differences in staining pattern
between cardiac muscle and Purkinje fibers despite the apparent
differences in mobility forms noted on Western blotting of these
tissues (Fig. 1). The staining pattern was identical to that
obtained with anti-cadherin antibody, which specifically labels the
fascia adherens at the intercalated discs in heart tissue (Fig. 2B). Immunostaining of human skeletal muscle with
anti-DM kinase antibody demonstrated specific localization to the motor
endplate of the neuromuscular junctions (Fig. 2D), by
double staining with a rhodamine-labeled -bungarotoxin (Fig. 2E).
Figure 2:
Immunolocalization of DM kinase in heart,
Purkinje fibers, and skeletal muscle. Longitudinal section of heart
tissue (A) and Purkinje fibers (C) showed DM kinase
was localized to the intercalated discs (whitearrows). As a control, anti-cadherin antibody stained
intercalated discs in a pattern identical to DM kinase (B). In
skeletal muscle DM kinase was localized to discrete membrane structures (D, white arrow). Control staining in the same
section with -bungarotoxin identified this structure as the motor
endplate of the neuromuscular junction (E, whitearrow). Bar, 30
µm.
We further analyzed the nature of DM kinase association with structures in the heart using the methods described by Green and Severs for partial purification of intercalated discs(20) . Most of the DM kinase was easily dissociated from the final pellet, but small amount of the total DM kinase remained in the crude membrane fraction containing intercalated discs (Fig. 3). Supernatants 1 and 2 from this preparation contained both DM kinase mobility forms noted in whole heart homogenates. High speed centrifugation of the supernatant in a sucrose gradient showed that both forms were found in the top layer and demonstrates that these are soluble proteins (data not shown). The predominance of soluble DM kinase in cell homogenate supernatant suggests that the putative transmembrane domain proposed by sequence analysis of DM kinase (21) does not confer a strong membrane interaction. This observation is consistent with the fact that most serine-threonine protein kinases are soluble and not membrane-bound. Interestingly, our preparations consistently contained more of the smaller 71-kDa DM kinase form in the supernatant, while the 80-kDa DM kinase form was the major form isolated in the crude membrane pellet. The affinity of DM kinase for these specialized structures may thus vary between the 80- and 71-kDa forms, yet the nature of these interactions are unknown. To control for the stability of the preparation, we reprobed the membrane with anti-cadherin antibody. Cadherin, which is localized to the intercalated discs, was almost exclusively found in the crude membrane pellet (Fig. 3B). Because the pellet contains other cellular particulate matter, our observations do not prove that the 80-kDa form is associated with the intercalated disc.
Figure 3: Isolation of crude cardiac membranes containing intercalated discs and DM kinase from human left ventricle. A, the crude membrane pellet co-purified the 80-kDa DM kinase form. A majority of DM kinase was easily extracted in the first supernatant fraction (Sup1), and this fraction contains proportionately more of the 71-kDa form. Supernatants 3 (Sup3) and 4 (Sup4) demonstrated complete removal of the soluble fraction of DM kinase from the pellet prior to analysis. B, cadherin immunoblotting of the stripped membrane in panelA demonstrated that almost all the intercalated disc associated protein cadherin (135 kDa) was confined to the pellet. Supernatants 1-4 and pellet contained 8, 1, 0.15, 0.85, and 8 µg of total protein loading, respectively.
To test whether DM kinase expression is altered in myotonic dystrophy, a few fresh frozen skeletal muscle samples from 2 DM patients (DM 1 and 2) were analyzed by Western blotting (Fig. 4). We found an obvious decrease in DM kinase abundance in affected skeletal muscle. Heart tissue from a DM patient (DM 3) also showed low abundance of both forms of DM kinase (Fig. 4). DM kinase was not diminished in cardiac tissue from other forms of cardiomyopathy. This preliminary observation suggests that, if DM is a disease of DM kinase deficiency, then haplotype insufficiency is the likely mechanism for autosomal dominant disease expression. A dominant disease expressed via haplotype insufficiency can occur in mutations of structural proteins or enzymes in critical metabolic pathways(22, 23) .
Figure 4: Immunoblotting of DM kinase in patients with DM. Left, DM kinase in skeletal muscle. DM subjects (DM1 and DM2) showed low abundance of DM kinase (DMK) when compared to control skeletal muscle (CON). Myosin abundance in each sample is demonstrated by Coomassie Blue stain of the gel in the lower panel. Right, DM kinase in heart. DM kinase (DMK) was decreased in heart tissue from a patient with DM (DM3), but not in heart specimens from normal control (CON), Becker muscular dystrophy (BMD), ischemic cardiomyopathy (ISC), or idiopathic dilated cardiomyopathy (IDC). Cadherin immunoblotting of the same membrane is shown in the lower panel as a control. 12 µg of total protein were loaded in skeletal muscle and heart lanes.
The Purkinje conduction system of the heart is a highly specialized group of cells, which exhibit a 4-fold increase in conduction velocity and a high number of intercellular connections, gap junctions, and desmosomes in the intercalated disc, compared to the disc structure in normal cardiac myocytes(24) . Electrophysiologic studies have shown that the His-Purkinje system is damaged in DM, and this conduction disturbance becomes progressively worse with age(25) . The relationship between the localization and expression of DM kinase and the observed clinical phenotype is not clear. One report has described reduced myocardial glucose phosphorylation in the hearts of DM subjects(26) , and recently another group has shown that increased CTG repeat length is correlated with the severity of conduction abnormalities(7) . In this report, we described a DM patient with severe bradycardia, who had decreased myocardial DM kinase expression as detected by Western blotting. Thus, the observation that DM kinase is expressed in the specialized conduction cells of the heart may be important to the common cardiac conduction defects found in DM patients.
The two major immunoreactive proteins of 80 and 71 kDa detected in normal tissues, and by recombinant protein synthesis in COS cells, are different from the putative DM kinase protein described by other investigators(8, 13, 14, 15) . The calculated molecular mass of DM kinase from the cDNA coding sequence is 69 kDa(12) , close to the 71-kDa form described in this report. It is thus tempting to speculate that the 71-kDa form is the nascent full-length DM kinase protein and that the 80-kDa protein form has been modified in the cell. The two immunoreactive proteins seen in normal tissues and transfected COS cells are not produced via alternative splicing since they were derived from a single cDNA construct transfected into COS cells. A recent study demonstrated an increase in molecular size of DM kinase fusion protein after phosphorylation(27) . Therefore, it is conceivable that post-translational protein modification or phosphorylation state may be responsible for the two different mobility forms detected in SDS-PAGE.
Numerous reports have attempted to define the cellular localization
of DM kinase. Because our data conflict in part with these reports,
several comments should be made. Van der Ven et al.(13) described immunolocalization of a 53-kDa protein at the
neuromuscular junctions, myotendinous junctions and the cytoplasm of
type 1 skeletal muscle fibers, and intercalated discs in the heart.
Interestingly, their antibody also detected a larger molecular size
protein of 80 kDa, which may represent activity against both DM
kinase and the 53-kDa protein. The fact that other studies have shown
similar immunolocalization data of a
50-54-kDa protein does
not detract from our results. Other proteins such as utrophin localize
to neuromuscular junctions and intercalated
discs(28, 29) , and this report demonstrates that the
71- and 80-kDa DM kinase proteins localize specifically to these
structures.
This study demonstrates that the protein product of the DM gene is translated as 71- and 80-kDa proteins in striated muscle tissues and displays tissue-specific variation in the expression of these two forms. DM kinase is localized to specialized cell structures in both heart and skeletal muscle that are associated with intercellular conduction and impulse transmission. However, because DM kinase remains uncharacterized biochemically and physiologically, no conclusions can be made regarding its role in the cell.