RESUMO
Cardiac stromal interaction molecule 1 (STIM1), a key mediator of store-operated Ca2+ entry (SOCE), is a known determinant of cardiomyocyte pathological growth in hypertrophic cardiomyopathy. We examined the role of STIM1 and SOCE in response to exercise-dependent physiological hypertrophy. Wild-type (WT) mice subjected to exercise training (WT-Ex) showed a significant increase in exercise capacity and heart weight compared with sedentary (WT-Sed) mice. Moreover, myocytes from WT-Ex hearts displayed an increase in length, but not width, compared with WT-Sed myocytes. Conversely, exercised cardiac-specific STIM1 knock-out mice (cSTIM1KO-Ex), although displaying significant increase in heart weight and cardiac dilation, evidenced no changes in myocyte size and displayed a decreased exercise capacity, impaired cardiac function, and premature death compared with sedentary cardiac-specific STIM1 knock-out mice (cSTIM1KO-Sed). Confocal Ca2+ imaging demonstrated enhanced SOCE in WT-Ex myocytes compared with WT-Sed myocytes with no measurable SOCE detected in cSTIM1KO myocytes. Exercise training induced a significant increase in cardiac phospho-Akt Ser473 in WT mice but not in cSTIM1KO mice. No differences were observed in phosphorylation of mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK) in exercised versus sedentary cSTIM1KO mice hearts. cSTIM1KO-Sed mice showed increased basal MAPK phosphorylation compared with WT-Sed that was not altered by exercise training. Finally, histological analysis revealed exercise resulted in increased autophagy in cSTIM1KO but not in WT myocytes. Taken together, our results suggest that adaptive cardiac hypertrophy in response to exercise training involves STIM1-mediated SOCE. Our results demonstrate that STIM1 is involved in and essential for the myocyte longitudinal growth and mTOR activation in response to endurance exercise training.NEW & NOTEWORTHY Store-operated Ca2+ entry (SOCE) has been implicated in pathological cardiac hypertrophy; however, its role in physiological hypertrophy is unknown. Here we report that SOCE is also essential for physiological cardiac hypertrophy and functional adaptations in response to endurance exercise. These adaptations were associated with activation of AKT/mTOR pathway and curtailed cardiac autophagy and degeneration. Thus, SOCE is a common mechanism and an important bifurcation point for signaling paths involved in physiological and pathological hypertrophy.
Assuntos
Canais de Cálcio , Miócitos Cardíacos , Camundongos , Animais , Miócitos Cardíacos/metabolismo , Canais de Cálcio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Cardiomegalia/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Camundongos Knockout , Cálcio/metabolismo , Sinalização do Cálcio , Mamíferos/metabolismoRESUMO
Dofetilide is a rapid delayed rectifier potassium current inhibitor widely used to prevent the recurrence of atrial fibrillation and flutter. The clinical use of this drug is associated with increases in QTc interval, which predispose patients to ventricular cardiac arrhythmias. The mechanisms involved in the disposition of dofetilide, including its movement in and out of cardiomyocytes, remain unknown. Using a xenobiotic transporter screen, we identified MATE1 (SLC47A1) as a transporter of dofetilide and found that genetic knockout or pharmacological inhibition of MATE1 in mice was associated with enhanced retention of dofetilide in cardiomyocytes and increased QTc prolongation. The urinary excretion of dofetilide was also dependent on the MATE1 genotype, and we found that this transport mechanism provides a mechanistic basis for previously recorded drug-drug interactions of dofetilide with various contraindicated drugs, including bictegravir, cimetidine, ketoconazole, and verapamil. The translational significance of these observations was examined with a physiologically-based pharmacokinetic model that adequately predicted the drug-drug interaction liabilities in humans. These findings support the thesis that MATE1 serves a conserved cardioprotective role by restricting excessive cellular accumulation and warrant caution against the concurrent administration of potent MATE1 inhibitors and cardiotoxic substrates with a narrow therapeutic window.
Assuntos
Antiarrítmicos , Fibrilação Atrial , Animais , Antiarrítmicos/farmacologia , Humanos , Camundongos , Fenetilaminas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
Secreted frizzled-related protein 2 (SFRP2) promotes the migration/invasion of metastatic osteosarcoma (OS) cells and tube formation by endothelial cells. However, its function on T-cells is unknown. We hypothesized that blocking SFRP2 with a humanized monoclonal antibody (hSFRP2 mAb) can restore immunity by reducing CD38 and PD-1 levels, ultimately overcoming resistance to PD-1 inhibitors. Treating two metastatic murine OS cell lines in vivo, RF420 and RF577, with hSFRP2 mAb alone led to a significant reduction in the number of lung metastases, compared to IgG1 control treatment. While PD-1 mAb alone had minimal effect, hSFRP2 mAb combination with PD-1 mAb had an additive antimetastatic effect. This effect was accompanied by lower SFRP2 levels in serum, lower CD38 levels in tumor-infiltrating lymphocytes and T-cells, and lower PD-1 levels in T-cells. In vitro data confirmed that SFRP2 promotes NFATc3, CD38 and PD-1 expression in T-cells, while hSFRP2 mAb treatment counteracts these effects and increases NAD+ levels. hSFRP2 mAb treatment further rescued the suppression of T-cell proliferation by tumor cells in a co-culture model. Finally, hSFRP2 mAb induced apoptosis in RF420 and RF577 OS cells but not in T-cells. Thus, hSFRP2 mAb therapy could potentially overcome PD-1 inhibitor resistance in metastatic osteosarcoma.
RESUMO
Heart failure (HF) is characterized by asymmetrical autonomic balance. Treatments to restore parasympathetic activity in human heart failure trials have shown beneficial effects. However, mechanisms of parasympathetic-mediated improvement in cardiac function remain unclear. The present study examined the effects and underpinning mechanisms of chronic treatment with the cholinesterase inhibitor, pyridostigmine (PYR), in pressure overload HF induced by transverse aortic constriction (TAC) in mice. TAC mice exhibited characteristic adverse structural (left ventricular hypertrophy) and functional remodelling (reduced ejection fraction, altered myocyte calcium (Ca) handling, increased arrhythmogenesis) with enhanced predisposition to arrhythmogenic aberrant sarcoplasmic reticulum (SR) Ca release, cardiac ryanodine receptor (RyR2) hyper-phosphorylation and up-regulated store-operated Ca entry (SOCE). PYR treatment resulted in improved cardiac contractile performance and rhythmic activity relative to untreated TAC mice. Chronic PYR treatment inhibited altered intracellular Ca handling by alleviating aberrant Ca release and diminishing pathologically enhanced SOCE in TAC myocytes. At the molecular level, these PYR-induced changes in Ca handling were associated with reductions of pathologically enhanced phosphorylation of RyR2 serine-2814 and STIM1 expression in HF myocytes. These results suggest that chronic cholinergic augmentation alleviates HF via normalization of both canonical RyR2-mediated SR Ca release and non-canonical hypertrophic Ca signaling via STIM1-dependent SOCE.
Assuntos
Arritmias Cardíacas/tratamento farmacológico , Cálcio/metabolismo , Inibidores da Colinesterase/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Brometo de Piridostigmina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Molécula 1 de Interação Estromal/antagonistas & inibidores , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell-derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.
Assuntos
Doxorrubicina/efeitos adversos , Traumatismos Cardíacos/induzido quimicamente , Traumatismos Cardíacos/tratamento farmacológico , Terapia de Alvo Molecular , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Animais , Criança , Regulação da Expressão Gênica , Traumatismos Cardíacos/fisiopatologia , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/deficiência , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Análise de Sequência de RNARESUMO
Post-translational modifications of proteins involved in calcium handling in myocytes, such as the cardiac ryanodine receptor (RyR2), critically regulate cardiac contractility. Recent studies have suggested that phosphorylation of RyR2 by protein kinase G (PKG) might contribute to the cardioprotective effects of cholinergic stimulation. However, the specific mechanisms underlying these effects remain unclear. Here, using murine ventricular myocytes, immunoblotting, proximity ligation as-says, and nitric oxide imaging, we report that phosphorylation of Ser-2808 in RyR2 induced by the muscarinic receptor agonist carbachol is mediated by a signaling axis comprising phosphoinositide 3-phosphate kinase, Akt Ser/Thr kinase, nitric oxide synthase 1, nitric oxide, soluble guanylate cyclase, cyclic GMP (cGMP), and PKG. We found that this signaling pathway is compartmentalized in myocytes, as it was distinct from atrial natriuretic peptide receptor-cGMP-PKG-RyR2 Ser-2808 signaling and independent of muscarinic-induced phosphorylation of Ser-239 in vasodilator-stimulated phosphoprotein. These results provide detailed insights into muscarinic-induced PKG signaling and the mediators that regulate cardiac RyR2 phosphorylation critical for cardiovascular function.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , FosforilaçãoRESUMO
Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor (RyR2). The transfer of Ca2+ from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca2+ leak, via altered SR-mitochondrial Ca2+ transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under ß-adrenergic stimulation. Expression of mitochondrial Ca2+ probe mtRCamp1h revealed diminished net mitochondrial [Ca2+] with enhanced SR Ca2+ leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca2+ overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca2+ release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca2+ uniporter (MCU) supported the hypothesis that SR-mitochondria Ca2+ transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca2+ handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Feminino , Cardiopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-DawleyRESUMO
AIMS: Bradycardia contributes to tachy-brady arrhythmias or sinus arrest during heart failure (HF). Sinoatrial node (SAN) adenosine A1 receptors (ADO A1Rs) are upregulated in HF, and adenosine is known to exert negative chronotropic effects on the SAN. Here, we investigated the role of A1R signaling at physiologically relevant ADO concentrations on HF SAN pacemaker cells. MAIN METHODS: Dogs with tachypacing-induced chronic HF and normal controls (CTL) were studied. SAN tissue was collected for A1R and GIRK mRNA quantification. SAN cells were isolated for perforated patch clamp recordings and firing rate (bpm), slope of slow diastolic depolarization (SDD), and maximum diastolic potential (MDP) were measured. Action potentials (APs) and currents were recorded before and after addition of 1 and 10⯵M ADO. To assess contributions of A1R and G protein-coupled Inward Rectifier Potassium Current (GIRK) to ADO effects, APs were measured after the addition of DPCPX (selective A1R antagonist) or TPQ (selective GIRK blocker). KEY FINDINGS: A1R and GIRK mRNA expression were significantly increased in HF. In addition, ADO induced greater rate slowing and membrane hyperpolarization in HF vs CTL (pâ¯<â¯0.05). DPCPX prevented ADO-induced rate slowing in CTL and HF cells. The ADO-induced inward rectifying current, IKado, was observed significantly more frequently in HF than in CTL. TPQ prevented ADO-induced rate slowing in HF. SIGNIFICANCE: An increase in A1R and GIRK expression enhances IKAdo, causing hyperpolarization, and subsequent negative chronotropic effects in canine chronic HF at relevant [ADO]. GIRK blockade may be a useful strategy to mitigate bradycardia in HF.
Assuntos
Agonistas do Receptor A1 de Adenosina/farmacologia , Adenosina/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/agonistas , Insuficiência Cardíaca/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Receptor A1 de Adenosina/metabolismo , Nó Sinoatrial/citologia , Nó Sinoatrial/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Antagonistas do Receptor A1 de Adenosina/farmacologia , Animais , Venenos de Abelha/farmacologia , Relógios Biológicos , Doença Crônica , Cães , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/efeitos dos fármacos , Técnicas In Vitro , Masculino , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Receptor A1 de Adenosina/efeitos dos fármacos , Xantinas/farmacologiaRESUMO
BACKGROUND: We previously reported that secreted frizzled-related protein-2 (SFRP2) is expressed in a variety of tumors, including sarcoma and breast carcinoma, and stimulates angiogenesis and inhibits tumor apoptosis. Therefore, we hypothesized that a humanized SFRP2 monoclonal antibody (hSFRP2 mAb) would inhibit tumor growth. METHODS: The lead hSFRP2 antibody was tested against a cohort of 22 healthy donors using a time course T-cell assay to determine the relative risk of immunogenicity. To determine hSFRP2 mAb efficacy, nude mice were subcutaneously injected with SVR angiosarcoma cells and treated with hSFRP2 mAb 4 mg/kg intravenously every 3 days for 3 weeks. We then injected Hs578T triple-negative breast cells into the mammary fat pad of nude mice and treated for 40 days. Control mice received an immunoglobulin (Ig) G1 control. The SVR and Hs578T tumors were then stained using a TUNEL assay to detect apoptosis. RESULTS: Immunogenicity testing of hSFRP2 mAb did not induce proliferative responses using a simulation index (SI) ≥ 2.0 (p < 0.05) threshold in any of the healthy donors. SVR angiosarcoma tumor growth was inhibited in vivo, evidenced by significant tumor volume reduction in the hSFRP2 mAb-treated group, compared with controls (n = 10, p < 0.001). Likewise, Hs578T triple-negative breast tumors were smaller in the hSFRP2 mAb-treated group compared with controls (n = 10, p < 0.001). The hSFRP2 mAb treatment correlated with an increase in tumor cell apoptosis (n = 11, p < 0.05). Importantly, hSFRP2 mAb treatment was not associated with any weight loss or lethargy. CONCLUSION: We present a novel hSFRP2 mAb with therapeutic potential in breast cancer and sarcoma that has no effect on immunogenicity.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Apoptose , Hemangiossarcoma/tratamento farmacológico , Proteínas de Membrana/imunologia , Neovascularização Patológica/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/biossíntese , Proliferação de Células , Feminino , Hemangiossarcoma/metabolismo , Hemangiossarcoma/patologia , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Store-operated Ca2+ entry (SOCE), a major Ca2+ signaling mechanism in non-myocyte cells, has recently emerged as a component of Ca2+ signaling in cardiac myocytes. Though it has been reported to play a role in cardiac arrhythmias and to be upregulated in cardiac disease, little is known about the fundamental properties of cardiac SOCE, its structural underpinnings or effector targets. An even greater question is how SOCE interacts with canonical excitation-contraction coupling (ECC). We undertook a multiscale structural and functional investigation of SOCE in cardiac myocytes from healthy mice (wild type; WT) and from a genetic murine model of arrhythmic disease (catecholaminergic ventricular tachycardia; CPVT). Here we provide the first demonstration of local, transient Ca2+ entry (LoCE) events, which comprise cardiac SOCE. Although infrequent in WT myocytes, LoCEs occurred with greater frequency and amplitude in CPVT myocytes. CPVT myocytes also evidenced characteristic arrhythmogenic spontaneous Ca2+ waves under cholinergic stress, which were effectively prevented by SOCE inhibition. In a surprising finding, we report that both LoCEs and their underlying protein machinery are concentrated at the intercalated disk (ID). Therefore, localization of cardiac SOCE in the ID compartment has important implications for SOCE-mediated signaling, arrhythmogenesis and intercellular mechanical and electrical coupling in health and disease.
Assuntos
Arritmias Cardíacas/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Acoplamento Excitação-Contração , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Proteína ORAI1/metabolismo , Retículo Sarcoplasmático/metabolismo , Molécula 1 de Interação Estromal/metabolismoRESUMO
Recent evidence suggests that neuronal Na+ channels (nNavs) contribute to catecholamine-promoted delayed afterdepolarizations (DADs) and catecholaminergic polymorphic ventricular tachycardia (CPVT). The newly identified overlap between CPVT and long QT (LQT) phenotypes has stoked interest in the cross-talk between aberrant Na+ and Ca2+ handling and its contribution to early afterdepolarizations (EADs) and DADs. Here, we used Ca2+ imaging and electrophysiology to investigate the role of Na+ and Ca2+ handling in DADs and EADs in wild-type and cardiac calsequestrin (CASQ2)-null mice. In experiments, repolarization was impaired using 4-aminopyridine (4AP), whereas the L-type Ca2+ and late Na+ currents were augmented using Bay K 8644 (BayK) and anemone toxin II (ATX-II), respectively. The combination of 4AP and isoproterenol prolonged action potential duration (APD) and promoted aberrant Ca2+ release, EADs, and DADs in wild-type cardiomyocytes. Similarly, BayK in the absence of isoproterenol induced the same effects in CASQ2-null cardiomyocytes. In vivo, it prolonged the QT interval and, upon catecholamine challenge, precipitated wide QRS polymorphic ventricular tachycardia that resembled human torsades de pointes. Treatment with ATX-II produced similar effects at both the cellular level and in vivo. Importantly, nNav inhibition with riluzole or 4,9-anhydro-tetrodotoxin reduced the incidence of ATX-II-, BayK-, or 4AP-induced EADs, DADs, aberrant Ca2+ release, and VT despite only modestly mitigating APD prolongation. These data reveal the contribution of nNaVs to triggered arrhythmias in murine models of LQT and CPVT-LQT overlap phenotypes. We also demonstrate the antiarrhythmic impact of nNaV inhibition, independent of action potential and QT interval duration, and provide a basis for a mechanistically driven antiarrhythmic strategy.
Assuntos
Síndrome do QT Longo/metabolismo , Potenciais da Membrana , Canais de Sódio/metabolismo , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Calsequestrina/genética , Células Cultivadas , Síndrome do QT Longo/genética , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologiaRESUMO
BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin (CASQ2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral-mediated delivery to alleviate arrhythmias in non-CaM-related CPVT. METHODS AND RESULTS: To that end, we have designed a CaM protein (GSH-M37Q; dubbed as therapeutic CaM or T-CaM) that exhibited a slowed N-terminal Ca dissociation rate and prolonged RyR2 refractoriness in permeabilized myocytes derived from CPVT mice carrying the CASQ2 mutation R33Q. This T-CaM was introduced to the heart of R33Q mice through recombinant adeno-associated viral vector serotype 9. Eight weeks postinfection, we performed confocal microscopy to assess Ca handling and recorded surface ECGs to assess susceptibility to arrhythmias in vivo. During catecholamine stimulation with isoproterenol, T-CaM reduced isoproterenol-promoted diastolic Ca waves in isolated CPVT cardiomyocytes. Importantly, T-CaM exposure abolished ventricular tachycardia in CPVT mice challenged with catecholamines. CONCLUSIONS: Our results suggest that gene transfer of T-CaM by adeno-associated viral vector serotype 9 improves myocyte Ca handling and alleviates arrhythmias in a calsequestrin-associated CPVT model, thus supporting the potential of a CaM-based antiarrhythmic approach as a therapeutic avenue for genetically distinct forms of CPVT.
Assuntos
Calmodulina/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Frequência Cardíaca , Taquicardia Ventricular/terapia , Animais , Sinalização do Cálcio , Calmodulina/biossíntese , Calsequestrina/deficiência , Calsequestrina/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Fenótipo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologiaRESUMO
TRAF3-interacting protein 3 (TRAF3IP3) is expressed in the immune system and participates in cell maturation, tissue development, and immune response. In a previous study, we reported that TRAF3IP3 levels were substantially increased in the vasculature of breast cancer tissues, suggesting a proangiogenic role. In this study, we investigated TRAF3IP3 tumorigenic function. TRAF3IP3 protein was present in several cancer cell lines, with highest levels in melanoma. In addition, tumor microarray analysis on 23 primary melanoma and nine positive lymph nodes revealed that 70% of human primary melanoma and 66% of lymph node metastases were positive for TRAF3IP3. Importantly, TRAF3IP3 downregulation correlated with an 83% reduction of tumor growth in a subcutaneous xenograft mouse model (n=10, P=0.005). Immunohistochemistry analysis of the tumors revealed that TRAF3IP3-shRNA tumors had increased apoptosis (n=4, P<0.01) and reduced microvascular density (n=4, P<0.002). In addition, TRAF3IP3 downregulation in malignant endothelial cells reduced tube formation in a Matrigel tube formation assay. In melanoma cells, decreased levels of TRAF3IP3 were also associated with reduced viability (n=4, P=0.03) and proliferation (n=3, P=0.03), together with increased sensitivity to ultraviolet-induced apoptosis (n=4, P=0.0004). Furthermore, TRAF3IP3 downregulation correlated with increased amounts of interferon-γ. Interferon-γ inhibits tumor growth and angiogenesis, thus suggesting a new pathway for TRAF3IP3 in cancer. Collectively, the association of TRAF3IP3 with malignant properties of melanoma suggest a clinical potential for targeted therapy.
Assuntos
Melanoma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Xenoenxertos , Imuno-Histoquímica , Interferon gama/metabolismo , Masculino , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Análise Serial de Tecidos , TransfecçãoRESUMO
Secreted frizzled-related protein 2 (SFRP2) is a pro-angiogenic factor expressed in the vasculature of a wide variety of human tumors, and modulates angiogenesis via the calcineurin-dependent nuclear factor of activated T-cells cytoplasmic 3 (NFATc3) pathway in endothelial cells. However, until now, SFRP2 receptor for this pathway was unknown. In the present study, we first used amino acid alignments and molecular modeling to demonstrate that SFRP2 interaction with frizzled-5 (FZD5) is typical of Wnt/FZD family members. To confirm this interaction, we performed co-immunofluorescence, co-immunoprecipitation, and ELISA binding assays, which demonstrated SFRP2/FZD5 binding. Functional knock-down studies further revealed that FZD5 is necessary for SFRP2-induced tube formation and intracellular calcium flux in endothelial cells. Using protein analysis on endothelial cell nuclear extracts, we also discovered that FZD5 is required for SFRP2-induced activation of NFATc3. Our novel findings reveal that FZD5 is a receptor for SFRP2 and mediates SFRP2-induced angiogenesis via calcineurin/NFATc3 pathway in endothelial cells.
Assuntos
Receptores Frizzled/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Transcrição NFATC/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Animais , Neoplasias da Mama/patologia , Cálcio/metabolismo , Linhagem Celular , Movimento Celular , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Espaço Intracelular/metabolismo , Camundongos , Ligação Proteica , Homologia Estrutural de ProteínaRESUMO
In heart failure (HF), dysregulated cardiac ryanodine receptors (RyR2) contribute to the generation of diastolic Ca2+ waves (DCWs), thereby predisposing adrenergically stressed failing hearts to life-threatening arrhythmias. However, the specific cellular, subcellular, and molecular defects that account for cardiac arrhythmia in HF remain to be elucidated. Patch-clamp techniques and confocal Ca2+ imaging were applied to study spatially defined Ca2+ handling in ventricular myocytes isolated from normal (control) and failing canine hearts. Based on their activation time upon electrical stimulation, Ca2+ release sites were categorized as coupled, located in close proximity to the sarcolemmal Ca2+ channels, and uncoupled, the Ca2+ channel-free non-junctional Ca2+ release units. In control myocytes, stimulation of ß-adrenergic receptors with isoproterenol (Iso) resulted in a preferential increase in Ca2+ spark rate at uncoupled sites. This site-specific effect of Iso was eliminated by the phosphatase inhibitor okadaic acid, which caused similar facilitation of Ca2+ sparks at coupled and uncoupled sites. Iso-challenged HF myocytes exhibited increased predisposition to DCWs compared to control myocytes. In addition, the overall frequency of Ca2+ sparks was increased in HF cells due to preferential stimulation of coupled sites. Furthermore, coupled sites exhibited accelerated recovery from functional refractoriness in HF myocytes compared to control myocytes. Spatially resolved subcellular Ca2+ mapping revealed that DCWs predominantly originated from coupled sites. Inhibition of CaMKII suppressed DCWs and prevented preferential stimulation of coupled sites in Iso-challenged HF myocytes. These results suggest that CaMKII- (and phosphatase)-dependent dysregulation of junctional Ca2+ release sites contributes to Ca2+-dependent arrhythmogenesis in HF.
Assuntos
Arritmias Cardíacas/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Frequência Cardíaca , Microdomínios da Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Função Ventricular Esquerda , Agonistas Adrenérgicos beta/farmacologia , Animais , Arritmias Cardíacas/fisiopatologia , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Estimulação Cardíaca Artificial , Diástole , Modelos Animais de Doenças , Cães , Feminino , Insuficiência Cardíaca/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Masculino , Potenciais da Membrana , Miócitos Cardíacos/efeitos dos fármacos , Período Refratário Eletrofisiológico , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sarcolema/metabolismo , Sus scrofa , Fatores de Tempo , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
BACKGROUND: Particulate matter (PM; PM2.5 [PM with diameters of <2.5 µm]) exposure during development is strongly associated with adverse cardiovascular outcomes at adulthood. In the present study, we tested the hypothesis that in utero PM2.5 exposure alone could alter cardiac structure and function at adulthood. METHODS AND RESULTS: Female FVB mice were exposed either to filtered air or PM2.5 at an average concentration of 73.61 µg/m3 for 6 h/day, 7 days/week throughout pregnancy. After birth, animals were analyzed at 12 weeks of age. Echocardiographic (n=9-10 mice/group) and pressure-volume loop analyses (n=5 mice/group) revealed reduced fractional shortening, increased left ventricular end-systolic and -diastolic diameters, reduced left ventricular posterior wall thickness, end-systolic elastance, contractile reserve (dP/dtmax/end-systolic volume), frequency-dependent acceleration of relaxation), and blunted contractile response to ß-adrenergic stimulation in PM2.5-exposed mice. Isolated cardiomyocyte (n=4-5 mice/group) function illustrated reduced peak shortening, ±dL/dT, and prolonged action potential duration at 90% repolarization. Histological left ventricular analyses (n=3 mice/group) showed increased collagen deposition in in utero PM2.5-exposed mice at adulthood. Cardiac interleukin (IL)-6, IL-1ß, collagen-1, matrix metalloproteinase (MMP) 9, and MMP13 gene expressions were increased at birth in in utero PM2.5-exposed mice (n=4 mice/group). In adult hearts (n=5 mice/group), gene expressions of sirtuin (Sirt) 1 and Sirt2 were decreased, DNA methyltransferase (Dnmt) 1, Dnmt3a, and Dnmt3b were increased, and protein expression (n=6 mice/group) of Ca2+-ATPase, phosphorylated phospholamban, and Na+/Ca2+ exchanger were decreased. CONCLUSIONS: In utero PM2.5 exposure triggers an acute inflammatory response, chronic matrix remodeling, and alterations in Ca2+ handling proteins, resulting in global adult cardiac dysfunction. These results also highlight the potential involvement of epigenetics in priming of adult cardiac disease.
Assuntos
Arritmias Cardíacas/induzido quimicamente , Remodelamento Atrial/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Insuficiência Cardíaca/induzido quimicamente , Exposição por Inalação/efeitos adversos , Exposição Materna/efeitos adversos , Material Particulado/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Função Ventricular Esquerda/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Fatores Etários , Animais , Animais Recém-Nascidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Feminino , Idade Gestacional , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Tamanho da Partícula , Fosforilação , Gravidez , Sirtuína 1/genética , Sirtuína 1/metabolismo , Sirtuína 2/genética , Sirtuína 2/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Remodelação Ventricular/efeitos dos fármacos , DNA Metiltransferase 3BRESUMO
Connexin 43 (Cx43) is a gap junction protein whose function in the development of breast cancer and in breast cancer progression remains unclear. Evidence suggests that Cx43 (GJA1) mRNA and protein expression is altered in breast tumors. However, reports indicate both increased and decreased Cx43 levels in human breast cancer samples. Studies also suggest that loss of Cx43 regulated gap junction intercellular communication is a common feature of breast malignancies that potentially correlates with histological stage. Further evidence suggests that Cx43 (GJA1) mRNA expression is negatively correlated with HER2 positivity but a relationship between Cx43 and HER2 in breast cancer is not well defined. Therefore, in this study, we sought to evaluate the relationship between Cx43 activity, HER2, and drug resistance. Using HER2+ breast cancer cell lines that are sensitive or resistant to HER2 inhibitor, we evaluated Cx43 gap junction function. We found that Cx43 gap junction activity is completely lost in drug resistant HER2-positive (HER2+) breast cancer cells, whereas Cx43 gap junction activity can be restored by Cx43 overexpression in drug sensitive HER2+ cells. Moreover, the dysregulation of Cx43 resulted in increased tumorigenic and migratory capacity of the HER2+ drug resistant breast cancer cells.
RESUMO
Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Muscarínicos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Células Cultivadas , Colinérgicos/farmacologia , Modelos Animais de Doenças , Cães , Acoplamento Excitação-Contração/fisiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: Over the last 40 years omega-3 polyunsaturated fatty acids (PUFAs) have been shown to be anti-arrhythmic or pro-arrhythmic depending on the method and duration of administration and model studied. We previously reported that omega-3 PUFAs do not confer anti-arrhythmic properties and are pro-arrhythmic in canine model of sudden cardiac death (SCD). Here, we evaluated the effects of chronic omega-3 PUFA treatment in post-MI animals susceptible (VF+) or resistant (VF-) to ventricular tachyarrhythmias. METHODS: Perforated patch clamp techniques were used to measure cardiomyocyte action potential durations (APD) at 50 and 90% repolarization and short term variability of repolarization. The early repolarizing transient outward potassium current Ito was also studied. RESULTS: Omega-3 PUFAs prolonged the action potential in VF- myocytes at both 50 and 90% repolarization. Short term variability of repolarization was increased in both untreated and treated VF- myocytes vs. CONTROLS: Ito was unaffected by omega-3 PUFA treatment. Omega-3 PUFA treatment attenuated the action potential prolongation in VF+ myocytes, but did not return repolarization to control values. CONCLUSIONS: Omega-3 PUFAs do not confer anti-arrhythmic properties in the setting of healed myocardial infarction in a canine model of SCD. In canines previously resistant to ventricular fibrillation (VF-), omega-3 PUFA treatment prolonged the action potential in VF- myocytes, and may contribute to pro-arrhythmic responses.
RESUMO
OBJECTIVES: To evaluate the clinical efficacy of magnetic resonance-guided focused ultrasound surgery in a Mexican mestizo population. METHODS: This retrospective study included 159 women (mean age 37 ± 6.4 years, range 22-53 years) from 2008 to 2010. Two hundred sixty-eight symptomatic uterine fibroids were treated using MR-guided focused ultrasound surgery. Parameters included initial perfused volume, final perfused volume, non-perfused volume (NPV), and treated volume ratio (TVR). Follow-up up to 15 months assessed treatment efficacy and symptomatic relief. Non-parametric statistics and the Kaplan-Meier method were performed. RESULTS: T2-weighted hypointense fibroids showed a frequency of 93.6%; isointense and hyperintense fibroids had frequencies of 5.60 and 1.1%. There was a negative correlation between NPV and age (r = -0.083, p = 0.307) and treatment time (r = -0.253, p = 0.001). Median TVR was 96.0% in small fibroids and 76.5% in large fibroids. Involution of 50% and 80% was achieved at months 6-7 and month 11, respectively. Relief of symptoms was significant (p < 0.05). CONCLUSIONS: Our data show that higher TVR attained immediately post-treatment of MRgFUS favours higher involution percentages at follow-up; however, careful patient selection and use of pretreatment imaging are important components for predicting success using MR-guided focused ultrasound surgery. KEY POINTS: ⢠Type 1 fibroids were the most common (93.2%). ⢠Age and treated volume were not correlated (r s = -0.215, p = 0.165). ⢠Small fibroids achieved a higher treated volume than large (96.0% vs. 76.5%). ⢠A 50% involution was achieved at 6-month follow-up for type-1 fibroid. ⢠A decrease of 80% was reached at 11 months for type-1 fibroids.