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Originally published In Press as doi:10.1074/jbc.M412845200 on December 13, 2004

J. Biol. Chem., Vol. 280, Issue 9, 8016-8021, March 4, 2005
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Myotonic Dystrophy Protein Kinase Phosphorylates Phospholamban and Regulates Calcium Uptake in Cardiomyocyte Sarcoplasmic Reticulum*

Perla Kaliman{ddagger}§, Daniele Catalucci¶, Jason T. Lam¶, Richard Kondo¶, José Carlos Paz Gutiérrez{ddagger}, Sita Reddy||, Manuel Palacín**, Antonio Zorzano{ddagger}, Kenneth R. Chien¶, and Pilar Ruiz-Lozano¶

From the Institute of Molecular Medicine, University of California, San Diego, California 92093, ||Institute for Genetic Medicine, University of Southern California, Los Angeles, California 90033, and **Institut de Recerca Biomèdica, Parc Científic de Barcelona, {ddagger}Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, E-08028 Barcelona, Spain

Received for publication, November 12, 2004 , and in revised form, December 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Myotonic dystrophy (DM) is caused by a CTG expansion in the 3'-untranslated region of a protein kinase gene (DMPK). Cardiovascular disease is one of the most prevalent causes of death in DM patients. Electrophysiological studies in cardiac muscles from DM patients and from DMPK-/- mice suggested that DMPK is critical to the modulation of cardiac contractility and to the maintenance of proper cardiac conduction activity. However, there are no data regarding the molecular signaling pathways involved in DM heart failure. Here we show that DMPK expression in cardiac myocytes is highly enriched in the sarcoplasmic reticulum (SR) where it colocalizes with the ryanodine receptor and phospholamban (PLN), a muscle-specific SR Ca2+-ATPase (SERCA2a) inhibitor. Coimmunoprecipitation studies showed that DMPK and PLN can physically associate. Furthermore, purified wild-type DMPK, but not a kinase-deficient mutant (K110A DMPK), phosphorylates PLN in vitro. Subsequent studies using the DMPK-/- mice demonstrated that PLN is hypo-phosphorylated in SR vesicles from DMPK-/- mice compared with wild-type mice both in vitro and in vivo. Finally, we show that Ca2+ uptake in SR is impaired in ventricular homogenates from DMPK-/- mice. Together, our data suggest the existence of a novel regulatory DMPK pathway for cardiac contractility and provide a molecular mechanism for DM heart pathology.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Myotonic muscular dystrophy (DM)1 is an autosomal, dominant inherited, neuromuscular disorder with an incidence of 1 in 8000 in European and North American populations. Clinical expression of DM is extremely variable; patients present with progressive muscular dystrophy associated with the inability to promote normal muscle relaxation (myotonia), cataracts, cardiac arrhythmia, testicular atrophy, and insulin resistance (1).

The DM1 mutation has been identified as the expansion of an unstable CTG repeat in the 3'-untranslated region of a gene encoding myotonic dystrophy protein kinase (DMPK) at chromosome 19q13.3. The age of onset and the severity of the disease correlate with the extent of expansion (2, 3). The dmpk gene product is a Ser/Thr protein kinase homologous to the MRCK p21-activated kinases (4) and the Rho family of kinases (5). Data obtained by using antibodies that detect specific isoforms of DMPK indicate that the most abundant isoform of DMPK is an 80-kDa protein expressed almost exclusively in smooth, skeletal, and cardiac muscles (6). This kinase exists both as a membrane-associated and as a soluble form in human left ventricular samples (7). The different C termini of DMPK that arise from alternative splicing determine its localization to the endoplasmic reticulum, mitochondria, or cytosol in transfected COS-1 cells (8). Among the substrates for DMPK proposed by in vitro studies are phospholemman, the dihydropyridine receptor, and the myosin phosphatase targeting subunit (9–11). However, an in vivo demonstration of the phosphorylation of these substrates by DMPK remains to be established, and a link between these substrates and the clinical manifestations of DM is unclear.

DMPK knock-out mice (DMPK-/-) display a cardiac phenotype that reproduces many cardiac conduction defects observed in DM patients, including first-, second-, and third-degree atrioventricular block (12–14). A cardiac phenotype is also observed in heterozygous DMPK-/+ mice, which develop first-degree heart block, a conduction defect strikingly similar to that observed in DM patients (15). DMPK is involved in the modulation of calcium homeostasis in skeletal muscle (16–19), and in cardiac myocytes (15) isolated from DMPK-/- mice, enhanced basal contractility and increased intracellular Ca2+ concentration have been described (20). However, the exact molecular mechanisms involved in the onset of heart failure in DM patients remain elusive (16–19).

In the heart, calcium cycling across the SR plays a key role in regulating contraction-relaxation cycles (21). When active, SR Ca2+-ATPase (SERCA2a) sequestrates cytoplasmic Ca2+, initiating cardiac relaxation. The activation of SERCA2a is tightly controlled by the SR membrane protein phospholamban (PLN) (22), and chronic PLN-SERCA2a interaction is the critical Ca2+ cycling defect in dilated cardiomyopathy (23). Cardiac phospholamban is a 52-amino acid phosphoprotein located in SR membranes with two adjacent residues, Ser-16 and Thr-17, identified as the phosphorylation sites for PKA and (Ca2+)/calmodulin-dependent kinase II, respectively (24–26). Unphosphorylated PLN inhibits SR Ca2+-ATPase, whereas phosphorylation of PLN reverses this inhibition (26). A previous report (20) suggested that PLN was hyperphosphorylated in cardiac homogenates obtained from DMPK-/- compared with control animals. However, the authors used specific anti-Ser(P)-16-PLN and anti-Thr(P)-17-PLN antibodies and showed a difference in mobility rather than the intensity of the bands, indicating that the modification of PLN that they observed in DMPK-/- samples was not Ser-16 or Thr-17 phosphorylation.

Here we report that DMPK is highly enriched in cardiomyocyte SR, and it colocalizes, interacts with, and phosphorylates PLN. We demonstrate that PLN is under-phosphorylated in SR vesicles from DMPK-/- mice compared with wild-type mice both in vitro and in vivo. Finally, we show that SR Ca2+ uptake is highly reduced in ventricular homogenates from DMPK-/- mice. The loss of DMPK phosphorylation of PLN may therefore represent an important determinant of ventricular dysfunction in DM.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Mouse full-length DMPK cDNA was kindly provided by Dr. B. Wieringa (University of Nijmegen, The Netherlands). Human DMPK cDNA subcloned into pRSETc vector was a kind gift from Dr. L. T. Timchenko (Baylor College of Medicine, Houston), and Myc-tagged hDMPK was kindly provided by Dr. M. B. Perryman (University of Colorado Health Sciences Center, Denver, CO).

Monoclonal antibodies against human DMPK (MANDM5, 3D10) were provided by Dr. G. E. Morris (MRIC Biochemistry Group, North East Wales Institute, UK). Anti-FLAG M2 monoclonal antibodies were from Sigma. Anti-phospholamban (clone A1) and anti-phospho-phospholamban (Ser-16) were from Upstate Biotechnology, Inc. (Lake Placid, NY). To detect mouse DMPK, we used the polyclonal anti-DMPK antibody from Zymed Laboratories Inc.. Anti-calsequestrin and anti-SERCA2a ATPase antibodies were from Affinity Bioreagents (Golden, CO). Monoclonal anti-Myc antibody 9E10 was obtained from the ATCC.

Preparation of SR Vesicles from Mouse Left Ventricle—Left ventricles were homogenized in ice-cold solution 1 (10 mM NaHCO3, 0.2 mM CaCl2, and protease inhibitors) (1.5 ml/ventricle) by three bursts of 10 s in a Polytron homogenizer at half-maximum speed. The homogenate was diluted in an equal volume of ice-cold solution 2 (500 mM sucrose, 300 mM KCl, 4 mM MgCl2, 60 mM histidine, pH 7.4) and centrifuged at 1000 x g for 20 min at 4 °C. The supernatant was recovered (total extract), diluted with 0.25 volumes of 3 M KCl, and centrifuged at 10,000 x g for 20 min at 4 °C. The supernatant was recovered and centrifuged at 100,000 x g for 30 min at 4 °C. The supernatant was recovered as cytosolic fraction. The pellet was washed in solution 3 (250 mM sucrose, 600 mM KCl, 3 mM MgCl2, 30 mM histidine pH 7.4) and centrifuged at 100,000 x g for 30 min at 4 °C. The pellet was finally resuspended in 200 µl of solution 4 (250 mM sucrose, 30 mM histidine pH 7.4) and repeatedly passed through a 25-gauge needle before storage at -80 °C. Proteins were measured by the method of Bradford (27).

Recombinant Adenovirus Generation and Neonatal Myocyte Gene Transfer—Recombinant adenoviruses expressing Myc-tagged human DMPK were generated by homologous recombination as described by Graham and Prevec (28). The cDNAs were cloned into the shuttle plasmid pAdl1/RSV and cotransfected with pJM17 into 293 cells to achieve homologous recombination as described previously (29).

Neonatal rat ventricular myocytes were prepared from hearts of 2–3-day-old Sprague-Dawley rat pups as described previously (30). Briefly, hearts were digested with collagenase, and myocytes were purified over a Percoll gradient. Myocytes were seeded on coverslips in 4:1 Dulbecco's modified Eagle's medium/medium 199 containing 10% horse serum, 5% fetal calf serum, and antibiotics (100 units/ml penicillin, 100 µg/ml streptomycin). Ten hours after isolation and culture, myocytes were infected for 2 h with either Myc-hDMPK (ad-myc-hDMPK) or green fluorescent protein (ad-GFP) as a control (data not shown) at a multiplicity of infection of 100, before the addition of a suitable volume of culture media. Twenty four hours later, the myocytes were washed and harvested for immunostaining.

Immunofluorescence and in Situ Hybridization—Cardiomyocytes plated on coverslips were fixed for 20 min with 3% paraformaldehyde in PBS, washed three times in PBS, and then treated as described previously (31). Images were obtained using a Leica TCS 4D laser confocal fluorescence microscope with a x40 objective. For in situ hybridization, 7-µm paraffin sections from mouse day 14 embryos were rehydrated, treated with 10 mg/ml proteinase K for 7 min 30 s, and hybridized with 10,000 cpm 35S riboprobe/ml in hybridization solution containing 50% formamide, 30 mM, NaCl, 20 mM EDTA, 10 mM NaH2PO4, 10% dextran sulfate, 1x Denhardt's, and 10 mM dithiothreitol (DTT). Hybridization was carried out for 14 h at 60 °C. Samples were subsequently washed at 60 °C in 5x SSC, 10 mM DTT, 2x SSC, 50% formamide, 10 mM DTT and digested with RNase A (10 µg/ml) for 30 min at 37 °C and dehydrated. The antisense RNA probe was transcribed in vitro at 37 °C for 2 h with T7 RNA polymerase after the subcloned plasmid was linearized with NotI. After in vitro transcription, the plasmid template was digested with RNase-free DNase. The probe was purified using an RNase free S-200 microspin column (Amersham Biosciences). Slides were dipped in LM-1 photographic emulsion (Amersham Biosciences), exposed for 10 days, and developed at 12 °C in Kodak D19 solution for 3 min 30 s. Counterstaining was performed in 0.02% toluidine blue, and slides were mounted in Permount solution. Images were taken using an Olympus microscope equipped with dark field condenser (32).

Transfections in HeLa Cells—Transient transfections were performed in HeLa cells by standard calcium phosphate precipitation in 10-cm diameter plates with a mixture of DNA containing 20 µg of total DNA (31). The GFP-encoding plasmid was included to monitor transfection efficiencies, which ranged from 60 to 90% as assessed by fluorescence-activated cell sorter analysis.

Generation and Purification of His6 Fusion Proteins—His6-hDMPK cDNA cloned into pRSETc vector (Invitrogen) was used as template to generate the K110A mutant hDMPK with the QuickChange site-directed mutagenesis kit (Stratagene), according to the manufacturer's protocol. The mutagenic oligonucleotide was 5'-CCAGGTGTATGCCATG(GC)GATCATGAACAAGTGGG-3' (sense strand, the mutated nucleotides 1521 and 1522 are indicated by parentheses). Proper construction of the mutated cDNA was confirmed by complete sequencing. Expression of fusion proteins in Escherichia coli (BL 21) was induced by adding 0.1 mM isopropylthiogalactoside for 3 h to the bacterial culture in the exponential phase of growth. The bacteria were pelleted, resuspended in PBS, pH 7.4, lysed by sonication, and pelleted to remove debris. The soluble proteins were purified using chelating Sepharose charged with Ni2+ ions (Amersham Biosciences) and visualized by silver staining. Kinase activity of the recombinant proteins was measured as described below by using 10 µg of the purified fractions.

Electrophoresis and Immunoblotting—Cells were lysed for 30 min at 4 °C in 50 mM Tris, pH 7.5, 120 mM NaCl, 1 mM EDTA, 6 mM EGTA, 15 mM Na4P2O7, 20 mM NaF, 0.1% phenylmethylsulfonyl fluoride, 0.1% aprotinin, 2 mM pepstatin, 2 mM leupeptin and supplemented with 1% Nonidet P-40. Cell extracts were centrifuged at 10,000 x g for 20 min at 4 °C, and 50 µg of the solubilized proteins was loaded. SDS-PAGE and immunoblot analysis were performed as described previously (33). Protein expression was quantified by scanning densitometry of at least three independent experiments for each condition.

Coimmunoprecipitation Assays—HeLa cells were cotransfected with Myc-hDMPK and FLAG-PLN. 24 h after transfection, cells were washed twice in PBS, scraped, and solubilized as described above. The supernatants (500 µg) were immunoprecipitated for 90 min at 4 °C with anti-Myc, anti-FLAG, or nonimmune control antibodies preadsorbed on protein G-Sepharose. The immunopellets were washed three times in solubilization buffer before being resuspended in SDS-PAGE sample buffer under reduction conditions and analyzed by Western blot using polyclonal antibodies against Myc or FLAG, as indicated.

Immunoprecipitation and Protein Kinase Activity—Cell lysates were immunoprecipitated for 2 h at 4 °C with protein G-bound anti-Myc monoclonal antibody. Immunopellets were rinsed three times in lysis buffer and once in kinase buffer (25 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 mM MgCl2, 5 mM {beta}-glycerophosphate, 2.5 mM DTT). Assays for protein kinase activity were carried out for 1 h at 37 °C in a volume of 40 µl of kinase buffer containing 0.1 µM ATP. Substrates used were FLAG- or Myc-tagged PLN expressed in HeLa cells and immunoprecipitated with anti-FLAG or anti-Myc antibodies, respectively. Reactions were stopped with Laemmli sample buffer, and samples were loaded onto 15% acrylamide SDS gels. SR vesicles (10 µg) were solubilized for 30 min at 4 °C with 1% Nonidet P-40 in the presence of protease inhibitors, and phosphorylation was carried out in the kinase buffer described above for 30 min at 37 °C, using 0.1 µM [{gamma}-32P]ATP (1.5 mCi/µmol) (Amersham Biosciences) and in the presence of 5 µM PKA peptide inhibitor, PKI (Calbiochem). Gels were dried and developed by autoradiography. Phosphorylation was quantified by scanning densitometry of three independent experiments.

Measurements of SR Ca2+ Uptake—Ventricular tissue from 3- to 4-month-old DMPK+/+ and DMPK-/- mouse hearts was homogenized at 4 °C in 2.5 ml of homogenizing solution (25 mM imidazole, pH 7.0) with a Teflon glass Thomas tissue grinder. SR Ca2+ uptake assays were performed in ventricular homogenates at room temperature based on a protocol modified from that of Pagani and Solaro (34). Aliquots (175 µl) of homogenates were transferred into tubes containing 1.4 ml of uptake buffer (100 mM KCl, 10 mM potassium oxalate, 40 mM imidazole, 10 mM sodium azide, 4 mM MgCl2, 1 µM ruthenium red) and 45Ca-EGTA buffer containing 0.185 µCi/ml 45Ca (PerkinElmer Life Sciences) and a given amount of free Ca2+ (20 or 200 nM), which was calculated on the basis of the amount of added EGTA. Ruthenium Red was used to block Ca2+ efflux via the ryanodine receptor. After 5 min of preincubation, the uptake reaction was initiated by the addition of 2.5 mM sodium ATP. Ca2+ uptake was terminated at various times (1, 3, and 5 min for 200 nM free Ca2+; 1, 10, and 20 min for 20 nM free Ca2+) by filtering 250-µl aliquots on 0.45-µm nitrocellulose membranes (Millipore-type MA), followed by two washes (5 ml) with uptake buffer without Ca2+ and ATP. The remaining radioactivity on the nitrocellulose filters was determined by liquid scintillation spectroscopy. Protein concentration was assayed with a DC protein assay kit (Bio-Rad). Ca2+ uptake was calculated from the slope of the linear regression analysis relating 45Ca2+ uptake/mg of protein to reaction time.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression Pattern and Cellular Distribution of DMPK— DMPK under-expression contributes to DM pathology; however, little is known regarding the specific cell types in which it is expressed. To obtain specific single cell resolution data on dmpk expression, we have performed radioactive in situ hybridization and immunohistochemistry analysis. In the developing mouse embryo, dmpk mRNA is primarily detected in striated muscle structures that include all major muscles in the skeletal structures (Fig. 1, A and B), cardiac muscle, and diaphragm (Fig. 1D). We also detected dmpk mRNA in the smooth muscle of the lung and gut. We do not detect dmpk expression in the epithelium of the gut at this developmental stage (Fig. 1C). In the heart, dmpk expression is restricted to the cardiomyocytes in the ventricle and atrium, and we do not detect a hybridization signal in the epicardium nor in the endocardium. In the myocardium, the dmpk hybridization signal is stronger in the compact muscular zone compared with the trabecular zone, and the hybridization signal is almost indistinguishable from background in the primordia of the papillary muscles. We also observed dmpk expression in the muscular portion of the outflow tract (Fig. 1D).



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  		FIG. 1.
Expression pattern of DMPK in embryonic day 14.5 mouse embryos. DMPK mRNA is detected at all major striated muscle tissues including all skeletal muscle in the back (A) and leg (B). DMPK is also expressed in the smooth muscle of the gut (C) and in the diaphragm and heart (D).

 
Previous work suggested that DMPK associates with the sarcoplasmic reticulum and gap junctions in rat cardiac muscle (35). However, those immunocytochemistry and electron microscopy data need to be redefined, because they were obtained using a polyclonal antibody that detected a 54-kDa band by Western blot, whereas it is now known that the main DMPK cardiac isoform is an 80-kDa protein (35). Accordingly, we subsequently performed immunofluorescence studies to characterize the subcellular distribution of DMPK in cardiomyocytes, using a specific anti-hDMPK monoclonal antibody (6). In adult human cardiomyocytes (Fig. 2, A–C), DMPK specifically colocalized with the ryanodine receptor (RyR), which has been characterized by ultrastructural studies as mainly confined to the SR membranes (36). Moreover, forced expression of Myc-tagged hDMPK by adenoviral gene transfer to rat neonatal cardiomyocytes showed that Myc-hDMPK was targeted to perinuclear structures where it colocalized with the SR proteins RyR and phospholamban (PLN) (Fig. 2, D–L). Control experiments with adenoviruses expressing recombinant GFP exhibited both cytosolic and nuclear localization (data not shown) but did not colocalize with endoplasmic reticulum markers.



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  		FIG. 2.
Double immunofluorescent staining of DMPK and RyR in cardiomyocytes. A–C, localization of DMPK in adult human myocytes. Simultaneous double-immunofluorescent pictures of DMPK and RyR (merge) show colocalization of both proteins. D–L, colocalization of DMPK in neonatal rat cardiomyocytes infected with adenovirus expressing Myc-tagged DMPK was analyzed by double immunofluorescent staining. RyR and PLN were used as the marker for sarcoplasmic reticulum, and {alpha}-actinin was used as a control. Adenoviruses expressing GFP were used as a control (data not shown). M, subcellular distribution of DMPK in adult rat left ventricles. After subcellular fractionation, the relative abundance of DMPK and PLN was analyzed in 10 µg of total extracts (total), cytosolic fraction (cytosol), and low density SR-enriched membranes (SR). Representative images from three independent experiments are shown.

 
As an approach to estimate the percentage of DMPK localized in the SR, we performed subcellular fractionation of mouse left ventricles, and by Western blot we analyzed the proteins obtained at different steps in the fractionation procedure (10 µg of total extract, cytosol, and SR-enriched low density membranes). By taking into account the relative abundance of DMPK in each fraction and the total amount of protein recovered in each fraction, we determined that 31 ± 4% (n = 3) of total left ventricle DMPK localized to the low density microsomal fraction. This fraction, although enriched in SR membranes, contains other subcellular membranes where DMPK may also be expressed. (Fig. 2M). Most interestingly, DMPK is distributed similarly to the SR protein PLN (Fig. 2M, lower panel).

DMPK Interacts with and Phosphorylates PLN—Because DMPK seems to be highly abundant in the SR of cardiomyocytes (Fig. 2M) and colocalizes with phospholamban in these cells (Fig. 2, A–L), and the phospholamban sequence contains several putative DMPK phosphorylation sites (7, 8), we hypothesized that PLN could be a substrate for DMPK. We generated a hDMPK mutated at the ATP-binding site (K110A), which is a kinase-deficient mutant as observed by using myelin basic protein as a substrate (data not shown). We purified bacterially expressed recombinant His6-hDMPK (WT) and a His6-(K110A)hDMPK (K110A). We observed that FLAG-PLN expressed in HeLa cells and immunoprecipitated with anti-FLAG antibody was phosphorylated at Ser-16 by wild-type but not by K110A-hDMPK (Fig. 3A). To rule out any effect because of improper folding of bacterially expressed DMPK, we also measured the kinase activity of Myc-hDMPK expressed in HeLa cells, observing the phosphorylation of PLN at Ser-16 by Myc-DMPK (Fig. 3B). No specific phosphorylation on PLN Thr-17 was detected under our experimental conditions (data not shown).



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  		FIG. 3.
DMPK phosphorylates PLN in vitro. A, FLAG-PLN was expressed in HeLa cells and immunoprecipitated with an anti-FLAG antibody, and the phosphorylation reaction was carried out in the presence of 10 µg of bacterially expressed recombinant hDMPK (WT), recombinant hDMPK(K110A), or a nonspecific preparation from non-transformed bacterial extract (NS). PLN phosphorylation was detected by Western blot using an anti-Ser(P)-16 PLN antibody. Equivalent amounts of FLAG-PLN were immunoprecipitated in each condition as observed after re-blotting with an anti-PLN antibody. B, HeLa cells were transfected with Myc-DMPK or Myc-PLN, and cell extracts from each condition (10 or 100 µg, respectively) were independently immunoprecipitated using anti-Myc antibody. The immunopellets were combined as indicated in the figure, and phosphorylation was carried out in the presence of kinase buffer. PLN phosphorylation was detected by Western blot by using an anti-Ser(P)-16 PLN antibody (blot, anti-P-Ser-16 PLN). The amount of total DMPK immunoprecipitated was analyzed with 3D10 monoclonal antibody to hDMPK (blot, DMPK). 10 µg of nontransfected cells were immunoprecipitated with anti-Myc antibody as nonspecific control for kinase activity. Data from representative experiments are shown.

 
DMPK and PLN physically interact, as shown by coimmunoprecipitation studies (Fig. 4). HeLa cells cotransfected with Myc-DMPK and FLAG-PLN were coimmunoprecipitated with either anti-Myc (Fig. 4A) or anti-FLAG (Fig. 4B) antibodies to immunoprecipitate DMPK or PLN, respectively, or to control nonimmune mouse IgGs. The association of DMPK and PLN was clearly evident in both conditions by assessing FLAG-PLN in the Myc-DMPK immunopellet and vice versa.



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  		FIG. 4.
Coimmunoprecipitation of DMPK and PLN. MycDMPK and FLAGPLN cDNAs were cotransfected in HeLa cells. Cell extracts were immunoprecipitated (IP) either with anti-Myc (A), anti-FLAG (B), or nonspecific immunoglobulins (ns Ig). The pellets were extensively washed and analyzed by Western blot using monoclonal antibodies to DMPK (3D10) or to PLN (cA1) to analyze their content in the immunopellets (pt) and the supernatants (snt). Data shown are representative of three independent experiments.

 
Decreased PLN Phosphorylation Activity in Left Ventricle SR Vesicles from DMPK-/- Mice—We analyzed the phosphorylation state of PLN in SR vesicles purified from 12-week-old male WT and DMPK -/- mice. In both groups of animals, we examined the relative abundance of Ser(P)-16 PLN and the expression of the SR proteins SERCA2a ATPase, PLN, and calsequestrin. Although the steady state levels of all the proteins analyzed was similar in SR vesicles from DMPK-/- and WT mice, the amount of Ser(P)-16 PLN decreased by 1.8 ± 0.2-fold (n = 4) in DMPK-/- mice compared with WT animals (Fig. 5A).



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  		FIG. 5.
Decreased PLN phosphorylation activity in SR vesicles from DMPK-/- mice. A, left ventricle SR vesicles were prepared from DMPK-/- or DMPK+/+ mice. Content of the indicated proteins in 10 µg of SR vesicles was analyzed by Western blot. Images shown are representative of four independent experiments. B, left ventricle SR vesicles from DMPK-/- or DMPK+/+ mice (10 µg) were incubated in a kinase reaction containing [{gamma}-32P]ATP and PKA peptide inhibitor (PKI) for 30 min at 37 °C. Reactions were stopped with Laemmli sample buffer, and samples were loaded onto SDS-15% acrylamide gels. Gels were dried and visualized by autoradiography. The image shown is representative of three independent experiments.

 
As described above (Fig. 3), DMPK phosphorylates PLN in vitro. To analyze whether this reaction occurs in cardiac myocyte SR, we measured the endogenous PLN phosphorylation activity of SR vesicles in the presence of kinase buffer and [{gamma}-32P]ATP in SR vesicles from WT and DMPK-/- mice (Fig. 5B). A strongly phosphorylated band was detected, which we identified as PLN considering that it is the only phosphoacceptor protein so far described in cardiomyocytes with a molecular mass of 6 kDa. PLN phosphorylation was 2.2 ± 0.4-fold (n = 3) higher in vesicles from DMPK+/+ animals than that observed in vesicles from DMPK-/- animals. These results provide evidence of a direct link between DMPK expression and PLN phosphorylation state.

Decreased Ca2+ Uptake in Left Ventricle SR from DMPK-/- Mice—We next explored the physiological relevance of our set of data as follows: (i) localization of DMPK in the SR; (ii) the interaction DMPK/PLN; and (iii) DMPK phosphorylation of PLN, by comparing the SR calcium uptake rates in left ventricles from the DMPK-/- and DMPK+/+ mice. Calcium uptake rates were markedly reduced in DMPK-/- mice observing a 48 and 84% decrease at 200 and 20 nM free calcium concentrations, respectively, compared with those of controls (Table I and Fig. 6). These results are concomitant with the decrease in PLN phosphorylation in DMPK-/- mice and cannot be attributed to alterations in the content of the SR Ca2+-cycling proteins SERCA2a and PLN that showed similar expression levels in both DMPK-/- and DMPK+/+ mice (Fig. 5A).


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  		TABLE I
SR Ca2+ uptake in DMPK+/+ and DMPK-/- mice

 



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  		FIG. 6.
Fig. 6. SR Ca2+ uptake in DMPK+/+ and DMPK-/- mice. SR Ca2+ uptake assays were performed in ventricular homogenates from DMPK+/+ and DMPK-/- mice at 200 and 20 nM free calcium and at room temperature (n = 3). SR Ca2+ uptake (nmol/min/mg protein) was calculated from the slope of the linear regression analysis relating 45Ca2+ uptake/mg of total protein to reaction time. Activity represents the percentage of DMPK+/+ SR Ca2+ uptake for each free calcium concentration. p < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cardiovascular disease is one of the most prevalent causes of death in DM patients, accounting for about 30% of fatalities. Cardiac mortality occurs because of progressive left ventricular dysfunction, ischemic heart disease, pulmonary embolism, or unexpected sudden death (37). Electrophysiological studies in skeletal and cardiac muscles from DM patients and from DMPK-/- mice showed defects in intracellular Ca2+ cycling (16–20). Here, by means of localization studies, biochemical analysis, and calcium uptake measurements, we describe a novel substrate for DMPK that provides a new mechanism for cardiac dysfunction in DM.

We provide evidence for a preferential muscle-restricted expression of dmpk during mouse development. Skeletal and cardiac muscle, as well as smooth muscle in the gut and lung, are positive for the dmpk signal in mouse embryos. This is in agreement with a recent report (38) describing the expression pattern of dmpk in adult tissues. In the developing heart, we did not detect a hybridization signal in the epicardium or in the endocardium, suggesting that DMPK is primarily restricted to the contractile muscle. This is of particular importance when addressing cardiomyopathy, because many cardiac phenotypes are the consequence of mutation in nonmuscular genes (39).

Our data also demonstrate that DMPK is enriched in cardiomyocyte SR. Most interestingly, following adenoviral gene transfer of Myc-tagged DMPK to ventricular cardiomyocytes from neonatal rats, the kinase is targeted to the SR where it colocalizes with PLN, showing a similar pattern of cellular distribution to that observed for the endogenous DMPK in human cardiac myocytes. The observation that DMPK adenoviral gene transfer results in a correct targeting of the kinase to SR suggests that DMPK gene-delivery strategies may become feasible for the improvement of the DM ventricular dysfunction.

The screening of a library of synthetic peptides has shown that the optimal DMPK substrate sequences should consist of three to four arginines (or lysines) at distinct positions N-terminal to the phosphoacceptor (8). Remarkably, the PLN sequence surrounding Ser-16 is similar to that of the DMPK-preferred Ser substrate (R/K)XXXRRf(S)Xf (where X is any amino acid, preferably not P or E; f is a hydrophobic residue; and boldface type indicate the phosphoacceptor). In contrast, PLN Thr-17 does not fit any of the defined consensus sequences as there is no basic residue at positions -1 or -2 from this Thr, which has been defined as one of the most important features of DMPK phosphorylation on Thr residues. Consistent with these analyses of phosphorylation consensus sequences in synthetic peptides, we demonstrate here that DMPK can phosphorylate PLN at Ser-16, whereas we found no evidence of any specific phosphorylation in threonine 17.

Moreover, in SR vesicles purified from DMPK-/- mice, endogenous PLN 32P incorporation was 2-fold decreased compared with control animals directly correlating a decreased PLN phosphorylation activity with the lack of DMPK expression. Under physiological conditions, PLN phosphorylation at Ser-16 by PKA is the predominant event so far described that leads to proportional increases in the rate of Ca2+ uptake into SR and accelerates ventricular relaxation (40, 41). DMPK phosphorylation of Ser-16-PLN emerges as a novel mechanism involved in the regulation of cardiac contractility that may be therapeutically relevant because of the preferential muscular expression pattern of DMPK. The proposed model is supported by our data on Ca2+ uptake in the DMPK-/- mouse. A decrease in Ca2+ uptake is the central feature of human and animal heart failure, and an increase in the amount of PLN associated with SERCA2a is an important determinant of SR dysfunction in the heart (21, 42, 43). Indeed, the finding that DMPK deficiency results in a marked decrease in SR Ca2+ uptake activity points to PLN phosphorylation by DMPK as a physiological relevant event. Most importantly, the decrease in SR Ca2+ uptake is detectable at 14 weeks of age, which is much earlier than the first detectable signs of muscle weakness (7–10 months) (12) in these animals, thus suggesting that a decrease in SR Ca2+ uptake is primary to cardiac dysfunction.

In conclusion, our data indicate that ventricular function can be regulated through DMPK phosphorylation of PLN and provide a molecular mechanism to the ventricular dysfunctions detected in both DM patients and DM animal models. Given that PLN is markedly under-phosphorylated in most acquired forms of heart failure, the identification of the molecular signaling pathways involving DMPK in the heart may provide crucial molecular tools to design new therapies for the treatment of DM and other cardiomyopathies.


   FOOTNOTES
 
* This work has been supported by National Institutes of Health Grants HL065484 (to P. R.-L.) and SAF2001-3500 from the Ministerio de Ciencia y Tecnología, Spain (to P. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, E-08028 Barcelona, Spain. Tel.: 34-93-4034700; Fax: 34-93-4021559; E-mail: pkaliman{at}ub.edu.

1 The abbreviations used are: DM, myotonic dystrophy; DMPK, myotonic dystrophy protein kinase; PLN, phospholamban; SR, sarcoplasmic reticulum; PKA, cAMP-dependent protein kinase; RyR, ryanodine receptor; WT, wild type; DTT, dithiothreitol; PKA, cAMP-dependent protein kinase; PBS, phosphate-buffered saline; GFP, green fluorescent protein; hDMPK, human DMPK. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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