RESUMEN
The clinical management of severe COVID-19 cases is not yet well resolved. Therefore, it is important to identify and characterize cell signaling pathways involved in virus pathogenesis that can be targeted therapeutically. Envelope (E) protein is a structural protein of the virus, which is known to be highly expressed in the infected host cell and is a key virulence factor; however, its role is poorly characterized. The E protein is a single-pass transmembrane protein that can assemble into a pentamer forming a viroporin, perturbing Ca2+ homeostasis. Because it is structurally similar to regulins such as, for example, phospholamban, that regulate the sarco/endoplasmic reticulum calcium ATPases (SERCA), we investigated whether the SARS-CoV-2 E protein affects the SERCA system as an exoregulin. Using FRET experiments we demonstrate that E protein can form oligomers with regulins, and thus can alter the monomer/multimer regulin ratio and consequently influence their interactions with SERCAs. We also confirm that a direct interaction between E protein and SERCA2b results in a decrease in SERCA-mediated ER Ca2+ reload. Structural modeling of the complexes indicates an overlapping interaction site for E protein and endogenous regulins. Our results reveal novel links in the host-virus interaction network that play an important role in viral pathogenesis and may provide a new therapeutic target for managing severe inflammatory responses induced by SARS-CoV-2.
Asunto(s)
COVID-19 , Señalización del Calcio , Proteínas de la Envoltura de Coronavirus , SARS-CoV-2 , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Humanos , SARS-CoV-2/metabolismo , COVID-19/virología , COVID-19/metabolismo , Proteínas de la Envoltura de Coronavirus/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Unión ProteicaRESUMEN
Brody disease (BD) is an "ultra-rare" human genetic disorder of skeletal muscle function due to defects in the atp2a1 gene causing deficiency of the SERCA protein, isoform1. The main clinical signs are exercise-induced stiffness and delayed muscular relaxation after physical exercises, even mild ones. No mouse model nor specific therapies exist for Brody myopathy, which is therefore considered an orphan disease. Bovine congenital pseudomyotonia (PMT) is a muscular disorder characterized by an impairment of muscle relaxation and is the only mammalian model of human BD. The pathogenetic mechanism underlying bovine PMT has been recently clarified. These findings prompted us to purpose a potential pharmacological approach addressing a specific population of BD patients who exhibit reduced expression but still exhibit activity of the SERCA1 pump. Preclinical research involving in vivo studies is essential and necessary before clinical trials can be pursued and SERCA protein shows a high degree of conservation among species. So far, the only animal models available to study BD in vivo are a group of zebrafish mutant lines known as accordion zebrafish (acc). In this paper, we focused on a comprehensive characterization of the "acctq206" zebrafish variant. Our aim was to use this mutant line as an experimental animal model for testing the novel therapeutic approach for BD.
Asunto(s)
Modelos Animales de Enfermedad , Mutación , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Pez Cebra , Animales , Pez Cebra/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Miotonía Congénita/genética , Miotonía Congénita/tratamiento farmacológico , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Cardiac metabolism ensures a continuous ATP supply, primarily using fatty acids in a healthy state and favoring glucose in pathological conditions. Pyruvate kinase muscle (PKM) controls the final step of glycolysis, with PKM1 being the main isoform in the heart. PKM2, elevated in various heart diseases, has been suggested to play a protective role in cardiac stress, but its function in basal cardiac metabolism remains unclear. We examined hearts from global PKM2 knockout (PKM2-/-) mice and found reduced intracellular glucose. Isotopic tracing of U-13C glucose revealed a shift to biosynthetic pathways in PKM2-/- cardiomyocytes. Total ATP content was two-thirds lower in PKM2-/- hearts, and functional analysis indicated reduced mitochondrial oxygen consumption. Total reactive oxygen species (ROS) and mitochondrial superoxide were also increased in PKM2-/- cardiomyocytes. Intriguingly, PKM2-/- hearts had preserved ejection fraction compared to controls. Mechanistically, increased calcium/calmodulin-dependent kinase II activity and phospholamban phosphorylation may contribute to higher sarcoendoplasmic reticulum calcium ATPase 2 pump activity in PKM2-/- hearts. Loss of PKM2 led to altered glucose metabolism, diminished mitochondrial function, and increased ROS in cardiomyocytes. These data suggest that cardiac PKM2 acts as an important rheostat to maintain ATP levels while limiting oxidative stress. Although loss of PKM2 did not impair baseline contractility, its absence may make hearts more sensitive to environmental stress or injury.
Asunto(s)
Miocitos Cardíacos , Estrés Oxidativo , Animales , Miocitos Cardíacos/metabolismo , Ratones , Ratones Noqueados , Glucosa/metabolismo , Masculino , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Mitocondrias Cardíacas/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Ratones Endogámicos C57BL , Piruvato Quinasa/metabolismo , Piruvato Quinasa/genética , Adenosina Trifosfato/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Miocardio/metabolismoRESUMEN
In this research, we employed the alchemical double-decoupling method alongside restraining potentials, coupled with the FEPMD method, to ascertain the standard binding free energy of a drug-like molecule termed BHQ and three analogous compounds engineered with progressive addition of bulky para-alkyl groups binding to SERCA (Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum). Integral transmembrane proteins represent crucial drug targets in numerous therapeutic interventions, presenting computational challenges due to their considerable system sizes. Our approach integrated the generalized born potential method and the spherical solvent boundary potential method, allowing us to explicitly focus on the active binding site while treating the remainder of the system implicitly. We evaluated contributions to the standard binding free energy from distinct interaction potentials: electrostatic, repulsive, dispersive, and restraining potentials, computed separately. The resulting absolute binding free energy of BHQ (11.63 kcal/mol) closely aligns with experimental measurements (10.56 kcal/mol). Notably, an accurate estimation of the absolute binding free energy was achieved for the simplest analog, created with the addition of a single para-methyl group. However, the analog with two para-methyl groups exhibited the highest binding free energy, which disagreed with experimental results. Determining the binding free energy of the BHQ analog engineered with three para-methyl groups presented challenges in convergence and resulted in the lowest free energy among the three.
Asunto(s)
Termodinámica , Unión Proteica , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Sitios de Unión , Simulación de Dinámica Molecular , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismoRESUMEN
BACKGROUND: Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca2+. METHODS: HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patch clamp, Ca2+, and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure. RESULTS: Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca2+ release in the cell interior. Systolic Ca2+, ICa-L, sarcoplasmic reticulum Ca2+ content, and sarcoendoplasmic reticulum Ca2+ ATPase function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca2+ transient, the rate of Ca2+ removal, and the peak L-type Ca2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria. CONCLUSIONS: We show that recovery from HF restores atrial t-tubules, and this promotes recovery of ICa-L, sarcoplasmic reticulum Ca2+ content, and systolic Ca2+. We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy.
Asunto(s)
Atrios Cardíacos , Insuficiencia Cardíaca , Miocitos Cardíacos , Animales , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Miocitos Cardíacos/ultraestructura , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Atrios Cardíacos/fisiopatología , Ovinos , Calcio/metabolismo , Señalización del Calcio , Ratas , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/ultraestructura , Retículo Sarcoplasmático/patología , Recuperación de la Función , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/ultraestructura , Mitocondrias Cardíacas/patología , Células Cultivadas , Sístole , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Ratas Sprague-Dawley , FemeninoRESUMEN
Aging is associated with cardiac contractile abnormalities, but the etiology of these contractile deficits is unclear. We hypothesized that cardiac contractile and regulatory protein expression is altered during aging. To investigate this possibility, left ventricular (LV) lysates were prepared from young (6 months) and old (24 months) Fischer344 rats. There are no age-related changes in SERCA2 expression or phospholamban phosphorylation. Additionally, neither titin isoform expression nor phosphorylation differed. However, there is a significant increase in ß-isoform of the myosin heavy chain (MyHC) expression and phosphorylation of TnI and MyBP-C during aging. In permeabilized strips of papillary muscle, force and Ca2+ sensitivity are reduced during aging, consistent with the increase in ß-MyHC expression and TnI phosphorylation. However, the increase in MyBP-C phosphorylation during aging may represent a mechanism to compensate for age-related contractile deficits. In isolated cardiomyocytes loaded with Fura-2, the peak of the Ca2+ transient is reduced, but the kinetics of the Ca2+ transient are not altered. Furthermore, the extent of shortening and the rates of both sarcomere shortening and re-lengthening are reduced. These results demonstrate that aging is associated with changes in contractile and regulatory protein expression and phosphorylation, which affect the mechanical properties of cardiac muscle.
Asunto(s)
Envejecimiento , Contracción Miocárdica , Miocitos Cardíacos , Ratas Endogámicas F344 , Animales , Masculino , Contracción Miocárdica/fisiología , Envejecimiento/metabolismo , Envejecimiento/fisiología , Ratas , Fosforilación , Miocitos Cardíacos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Conectina/metabolismo , Troponina I/metabolismo , Calcio/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Proteínas PortadorasRESUMEN
The natural product curcumin and some of its analogs are known inhibitors of the transmembrane enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). Despite their widespread use, the curcuminoids' binding site in SERCA and their relevant interactions with the enzyme remain elusive. This lack of knowledge has prevented the development of curcuminoids into valuable experimental tools or into agents of therapeutic value. We used the crystal structures of SERCA in its E1 conformation in conjunction with computational tools such as docking and surface screens to determine the most likely curcumin binding site, along with key enzyme/inhibitor interactions. Additionally, we determined the inhibitory potencies and binding affinities for a small set of curcumin analogs. The predicted curcumin binding site is a narrow cleft in the transmembrane section of SERCA, close to the transmembrane/cytosol interface. In addition to pronounced complementarity in shape and hydrophobicity profiles between curcumin and the binding pocket, several hydrogen bonds were observed that were spread over the entire curcumin scaffold, involving residues on several transmembrane helices. Docking-predicted interactions were compatible with experimental observations for inhibitory potencies and binding affinities. Based on these findings, we propose an inhibition mechanism that assumes that the presence of a curcuminoid in the binding site arrests the catalytic cycle of SERCA by preventing it from converting from the E1 to the E2 conformation. This blockage of conformational change is accomplished by a combination of steric hinderance and hydrogen-bond-based cross-linking of transmembrane helices that require flexibility throughout the catalytic cycle.
Asunto(s)
Curcumina , Simulación del Acoplamiento Molecular , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Curcumina/química , Curcumina/farmacología , Curcumina/análogos & derivados , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Sitios de Unión , Animales , Enlace de Hidrógeno , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Unión ProteicaRESUMEN
Darier disease (DD) is a rare severe acantholytic skin disease caused by mutations in the ATP2A2 gene that encodes for the sarco/endoplasmic reticulum calcium ATPase isoform 2 (SERCA2). SERCA2 maintains endoplasmic reticulum calcium homeostasis by pumping calcium into the ER, critical for regulating cellular calcium dynamics and cellular function. To date, there is no treatment that specifically targets the disease mechanisms in DD. Dantrolene sodium (Dl) is a ryanodine receptor antagonist that inhibits calcium release from ER to increase ER calcium levels and is currently used for non-dermatological indications. In this study, we first identified dysregulated genes and molecular pathways in DD patient skin, demonstrating downregulation of cell adhesion and calcium homeostasis pathways, as well as upregulation of ER stress and apoptosis. We then show in various in vitro models of DD and SERCA2 inhibition that Dl aided in the retention of ER calcium and promoted cell adhesion. In addition, Dl treatment reduced ER stress and suppressed apoptosis. Our findings suggest that Dl specifically targets pathogenic mechanisms of DD and may be a potential treatment.
Asunto(s)
Calcio , Dantroleno , Enfermedad de Darier , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Dantroleno/farmacología , Dantroleno/uso terapéutico , Enfermedad de Darier/tratamiento farmacológico , Enfermedad de Darier/metabolismo , Humanos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Calcio/metabolismo , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Piel/patología , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
Chronic heart failure is one of the most common reasons for hospitalization. Current risk stratification is based on ejection fraction, whereas many arrhythmic events occur in patients with relatively preserved ejection fraction. We aim to investigate the mechanistic link between proarrhythmic abnormalities, reduced contractility and diastolic dysfunction in heart failure, using electromechanical modelling and simulations of human failing cardiomyocytes. We constructed, calibrated and validated populations of human electromechanical models of failing cardiomyocytes, that were able to reproduce the prolonged action potential, reduced contractility and diastolic dysfunction as observed in human data, as well as increased propensity to proarrhythmic incidents such as early afterdepolarization and beat-to-beat alternans. Our simulation data reveal that proarrhythmic incidents tend to occur in failing myocytes with lower diastolic tension, rather than with lower contractility, due to the relative preserved SERCA and sodium calcium exchanger current. These results support the inclusion of end-diastolic volume to be potentially beneficial in the risk stratifications of heart failure patients.
Asunto(s)
Potenciales de Acción , Arritmias Cardíacas , Insuficiencia Cardíaca , Miocitos Cardíacos , Humanos , Miocitos Cardíacos/metabolismo , Insuficiencia Cardíaca/fisiopatología , Arritmias Cardíacas/fisiopatología , Arritmias Cardíacas/etiología , Diástole/fisiología , Modelos Cardiovasculares , Contracción Miocárdica/fisiología , Simulación por Computador , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Phospholamban (PLN) is a 52 amino acid regulin that allosterically modulates the activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) in the heart muscle. In its unphosphorylated form, PLN binds SERCA within its transmembrane (TM) domains, approximately 20 Å away from the Ca2+ binding site, reducing SERCA's apparent Ca2+ affinity (pKCa) and decreasing cardiac contractility. During the enzymatic cycle, the inhibitory TM domain of PLN remains anchored to SERCA, whereas its cytoplasmic region transiently binds the ATPase's headpiece. Phosphorylation of PLN at Ser16 by protein kinase A increases the affinity of its cytoplasmic domain to SERCA, weakening the TM interactions with the ATPase, reversing its inhibitory function, and augmenting muscle contractility. How the structural changes caused by pathological mutations in the PLN cytoplasmic region are transmitted to its inhibitory TM domain is still unclear. Using solid-state NMR spectroscopy and activity assays, we analyzed the structural and functional effects of a series of mutations and their phosphorylated forms located in the PLN cytoplasmic region and linked to dilated cardiomyopathy. We found that these missense mutations affect the overall topology and dynamics of PLN and ultimately modulate its inhibitory potency. Also, the changes in the TM tilt angle and cytoplasmic dynamics of PLN caused by these mutations correlate well with the extent of SERCA inhibition. Our study unveils new molecular determinants for designing variants of PLN that outcompete endogenous PLN to regulate SERCA in a tunable manner.
Asunto(s)
Proteínas de Unión al Calcio , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Humanos , Fosforilación/genética , Mutación/genética , Calcio/metabolismo , Citoplasma/metabolismo , Citoplasma/genética , Animales , Sitios de Unión/genética , Unión ProteicaAsunto(s)
Insuficiencia Cardíaca , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/enzimología , Humanos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Etiocolanolona/análogos & derivados , Etiocolanolona/farmacología , Etiocolanolona/uso terapéutico , Enfermedad Crónica , Activadores de Enzimas/farmacología , Activadores de Enzimas/uso terapéuticoRESUMEN
Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.
Asunto(s)
Retículo Endoplásmico , Respuesta al Choque Térmico , Termogénesis , Humanos , Animales , Retículo Endoplásmico/metabolismo , Ratones , Respuesta de Proteína Desplegada , Línea Celular Tumoral , Estrés del Retículo Endoplásmico , Hipertermia/metabolismo , Hipertermia/patología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Fibroblastos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
BACKGROUND & AIMS: The treatment of cardiovascular diseases (CVD) could greatly benefit from using nitric oxide (NO) donors. This study aimed to investigate the mechanisms of action of NONO2P that contribute to the observed responses in the mesenteric artery. The hypothesis was that NONO2P would have similar pharmacological actions to sodium nitroprusside (SNP) and NO. METHODS: Male Wistar rats were euthanized to isolate the superior mesenteric artery for isometric tension recordings. NO levels were measured using the DAF-FM/DA dye, and cyclic guanosine monophosphate (cGMP) levels were determined using a cGMP-ELISA Kit. RESULTS: NONO2P presented a similar maximum efficacy to SNP. The free radical of NO (NOâ¢) scavengers (PTIO; 100 µM and hydroxocobalamin; 30 µM) and nitroxyl anion (NO-) scavenger (L-cysteine; 3 mM) decreased relaxations promoted by NONO2P. The presence of the specific soluble guanylyl cyclase (sGC) inhibitor (ODQ; 10 µM) nearly abolished the vasorelaxation. The cGMP-dependent protein kinase (PKG) inhibition (KT5823; 1 µM) attenuated the NONO2P relaxant effect. The vasorelaxant response was significantly attenuated by blocking inward rectifying K+ channels (Kir), voltage-operated K+ channels (KV), and large conductance Ca2+-activated K+ channels (BKCa). NONO2P-induced relaxation was attenuated by cyclopiazonic acid (10 µM), indicating that sarcoplasmic reticulum Ca2+-ATPase (SERCA) activation is involved in this relaxation. Moreover, NONO2P increased NO levels in endothelial cells and cGMP production. CONCLUSIONS: NONO2P induces vasorelaxation with the same magnitude as SNP, releasing NO⢠and NO-. Its vasorelaxant effect involves sGC, PKG, K+ channels opening, and SERCA activation, suggesting its potential as a therapeutic option for CVD.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico , GMP Cíclico , Donantes de Óxido Nítrico , Óxido Nítrico , Canales de Potasio , Ratas Wistar , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Transducción de Señal , Guanilil Ciclasa Soluble , Vasodilatación , Animales , Masculino , Vasodilatación/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/metabolismo , Guanilil Ciclasa Soluble/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Ratas , Canales de Potasio/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Transducción de Señal/efectos de los fármacos , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Guanilato Ciclasa/metabolismo , Activación Enzimática/efectos de los fármacosRESUMEN
Cardiac arrhythmias remain a significant concern with Ibrutinib (IBR), a first-generation Bruton's tyrosine kinase inhibitor (BTKi). Acalabrutinib (ABR), a next-generation BTKi, is associated with reduced atrial arrhythmia events. However, the role of ABR in ventricular arrhythmia (VA) has not been adequately evaluated. Our study aimed to investigate VA vulnerability and ventricular electrophysiology following chronic ABR therapy in male Sprague-Dawley rats utilizing epicardial optical mapping for ventricular voltage and Ca2+ dynamics and VA induction by electrical stimulation in ex-vivo perfused hearts. Ventricular tissues were snap-frozen for protein analysis for sarcoplasmic Ca2+ and metabolic regulatory proteins. The results show that both ABR and IBR treatments increased VA vulnerability, with ABR showing higher VA regularity index (RI). IBR, but not ABR, is associated with the abbreviation of action potential duration (APD) and APD alternans. Both IBR and ABR increased diastolic Ca2+ leak and Ca2+ alternans, reduced conduction velocity (CV), and increased CV dispersion. Decreased SERCA2a expression and AMPK phosphorylation were observed with both treatments. Our results suggest that ABR treatment also increases the risk of VA by inducing proarrhythmic changes in Ca2+ signaling and membrane electrophysiology, as seen with IBR. However, the different impacts of these two BTKi on ventricular electrophysiology may contribute to differences in VA vulnerability and distinct VA characteristics.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa , Arritmias Cardíacas , Benzamidas , Piperidinas , Ratas Sprague-Dawley , Animales , Benzamidas/farmacología , Benzamidas/uso terapéutico , Masculino , Ratas , Agammaglobulinemia Tirosina Quinasa/metabolismo , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/inducido químicamente , Piperidinas/farmacología , Piperidinas/uso terapéutico , Potenciales de Acción/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Calcio/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Adenina/efectos adversos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Pirimidinas/farmacología , Señalización del Calcio/efectos de los fármacos , Pirazoles/farmacologíaRESUMEN
BACKGROUND: Chronic sympathetic stimulation drives desensitization and downregulation of ß1 adrenergic receptor (ß1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of ß1AR signaling in the heart. METHODS AND RESULTS: We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine ß1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular ß1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced ß1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P<0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P<0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P<0.05). However, the ß1AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P<0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the ß1AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced ß1AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening. CONCLUSIONS: This study reveals distinct mechanisms for the downregulation of subcellular ß1AR signaling in the heart under chronic adrenergic stimulation.
Asunto(s)
Canales de Calcio Tipo L , Proteínas Quinasas Dependientes de AMP Cíclico , Regulación hacia Abajo , Isoproterenol , Ratones Endogámicos C57BL , Miocitos Cardíacos , Receptores Adrenérgicos beta 1 , Canal Liberador de Calcio Receptor de Rianodina , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Transducción de Señal , Animales , Receptores Adrenérgicos beta 1/metabolismo , Masculino , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Isoproterenol/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/efectos de los fármacos , Modelos Animales de Enfermedad , Ratones , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/fisiopatología , Cardiomiopatías/metabolismo , Cardiomiopatías/inducido químicamente , Transferencia Resonante de Energía de FluorescenciaRESUMEN
Risk of cardiovascular disease mortality rises in women after menopause. While increased cardiovascular risk is largely attributed to postmenopausal declines in estrogens, the molecular changes in the heart that contribute to risk are poorly understood. Disruptions in intracellular calcium handling develop in ovariectomized mice and have been implicated in cardiac dysfunction. Using a mouse model of menopause in which ovarian failure occurs over 120 days, we sought to determine if perimenopause impacted calcium removal mechanisms in the heart and identify the molecular mechanisms. Mice were injected with 4-vinylcyclohexene diepoxide (VCD) to induce ovarian failure over 120 days, mimicking perimenopause. Hearts were removed at 60 and 120 days after VCD injections, representing the middle and end of perimenopause. SERCA2a function was significantly diminished at the end of perimenopause. Neither SERCA2a nor phospholamban expression changed at either time point, but phospholamban phosphorylation at S16 and T17 was dynamically altered. Intrinsic SERCA inhibitors sarcolipin and myoregulin increased >4-fold at day 60, as did the native activator DWORF. At the end of perimenopause, sarcolipin and myoregulin returned to baseline levels while DWORF was significantly reduced below controls. Sodium-calcium exchanger expression was significantly increased at the end of perimenopause. These results show that the foundation for increased cardiovascular disease mortality develops in the heart during perimenopause and that regulators of calcium handling exhibit significant fluctuations over time. Understanding the temporal development of cardiovascular risk associated with menopause and the underlying mechanisms is critical to developing interventions that mitigate the rise in cardiovascular mortality that arises after menopause.
Asunto(s)
Modelos Animales de Enfermedad , Perimenopausia , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Animales , Femenino , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Ratones , Perimenopausia/metabolismo , Compuestos de Vinilo/farmacología , Miocardio/metabolismo , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Insuficiencia Ovárica Primaria/metabolismo , Ciclohexenos/farmacología , Ratones Endogámicos C57BL , FosforilaciónRESUMEN
In cardiac myocytes, the type 2a sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) plays a key role in intracellular Ca regulation. Due to its critical role in heart function, SERCA2a activity is tightly regulated by different mechanisms, including micropeptides. While phospholamban (PLB) is a well-known SERCA2a inhibitor, dwarf open reading frame (DWORF) is a recently identified SERCA2a activator. Since PLB phosphorylation is the most recognized mechanism of SERCA2a activation during adrenergic stress, we studied whether PLB phosphorylation also affects SERCA2a regulation by DWORF. By using confocal Ca imaging in a HEK293 expressing cell system, we analyzed the effect of the co-expression of PLB and DWORF using a bicistronic construct on SERCA2a-mediated Ca uptake. Under these conditions of matched expression of PLB and DWORF, we found that SERCA2a inhibition by non-phosphorylated PLB prevails over DWORF activating effect. However, when PLB is phosphorylated at PKA and CaMKII sites, not only PLB's inhibitory effect was relieved, but SERCA2a was effectively activated by DWORF. Förster resonance energy transfer (FRET) analysis between SERCA2a and DWORF showed that DWORF has a higher relative affinity for SERCA2a when PLB is phosphorylated. Thus, SERCA2a regulation by DWORF responds to the PLB phosphorylation status, suggesting that DWORF might contribute to SERCA2a activation during conditions of adrenergic stress.
Asunto(s)
Proteínas de Unión al Calcio , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Humanos , Fosforilación , Células HEK293 , Sistemas de Lectura Abierta/genética , Calcio/metabolismo , Activación Enzimática , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismoRESUMEN
Abnormal calcium signaling is associated with non-small cell lung cancer (NSCLC) malignant progression, poor survival and chemotherapy resistance. Targeting endoplasmic reticulum (ER) Ca2+ channels or pumps to block calcium uptake in the ER induces ER stress and concomitantly promotes mitochondrial calcium uptake, leading to mitochondrial dysfunction and ultimately inducing cell death. Here, we identified Diphyllin was a potential specific inhibitor of endoplasmic reticulum (ER) calcium-importing protein sarco/endoplasmic-reticulum Ca2+ ATPase 2 (SERCA2). In vitro and in vivo studies showed that Diphyllin increased NSCLC cell apoptosis, along with inhibition of cell proliferation and migration. Mechanistically, Diphyllin promoted ER stress by directly inhibiting SERCA2 activity and decreasing ER Ca2+ levels. At the same time, the accumulated Ca2+ in cytoplasm flowed into mitochondria to increase reactive oxygen species (ROS) and decrease mitochondrial membrane potential (MMP), leading to cytochrome C (Cyto C) release and mitochondrial dysfunction. In addition, we found that Diphyllin combined with cisplatin could have a synergistic anti-tumor effect in vitro and in vivo. Taken together, our results suggested that Diphyllin, as a potential novel inhibitor of SERCA2, exerts anti-tumor effects by blocking ER Ca2+ uptake and thereby promoting ER stress and mitochondrial dysfunction.
Asunto(s)
Apoptosis , Carcinoma de Pulmón de Células no Pequeñas , Proliferación Celular , Estrés del Retículo Endoplásmico , Neoplasias Pulmonares , Mitocondrias , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Estrés del Retículo Endoplásmico/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Animales , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ratones , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , Calcio/metabolismo , Células A549 , Sinergismo Farmacológico , Ratones Desnudos , Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Cisplatino/farmacología , Ratones Endogámicos BALB C , Señalización del Calcio/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismoRESUMEN
The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) transports two Ca2+ ions per ATP hydrolyzed from the cytoplasm to the lumen. However, how the ATP hydrolysis remotely drives the Ca2+ transport is unclear. In the SERCA1a crystal structures, the ATP hydrolysis is accompanied by the notably increasing tilting angle of the central core (CC) and the Ca2+ transport, and the CC tilting angle dramatically decreases in the E2 to E1 transition. We demonstrated that the significantly increasing tilting motion of the CC drove the Ca2+ release in the molecular dynamics simulation of the R836A variant, and the dramatic spontaneous decrease in the CC tilting angle of the E2 state triggers the restart of the SERCA1a's transport cycle. The repulsion between the phosphorylated D351 and the phosphate groups in ADP triggers the release of ADP from the SERCA1a headpiece. We proposed a novel SERCA transport mechanism in which ATP hydrolysis drives a significant tilting motion of the CC, which drives Ca2+ transport and the A domain rotational motion in the E1P-ADP-2Ca2+ to E2P transition. The dramatic spontaneous decrease in the CC tilting angle of the E2 state drives the restart of the transport cycle.
Asunto(s)
Adenosina Trifosfato , Calcio , Simulación de Dinámica Molecular , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , Calcio/metabolismo , Adenosina Trifosfato/metabolismo , Hidrólisis , Adenosina Difosfato/metabolismo , Humanos , Transporte BiológicoRESUMEN
Studying the calcium dynamics within a fibroblast cell individually has provided only a restricted understanding of its functions. However, research efforts focusing on systems biology approaches for such investigations have been largely neglected by researchers until now. Fibroblast cells rely on signaling from calcium ( C a 2 + ) and nitric oxide (NO) to maintain their physiological functions and structural stability. Various studies have demonstrated the correlation between NO and the control of C a 2 + dynamics in cells. However, there is currently no existing model to assess the disruptions caused by various factors in regulatory dynamics, potentially resulting in diverse fibrotic disorders. A mathematical model has been developed to investigate the effects of changes in parameters such as buffer, receptor, sarcoplasmic endoplasmic reticulum C a 2 + -ATPase (SERCA) pump, and source influx on the regulation and dysregulation of spatiotemporal calcium and NO dynamics in fibroblast cells. This model is based on a system of reaction-diffusion equations, and numerical simulations are conducted using the finite element method. Disturbances in key processes related to calcium and nitric oxide, including source influx, buffer mechanism, SERCA pump, and inositol trisphosphate ( I P 3 ) receptor, may contribute to deregulation in the calcium and NO dynamics within fibroblasts. The findings also provide new insights into the extent and severity of disorders resulting from alterations in various parameters, potentially leading to deregulation and the development of fibrotic disease.