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The transmembrane proteoglycan syndecan-4 is known to be involved in the hypertrophic response to pressure overload. Although multiple downstream signaling pathways have been found to be involved in this response in a syndecan-4-dependent manner, there are likely more signaling components involved. As part of a larger syndecan-4 interactome screening, we have previously identified MLP as a binding partner to the cytoplasmic tail of syndecan-4. Interestingly, many human MLP mutations have been found in patients with hypertrophic (HCM) and dilated cardiomyopathy (DCM). To gain deeper insight into the role of the syndecan-4-MLP interaction and its potential involvement in MLP-associated cardiomyopathy, we have here investigated the syndecan-4-MLP interaction in primary adult rat cardiomyocytes and the H9c2 cell line. The binding of syndecan-4 and MLP was analyzed in total lysates and subcellular fractions of primary adult rat cardiomyocytes, and baseline and differentiated H9c2 cells by immunoprecipitation. MLP and syndecan-4 localization were determined by confocal microscopy, and MLP oligomerization was determined by immunoblotting under native conditions. Syndecan-4-MLP binding, as well as MLP self-association, were also analyzed by ELISA and peptide arrays. Our results showed that MLP-WT and syndecan-4 co-localized in many subcellular compartments; however, their binding was only detected in nuclear-enriched fractions of isolated adult cardiomyocytes. In vitro, syndecan-4 bound to MLP at three sites, and this binding was reduced in some HCM-associated MLP mutations. While MLP and syndecan-4 also co-localized in many subcellular fractions of H9c2 cells, these proteins did not bind at baseline or after differentiation into cardiomyocyte-resembling cells. Independently of syndecan-4, mutated MLP proteins had an altered subcellular localization in H9c2 cells, compared to MLP-WT. The DCM- and HCM-associated MLP mutations, W4R, L44P, C58G, R64C, Y66C, K69R, G72R, and Q91L, affected the oligomerization of MLP with an increase in monomeric at the expense of trimeric and tetrameric recombinant MLP protein. Lastly, two crucial sites for MLP self-association were identified, which were reduced in most MLP mutations. Our data indicate that the syndecan-4-MLP interaction was present in nuclear-enriched fractions of isolated adult cardiomyocytes and that this interaction was disrupted by some HCM-associated MLP mutations. MLP mutations were also linked to changes in MLP oligomerization and self-association, which may be essential for its interaction with syndecan-4 and a critical molecular mechanism of MLP-associated cardiomyopathy.
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Miocitos Cardíacos , Unión Proteica , Sindecano-4 , Animales , Humanos , Ratas , Línea Celular , Miocitos Cardíacos/metabolismo , Sindecano-4/metabolismo , Sindecano-4/genéticaRESUMEN
Introduction: The skeletal muscle deformity of commercial chickens (Gallus gallus), known as the wooden breast (WB), is associated with fibrotic myopathy of unknown etiology. For future breeding strategies and genetic improvements, it is essential to identify the molecular mechanisms underlying the phenotype. The pathophysiological hallmarks of WB include severe skeletal muscle fibrosis, inflammation, myofiber necrosis, and multifocal degeneration of muscle tissue. The transmembrane proteoglycans syndecans have a wide spectrum of biological functions and are master regulators of tissue homeostasis. They are upregulated and shed (cleaved) as a regulatory mechanism during tissue repair and regeneration. During the last decades, it has become clear that the syndecan family also has critical functions in skeletal muscle growth, however, their potential involvement in WB pathogenesis is unknown. Methods: In this study, we have categorized four groups of WB myopathy in broiler chickens and performed a comprehensive characterization of the molecular and histological profiles of two of them, with a special focus on the role of the syndecans and remodeling of the extracellular matrix (ECM). Results and discussion: Our findings reveal differential expression and shedding of the four syndecan family members and increased matrix metalloproteinase activity. Additionally, we identified alterations in key signaling pathways such as MAPK, AKT, and Wnt. Our work provides novel insights into a deeper understanding of WB pathogenesis and suggests potential therapeutic targets for this condition.
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Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg9 and Arg11 exert off-target effects on cellular Ca2+ homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca2+ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca2+ leak remained unchanged, indicating that protracted Ca2+ removal from the cytosol stemmed from inhibition of the SR Ca2+ ATPase 2 (SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as SERCA2 is expressed in diverse cell types, the wide-ranging consequences of SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings.
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Péptidos de Penetración Celular , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/metabolismo , Músculo Esquelético/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.
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Dinamina II , Miocitos Cardíacos , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Dinamina II/genética , Dinamina II/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca2+ ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators. METHODS: Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays. RESULTS: PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding. CONCLUSIONS: Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.
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Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Insuficiencia Cardíaca , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Animales , Humanos , Ratones , Calcio/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Insuficiencia Cardíaca/metabolismo , Células HEK293 , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Background: In cardiac muscle, the ubiquitously expressed proteoglycan syndecan-4 is involved in the hypertrophic response to pressure overload. Protein kinase Akt signaling, which is known to regulate hypertrophy, has been found to be reduced in the cardiac muscle of exercised male syndecan-4-/- mice. In contrast, we have recently found that pSer473-Akt signaling is elevated in the skeletal muscle (tibialis anterior, TA) of female syndecan-4-/- mice. To determine if the differences seen in Akt signaling are sex specific, we have presently investigated Akt signaling in the cardiac muscle of sedentary and exercised female syndecan-4-/- mice. To get deeper insight into the female syndecan-4-/- heart, alterations in cardiomyocyte size, a wide variety of different extracellular matrix components, well-known syndecan-4 binding partners and associated signaling pathways have also been investigated. Methods: Left ventricles (LVs) from sedentary and exercise trained female syndecan-4-/- and WT mice were analyzed by immunoblotting and real-time PCR. Cardiomyocyte size and phosphorylated Ser473-Akt were analyzed in isolated adult cardiomyocytes from female syndecan-4-/- and WT mice by confocal imaging. LV and skeletal muscle (TA) from sedentary male syndecan-4-/- and WT mice were immunoblotted with Akt antibodies for comparison. Glucose levels were measured by a glucometer, and fasting blood serum insulin and C-peptide levels were measured by ELISA. Results: Compared to female WT hearts, sedentary female syndecan-4-/- LV cardiomyocytes were smaller and hearts had higher levels of pSer473-Akt and its downstream target pSer9-GSK-3ß. The pSer473-Akt inhibitory phosphatase PHLPP1/SCOP was lowered, which may be in response to the elevated serum insulin levels found in the female syndecan-4-/- mice. We also observed lowered levels of pThr308-Akt/Akt and GLUT4 in the female syndecan-4-/- heart and an increased LRP6 level after exercise. Otherwise, few alterations were found. The pThr308-Akt and pSer473-Akt levels were unaltered in the cardiac and skeletal muscles of sedentary male syndecan-4-/- mice. Conclusion: Our data indicate smaller cardiomyocytes, an elevated insulin/pSer473-Akt/pSer9-GSK-3ß signaling pathway, and lowered SCOP, pThr308-Akt/Akt and GLUT4 levels in the female syndecan-4-/- heart. In contrast, cardiomyocyte size, and Akt signaling were unaltered in both cardiac and skeletal muscles from male syndecan-4-/- mice, suggesting important sex differences.
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BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Sitios de Unión , Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Células Cultivadas , Células HEK293 , Humanos , Miocitos Cardíacos/metabolismo , Unión Proteica , Ratas , Ratas WistarRESUMEN
The cardiac sodium-calcium exchanger (NCX1) is important for normal Na+- and Ca2+-homeostasis and cardiomyocyte relaxation and contraction. It has been suggested that NCX1 activity is reduced by phosphorylated phospholemman (pSer68-PLM); however its direct interaction with PLM is debated. Disruption of the potentially inhibitory pSer68-PLM-NCX1 interaction might be a therapeutic strategy to increase NCX1 activity in cardiac disease. In the present study, we aimed to analyze the binding affinities and kinetics of the PLM-NCX1 and pSer68-PLM-NCX1 interactions by surface plasmon resonance (SPR) and to develop a proteolytically stable NCX1 activator peptide for future in vivo studies. The cytoplasmic parts of PLM (PLMcyt) and pSer68-PLM (pSer68-PLMcyt) were found to bind strongly to the intracellular loop of NCX1 (NCX1cyt) with similar K D values of 4.1 ± 1.0 nM and 4.3 ± 1.9 nM, but the PLMcyt-NCX1cyt interaction showed higher on/off rates. To develop a proteolytically stable NCX1 activator, we took advantage of a previously designed, high-affinity PLM binding peptide (OPT) that was derived from the PLM binding region in NCX1 and that reverses the inhibitory PLM (S68D)-NCX1 interaction in HEK293. We performed N- and C-terminal truncations of OPT and identified PYKEIEQLIELANYQV as the minimum sequence required for pSer68-PLM binding. To increase peptide stability in human serum, we replaced the proline with an N-methyl-proline (NOPT) after identification of N-terminus as substitution tolerant by two-dimensional peptide array analysis. Mass spectrometry analysis revealed that the half-life of NOPT was increased 17-fold from that of OPT. NOPT pulled down endogenous PLM from rat left ventricle lysate and exhibited direct pSer68-PLM binding in an ELISA-based assay and bound to pSer68-PLMcyt with a K D of 129 nM. Excess NOPT also reduced the PLMcyt-NCX1cyt interaction in an ELISA-based competition assay, but in line with that NCX1 and PLM form oligomers, NOPT was not able to outcompete the physical interaction between endogenous full length proteins. Importantly, cell-permeable NOPT-TAT increased NCX1 activity in cardiomyocytes isolated from both SHAM-operated and aorta banded heart failure (HF) mice, indicating that NOPT disrupted the inhibitory pSer68-PLM-NCX1 interaction. In conclusion, we have developed a proteolytically stable NCX1-derived PLM binding peptide that upregulates NCX1 activity in SHAM and HF cardiomyocytes.
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The extracellular matrix (ECM) is important in cardiac remodeling and syndecans have gained increased interest in this process due to their ability to convert changes in the ECM to cell signaling. In particular, syndecan-4 has been shown to be important for cardiac remodeling, whereas the role of its close relative syndecan-2 is largely unknown in the heart. To get more insight into the role of syndecan-2, we here sought to identify interaction partners of syndecan-2 in rat left ventricle. By using three different affinity purification methods combined with mass spectrometry (MS) analysis, we identified 30 novel partners and 9 partners previously described in the literature, which together make up the first cardiac syndecan-2 interactome. Eleven of the novel partners were also verified in HEK293 cells (i.e., AP2A2, CAVIN2, DDX19A, EIF4E, JPH2, MYL12A, NSF, PFDN2, PSMC5, PSMD11, and RRAD). The cardiac syndecan-2 interactome partners formed connections to each other and grouped into clusters mainly involved in cytoskeletal remodeling and protein metabolism, but also into a cluster consisting of a family of novel syndecan-2 interaction partners, the CAVINs. MS analyses revealed that although syndecan-2 was significantly enriched in fibroblast fractions, most of its partners were present in both cardiomyocytes and fibroblasts. Finally, a comparison of the cardiac syndecan-2 and -4 interactomes revealed surprisingly few protein partners in common.
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BACKGROUND: Extracellular matrix (ECM) remodeling is essential for skeletal muscle development and adaption in response to environmental cues such as exercise and injury. The cell surface proteoglycan syndecan-4 has been reported to be essential for muscle differentiation, but few molecular mechanisms are known. Syndecan-4-/- mice are unable to regenerate damaged muscle, and display deficient satellite cell activation, proliferation, and differentiation. A reduced myofiber basal lamina has also been reported in syndecan-4-/- muscle, indicating possible defects in ECM production. To get a better understanding of the underlying molecular mechanisms, we have here investigated the effects of syndecan-4 genetic ablation on molecules involved in ECM remodeling and muscle growth, both under steady state conditions and in response to exercise. METHODS: Tibialis anterior (TA) muscles from sedentary and exercised syndecan-4-/- and WT mice were analyzed by immunohistochemistry, real-time PCR and western blotting. RESULTS: Compared to WT, we found that syndecan-4-/- mice had reduced body weight, reduced muscle weight, muscle fibers with a smaller cross-sectional area, and reduced expression of myogenic regulatory transcription factors. Sedentary syndecan-4-/- had also increased mRNA levels of syndecan-2, decorin, collagens, fibromodulin, biglycan, and LOX. Some of these latter ECM components were reduced at protein level, suggesting them to be more susceptible to degradation or less efficiently translated when syndecan-4 is absent. At the protein level, TRPC7 was reduced, whereas activation of the Akt/mTOR/S6K1 and Notch/HES-1 pathways were increased. Finally, although exercise induced upregulation of several of these components in WT, a further upregulation of these molecules was not observed in exercised syndecan-4-/- mice. CONCLUSION: Altogether our data suggest an important role of syndecan-4 in muscle development.
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Costameres are signaling hubs at the sarcolemma and important contact points between the extracellular matrix and cell interior, sensing and transducing biomechanical signals into a cellular response. The transmembrane proteoglycan syndecan-4 localizes to these attachment points and has been shown to be important in the initial stages of cardiac remodeling, but its mechanistic function in the heart remains insufficiently understood. Here, we sought to map the cardiac interactome of syndecan-4 to better understand its function and downstream signaling mechanisms. By combining two different affinity purification methods with MS analysis, we found that the cardiac syndecan-4 interactome consists of 21 novel and 29 previously described interaction partners. Nine of the novel partners were further validated to bind syndecan-4 in HEK293 cells (i.e. CAVIN1/PTRF, CCT5, CDK9, EIF2S1, EIF4B, MPP7, PARVB, PFKM, and RASIP). We also found that 19 of the 50 interactome partners bind differently to syndecan-4 in the left ventricle lysate from aortic-banded heart failure (ABHF) rats compared with SHAM-operated animals. One of these partners was the well-known mechanotransducer muscle LIM protein (MLP), which showed direct and increased binding to syndecan-4 in ABHF. Nuclear translocation is important in MLP-mediated signaling, and we found less MLP in the nuclear-enriched fractions from syndecan-4-/- mouse left ventricles but increased nuclear MLP when syndecan-4 was overexpressed in a cardiomyocyte cell line. In the presence of a cell-permeable syndecan-4-MLP disruptor peptide, the nuclear MLP level was reduced. These findings suggest that syndecan-4 mediates nuclear translocation of MLP in the heart.
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Núcleo Celular/metabolismo , Ventrículos Cardíacos/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteínas Musculares/metabolismo , Sindecano-4/metabolismo , Animales , Línea Celular , Células HEK293 , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Humanos , Proteínas con Dominio LIM/química , Ratones , Ratones Noqueados , Proteínas Musculares/química , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Dominios PDZ , Mapas de Interacción de Proteínas , Transporte de Proteínas , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Wistar , Transducción de Señal , Sindecano-4/química , Sindecano-4/genéticaRESUMEN
BACKGROUND: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. METHODS: Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes. RESULTS: SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. CONCLUSIONS: SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.
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Paro Cardíaco/metabolismo , Neuropéptidos/metabolismo , Secretogranina II/metabolismo , Taquicardia Ventricular/metabolismo , Animales , Biomarcadores/metabolismo , Calcio/metabolismo , Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Paro Cardíaco/fisiopatología , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Péptido Natriurético Encefálico/metabolismo , Técnicas de Placa-Clamp , Fragmentos de Péptidos/metabolismo , Fosforilación , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/fisiopatología , Troponina T/metabolismo , Regulación hacia ArribaRESUMEN
The sodium (Na+ )-calcium (Ca2+ ) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca2+ homeostasis, serving as the primary mechanism for Ca2+ extrusion during relaxation. Dysregulation of NCX1 is observed in end-stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti-NCX1 against endogenous NCX1 and (2) anti-His (where His is histidine) with His-trigger factor-NCX1cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein-protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where "cell communication" and "signal transduction" formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in "cardiovascular disease" which can be explored as novel drug targets in future research.
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Ventrículos Cardíacos/metabolismo , Corazón/fisiología , Mapeo de Interacción de Proteínas/métodos , Proteómica/métodos , Intercambiador de Sodio-Calcio/metabolismo , Animales , Calcio/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Ratas , Ratas Wistar , Transducción de Señal , Sodio/metabolismoRESUMEN
NCX1 (Na(+)/Ca(2+) exchanger 1) is an important regulator of intracellular Ca(2+) and a potential therapeutic target for brain ischaemia and for diastolic heart failure with preserved ejection fraction. PLM (phospholemman), a substrate for protein kinases A and C, has been suggested to regulate NCX1 activity. However, although several studies have demonstrated that binding of phosphorylated PLM (pSer(68)-PLM) leads to NCX1 inhibition, other studies have failed to demonstrate a functional interaction of these proteins. In the present study, we aimed to analyse the biological function of the pSer(68)-PLM-NCX1 interaction by developing high-affinity blocking peptides. PLM was observed to co-fractionate and co-immunoprecipitate with NCX1 in rat left ventricle, and in co-transfected HEK (human embryonic kidney)-293 cells. For the first time, the NCX1-PLM interaction was also demonstrated in the brain. PLM binding sites on NCX1 were mapped to two regions by peptide array assays, containing the previously reported PASKT and QKHPD motifs. Conversely, the two NCX1 regions bound identical sequences in the cytoplasmic domain of PLM, suggesting that NCX1-PASKT and NCX1-QKHPD might bind to each PLM monomer. Using two-dimensional peptide arrays of the native NCX1 sequence KHPDKEIEQLIELANYQVLS revealed that double substitution of tyrosine for positions 1 and 4 (K1Y and D4Y) enhanced pSer(68)-PLM binding 8-fold. The optimized peptide blocked binding of NCX1-PASKT and NCX1-QKHPD to PLM and reversed PLM(S68D) inhibition of NCX1 activity (both forward and reverse mode) in HEK-293 cells. Altogether our data indicate that PLM interacts directly with NCX1 and inhibits NCX1 activity when phosphorylated at Ser(68).
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Proteínas de la Membrana/farmacología , Péptidos/farmacología , Fosfoproteínas/farmacología , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Animales , Sitios de Unión , Encéfalo/metabolismo , Células HEK293 , Humanos , Miocardio/metabolismo , Fosforilación , Ratas , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.
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Modelos Animales de Enfermedad , Insuficiencia Cardíaca/metabolismo , Proteínas de la Membrana/metabolismo , Miocitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatasa 1/metabolismo , Procesamiento Proteico-Postraduccional , Intercambiador de Sodio-Calcio/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Biología Computacional , Células HEK293 , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/patología , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/genética , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Intercambiador de Sodio-Calcio/química , Intercambiador de Sodio-Calcio/genética , Especificidad por SustratoRESUMEN
OBJECTIVE: The aim of this study was to evaluate the predictive validity of the apathy subsyndrome in patients with therapy-resistant depression in the dose-remission study with transcranial pulsating electromagnetic fields (T-PEMF). METHODS: The apathy subsyndrome consists of the symptoms of fatigue, concentration and memory problems, lack of interests, difficulties in making decisions, and sleep problems. We evaluated 65 patients with therapy-resistant depression. In total, 34 of these patients received placebo T-PEMF in the afternoon and active T-PEMF in the morning, that is, one daily dose. The remaining 31 patients received active T-PEMF twice daily. Duration of treatment was 8 weeks in both groups. The Hamilton Depression Scale (HAM-D17) and the Bech-Rafaelsen Melancholia Scale (MES) were used to measure remission. We also focused on the Diagnostic Apathia Scale, which is based on a mixture of items from the MINI and the HAM-D17/MES. RESULTS: In patients without apathy, the remission rate after T-PEMF was 83.9% versus 58.8% in patients with apathy (p≤0.05). In patients without apathy receiving one active dose daily 94.4% remitted versus 50% for patients with apathy (p≤0.05). In patients without apathy who received two active doses 69.9% remitted versus 66.7% for patients with apathy (p≤0.05). CONCLUSION: Taking the baseline diagnosis of the apathy syndrome into consideration, we found that in patients without apathy one daily dose of T-PEMF is sufficient, but in patients with apathy two daily doses are necessary. Including the apathy syndrome as predictor in future studies would seem to be clinically relevant.
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Apatía , Trastorno Depresivo Resistente al Tratamiento/psicología , Trastorno Depresivo Resistente al Tratamiento/terapia , Escalas de Valoración Psiquiátrica , Estimulación Magnética Transcraneal , Humanos , Valor Predictivo de las Pruebas , Inducción de Remisión/métodos , Reproducibilidad de los Resultados , SíndromeRESUMEN
Cardiac sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is central to the maintenance of normal Ca(2+) homeostasis and contraction. Studies indicate that the Ca(2+)-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca(2+) binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met(369) antibody identified a novel calpain cleavage site at Met(369). Engineering NCX1-Met(369) into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met(369) inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met(369) cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.
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Estenosis de la Válvula Aórtica/metabolismo , Calpaína/metabolismo , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Anciano , Secuencia de Aminoácidos , Animales , Estenosis de la Válvula Aórtica/genética , Estenosis de la Válvula Aórtica/patología , Sitios de Unión , Calpaína/genética , Femenino , Células HEK293 , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Ventrículos Cardíacos/patología , Humanos , Masculino , Datos de Secuencia Molecular , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/patología , Cultivo Primario de Células , Unión Proteica , Proteolisis , Ratas , Ratas Wistar , Intercambiador de Sodio-Calcio/química , Intercambiador de Sodio-Calcio/genéticaRESUMEN
OBJECTIVE: To evaluate to what extent a twice daily dose of Transcranial Pulsating ElectroMagnetic Fields (T-PEMF) was superior to once daily in patients with treatment-resistant depression as to obtaining symptom remission after 8 weeks of augmentation therapy. METHODS: A self-treatment set-up of the T-PEMF device was used allowing self-administration by patients in own homes. All patients were treated for 30 min per T-PEMF session. The antidepressant medication the patients were receiving at baseline remained unchanged during the trial. The patients were randomised to either one T-PEMF dose (active dose in the morning and sham in the afternoon) or two T-PEMF doses (active dose both morning and afternoon) in a double-blind procedure. A score of 7 or less on the Hamilton Depression Scale (HAM-D17) was the criterion of remission. RESULTS: In total 34 patients received active T-PEMF once a day and 31 patients twice daily. After 5 weeks of therapy remission was obtained in 26.5% and 32.3% on one dose and two doses of T-PEMF, respectively. After 8 weeks the rate of remission was 73.5% and 67.7%, respectively. The side effects as measured by the Udvalget for Kliniske Undersøgelser scale showed a better toleration of the antidepresssive medication in both treatment groups, which was reflected by the WHO-5 well-being scale with increased scores in both groups of patients. CONCLUSION: The high remission rate obtained by the T-PEMF augmentation was not a dose effect (one versus two daily T-PEMF sessions) but was explained by the extension of the treatment period from 5 to 8 weeks.
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Trastorno Depresivo Resistente al Tratamiento/terapia , Estimulación Magnética Transcraneal , Adulto , Antidepresivos/uso terapéutico , Terapia Combinada , Trastorno Depresivo Resistente al Tratamiento/tratamiento farmacológico , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Autoadministración , Resultado del TratamientoRESUMEN
OBJECTIVE: The Melancholia Scale (MES) consists of the psychic core items of the Hamilton Depression Scale (HAM-D6) (depressed mood, interests, psychic anxiety, general somatic, guilt feelings, and psychomotor retardation) and the neuropsychiatric items of the Cronholm-Ottossen Depression Scale. Patients resistant to anti-depressant medication (therapy-resistant depression) have participated in our trials with non-pharmacological augmentation. On the basis of these trials, we have evaluated to what extent the neuropsychiatric subscale of the MES (concentration difficulties, fatigability, emotional introversion, sleep problems, and decreased verbal communication) is a measure of severity of apathia when compared with the HAM-D6 subscale of the MES. METHODS: We have focused on rating sessions at baseline (week 0) and after 2 and 4 weeks of therapy in four clinical trials on therapy-resistant depression with the following augmentations: electroconvulsive therapy, bright light therapy, transcranial magnetic stimulation or pulsed electromagnetic fields, and wake therapy. The item response theory model constructed by Mokken has been used as the psychometric validation of unidimensionality. For the numerical evaluation of transferability, we have tested item ranks across the rating weeks. RESULTS: In the Mokken analysis, the coefficient of homogeneity was above 0.40 for both the HAM-D subscale and the apathia subscale at week 4. The numerical transferability across the weeks was statistically significant (p < 0.05) for both subscales. CONCLUSION: The apathia subscale is a unidimensional scale with acceptable transferability for the measurement of treatment-resistant symptoms, analogue to the psychic core subscale (HAM-D6).
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Trastorno Depresivo Resistente al Tratamiento/diagnóstico , Escalas de Valoración Psiquiátrica , Adulto , Anciano , Ensayos Clínicos como Asunto , Trastorno Depresivo Resistente al Tratamiento/terapia , Terapia Electroconvulsiva , Femenino , Humanos , Magnetoterapia , Masculino , Persona de Mediana Edad , Fototerapia , Psicometría , Índice de Severidad de la Enfermedad , Estimulación Magnética Transcraneal , Resultado del TratamientoRESUMEN
Background Multiple chemical sensitivity (MCS) is a chronic, disabling condition characterized by recurrent multisystem symptoms triggered by common airborne chemicals. Evidence points towards abnormal sensory processing in the central nervous system (CNS) as a likely pathophysiological mechanism. No effective treatment has yet been reported, but clinical observations suggest that as pulsed electromagnetic fields (PEMF) is a treatment for some CNS disorders (depression and chronic pain), it may also be a treatment modality for MCS. Methods In an open case study, the effects of PEMF were assessed in three MCS patients. All cases received 30 min daily treatment 5 days a week for 8 consecutive weeks. Symptoms and functional impairments related to MCS, depressive symptoms, and capsaicin-induced secondary punctate hyperalgesia were assessed at baseline and weekly until an 18-week follow-up. Results Two of the three cases showed considerable improvement on all measures of symptoms and functional impairments related to MCS in response to PEMF therapy. One case showed no improvement and during the treatment period was unexpectedly diagnosed with depression. Conclusion Our findings indicate potential benefits of PEMF therapy in MCS. Implication The therapeutic effect of PEMF in MCS needs to be investigated by a randomized placebo-controlled trial.