RESUMO
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.
Assuntos
Envelhecimento , Contração Miocárdica , Miócitos Cardíacos , Ratos Endogâmicos F344 , Animais , Masculino , Contração Miocárdica/fisiologia , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Ratos , Fosforilação , Miócitos Cardíacos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Conectina/metabolismo , Troponina I/metabolismo , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de TransporteRESUMO
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.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico , GMP Cíclico , Doadores de Óxido Nítrico , Óxido Nítrico , Canais de Potássio , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Transdução de Sinais , Guanilil Ciclase Solúvel , Vasodilatação , Animais , Masculino , Vasodilatação/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Guanilil Ciclase Solúvel/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Ratos , Canais de Potássio/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Guanilato Ciclase/metabolismo , Ativação Enzimática/efeitos dos fármacosRESUMO
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.
Assuntos
Potenciais de Ação , Arritmias Cardíacas , Insuficiência Cardíaca , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/metabolismo , Insuficiência Cardíaca/fisiopatologia , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/etiologia , Diástole/fisiologia , Modelos Cardiovasculares , Contração Miocárdica/fisiologia , Simulação por Computador , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
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.
Assuntos
Retículo Endoplasmático , Resposta ao Choque Térmico , Termogênese , Humanos , Animais , Retículo Endoplasmático/metabolismo , Camundongos , Resposta a Proteínas não Dobradas , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático , Hipertermia/metabolismo , Hipertermia/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fibroblastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoAssuntos
Insuficiência Cardíaca , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/enzimologia , Humanos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Etiocolanolona/análogos & derivados , Etiocolanolona/farmacologia , Etiocolanolona/uso terapêutico , Doença Crônica , Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/uso terapêuticoRESUMO
Several errors (shown with underlines) in the following list appeared in the article "B-Type Natriuretic Peptide Inhibits the Expression and Function of SERCA2a in Heart Failure" by Yuting Zhai, Junhong Chen, Rongsheng Kan, Haochen Xuan, Chaofan Wang, Dongye Li, Tongda Xu (Vol. 65 No.2, 292-299, 2024).
Assuntos
Insuficiência Cardíaca , Peptídeo Natriurético Encefálico , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/tratamento farmacológico , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Humanos , Peptídeo Natriurético Encefálico/metabolismoRESUMO
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.
Assuntos
Canais de Cálcio Tipo L , Proteínas Quinases Dependentes de AMP Cíclico , Regulação para Baixo , Isoproterenol , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Receptores Adrenérgicos beta 1 , Canal de Liberação de Cálcio do Receptor de Rianodina , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Transdução de Sinais , Animais , Receptores Adrenérgicos beta 1/metabolismo , Masculino , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Isoproterenol/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Modelos Animais de Doenças , Camundongos , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/fisiopatologia , Cardiomiopatias/metabolismo , Cardiomiopatias/induzido quimicamente , Transferência Ressonante de Energia de FluorescênciaRESUMO
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.
Assuntos
Modelos Animais de Doenças , Perimenopausa , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , Feminino , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Camundongos , Perimenopausa/metabolismo , Compostos de Vinila/farmacologia , Miocárdio/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Insuficiência Ovariana Primária/metabolismo , Cicloexenos/farmacologia , Camundongos Endogâmicos C57BL , FosforilaçãoRESUMO
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.
Assuntos
Proteínas de Ligação ao Cálcio , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Fosforilação , Células HEK293 , Fases de Leitura Aberta/genética , Cálcio/metabolismo , Ativação Enzimática , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismoRESUMO
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.
Assuntos
Tirosina Quinase da Agamaglobulinemia , Arritmias Cardíacas , Benzamidas , Piperidinas , Ratos Sprague-Dawley , Animais , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Masculino , Ratos , Tirosina Quinase da Agamaglobulinemia/metabolismo , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/induzido quimicamente , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Potenciais de Ação/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Cálcio/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Adenina/efeitos adversos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Pirimidinas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Pirazóis/farmacologiaRESUMO
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.
Assuntos
Apoptose , Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Estresse do Retículo Endoplasmático , Neoplasias Pulmonares , Mitocôndrias , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Animais , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Camundongos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Cálcio/metabolismo , Células A549 , Sinergismo Farmacológico , Camundongos Nus , Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Camundongos Endogâmicos BALB C , Sinalização do Cálcio/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismoRESUMO
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.
Assuntos
Cálcio , Fibroblastos , Fibrose , Modelos Biológicos , Óxido Nítrico , Fibroblastos/metabolismo , Óxido Nítrico/metabolismo , Cálcio/metabolismo , Transdução de Sinais , Humanos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sinalização do CálcioRESUMO
Myocardial sarcoendoplasmic reticulum calcium ATPase 2 (SERCA2) activity is critical for heart function. We have demonstrated that inhaled halogen (chlorine or bromine) gases inactivate SERCA2, impair calcium homeostasis, increase proteolysis, and damage the myocardium ultimately leading to cardiac dysfunction. To further elucidate the mechanistic role of SERCA2 in halogen-induced myocardial damage, we used bromine-exposed cardiac-specific SERCA2 knockout (KO) mice [tamoxifen-administered SERCA2 (flox/flox) Tg (αMHC-MerCreMer) mice] and compared them to the oil-administered controls. We performed echocardiography and hemodynamic analysis to investigate cardiac function 24 hours after bromine (600 ppm for 30 minutes) exposure and measured cardiac injury markers in plasma and proteolytic activity in cardiac tissue and performed electron microscopy of the left ventricle (LV). Cardiac-specific SERCA2 knockout mice demonstrated enhanced toxicity to bromine. Bromine exposure increased ultrastructural damage, perturbed LV shape geometry, and demonstrated acutely increased phosphorylation of phospholamban in the KO mice. Bromine-exposed KO mice revealed significantly enhanced mean arterial pressure and sphericity index and decreased LV end diastolic diameter and LV end systolic pressure when compared with the bromine-exposed control FF mice. Strain analysis showed loss of synchronicity, evidenced by an irregular endocardial shape in systole and irregular vector orientation of contractile motion across different segments of the LV in KO mice, both at baseline and after bromine exposure. These studies underscore the critical role of myocardial SERCA2 in preserving cardiac ultrastructure and function during toxic halogen gas exposures. SIGNIFICANCE STATEMENT: Due to their increased industrial production and transportation, halogens such as chlorine and bromine pose an enhanced risk of exposure to the public. Our studies have demonstrated that inhalation of these halogens leads to the inactivation of cardiopulmonary SERCA2 and results in calcium overload. Using cardiac-specific SERCA2 KO mice, these studies further validated the role of SERCA2 in bromine-induced myocardial injury. These studies highlight the increased susceptibility of individuals with pathological loss of cardiac SERCA2 to the effects of bromine.
Assuntos
Bromo , Camundongos Knockout , Miocárdio , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Masculino , Camundongos Endogâmicos C57BL , Administração por Inalação , Proteínas de Ligação ao CálcioRESUMO
Metabolic dysfunction-associated steatotic liver disease is the most common form of liver disease and poses significant health risks to patients who progress to metabolic dysfunction-associated steatohepatitis. Fatty acid overload alters endoplasmic reticulum (ER) calcium stores and induces mitochondrial oxidative stress in hepatocytes, leading to hepatocellular inflammation and apoptosis. Obese mice have impaired liver sarco/ER Ca2+-ATPase (SERCA) function, which normally maintains intracellular calcium homeostasis by transporting Ca2+ ions from the cytoplasm to the ER. We hypothesized that restoration of SERCA activity would improve diet-induced steatohepatitis in mice by limiting ER stress and mitochondrial dysfunction. WT and melanocortin-4 receptor KO (Mc4r-/-) mice were placed on either chow or Western diet (WD) for 8 weeks. Half of the WD-fed mice were administered CDN1163 to activate SERCA, which reduced liver fibrosis and inflammation. SERCA activation also restored glucose tolerance and insulin sensitivity, improved histological markers of metabolic dysfunction-associated steatohepatitis, increased expression of antioxidant enzymes, and decreased expression of oxidative stress and ER stress genes. CDN1163 decreased hepatic citric acid cycle flux and liver pyruvate cycling, enhanced expression of mitochondrial respiratory genes, and shifted hepatocellular [NADH]/[NAD+] and [NADPH]/[NADP+] ratios to a less oxidized state, which was associated with elevated PUFA content of liver lipids. In sum, the data demonstrate that pharmacological SERCA activation limits metabolic dysfunction-associated steatotic liver disease progression and prevents metabolic dysfunction induced by WD feeding in mice.
Assuntos
Fígado , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Camundongos , Fígado/metabolismo , Fígado/patologia , Masculino , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Estresse do Retículo Endoplasmático , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Dieta Ocidental/efeitos adversos , Camundongos KnockoutRESUMO
High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17ß-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases. In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases. In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17ß-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.
Assuntos
Cálcio , Estradiol , Simulação de Acoplamento Molecular , ATPases Transportadoras de Cálcio da Membrana Plasmática , Animais , Cobaias , Estradiol/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Cálcio/metabolismo , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Masculino , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Contração Muscular/efeitos dos fármacos , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Carbacol/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismoRESUMO
Regional endothermy is the ability of an animal to elevate the temperature of specific regions of the body above that of the surrounding environment and has evolved independently among several fish lineages. Sarcolipin (SLN) is a small transmembrane protein that uncouples the sarcoplasmic reticulum calcium ATPase pump (SERCA1b) resulting in futile Ca2+ cycling and is thought to play a role in non-shivering thermogenesis (NST) in cold-challenged mammals and possibly some fishes. This study investigated the relative expression of sln and serca1 transcripts in three regionally-endothermic fishes (the skipjack, Katsuwonus pelamis, and yellowfin tuna, Thunnus albacares, both of which elevate the temperatures of their slow-twitch red skeletal muscle (RM) and extraocular muscles (EM), as well as the cranial endothermic swordfish, Xiphias gladius), and closely related ectothermic scombrids (the Eastern Pacific bonito, Sarda chiliensis, and Pacific chub mackerel, Scomber japonicus). Using Reverse Transcription quantitative PCR (RT-qPCR) and species-specific primers, relative sln expression trended higher in both the RM and EM for all four scombrid species compared to white muscle. In addition, relative serca1 expression was found to be higher in RM of skipjack and yellowfin tuna in comparison to white muscle. However, neither sln nor serca1 transcripts were higher in swordfish RM, EM or cranial heater tissue in comparison to white muscle. A key phosphorylation site in sarcolipin, threonine 5, is conserved in the swordfish, but is mutated to alanine or valine in tunas and the endothermic smalleye Pacific opah, Lampris incognitus, which should result in increased uncoupling of the SERCA pump. Our results support the role of potential SLN-NST in endothermic tunas and the lack thereof for swordfish.
Assuntos
Cálcio , Proteínas Musculares , Proteolipídeos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Termogênese , Animais , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteolipídeos/genética , Proteolipídeos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Termogênese/genética , Cálcio/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Perciformes/genética , Perciformes/fisiologia , Perciformes/metabolismo , Atum/genética , Atum/metabolismo , Atum/fisiologiaRESUMO
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.
Assuntos
Trifosfato de Adenosina , Cálcio , Simulação de Dinâmica Molecular , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , Cálcio/metabolismo , Trifosfato de Adenosina/metabolismo , Hidrólise , Difosfato de Adenosina/metabolismo , Humanos , Transporte BiológicoRESUMO
Diabetes is commonly associated with an elevated level of reactive carbonyl species due to alteration of glucose and fatty acid metabolism. These metabolic changes cause an abnormality in cardiac Ca2+ regulation that can lead to cardiomyopathies. In this study, we explored how the reactive α-dicarbonyl methylglyoxal (MGO) affects Ca2+ regulation in mouse ventricular myocytes. Analysis of intracellular Ca2+ dynamics revealed that MGO (200 µM) increases action potential (AP)-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load, with a limited effect on L-type Ca2+ channel-mediated Ca2+ transients and SERCA-mediated Ca2+ uptake. At the same time, MGO significantly slowed down cytosolic Ca2+ extrusion by Na+/Ca2+ exchanger (NCX). MGO also increased the frequency of Ca2+ waves during rest and these Ca2+ release events were abolished by an external solution with zero [Na+] and [Ca2+]. Adrenergic receptor activation with isoproterenol (10 nM) increased Ca2+ transients and SR Ca2+ load, but it also triggered spontaneous Ca2+ waves in 27% of studied cells. Pretreatment of myocytes with MGO increased the fraction of cells with Ca2+ waves during adrenergic receptor stimulation by 163%. Measurements of intracellular [Na+] revealed that MGO increases cytosolic [Na+] by 57% from the maximal effect produced by the Na+-K+ ATPase inhibitor ouabain (20 µM). This increase in cytosolic [Na+] was a result of activation of a tetrodotoxin-sensitive Na+ influx, but not an inhibition of Na+-K+ ATPase. An increase in cytosolic [Na+] after treating cells with ouabain produced similar effects on Ca2+ regulation as MGO. These results suggest that protein carbonylation can affect cardiac Ca2+ regulation by increasing cytosolic [Na+] via a tetrodotoxin-sensitive pathway. This, in turn, reduces Ca2+ extrusion by NCX, causing SR Ca2+ overload and spontaneous Ca2+ waves.
Assuntos
Cálcio , Miócitos Cardíacos , Carbonilação Proteica , Retículo Sarcoplasmático , Sódio , Animais , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Camundongos , Cálcio/metabolismo , Sódio/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Trocador de Sódio e Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Ventrículos do Coração/citologia , Aldeído Pirúvico/farmacologia , Aldeído Pirúvico/metabolismo , Sinalização do Cálcio/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Potenciais de Ação/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Células Cultivadas , MasculinoRESUMO
Cardiac conduction regulatory RNA (CCRR) has been documented as an antiarrhythmic lncRNA in our earlier investigation. This study aimed to evaluate the effects of CCRR on SERCA2a and the associated Ca2+ homeostasis in myocardial infarction (MI). Overexpression of CCRR via AAV9-mediated delivery not only partially reversed ischemia-induced contractile dysfunction but also alleviated abnormal Ca2+ homeostasis and reduced the heightened methylation level of SERCA2a following MI. These effects were also observed in CCRR over-expressing transgenic mice. A conserved sequence domain of CCRR mimicked the protective function observed with the full length. Furthermore, silencing CCRR in healthy mice led to intracellular Ca2+ overloading of cardiomyocytes. CCRR increased SERCA2a protein stability by upregulating FTO expression. The direct interaction between CCRR and FTO protein was characterized by RNA-binding protein immunoprecipitation (RIP) analysis and RNA pulldown experiments. Activation of NFATc3 was identified as an upstream mechanism responsible for CCRR downregulation in MI. This study demonstrates that CCRR is a protective lncRNA that acts by maintaining the function of FTO, thereby reducing the m6A RNA methylation level of SERCA2a, ultimately preserving calcium homeostasis for myocardial contractile function in MI. Therefore, CCRR may be considered a promising therapeutic strategy with a beneficial role in cardiac pathology.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato , Cálcio , Homeostase , Infarto do Miocárdio , Miócitos Cardíacos , RNA Longo não Codificante , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Camundongos , Cálcio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Miócitos Cardíacos/metabolismo , Masculino , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Metilação , HumanosRESUMO
Calcium homeostasis imbalance is one of the important pathological mechanisms in heart failure. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a), a calcium ATPase on the sarcoplasmic reticulum in cardiac myocytes, is a myocardial systolic-diastolic Ca2â +â homeostasis regulating enzyme that is not only involved in cardiac diastole but also indirectly affects cardiac myocyte contraction. SERCA2a expression was found to be decreased in myocardial tissue in heart failure, however, there are few reports on serum SERCA2a expression in patients with heart failure, and this study was designed to investigate whether serum SERCA2a levels are associated with the occurrence of adverse events after discharge in patients hospitalized with heart failure. Patients with heart failure hospitalized in the cardiovascular department of the Second Affiliated Hospital of Guangdong Medical University, China, from July 2018 to July 2019 were included in this study, and serum SERCA2a concentrations were measured; each enrolled patient was followed up by telephone after 6 months (6â ±â 1 months) for general post-discharge patient status. The correlation between serum SERCA2a levels and the occurrence of adverse events (death or readmission due to heart failure) after hospital discharge was assessed using multiple analysis and trend analysis. Seventy-one patients with heart failure were finally included in this study, of whom 38 (53.5%) were men and 33 (46.5%) were women (All were postmenopausal women). Multiple analysis revealed no correlation between serum SERCA2a levels and the occurrence of adverse events in the total study population and in male patients, but serum SERCA2a levels were associated with the occurrence of adverse outcome events after hospital discharge in female patients (ORâ =â 1.02, Pâ =â .047). Further analysis using a trend analysis yielded a 4.0% increase in the risk of adverse outcomes after hospital discharge for each unit increase in SERCA2a in female patients (ORâ =â 1.04; Pâ =â .02), while no significant difference was seen in men. This study suggests that serum SERCA2a levels at admission are associated with the occurrence of post-discharge adverse events in postmenopausal female patients hospitalized with heart failure.