RESUMO
The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current INa and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on INa We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.
Assuntos
Endossomos/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Potenciais de Ação , Animais , Estudo de Associação Genômica Ampla , Humanos , Miócitos Cardíacos/citologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Proteínas Nucleares/genética , Ligação Proteica , Transporte Proteico , Coelhos , Fatores de Transcrição/genética , Ubiquitinação , Peixe-ZebraRESUMO
The QT interval is an important diagnostic feature on surface electrocardiograms because it reflects the duration of the ventricular action potential. A previous genome-wide association study has reported a significant linkage between a single-nucleotide polymorphism â¼11.7 kb downstream of the gene encoding the RING finger ubiquitin ligase rififylin (RFFL) and variability in the QT interval. This, along with results in animal studies, suggests that RFFL may have effects on cardiac repolarization. Here, we sought to determine the role of RFFL in cardiac electrophysiology. Adult rabbit cardiomyocytes with adenovirus-expressed RFFL exhibited reduced rapid delayed rectifier current (IKr). Neonatal rabbit cardiomyocytes transduced with RFFL-expressing adenovirus exhibited reduced total expression of the potassium channel ether-a-go-go-related gene (rbERG). Using transfections of 293A cells and Western blotting experiments, we observed that RFFL and the core-glycosylated form of the human ether-a-go-go-related gene (hERG) potassium channel interact. Furthermore, RFFL overexpression led to increased polyubiquitination and proteasomal degradation of hERG protein and to an almost complete disappearance of IKr, which depended on the intact RING domain of RFFL. Blocking the ER-associated degradation (ERAD) pathway with a dominant-negative form of the ERAD core component, valosin-containing protein (VCP), in 293A cells partially abolished RFFL-mediated hERG degradation. We further substantiated the link between RFFL and ERAD by showing an interaction between RFFL and VCP in vitro We conclude that RFFL is an important regulator of voltage-gated hERG potassium channel activity and therefore cardiac repolarization and that this ubiquitination-mediated regulation requires parts of the ERAD pathway.
Assuntos
Canal de Potássio ERG1/metabolismo , Degradação Associada com o Retículo Endoplasmático , Miócitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Canal de Potássio ERG1/genética , Células HEK293 , Humanos , Transporte Proteico , Coelhos , Ubiquitina-Proteína Ligases/genéticaRESUMO
Two recent studies (Newton-Cheh, C. et al. (2009) Common variants at ten loci influence QT interval duration in the QTGEN Study. Nat. Genet. 41, 399-406 and Pfeufer, A. et al. (2009) Common variants at ten loci modulate the QT interval duration in the QTSCD Study. Nat. Genet. 41, 407-414) identified an association, with genome-wide significance, between a single nucleotide polymorphism within the gene encoding RING finger protein 207 (RNF207) and the QT interval. We sought to determine the role of RNF207 in cardiac electrophysiology. Morpholino knockdown of RNF207 in zebrafish embryos resulted in action potential duration prolongation, occasionally a 2:1 atrioventricular block, and slowing of conduction velocity. Conversely, neonatal rabbit cardiomyocytes infected with RNF207-expressing adenovirus exhibited shortened action potential duration. Using transfections of U-2 OS and HEK293 cells, Western blot analysis and immunocytochemistry data demonstrate that RNF207 and the human ether-a-go-go-related gene (HERG) potassium channel interact and colocalize. Furthermore, RNF207 overexpression significantly elevated total and membrane HERG protein and HERG-encoded current density by â¼30-50%, which was dependent on the intact N-terminal RING domain of RNF207. Finally, coexpression of RNF207 and HSP70 increased HERG expression compared with HSP70 alone. This effect was dependent on the C terminus of RNF207. Taken together, the evidence is strong that RNF207 is an important regulator of action potential duration, likely via effects on HERG trafficking and localization in a heat shock protein-dependent manner.
Assuntos
Bloqueio Atrioventricular/genética , Canais de Potássio Éter-A-Go-Go/genética , Proteínas de Choque Térmico HSP70/genética , Coração/fisiologia , Miócitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligases/genética , Potenciais de Ação/genética , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Bloqueio Atrioventricular/metabolismo , Bloqueio Atrioventricular/fisiopatologia , Canal de Potássio ERG1 , Embrião não Mamífero , Canais de Potássio Éter-A-Go-Go/metabolismo , Acoplamento Excitação-Contração , Regulação da Expressão Gênica , Vetores Genéticos , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Coração/embriologia , Coração/fisiopatologia , Humanos , Dados de Sequência Molecular , Morfolinos , Miócitos Cardíacos/patologia , Estrutura Terciária de Proteína , Coelhos , Ubiquitina-Proteína Ligases/metabolismo , Peixe-ZebraRESUMO
We recently showed that progesterone treatment abolished arrhythmias and sudden cardiac death in a transgenic rabbit model of long QT syndrome type 2 (LQT2). Moreover, levels of cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase type 2a (SERCA2a) were upregulated in LQT2 heart extracts. We hypothesized that progesterone treatment upregulated SERCA2a expression, thereby reducing Ca(2+)-dependent arrhythmias in LQT2 rabbits. We therefore investigated the effect of progesterone on SERCA2a regulation in isolated cardiomyocytes. Cardiomyocytes from neonatal (3- to 5-day-old) rabbits were isolated, cultured, and treated with progesterone and other pharmacological agents. Immunoblotting was performed on total cell lysates and sarcoplasmic reticulum-enriched membrane fractions for protein abundance, and mRNA transcripts were quantified using real-time PCR. The effect of progesterone on baseline Ca(2+) transients and Ca(2+) clearance was determined using digital imaging. Progesterone treatment increased the total pool of SERCA2a protein by slowing its degradation. Using various pharmacological inhibitors of degradation pathways, we showed that progesterone-associated degradation of SERCA2a involves ubiquitination, and progesterone significantly decreases the levels of ubiquitin-tagged SERCA2a polypeptides. Our digital imaging data revealed that progesterone significantly shortened the decay and duration of Ca(2+) transients. Progesterone treatment increases protein levels and activity of SERCA2a. Progesterone stabilizes SERCA2a, in part, by decreasing the ubiquitination level of SERCA2a polypeptides.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Progesterona/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Ubiquitinação/efeitos dos fármacosRESUMO
Necrotizing enterocolitis (NEC) is a complex, multifactorial gastrointestinal disorder predominantly affecting preterm infants. The pathogenesis of this condition involves a complex interplay between intestinal barrier dysfunction, microbial dysbiosis, and an altered immune response. This study investigates the potential role of endogenous hyaluronan (HA) in both the early phases of intestinal development and in the context of NEC-like intestinal injury. We treated neonatal CD-1 mouse pups with PEP1, a peptide inhibiting HA receptor interactions, from postnatal days 8 to 12. We evaluated postnatal intestinal developmental indicators, such as villi length, crypt depth, epithelial cell proliferation, crypt fission, and differentiation of goblet and Paneth cells, in PEP1-treated animals compared with those treated with scrambled peptide. PEP1 treatment significantly impaired intestinal development, as evidenced by reductions in villi length, crypt depth, and epithelial cell proliferation, along with a decrease in crypt fission activity. These deficits in PEP1-treated animals correlated with increased susceptibility to NEC-like injuries, including higher mortality rates, and worsened histological intestinal injury. These findings highlight the role of endogenous HA in supporting intestinal development and protecting against NEC.
Assuntos
Enterocolite Necrosante , Homeostase , Ácido Hialurônico , Intestinos , Animais , Ácido Hialurônico/farmacologia , Ácido Hialurônico/metabolismo , Enterocolite Necrosante/patologia , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/tratamento farmacológico , Camundongos , Homeostase/efeitos dos fármacos , Intestinos/patologia , Intestinos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Animais Recém-Nascidos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos dos fármacos , Modelos Animais de DoençasRESUMO
BACKGROUND AND PURPOSE: In human airway smooth muscle (hASM) cells, not all receptors stimulating cAMP production elicit the same effects. This can only be explained if cAMP movement throughout the cell is restricted, yet the mechanisms involved are not fully understood. Phosphodiesterases (PDEs) contribute to compartmentation of many cAMP responses, but PDE activity alone is predicted to be insufficient if cAMP is otherwise freely diffusible. We tested the hypothesis that buffering of cAMP by protein kinase A (PKA) associated with A kinase anchoring proteins (AKAPs) slows cAMP diffusion and that this contributes to receptor-mediated, compartmentalized responses. EXPERIMENTAL APPROACH: Raster image correlation spectroscopy (RICS) was used to measure intracellular cAMP diffusion coefficients and evaluate the contribution of PKA-AKAP interactions. Western blotting and immunocytochemistry were used to identify the AKAPs involved. RNA interference was used to down-regulate AKAP expression and determine its effects on cAMP diffusion. Compartmentalized cAMP responses were measured using fluorescence resonance energy transfer (FRET) based biosensors. KEY RESULTS: Cyclic AMP movement was significantly slower than that of free-diffusion in hASM cells, and disrupting PKA-AKAP interactions significantly increased the diffusion coefficient. PKA associated with the outer mitochondrial membrane appears to play a prominent role in this effect. Consistent with this idea, knocking down expression of D-AKAP2, the primary mitochondrial AKAP, increased cAMP diffusion and disrupted compartmentation of receptor-mediated responses. CONCLUSION AND IMPLICATIONS: Our results confirm that AKAP-anchored PKA contributes to the buffering of cAMP and is consequential in the compartmentation of cAMP responses in hASM cells.
Assuntos
Proteínas de Ancoragem à Quinase A , Proteínas Quinases Dependentes de AMP Cíclico , AMP Cíclico , Miócitos de Músculo Liso , Transdução de Sinais , Humanos , AMP Cíclico/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miócitos de Músculo Liso/metabolismo , Células Cultivadas , Difusão , Transferência Ressonante de Energia de FluorescênciaRESUMO
Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes.
Assuntos
Western Blotting , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Lentivirus , RNA Interferente Pequeno , Humanos , Lentivirus/genética , Citometria de Fluxo/métodos , Técnicas de Silenciamento de Genes/métodos , RNA Interferente Pequeno/genética , Organoides/metabolismo , Vetores Genéticos/genéticaRESUMO
KCNQ1 and hERG encode the voltage-gated potassium channel α-subunits of the cardiac repolarizing currents I(Ks) and I(Kr), respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs), and loss-of-function mutations in these channels manifest clinically as long QT syndrome, characterized by the prolongation of the QT interval, polymorphic ventricular tachycardia, and sudden cardiac death. Previous cellular electrophysiology experiments in transgenic rabbit cardiomyocytes and heterologous cell lines demonstrated functional downregulation of complementary repolarizing currents. Biochemical assays indicated direct, protein-protein interactions between KCNQ1 and hERG may underlie the interplay between I(Ks) and I(Kr). Our objective was to investigate hERG-KCNQ1 interactions in the intact cellular environment primarily through acceptor photobleach FRET (apFRET) experiments. We quantitatively assessed the extent of interactions based on fluorophore location and the potential regulation of interactions by physiologically relevant signals. apFRET experiments established specific hERG-KCNQ1 associations in both heterologous and primary cardiomyocytes. The largest FRET efficiency (E(f); 12.0 ± 5.2%) was seen between ion channels with GFP variants fused to the COOH termini. Acute treatment with forskolin + IBMX or a membrane-permeable cAMP analog significantly and specifically reduced the extent of hERG-KCNQ1 interactions (by 41 and 38%, respectively). Our results demonstrate direct interactions between KCNQ1 and hERG occur in both intact heterologous cells and primary cardiomyocytes and are mediated by their COOH termini. Furthermore, this interplay between channel proteins is regulated by intracellular cAMP.
Assuntos
AMP Cíclico/química , Canais de Potássio Éter-A-Go-Go/química , Canal de Potássio KCNQ1/química , 1-Metil-3-Isobutilxantina/administração & dosagem , Potenciais de Ação/fisiologia , Animais , Células CHO , Células Cultivadas , Colforsina/administração & dosagem , Cricetinae , Cricetulus , AMP Cíclico/agonistas , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/fisiologia , Feminino , Células HEK293 , Coração/efeitos dos fármacos , Coração/fisiologia , Humanos , Canal de Potássio KCNQ1/fisiologia , Masculino , Inibidores de Fosfodiesterase/administração & dosagem , CoelhosRESUMO
[This corrects the article DOI: 10.3389/fphys.2021.672360.].
RESUMO
Activation of different receptors that act by generating the common second messenger cyclic adenosine monophosphate (cAMP) can elicit distinct functional responses in cardiac myocytes. Selectively sequestering cAMP activity to discrete intracellular microdomains is considered essential for generating receptor-specific responses. The processes that control this aspect of compartmentalized cAMP signaling, however, are not completely clear. Over the years, technological innovations have provided critical breakthroughs in advancing our understanding of the mechanisms underlying cAMP compartmentation. Some of the factors identified include localized production of cAMP by differential distribution of receptors, localized breakdown of this second messenger by targeted distribution of phosphodiesterase enzymes, and limited diffusion of cAMP by protein kinase A (PKA)-dependent buffering or physically restricted barriers. The aim of this review is to provide a discussion of our current knowledge and highlight some of the gaps that still exist in the field of cAMP compartmentation in cardiac myocytes.
Assuntos
AMP Cíclico , Miócitos Cardíacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Compartmentation of cAMP signaling is a critical factor for maintaining the integrity of receptor-specific responses in cardiac myocytes. This phenomenon relies on various factors limiting cAMP diffusion. Our previous work in adult rat ventricular myocytes (ARVMs) indicates that PKA regulatory subunits anchored to the outer membrane of mitochondria play a key role in buffering the movement of cytosolic cAMP. PKA can be targeted to discrete subcellular locations through the interaction of both type I and type II regulatory subunits with A-kinase anchoring proteins (AKAPs). The purpose of this study is to identify which AKAPs and PKA regulatory subunit isoforms are associated with mitochondria in ARVMs. Quantitative PCR data demonstrate that mRNA for dual specific AKAP1 and 2 (D-AKAP1 & D-AKAP2), acyl-CoA-binding domain-containing 3 (ACBD3), optic atrophy 1 (OPA1) are most abundant, while Rab32, WAVE-1, and sphingosine kinase type 1 interacting protein (SPHKAP) were barely detectable. Biochemical and immunocytochemical analysis suggests that D-AKAP1, D-AKAP2, and ACBD3 are the predominant mitochondrial AKAPs exposed to the cytosolic compartment in these cells. Furthermore, we show that both type I and type II regulatory subunits of PKA are associated with mitochondria. Taken together, these data suggest that D-AKAP1, D-AKAP2, and ACBD3 may be responsible for tethering both type I and type II PKA regulatory subunits to the outer mitochondrial membrane in ARVMs. In addition to regulating PKA-dependent mitochondrial function, these AKAPs may play an important role by buffering the movement of cAMP necessary for compartmentation.
Assuntos
Proteínas de Ancoragem à Quinase A/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , Ventrículos do Coração/enzimologia , Mitocôndrias/enzimologia , Miócitos Cardíacos/enzimologia , Animais , Células Cultivadas , Ventrículos do Coração/citologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Cardiac arrhythmias significantly contribute to cardiovascular morbidity and mortality. The rabbit heart serves as an accepted model system for studying cardiac cell excitation and arrhythmogenicity. Accordingly, primary cultures of adult rabbit ventricular cardiomyocytes serve as a preferable model to study molecular mechanisms of human cardiac excitation. However, the use of adult rabbit cardiomyocytes is often regarded as excessively costly. Therefore, we developed and characterized a novel low-cost rabbit cardiomyocyte model, namely, 3-week-old ventricular cardiomyocytes (3wRbCMs). Ventricular myocytes were isolated from whole ventricles of 3-week-old New Zealand White rabbits of both sexes by standard enzymatic techniques. Using wheat germ agglutinin, we found a clear T-tubule structure in acutely isolated 3wRbCMs. Cells were adenovirally infected (multiplicity of infection of 10) to express Green Fluorescent Protein (GFP) and cultured for 48 h. The cells showed action potential duration (APD90 = 253 ± 24 ms) and calcium transients similar to adult rabbit cardiomyocytes. Freshly isolated and 48-h-old-cultured cells expressed critical ion channel proteins: calcium voltage-gated channel subunit alpha1 C (Cavα1c), sodium voltage-gated channel alpha subunit 5 (Nav1.5), potassium voltage-gated channel subfamily D member 3 (Kv4.3), and subfamily A member 4 (Kv1.4), and also subfamily H member 2 (RERG. Kv11.1), KvLQT1 (K7.1) protein and inward-rectifier potassium channel (Kir2.1). The cells displayed an appropriate electrophysiological phenotype, including fast sodium current (I Na), transient outward potassium current (I to), L-type calcium channel peak current (I Ca,L), rapid and slow components of the delayed rectifier potassium current (I Kr and I Ks), and inward rectifier (I K1). Although expression of the channel proteins and some currents decreased during the 48 h of culturing, we conclude that 3wRbCMs are a new, low-cost alternative to the adult-rabbit-cardiomyocytes system, which allows the investigation of molecular mechanisms of cardiac excitation on morphological, biochemical, genetic, physiological, and biophysical levels.
RESUMO
BACKGROUND AND PURPOSE: In cardiac myocytes, cyclic AMP (cAMP) produced by both ß1 - and ß2 -adrenoceptors increases L-type Ca2+ channel activity and myocyte contraction. However, only cAMP produced by ß1 -adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of ß2 -adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation. EXPERIMENTAL APPROACH: The cAMP responses produced by ß1 -and ß2 -adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex. KEY RESULTS: Selective activation of ß1 - or ß2 -adrenoceptors produced cAMP responses detected by Epac2-camps. However, only stimulation of ß1 -adrenoceptors produced a cAMP response detected by Epac2-αKAP. Yet, stimulation of ß2 -adrenoceptors was able to produce a cAMP signal detected by Epac2-αKAP in the presence of selective inhibitors of PDE2 or PDE3, but not PDE4. CONCLUSION AND IMPLICATIONS: These results support the conclusion that cAMP produced by ß2 -adrenoceptor stimulation was not able to reach subcellular locations where the SERCA2 pump is located. Furthermore, this compartmentalized response is due at least in part to PDE2 and PDE3 activity. This discovery could lead to novel PDE-based therapeutic treatments aimed at correcting cardiac relaxation defects associated with certain forms of heart failure.
Assuntos
AMP Cíclico , Miócitos Cardíacos , Animais , Ventrículos do Coração , Diester Fosfórico Hidrolases , Ratos , Receptores Adrenérgicos beta 1 , Receptores Adrenérgicos beta 2RESUMO
BACKGROUND: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. METHODS: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (ICa,L) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. RESULTS: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. CONCLUSIONS: LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Proteínas de Ligação a DNA/genética , Coração/embriologia , Humanos , Proteínas de Membrana/genética , Miócitos Cardíacos/enzimologia , Ubiquitina-Proteína Ligases Nedd4/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coelhos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinação , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase-9 (MMP-9). Cytochrome P450 (CYP) epoxygenase-2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF-kappabeta. However, the underlying molecular mechanisms for MMP-9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)-induced MMP-9 activation. CYP2J2 was over-expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF-kappabeta-mediated MMP-9 regulation were evaluated using Western blot, in-gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K-dependent AKT signal. Hcy induced the nuclear translocation of NF-kappabeta via downregulation of IKbetaalpha (endogenous cytoplasmic inhibitor of NF-kappabeta). Hcy induced MMP-9 activation by increasing NF-kappabeta-DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9-EET phosphorylated the AKT and attenuated Hcy-induced MMP-9 activation. This occurred, in part, by the inhibition of NF-kappabeta nuclear translocation, NF-kappabeta-DNA binding and activation of IKbetaalpha. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP-9 signal.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hiper-Homocisteinemia/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/farmacologia , Animais , Células Cultivadas , Citocromo P-450 CYP2J2 , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Homocisteína/farmacologia , Hiper-Homocisteinemia/enzimologia , Proteínas I-kappa B/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Inibidor de NF-kappaB alfa , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição RelA/metabolismo , TransfecçãoRESUMO
Chronic volume/pressure overload-induced heart failure augments oxidative stress and activates matrix metalloproteinase which causes endocardial endothelial-myocyte (EM) uncoupling eventually leading to decline in myocardial systolic and diastolic function. The elevated levels of homocysteine (Hcy), hyperhomocysteinemia (HHcy), are associated with decline in cardiac performance. Hcy impairs the EM functions associated with the induction of ventricular hypertrophy leading to cardiac stiffness and diastolic heart failure. Hcy-induced neurological defects are mediated by the NMDA-R (N-methyl-D-aspartate (NMDA) receptor) activation. NMDA-R is expressed in the heart. However, the role of NMDA-R on cardiac function during HHcy is still in its infancy. The blockade of NMDA-R attenuates NMDA-agonist-induced increase in the heart rate. Hcy increases intracellular calcium and activates calpain and calpain-associated mitochondrial (mt) abnormalities have been identified in HHcy. Mitochondrial permeabilization and uncoupling in the pathological setting is fueled by redox stress and calcium mishandling. Recently the role of cyclophilin D, a component of the mitochondrial membrane permeability transition pore, has been identified in cardiac-ischemia. Mechanisms underlying the potentiation between NMDA-R activation and mitochondrial defects leading to cardiac dysfunction during HHcy remain to be elucidated. This review addresses the mitochondrial mechanism by which Hcy contributes to the decline in mechano-electrical function and arrhythmogenesis via agonizing NMDA-R. The putative role of mitochondrial MMP activation, protease stress and mitochondrial permeability transition in cardiac conduction during HHcy is discussed. The review suggests that Hcy increases calcium overload and oxidative stress in the mitochondria and amplifies the activation of mtMMP, causing the opening of mitochondrial permeability transition pore leading to mechano-electrical dysfunction.
Assuntos
Arritmias Cardíacas/metabolismo , Insuficiência Cardíaca/metabolismo , Hiper-Homocisteinemia/metabolismo , Metaloproteinases da Matriz/metabolismo , Mitocôndrias Cardíacas/fisiologia , Animais , Arritmias Cardíacas/complicações , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Ativação Enzimática , Matriz Extracelular/fisiologia , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/fisiopatologia , Homocisteína/fisiologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hiper-Homocisteinemia/complicações , Hiper-Homocisteinemia/fisiopatologia , Mitocôndrias Cardíacas/enzimologia , Miócitos Cardíacos/fisiologia , Estresse Oxidativo , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de SinaisRESUMO
An elevated level of Homocysteine (Hcy) is a risk factor for vascular dementia and stroke. Cysthathionine beta Synthase (CBS) gene is involved in the clearance of Hcy. Homozygous individuals for (CBS-/-) die early, but heterozygous for (CBS-/+) survive with high levels of Hcy. The gamma-Amino Butyric Acid (GABA) presents in the central nervous system (CNS) and functions as an inhibitory neurotransmitter. Hcy competes with GABA at the GABA(A) receptor and affects the CNS function. We hypothesize that Hcy causes a decrease in blood flow to the brain due to increase in vascular resistance (VR) because of arterial remodeling in the carotid artery (CA). Blood pressure and blood flow in CA of wild type (WT), CBS-/+, CBS-/+ GABA(A)-/- double knockout, and GABA(A)-/- were measured. CA was stained with trichrome, and the brain permeability was measured. Matrix Metalloproteinases (MMP-2 and MMP-9), tissue inhibitor of metalloproteinase (TIMP-3, TIMP-4), elastin, and collagen-III expression were measured by real-time polymerase chain reaction (RT-PCR). Results showed an increase in VR in CBS-/+/GABA(A)-/-double knockout>CBS-/+/>GABA(A)-/- compared to WT mice. Increased MMP-2, MMP-9, collagen-III and TIMP-3 mRNA levels were found in GABA(A)-/-, CBS-/+, CBS-/+/GABA(A) double knockout compared to WT. The levels of TIMP-4 and elastin were decreased, whereas the levels of MMP-2, MMP-9 and TIMP-3 increased, which indirectly reflected the arterial resistance. These results suggested that Hcy caused arterial remodeling in part, by increase in collagen/elastin ratio thereby increasing VR leading to the decrease in CA blood flow.
Assuntos
Isquemia Encefálica/fisiopatologia , Artérias Carótidas/fisiopatologia , Circulação Cerebrovascular/fisiologia , Colágeno/metabolismo , Elastina/metabolismo , Homocisteína/metabolismo , Animais , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/metabolismo , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Circulação Cerebrovascular/efeitos dos fármacos , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Cistationina beta-Sintase/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Elastina/genética , Homocisteína/toxicidade , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de GABA-A/genética , Inibidores Teciduais de Metaloproteinases/genética , Inibidores Teciduais de Metaloproteinases/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Resistência Vascular , Ácido gama-Aminobutírico/metabolismoRESUMO
Individuals with homozygous deficiency in cystathionine-beta-synthase (CBS) develop high levels of homocysteine in plasma, a condition known as homocysteinuria. Mental retardation ensues with death in teens; the heterozygous live normally but develop vascular dementia and Alzheimer's disease (AD) in later part of life. The treatment with muscimol, a gamma amino butyric acid receptor-A (GABA(A)) agonist, mitigates the AD syndrome and vascular dementia. We tested the hypothesis that homocysteine (Hcy) antagonizes the GABA(A) receptor and behaves as an excitotoxic neurotransmitter that causes blood brain barrier (BBB) permeability and vascular dementia. The BBB permeability was measured by infusing Evan's blue dye (2% in saline 5 ml/kg concentration) in CBS-/+, GABA(A)-/-, CBS-/+/GABA(A)-/- double knockout, CBS-/+ mice treated with muscimol and wild type (WT) mice. Matrix Metalloproteinase (MMP-2, MMP-9), Tissue Inhibitor of Matrix Metalloproteinase (TIMP-3, TIMP-4), collagen-III and elastin levels were measured in whole brain by Western blot. These results suggested an increase in Evan's blue permeability: CBS-/+Assuntos
Barreira Hematoencefálica/metabolismo
, Circulação Cerebrovascular/efeitos dos fármacos
, Demência Vascular/tratamento farmacológico
, Agonistas de Receptores de GABA-A
, Homocisteína/metabolismo
, Homocistinúria/complicações
, Animais
, Barreira Hematoencefálica/efeitos dos fármacos
, Barreira Hematoencefálica/fisiopatologia
, Edema Encefálico/tratamento farmacológico
, Edema Encefálico/etiologia
, Edema Encefálico/fisiopatologia
, Artérias Cerebrais/efeitos dos fármacos
, Artérias Cerebrais/metabolismo
, Artérias Cerebrais/fisiopatologia
, Corantes/farmacocinética
, Demência Vascular/etiologia
, Demência Vascular/metabolismo
, Modelos Animais de Doenças
, Regulação para Baixo/efeitos dos fármacos
, Regulação para Baixo/fisiologia
, Azul Evans/farmacocinética
, Proteínas da Matriz Extracelular/efeitos dos fármacos
, Proteínas da Matriz Extracelular/metabolismo
, Feminino
, Agonistas GABAérgicos/farmacologia
, Homocistinúria/metabolismo
, Homocistinúria/fisiopatologia
, Masculino
, Camundongos
, Camundongos Endogâmicos C57BL
, Camundongos Knockout
, Muscimol/farmacologia
, Muscimol/uso terapêutico
, Receptores de GABA-A/genética
, Receptores de GABA-A/metabolismo
, Regulação para Cima/efeitos dos fármacos
, Regulação para Cima/fisiologia
RESUMO
Elevated levels of homocysteine (Hcy) (known as hyperhomocysteinemia HHcy) are involved in dilated cardiomyopathy. Hcy chelates copper and impairs copper-dependent enzymes. Copper deficiency has been linked to cardiovascular disease. We tested the hypothesis that copper supplement regresses left ventricular hypertrophy (LVH), fibrosis and endothelial dysfunction in pressure overload DCM mice hearts. The mice were grouped as sham, sham + Cu, aortic constriction (AC), and AC + Cu. Aortic constriction was performed by transverse aortic constriction. The mice were treated with or without 20 mg/kg copper supplement in the diet for 12 weeks. The cardiac function was assessed by echocardiography and electrocardiography. The matrix remodeling was assessed by measuring matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinases (TIMPs), and lysyl oxidase (LOX) by Western blot analyses. The results suggest that in AC mice, cardiac function was improved with copper supplement. TIMP-1 levels decreased in AC and were normalized in AC + Cu. Although MMP-9, TIMP-3, and LOX activity increased in AC and returned to baseline value in AC + Cu, copper supplement showed no significant effect on TIMP-4 activity after pressure overload. In conclusion, our data suggest that copper supplement helps improve cardiac function in a pressure overload dilated cardiomyopathic heart.
Assuntos
Cardiomiopatia Dilatada/tratamento farmacológico , Cobre/administração & dosagem , Suplementos Nutricionais , Insuficiência Cardíaca/tratamento farmacológico , Hemodinâmica/efeitos dos fármacos , Homocisteína/metabolismo , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Miocárdio/metabolismo , Animais , Aorta/cirurgia , Pressão Sanguínea , Western Blotting , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Constrição , Cobre/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Eletrocardiografia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Fibrose , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Homocisteína/sangue , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/enzimologia , Miocárdio/patologia , Proteína-Lisina 6-Oxidase/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Remodelação Ventricular/efeitos dos fármacosRESUMO
Although mitochondrial reduction-oxidation (redox) stress and increase in membrane permeability play an important role in diabetic-associated renal microvasculopathies, it is unclear whether the intra-renal mitochondrial oxidative stress induces mitochondrial protein modifications, leading to increase mitochondrial membrane permeability. The hypothesis is that mitochondrial oxidative stress induces mitochondrial protein modification and leakage in the mitochondrial membrane in type-2 diabetes. The present study was conducted to determine the involvement of intra-renal mitochondrial oxidative stress in mitochondrial protein modifications and modulation of membrane permeability in the setting of type-2 diabetes. Diabetes was induced by 6-week regimen of a high calorie and fat diet in C57BL/6J mice (Am J Physiol 291:F694-F701, 2006). Subcellular fractionation was carried out in kidney tissue from wild type and diabetic mice. All fractions were highly enriched in their corresponding marker enzyme. Subcellular protein modifications were determined by Western blot and 2-D proteomics. The results suggest that diabetes-induced oxidative stress parallels an increase in NADPH oxidase-4 (NOX-4) and decrease in superoxide dismutase-1, 2 (SOD-1, 2) expression, in mitochondrial compartment. We observed loss of mitochondrial membrane permeability as evidenced by leakage of mitochondrial cytochrome c and prohibitin to the cytosol. However, there was no loss in control tissue. The 2-D Western blots for mitochondrial post-translational modification showed an increase in nitrotyrosine generation in diabetes. We conclude that diabetes-induced intra-renal mitochondrial oxidative stress is reflected by an increase in mitochondrial membrane permeability and protein modifications by nitrotyrosine generation.