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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: 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
BACKGROUND AND OBJECTIVE: A number of studies have highlighted muscle-specific mechanisms of thermogenesis involving futile cycling of Ca2+ driven by sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA) and generating heat from ATP hydrolysis to be a promising strategy to counteract obesity and metabolic dysfunction. However, to the best of our knowledge, no experimental studies concerning the metabolic effects of pharmacologically targeting SERCA in human skeletal muscle cells have been reported. Thus, in the present study, we aimed to explore the effects of SERCA-activating compound, CDN1163, on energy metabolism in differentiated human skeletal muscle cells (myotubes). METHODS: In this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively. RESULTS: Both acute (4 âh) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid ß-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase (CPT) 1B, pyruvate dehydrogenase kinase (PDK) 4, as well as increased AMP-activated protein kinase (AMPK)Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase (SCD) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA. CONCLUSION: Altogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus.
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Subjects with functionally univentricular circulation who have completed staged single ventricle palliation, with the final stage culminating in the Fontan procedure, are often living into adulthood. However, high morbidity and mortality remain prevalent in these patients, as diastolic and systolic dysfunction of the single systemic ventricle are linked to Fontan circulatory failure. We presently investigated the effects of probenecid in post-Fontan patients. Used for decades for the treatment of gout, probenecid has been shown in recent years to positively influence cardiac function via effects on the Transient Receptor Potential Vanilloid 2 (TRPV2) channel in cardiomyocytes. Indeed, we observed that probenecid improved cardiac function and exercise performance in patients with a functionally univentricular circulation. This was consistent with our findings from a retrospective cohort of patients with single ventricle physiology where TRPV2 expression was increased. Experiments in isolated cardiomyocytes associated these positive actions to augmentation of diastolic calcium homeostasis.
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Agonistas de los Canales de Calcio/uso terapéutico , Procedimiento de Fontan/métodos , Cardiopatías Congénitas/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Probenecid/uso terapéutico , Administración Oral , Adolescente , Adulto , Calcio/metabolismo , Niño , Prueba de Esfuerzo , Femenino , Cardiopatías Congénitas/cirugía , Ventrículos Cardíacos/anomalías , Ventrículos Cardíacos/cirugía , Homeostasis/efectos de los fármacos , Humanos , Masculino , Miocitos Cardíacos/metabolismo , Estudios Retrospectivos , Canales Catiónicos TRPV/metabolismo , Resultado del Tratamiento , Adulto JovenRESUMEN
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
Reduced cardiac contractility during heart failure (HF) is linked to impaired Ca2+ release from Ryanodine Receptors (RyRs). We investigated whether this deficit can be traced to nanoscale RyR reorganization. Using super-resolution imaging, we observed dispersion of RyR clusters in cardiomyocytes from post-infarction HF rats, resulting in more numerous, smaller clusters. Functional groupings of RyR clusters which produce Ca2+ sparks (Ca2+ release units, CRUs) also became less solid. An increased fraction of small CRUs in HF was linked to augmented 'silent' Ca2+ leak, not visible as sparks. Larger multi-cluster CRUs common in HF also exhibited low fidelity spark generation. When successfully triggered, sparks in failing cells displayed slow kinetics as Ca2+ spread across dispersed CRUs. During the action potential, these slow sparks protracted and desynchronized the overall Ca2+ transient. Thus, nanoscale RyR reorganization during HF augments Ca2+ leak and slows Ca2+ release kinetics, leading to weakened contraction in this disease.
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Calcio/metabolismo , Insuficiencia Cardíaca/patología , Infarto del Miocardio/patología , Miocitos Cardíacos/patología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Potenciales de Acción , Animales , Cationes Bivalentes/metabolismo , Modelos Animales de Enfermedad , Microscopía Fluorescente , RatasRESUMEN
Protein O-GlcNAcylation has emerged as an important intracellular signaling system with both physiological and pathophysiological functions, but the role of protein O-GlcNAcylation in skeletal muscle remains elusive. In this study, we tested the hypothesis that protein O-GlcNAcylation is a dynamic signaling system in skeletal muscle in exercise and disease. Immunoblotting showed different protein O-GlcNAcylation pattern in the prototypical slow twitch soleus muscle compared to fast twitch EDL from rats, with greater O-GlcNAcylation level in soleus associated with higher expression of the modulating enzymes O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine fructose-6-phosphate amidotransferase isoforms 1 and 2 (GFAT1, GFAT2). Six weeks of exercise training by treadmill running, but not an acute exercise bout, increased protein O-GlcNAcylation in rat soleus and EDL There was a striking increase in O-GlcNAcylation of cytoplasmic proteins ~50 kDa in size that judged from mass spectrometry analysis could represent O-GlcNAcylation of one or more key metabolic enzymes. This suggests that cytoplasmic O-GlcNAc signaling is part of the training response. In contrast to exercise training, postinfarction heart failure (HF) in rats and humans did not affect skeletal muscle O-GlcNAcylation level, indicating that aberrant O-GlcNAcylation cannot explain the skeletal muscle dysfunction in HF Human skeletal muscle displayed extensive protein O-GlcNAcylation that by large mirrored the fiber-type-related O-GlcNAcylation pattern in rats, suggesting O-GlcNAcylation as an important signaling system also in human skeletal muscle.
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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
Myosin light chain 2 (MLC2) is a small protein in the myosin complex, regulating muscle contractile function by modulating Ca(2+) sensitivity of myofilaments. MLC2 can be modified by phosphorylation and O-GlcNAcylation, two reversible and dynamic posttranslational modifications. The slow isoform of MLC2 (sMLC2) is dephosphorylated in soleus muscle during in situ loaded shortening contractions, which correlates with reduction in shortening capacity. Here, we hypothesize that exhausting in vivo treadmill running induces dephosphorylation of MLC2 in slow twitch soleus, but not in fast twitch EDL muscle, and that there are reciprocal changes in MLC2 O-GlcNAcylation. At rest, both phosphorylation and O-GlcNAcylation of MLC2 were lower in slow than fast twitch muscles. One bout of exhausting treadmill running induced dephosphorylation of sMLC2 in soleus, paralleled by reduced levels of the kinase MLCK2 associated to myofilaments, suggesting that the acute reduction in phosphorylation is mediated by dissociation of MLCK2 from myofilaments. O-GlcNAcylation of MLC2 did not change significantly, and seems of limited importance in the regulation of MLC2 phosphorylation during in vivo running. After 6 weeks of treadmill running, the dephosphorylation of sMLC2 persisted in soleus along with reduction in MLCK2 both in myofilament- and total protein fraction. In EDL on the contrary, phosphorylation of MLC2 was not altered after one exercise bout or after 6 weeks of treadmill running. Thus, in contrast to fast twitch muscle, MLC2 dephosphorylation occurs in slow twitch muscle during in vivo exercise and may be linked to reduced myofilament-associated MLCK2 and reduced shortening capacity.
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Fatigue in muscles that shorten might have other causes than fatigue during isometric contractions, since both cross-bridge cycling and energy demand are different in the two exercise modes. While isometric contractions are extensively studied, the causes of fatigue in shortening contractions are poorly mapped. Here, we investigate fatigue mechanisms during shortening contractions in slow twitch skeletal muscle in near physiological conditions. Fatigue was induced in rat soleus muscles with maintained blood supply by in situ shortening contractions at 37°C. Muscles were stimulated repeatedly (1 s on/off at 30 Hz) for 15 min against a constant load, allowing the muscle to shorten and perform work. Fatigue and subsequent recovery was examined at 20 s, 100 s and 15 min exercise. The effects of prior exercise were investigated in a second exercise bout. Fatigue developed in three distinct phases. During the first 20 s the regulatory protein Myosin Light Chain-2 (slow isoform, MLC-2s) was rapidly dephosphorylated in parallel with reduced rate of force development and reduced shortening. In the second phase there was degradation of high-energy phosphates and accumulation of lactate, and these changes were related to slowing of muscle relengthening and relaxation, culminating at 100 s exercise. Slowing of relaxation was also associated with increased leak of calcium from the SR. During the third phase of exercise there was restoration of high-energy phosphates and elimination of lactate, and the slowing of relaxation disappeared, whereas dephosphorylation of MLC-2s and reduced shortening prevailed. Prior exercise improved relaxation parameters in a subsequent exercise bout, and we propose that this effect is a result of less accumulation of lactate due to more rapid onset of oxidative metabolism. The correlation between dephosphorylation of MLC-2s and reduced shortening was confirmed in various experimental settings, and we suggest MLC-2s as an important regulator of muscle shortening.
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Contracción Muscular/fisiología , Fatiga Muscular/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Animales , Calcio/metabolismo , Miosinas Cardíacas/metabolismo , Contracción Isométrica/fisiología , Lactatos/metabolismo , Masculino , Metaboloma , Fibras Musculares de Contracción Lenta/metabolismo , Relajación Muscular , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Condicionamiento Físico Animal , Ratas , Ratas Wistar , Retículo Sarcoplasmático/metabolismo , Factores de TiempoRESUMEN
Sarcoplasmic reticulum Ca(2+) ATPases (SERCAs) play a major role in muscle contractility by pumping Ca(2+) from the cytosol into the sarcoplasmic reticulum (SR) Ca(2+) store, allowing muscle relaxation and refilling of the SR with releasable Ca(2+). Decreased SERCA function has been shown to result in impaired muscle function and disease in human and animal models. In this study, we present a new mouse model with targeted disruption of the Serca2 gene in skeletal muscle (skKO) to investigate the functional consequences of reduced SERCA2 expression in skeletal muscle. SkKO mice were viable and basic muscle structure was intact. SERCA2 abundance was reduced in multiple muscles, and by as much as 95% in soleus muscle, having the highest content of slow-twitch fibres (40%). The Ca(2+) uptake rate was significantly reduced in SR vesicles in total homogenates. We did not find any compensatory increase in SERCA1 or SERCA3 abundance, or altered expression of several other Ca(2+)-handling proteins. Ultrastructural analysis revealed generally well-preserved muscle morphology, but a reduced volume of the longitudinal SR. In contracting soleus muscle in vitro preparations, skKO muscles were able to fully relax, but with a significantly slowed relaxation time compared to controls. Surprisingly, the maximal force and contraction rate were preserved, suggesting that skKO slow-twitch fibres may be able to contribute to the total muscle force despite loss of SERCA2 protein. Thus it is possible that SERCA-independent mechanisms can contribute to muscle contractile function.
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Relajación Muscular/fisiología , Músculo Esquelético/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/fisiología , Animales , Calcio/metabolismo , Ratones , Ratones Noqueados , Contracción Muscular/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/fisiología , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/deficiencia , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
During isometric contractions, slow twitch soleus muscles (SOL) from rats with chronic heart failure (chf) are more fatigable than those of sham animals. However, a muscle normally shortens during activity and fatigue development is highly task dependent. Therefore, we examined the development of skeletal muscle fatigue during shortening (isotonic) contractions in chf and sham-operated rats. Six weeks following coronary artery ligation, infarcted animals were classified as failing (chf) if left ventricle end diastolic pressure was >15 mmHg. During isoflurane anaesthesia, SOL with intact blood supply was stimulated (1s on 1s off) at 30 Hz for 15 min and allowed to shorten isotonically against a constant afterload. Muscle temperature was maintained at 37°C. In resting muscle, maximum isometric force (F(max)) and the concentrations of ATP and CrP were not different in the two groups. During stimulation, F(max) and the concentrations declined in parallel sham and chf. Fatigue, which was evident as reduced shortening during stimulation, was also not different in the two groups. The isometric force decline was fitted to a bi-exponential decay equation. Both time constants increased transiently and returned to initial values after approximately 200 s of the fatigue protocol. This resulted in a transient rise in baseline tension between stimulations, although this effect which was less prominent in chf than sham. Myosin light chain 2s phosphorylation declined in both groups after 100 s of isotonic contractions, and remained at this level throughout 15 min of stimulation. In spite of higher energy demand during isotonic than isometric contractions, both shortening capacity and rate of isometric force decline were as well or better preserved in fatigued SOL from chf rats than in sham. This observation is in striking contrast to previous reports which have employed isometric contractions to induce fatigue.
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Insuficiencia Cardíaca/fisiopatología , Fatiga Muscular , Músculo Esquelético/fisiopatología , Condicionamiento Físico Animal , Animales , Miosinas Cardíacas/metabolismo , Enfermedad Crónica , Insuficiencia Cardíaca/metabolismo , Contracción Isométrica , Masculino , Músculo Esquelético/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Ratas , Ratas WistarRESUMEN
The cardiac Na(+)/Ca(2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-(32)P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Miocardio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Agonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Colforsina/farmacología , Biología Computacional/métodos , Células HEK293 , Corazón/efectos de los fármacos , Humanos , Isoproterenol/farmacología , Ratones , Datos de Secuencia Molecular , Fosforilación , Ratas , Homología de Secuencia de Aminoácido , Intercambiador de Sodio-Calcio/genéticaRESUMEN
PURPOSE: Patients with chronic heart failure (CHF) typically complain about skeletal muscle fatigue. In rat experiments, reduced intracellular calcium release seems to be related to fatigue development in normal skeletal muscle but not in muscle from rats with CHF. We therefore hypothesize that training may not improve intracellular calcium cycling to the same extent in muscles from patients with CHF compared with healthy controls (HC). METHODS: Thirteen HC and 11 CHF patients performed 6 wk of unilateral knee extensor endurance training. Computed tomographic examinations of the thigh and biopsies of vastus lateralis were obtained bilaterally before and after the training period. RESULTS: Peak power of the trained leg was 10% and 14% greater than that in the untrained leg in HC and CHF, respectively. For the HC, training resulted in a higher Ca2+ release rate and a lower leak in the trained leg associated with a tendency of increased ryanodine receptor (RyR) content with reduced phosphorylation level. In the trained leg of CHF patients, RyR content was reduced without associated changes of either Ca2+ leak or release rate. CONCLUSIONS: Training in HC has an effect on Ca2+ leak and release of the sarcoplasmic reticulum, but in CHF patients, training is achieved without such changes. Thus, calcium handling seems not to be the site of decreased exercise tolerance in CHF.
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Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Músculo Esquelético/metabolismo , Aptitud Física/fisiología , Anciano , Ejercicio Físico/fisiología , Humanos , Pierna/fisiología , Contracción Muscular/fisiología , Retículo Sarcoplasmático/metabolismoRESUMEN
Skeletal muscle fatigue is most often studied in vitro at room temperature and is classically defined as a decline in maximum force production or power output, exclusively linked to repeated isometric contractions. However, most muscles shorten during normal use, and we propose that both the functional correlate of fatigue, as well as the fatigue mechanism, will be different during dynamic contractions compared with static contractions. Under isoflurane anesthesia, fatigue was induced in rat soleus muscles in situ by isotonic shortening contractions at 37 degrees C. Muscles were stimulated repeatedly for 1 s at 30 Hz every 2 s for a total of 15 min. The muscles were allowed to shorten isotonically against a load corresponding to one-third of maximal isometric force. Maximal unloaded shortening velocity (V(0)), maximum force production (F(max)), and isometric relaxation rate (-dF/dt) was reduced after 100 s but returned to almost initial values at the end of the stimulation protocol. Likewise, ATP and creatine phosphate (CrP) were reduced after 100 s, but the level of CrP was partially restored to initial values after 15 min. The rate of isometric force development, the velocity of shortening, and isotonic shortening were also reduced at 100 s, but in striking contrast, did not recover during the remainder of the stimulation protocol. The regulatory myosin light chain (MLC2s) was dephosphorylated after 100 s and did not recover. Although metabolic changes may account for the changes of F(max), -dF/dt, and V(0), dephosphorylation of MLC2s may be involved in the fatigue seen as sustained slower contraction velocities and decreased muscle shortening.
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Contracción Isotónica , Fatiga Muscular , Fibras Musculares de Contracción Lenta/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Miosinas Cardíacas/metabolismo , Estimulación Eléctrica , Masculino , Cadenas Ligeras de Miosina/metabolismo , Consumo de Oxígeno , Fosfocreatina/metabolismo , Fosforilación , Ratas , Ratas Wistar , Recuperación de la Función , Temperatura , Factores de TiempoRESUMEN
Rats with congestive heart failure (CHF) develop ventricular inotropic responsiveness to serotonin (5-HT), mediated through 5-HT(2A) and 5-HT(4) receptors. Human ventricle is similarly responsive to 5-HT through 5-HT(4) receptors. We studied isolated ventricular cardiomyocytes to clarify the effects of 5-HT on intracellular Ca(2+) handling. Left-ventricular cardiomyocytes were isolated from male Wistar rats 6 wk after induction of postinfarction CHF. Contractile function and Ca(2+) transients were measured in field-stimulated cardiomyocytes, and L-type Ca(2+) current (I(Ca,L)) and sarcoplasmic reticulum (SR) Ca(2+) content were measured in voltage-clamped cells. Protein phosphorylation was measured by Western blotting or phosphoprotein gel staining. 5-HT(4)- and 5-HT(2A)-receptor stimulation induced a positive inotropic response of 33 and 18% (both P < 0.05) and also increased the Ca(2+) transient (44 and 6%, respectively; both P < 0.05). I(Ca,L) and SR Ca(2+) content increased only after 5-HT(4)-receptor stimulation (57 and 65%; both P < 0.05). Phospholamban serine(16) (PLB-Ser(16)) and troponin I phosphorylation increased by 26 and 13% after 5-HT(4)-receptor stimulation (P < 0.05). 5-HT(2A)-receptor stimulation increased the action potential duration and did not significantly change the phosphorylation of PLB-Ser(16) or troponin I, but it increased myosin light chain 2 (MLC2) phosphorylation. In conclusion, the positive inotropic response to 5-HT(4) stimulation results from increased I(Ca,L) and increased phosphorylation of PLB-Ser(16), which increases the SR Ca(2+) content. 5-HT(4) stimulation is thus, like beta-adrenoceptor stimulation, possibly energetically unfavorable in CHF. 5-HT(2A)-receptor stimulation, previously studied in acute CHF, induces a positive inotropic response also in chronic CHF, probably mediated by MLC2 phosphorylation.
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Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Cardiotónicos/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Receptores de Serotonina 5-HT4/metabolismo , Retículo Sarcoplasmático/metabolismo , Serotonina/metabolismo , Función Ventricular , Potenciales de Acción , Agonistas Adrenérgicos beta/farmacología , Animales , Canales de Calcio Tipo L/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Miosinas Cardíacas , Vasos Coronarios/cirugía , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/fisiopatología , Indoles/farmacología , Isoproterenol/farmacología , Ketanserina/farmacología , Ligadura , Masculino , Contracción Miocárdica , Infarto del Miocardio/complicaciones , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/efectos de los fármacos , Cadenas Ligeras de Miosina , Fosforilación , Ratas , Ratas Wistar , Receptor de Serotonina 5-HT2A/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Antagonistas del Receptor de Serotonina 5-HT2 , Antagonistas del Receptor de Serotonina 5-HT4 , Antagonistas de la Serotonina/farmacología , Sulfonamidas/farmacología , Factores de Tiempo , Troponina I/metabolismo , Función Ventricular/efectos de los fármacosRESUMEN
Skeletal muscle weakness and decreased exercise capacity are major symptoms reported by patients with congestive heart failure (CHF). Intriguingly, these skeletal muscle symptoms do not correlate with the decreased heart function. This suggests that CHF leads to maladaptive changes in skeletal muscles, and as reported most markedly in slow-twitch muscles. We used rats at 6 weeks after infarction to measure expression of key proteins involved in SR Ca(2+) release and uptake in slow-twitch soleus muscles. We also measured force and myoplasmic free [Ca(2+)] ([Ca(2+)](i)) in intact single fibers of soleus muscles. CHF rats showed clear signs of severe cardiac dysfunction with marked increases in heart weight and left ventricular end-diastolic pressure compared with sham operated rats (Sham). There were small, but significant, changes in the content of proteins involved in cellular Ca(2+) handling in CHF muscles: slight increases in SR Ca(2+) release channels (ie, the ryanodine receptors) and in SR Ca(2+)-ATPase. Tetanic force and [Ca(2+)](i) were not significantly different between CHF and Sham soleus fibers under resting conditions. However, during the stimulation period there was a decrease in tetanic force without changes in [Ca(2+)](i) in CHF fibers that was not observed in Sham fibers. The fatigue-induced changes recovered rapidly. We conclude that CHF soleus fibers fatigue more rapidly than Sham fibers because of a reversible fatigue-induced decrease in myofibrillar function.
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Calcio/metabolismo , Insuficiencia Cardíaca/fisiopatología , Fatiga Muscular , Músculo Esquelético/fisiopatología , Animales , ATPasas Transportadoras de Calcio/metabolismo , Estimulación Eléctrica , Insuficiencia Cardíaca/metabolismo , Membranas Intracelulares/metabolismo , Contracción Muscular , Fibras Musculares de Contracción Lenta/metabolismo , Proteínas Musculares/metabolismo , Concentración Osmolar , Ratas , Ratas Wistar , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Although increased levels of circulating interleukin (IL)-18 have been demonstrated in patients with cardiovascular diseases, the functional consequences of chronically increased circulating IL-18 with respect to myocardial function have not been defined. Thus we aimed to examine the effects of chronic IL-18 exposure on left ventricular (LV) function in healthy mice. Moreover, to clarify whether IL-18 has direct effects on the cardiomyocyte, we examined effects of IL-18 on cardiomyocytes in vitro. After 7 days of daily intraperitoneal injections of 0.5 microg IL-18 in healthy mice, a 40% (P < 0.05) reduction in the LV maximal positive derivative, a 25% (P < 0.05) reduction in the LV maximal rate of pressure decay, and a 2.8-fold (P < 0.001) increase in the LV end-diastolic pressure were measured, consistent with myocardial dysfunction. Furthermore, we measured a 75% (P < 0.05) reduction in beta-adrenergic responsiveness to isoproterenol. IL-18 induced myocardial hypertrophy, and there was a 2.9-fold increase (P < 0.05) in atrial natriuretic peptide mRNA expression in the LV myocardium. In vitro examinations of isolated adult rat cardiomyocytes being stimulated with IL-18 (0.1 microg/ml) exhibited an increase in peak Ca2+ transients (P < 0.05) and in diastolic Ca2+ concentrations (P < 0.05). In conclusion, this study shows that daily administration of IL-18 in healthy mice causes LV myocardial dysfunction and blunted beta-adrenergic responsiveness to isoproterenol. A direct effect of IL-18 on the cardiomyocyte in vitro was demonstrated, suggesting that IL-18 reduces the responsiveness of the myofilaments to Ca2+. Finally, induction of myocardial hypertrophy by IL-18 indicates a role for this cytokine in myocardial remodeling.