ARTICLE |
Correspondence to: Sergio Salvatori, Dept. of Biomedical Sciences, University of Padova, Viale Giuseppe Colombo 3 (ex Via Trieste 75), 35121 Padova, Italy.
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Summary |
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Myotonic dystrophy (DM) is one of the most prevalent muscular diseases in adults. The molecular basis of this autosomal disorder has been identified as the expansion of a CTG repeat in the 3' untranslated region of a gene encoding a protein kinase (DMPK). The pathophysiology of the disease and the role of DMPK are still obscure. It has been previously demonstrated that DMPK is localized at neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum (SR), in the skeletal muscle, and at intercalated discs in the cardiac muscle. We report here new findings about specific localization of DMPK in the heart. Polyclonal antibodies raised against a peptide sequence of the human DMPK were used to analyze the subcellular distribution of the protein in rat papillary muscles. Confocal laser microscopy revealed a strong although discontinuous reactivity at intercalated discs, together with transverse banding on the sarcoplasm. At higher resolution with immunogold electron microscopy, we observed that DMPK is localized at the cytoplasmic surface of junctional and extended junctional sarcoplasmic reticulum, suggesting that DMPK is involved in the regulation of excitationcontraction coupling. Along the intercalated disc, DMPK was found associated with gap junctions, whereas it was absent in the two other kinds of junctional complexes (fasciae adherentes and desmosomes). Immunogold labeling of gap junction purified fractions showed that DMPK co-localized with connexin 43, the major component of this type of intercellular junctions, suggesting that DMPK plays a regulatory role in the transmission of signals between myocytes. (J Histochem Cytochem 47:383392, 1999)
Key Words: myotonic dystrophy protein kinase, DMPK, immunoelectron localization, cardiac muscle, gap junctions, connexin 43, terminal cisternae, sarcoplasmic reticulum, excitationcontraction coupling
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Introduction |
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Myotonic dystrophy (DM), or Steinert's disease, is a dominantly inherited disease characterized by myotonia and progressive muscle wasting, arrhythmia and cardiac conduction defects, mental retardation, cataract, and disorders of the endocrine system. The molecular lesion has been found in the 3' untranslated region of the DM gene as a trinucleotide-repeat amplification. On the basis of the primary structure deduced from the cDNA sequence, it has been assumed that the gene product of the human DM locus is a serine/threonine kinase or, alternatively, a tyrosine kinase, and was accordingly named myotonic dystrophy protein kinase (DMPK).
The disease mechanism and the role of DMPK are obscure at present. In the past few years, a number of studies have been devoted to the identification of DM gene transcripts in the muscle. We and others have found that DMPK is associated with the sarcoplasmic reticulum (
The microscopic anatomy of IDs is complex, in that different specialized regions can be distinguished, i.e., desmosomes, fasciae adherentes, and gap junctions. Each of these regions accomplishes a different function. Fasciae adherentes and desmosomes are mainly involved in end-to-end joining and lateral adhesion of myocytes (
GJs are constituted of clusters of connexons which, in turn, are composed of transmembrane proteins called connexins. Connexins are encoded by at least 13 different genes (for review see
GJ abnormalities, such as alteration in GJ distribution and reduced synthesis of Cx43, have been reported in association with ischemic heart disease (
To aid in characterizing the precise subcellular localization of DMPK and thus contribute to the identification of its substrate(s) in tissues relevant to the disease, we focused our attention on cardiac myofibers and further analyzed the subcellular distribution of the protein by confocal laser scanning microscopy and immunoelectron microscopy. This article presents ultrastructural evidence that, in rat cardiac muscle, DMPK is distributed both at the cytoplasmic surface of sarcoplasmic reticulum elements and at gap junctions. Some of the results have been partially reported elsewhere (
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Materials and Methods |
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Animal Tissues and Gap Junction Preparation
Young adult male Wistar rats were used as a source of cardiac and skeletal muscle for morphological and biochemical studies. In particular, papillary muscles were used for ultrastructural observations. Isolated GJs were prepared from whole hearts. After tissue homogenization, Kensler and Goodenough's four-step isolation procedure (1980) was executed as follows: (a) removal of cytoplasmic soluble elements and fragments of sarcoplasmic reticulum by differential centrifugation in the presence of 1 mM NaHCO3, pH 8.2; (b) removal of myofibrillar proteins by treatment with 0.6 M KI; (c) fractionation of the KI-resistant fraction and removal of fragments derived from adhesive junctions by sucrose density gradient centrifugation; and (d) solubilization of the great majority of nonjunctional membranes with Sarkosyl and Tween 20. In some experiments, partially purified GJs were further treated with 0.3% DOC (w/v) and fractioned by sucrose density gradient centrifugation. For electrophoresis and Western blotting analyses, after Sarkosyl/Tween treatment aliquots were taken and used from resuspended pellets and from fractions either layered at the 43%/30% sucrose interface or floated at the 30% sucrose layer. Fractions layered at the 43%/30% sucrose interface both after Sarkosyl/Tween and after 0.3% DOC treatment were used for ultrastructural studies. Small aliquots were taken and centrifuged for 20 min at 105,000 x g in a high-speed Airfuge centrifuge. The pellets were then processed for immunoelectron microscopy.
Protein concentration of samples was measured by the method of
Antibodies
Primary antibodies used were polyclonal antibodies developed in a young female New Zealand rabbit using a synthetic 20-mer peptide chosen within the predicted DMPK sequence (IREGAPLGVHLPFVGYSYSC). Preparation of antisera and affinity purification of peptide-specific IgG were carried out according to previously reported procedures (
Electrophoresis and Western Blotting
To examine the immunoreactive protein pattern of skeletal and cardiac muscles, homogenates were prepared as previously described (
Immunocytochemistry
Immunofluorescence by confocal laser microscopy was performed on small bundles of fibers that were prepared from unfixed papillary muscles as previously described (
Electron Microscopy
Gold immunolabeling was carried out either on thin bundles of fibers taken from papillary muscles or on high-speed pellets of partially purified GJs. After a 30-min treatment with cold 0.3 M ethylacetimidate in 0.1 M phosphate buffer, pH 7.2, the samples were lightly fixed in cold 3% p-formaldehyde in the same buffer. Then the samples were thoroughly washed in PBS with the addition of 1% BSA and a cocktail of antiproteases made up of 0.2 mM 4-(2-aminoethyl)benzenesulfonil fluoride (Calbiochem; La Jolla, CA), 1 mM benzamidine, 1 mM EGTA, and 5 mM leupeptin (all obtained from Sigma; St Louis, MO). This medium was also used for dilution of antibodies. After a preincubation with 5% nonimmune goat serum, the samples were overlain (overnight at 4C) with primary antibodies which were detected with anti-rabbit or with anti-mouse IgGgold conjugates (EM Grade; BioCell, Cardiff, UK). All incubations were followed by a number of washes (PBS with added BSA and anti-proteases) which lasted from 4 to 7 hr. In controls, primary antibodies were omitted. Double labeling experiments were carried out on high-speed pellets of partially purified GJs. After incubation with primary antibodies, the samples were incubated with biotinylated secondary antibodies which were detected with 15-nm streptavidingold or anti-biotingold conjugates (EM grade; BioCell). Then the samples were overlain (overnight at 4C) with second primary antibodies which were revealed using 5-nm anti-rabbit or anti-mousegold conjugates (EM grade; BioCell).
Finally, the samples were fixed with 2% glutaraldehyde in phosphate buffer (0.1 M, pH 7.2), postfixed in 1% osmium tetroxide in the same buffer, dehydrated, and embedded in Epon. Ultrathin plastic sections stained with uranyl acetate and lead citrate were observed in a Philips EM 301 electron microscope.
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Results |
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Immunofluorescence Studies
Thin fascicles of fibers obtained from rat papillary muscles were incubated with anti-DMPK antibodies and studied by confocal laser scanning microscopy. As already shown in a previous report (
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Immunoelectron Microscopic Localization of DMPK in Cardiac Tissue
Immunogold electron microscopy allowed a high-resolution analysis of DMPK distribution. In papillary muscles, a large amount of protein was found associated with sarcoplasmic reticulum elements, whereas the plasma membrane and the nuclear membrane were devoid of protein. In mammalian ventricular muscle, the organization of the sarcoplasmic reticulum (SR) is characterized by the presence of a single network of tubules surrounding the myofibrils and in register with the sarcomere, whereas T-tubules mostly run transversely to the longitudinal axis of the fiber at the Z-line level. DMPK immunogold particles were recognized around the network of thin longitudinal tubules but they were particularly abundant and regularly distributed around expanded portions of SR containing electron-dense material (Figure 2). Such vesicular structures were numerous and were observed either in connection with longitudinal tubules (Figure 2A) or in close proximity to and/or apposition with T-tubules (Figure 2B and Figure 2C), but also at the periphery of the fibers in subsarcolemmal accumulations of membranous structures (Figure 2D).
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In the cytoplasm of the myocardiocytes, the only other localization of DMPK was found at GJs (Figure 3A and Figure 3B) in the intercalated disc, where Cx43 is known to be present (Figure 3C). The distribution of the protein kinase was strictly limited to the GJs and was never observed at either fasciae adherentes or desmosomes (Figure 3D).
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To better analyze such a specific localization, we studied the presence of DMPK in partially purified fractions of GJs isolated from whole rat hearts. Immunogold staining confirmed that the protein exists at GJs (Figure 4A and Figure 4B), where it also co-localizes with Cx43 (Figure 4D and Figure 4E). Moreover, DMPK was still demonstrable in the gap junction after treatment with 0.3% DOC, pH 10 (Figure 4G and Figure 4H), and its distribution remained comparable to that of Cx43 (Figure 4F).
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The amount of protein kinase revealed by immunogold staining along single junctions varied from large (Figure 4A) to small, or was seen in a limited part of the structure (Figure 4B). Very likely such a variable distribution of DMPK was mainly due to technical reasons or to steric hindrance of gold immunoconjugates. The same variability was seen when anti-Cx43 antibodies were used (Figure 4C). On the other hand, the density of particles was much higher when 5-nm immunogold (Figure 4G and Figure 4H) was used but was very low with 15-nm immunogold conjugates (Figure 4D and Figure 4E).
Western Immunoblot Analyses
Muscle homogenates obtained both from soleus and cardiac muscles were analyzed by Western blotting. As already shown (
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Partially purified GJs from ventricular muscle exhibited strong immunoreactivity corresponding to the 54-kD band (Figure 5, Lanes 3 and 4). In the same fractions, Cx43 was revealed by monoclonal anti-Cx43 antibodies (Figure 5, Lanes 6 and 7). In contrast, a DMPK content was barely visible and Cx43 was completely absent in fractions floating at the 30% sucrose layer (Figure 5, Lanes 5 and 8), where only empty vescicular structures were found (results not shown). These biochemical results confirmed that Cx43 and DMPK co-localize in the same structures, i.e., cardiac gap junctions, and that such an association is resistant to detergent treatment.
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Discussion |
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The data presented here demonstrate that DMPK, the product of the DM gene, in rat cardiac muscle is associated with precise and highly specialized subcellular structures, i.e., junctional SR and GJs.
The immunofluorescence observations parallel the findings of
The results presented here on immunogold labeling (Figure 2) provide for the first time in rat papillary muscles a precise identification at the ultrastructural level of SR elements with which DMPK is associated, i.e., junctional and extended junctional SR. As in skeletal muscles, two components of sarcoplasmic reticulum can be distinguished in mammalian ventricular muscle, i.e., junctional and nonjunctional SR. The former, however, is more complicated in cardiac than in skeletal muscle. In addition to terminal cisternae proper, which make direct contact with T-tubules, two other forms have been included in the junctional types of SR: corbular and peripheral SR. Corbular SR is preferentially seen near the Z-band, whereas peripheral SR forms a multilayered network in subsarcolemmal spaces (
In human cardiac myofibers, DMPK has been localized at various distances from the intercalated discs (
In the past few years, several reports have shed light on the protein composition of ID and the different specific proteins found in its different regions. Desmosomes are characterized by the presence of desmin filaments, desmogleins and desmocollins (
Since the first report on Cx32 phosphorylation in hepatocytes ( from the cytoplasm to the sarcolemma and intercalated discs. It would be worthwhile to investigate in greater detail if DMPK and some of the PKC isoforms may have differential or synergistic effects on signal transduction mechanisms.
The crucial role played by the phosphorylation of Cx43 is dramatically demonstrated by a complex familial syndrome characterized by heart malformations and defects of laterality (visceroatrial heterotaxia) due to mutations at one or more phosphorylatable serine or threonine residues of the C-terminal tail of the protein (
Mice lacking the DMPK gene (
As mentioned before, Cx43 phosphorylation has been implicated in channel formation (
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Acknowledgments |
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Supported by institutional funds from Consiglio Nazionale delle Ricerche and by grant funds (to SS) from Telethon Italy (project No. 913).
The technical assistance of Mr V. Gobbo is gratefully acknowledged.
Received for publication June 18, 1998; accepted October 20, 1998.
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