Myotonic Dystrophy Protein Kinase Phosphorylates Phospholamban and Regulates Calcium Uptake in Cardiomyocyte Sarcoplasmic Reticulum

Abstract

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 Ca 2؉ -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 Ca 2؉ 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. 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 3Ј-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 an...

Myotonic Dystrophy Protein Kinase: Structure, Function and Its Possible Role in the Pathogenesis of Myotonic Dystrophy Type 1

“…In addition, multiple subcellular localizations have been assigned to DMPK, including neuromuscular and myotendinous junctions Berul et al, 2000;Shimokawa et al, 1997) and terminal cisternae and intercalated discs of skeletal muscle sarcoplasmic reticulum (Benders et al, 1997;Pall et al, 2003;Saba et al, 1999). Furthermore, DMPK localization to gap junctions of the intercalated discs has been reported in the heart and Purkinje fibers (Kaliman et al, 2005;Mounsey et al, 2000b).…”

“…In addition, DMPK -/-mice with cardiac contractility deregulation exhibit an impaired SERCA2a function. This ATPase is regulated by PLN protein, which is phosphorylated by DMPK (Kaliman et al, 2005). Furthermore, cardiac dysfunction, hypertrophic cardiomyopathy, propensity for dysrhythmia and enhanced neonatal mortality has been reported in the DMPK mice (O'Cochlain et al, 2004).…”

“…DMPK deficit also cause an enhanced basal contractility of single cardiomyocytes with associated increase in intracellular Ca 2+ , suggesting that DMPK has a modulator role in the control of intracellular Ca 2+ concentration (Benders et al, 1997;Pall et al, 2003). In addition, hypophosphorylation of phospholamban (PLN), a muscle-specific sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) inhibitor, causes a deregulation of Ca 2+ uptake in sarcoplasmic reticulum vesicles of ventricular homogenates from DMPK -/-mice (Kaliman et al, 2005). An effect on Na + current amplitude in DMPK -/-myocytes because of reduced channel number is also observed (Mounsey et al, 2000b;Reddy et al, 2002).…”

Myotonic Dystrophy Protein Kinase Is Critical for Nuclear Envelope Integrity

“…These analyses revealed specific localization of DMPK at the NE in interphase HeLa cells. NE localization of DMPK was reported previously in C2C12 mouse myoblasts and neonatal rat cardiac myocytes (19,35). DMPK Overexpression Causes Nuclear Fragmentation-We investigated the role of DMPK in human epithelial cells by overexpressing DMPK in HeLa cells.…”

Congenital Myotonic Dystrophy

“…The normal protein localizes to neuromuscular junctions [4], sarcoplasmic reticulum [5] and intercalated discs in the heart myocyte [6]. The functions of DMPK are manifold including: cell shape determination [4,7] and effects on excitation coupling through calcium homeostasis [8].…”