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1.
Nature ; 618(7964): 374-382, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37225988

RESUMO

Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.


Assuntos
Vesículas Extracelulares , Ácidos Graxos , Fígado Gorduroso , Fígado , Neoplasias Pancreáticas , Animais , Camundongos , Sistema Enzimático do Citocromo P-450/genética , Vesículas Extracelulares/metabolismo , Ácidos Graxos/metabolismo , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias Hepáticas/secundário , Humanos , Inflamação/metabolismo , Ácido Palmítico/metabolismo , Células de Kupffer , Fosforilação Oxidativa , Proteínas rab27 de Ligação ao GTP/deficiência
2.
Nature ; 577(7792): 695-700, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969708

RESUMO

Increased cardiac contractility during the fight-or-flight response is caused by ß-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and ß subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of ß-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which ß-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or ß2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during ß-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for ß subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteômica , Receptores Adrenérgicos beta/metabolismo , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo N/metabolismo , Microambiente Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Masculino , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Miocárdio/metabolismo , Fosforilação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transdução de Sinais , Proteínas ras/química , Proteínas ras/metabolismo
3.
Annu Rev Physiol ; 83: 183-203, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33106102

RESUMO

The identification of a gain-of-function mutation in CACNA1C as the cause of Timothy syndrome, a rare disorder characterized by cardiac arrhythmias and syndactyly, highlighted roles for the L-type voltage-gated Ca2+ channel CaV1.2 in nonexcitable cells. Previous studies in cells and animal models had suggested that several voltage-gated Ca2+ channels (VGCCs) regulated critical signaling events in various cell types that are not expected to support action potentials, but definitive data were lacking. VGCCs occupy a special position among ion channels, uniquely able to translate membrane excitability into the cytoplasmic Ca2+ changes that underlie the cellular responses to electrical activity. Yet how these channels function in cells not firing action potentials and what the consequences of their actions are in nonexcitable cells remain critical questions. The development of new animal and cellular models and the emergence of large data sets and unbiased genome screens have added to our understanding of the unanticipated roles for VGCCs in nonexcitable cells. Here, we review current knowledge of VGCC regulation and function in nonexcitable tissues and cells, with the goal of providing a platform for continued investigation.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Potenciais de Ação/fisiologia , Animais , Transtorno Autístico/metabolismo , Humanos , Síndrome do QT Longo/metabolismo , Transdução de Sinais/fisiologia , Sindactilia/metabolismo
4.
FASEB J ; 37(7): e23007, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37261735

RESUMO

Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone, and cartilage development and homeostasis, knowledge about Ca2+ signaling and the source of Ca2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca2+ signaling through CaV 1.2 voltage-gated Ca2+ channel in tendon formation. Using a reporter mouse, we found that CaV 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;CaV 1.2TS mice that express a gain-of-function mutant CaV 1.2 in tendon. We found that mutant tendons were hypertrophic, with more tendon fibroblasts but decreased cell density. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendons. Biomechanical testing revealed that the hypertrophic tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomic analysis showed no significant difference in the abundance of type I and III collagens, but mutant tendons had about two-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2, and cathepsin K. Taken together, these data highlight roles for increased Ca2+ signaling through CaV 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.


Assuntos
Mecanotransdução Celular , Miostatina , Animais , Camundongos , Fenômenos Biomecânicos , Colágeno/metabolismo , Miostatina/metabolismo , Proteômica , Tendões/metabolismo
5.
Circ Res ; 130(2): 273-287, 2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-35050691

RESUMO

Rapidly changing and transient protein-protein interactions regulate dynamic cellular processes in the cardiovascular system. Traditional methods, including affinity purification and mass spectrometry, have revealed many macromolecular complexes in cardiomyocytes and the vasculature. Yet these methods often fail to identify in vivo or transient protein-protein interactions. To capture these interactions in living cells and animals with subsequent mass spectrometry identification, enzyme-catalyzed proximity labeling techniques have been developed in the past decade. Although the application of this methodology to cardiovascular research is still in its infancy, the field is developing rapidly, and the promise is substantial. In this review, we outline important concepts and discuss how proximity proteomics has been applied to study physiological and pathophysiological processes relevant to the cardiovascular system.


Assuntos
Miocárdio/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteômica/métodos , Animais , Humanos , Proteoma/genética , Proteoma/metabolismo
6.
Circ Res ; 130(7): 963-977, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35255712

RESUMO

BACKGROUND: Increasing evidence suggests that cardiac arrhythmias are frequent clinical features of coronavirus disease 2019 (COVID-19). Sinus node damage may lead to bradycardia. However, it is challenging to explore human sinoatrial node (SAN) pathophysiology due to difficulty in isolating and culturing human SAN cells. Embryonic stem cells (ESCs) can be a source to derive human SAN-like pacemaker cells for disease modeling. METHODS: We used both a hamster model and human ESC (hESC)-derived SAN-like pacemaker cells to explore the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the pacemaker cells of the heart. In the hamster model, quantitative real-time polymerase chain reaction and immunostaining were used to detect viral RNA and protein, respectively. We then created a dual knock-in SHOX2:GFP;MYH6:mCherry hESC reporter line to establish a highly efficient strategy to derive functional human SAN-like pacemaker cells, which was further characterized by single-cell RNA sequencing. Following exposure to SARS-CoV-2, quantitative real-time polymerase chain reaction, immunostaining, and RNA sequencing were used to confirm infection and determine the host response of hESC-SAN-like pacemaker cells. Finally, a high content chemical screen was performed to identify drugs that can inhibit SARS-CoV-2 infection, and block SARS-CoV-2-induced ferroptosis. RESULTS: Viral RNA and spike protein were detected in SAN cells in the hearts of infected hamsters. We established an efficient strategy to derive from hESCs functional human SAN-like pacemaker cells, which express pacemaker markers and display SAN-like action potentials. Furthermore, SARS-CoV-2 infection causes dysfunction of human SAN-like pacemaker cells and induces ferroptosis. Two drug candidates, deferoxamine and imatinib, were identified from the high content screen, able to block SARS-CoV-2 infection and infection-associated ferroptosis. CONCLUSIONS: Using a hamster model, we showed that primary pacemaker cells in the heart can be infected by SARS-CoV-2. Infection of hESC-derived functional SAN-like pacemaker cells demonstrates ferroptosis as a potential mechanism for causing cardiac arrhythmias in patients with COVID-19. Finally, we identified candidate drugs that can protect the SAN cells from SARS-CoV-2 infection.


Assuntos
COVID-19 , Ferroptose , Humanos , Miócitos Cardíacos/metabolismo , SARS-CoV-2 , Nó Sinoatrial/metabolismo
7.
J Neurosci ; 2022 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-35672149

RESUMO

Brain enriched voltage-gated sodium channel (VGSC) Nav1.2 and Nav1.6 are critical for electrical signaling in the central nervous system. Previous studies have extensively characterized cell-type specific expression and electrophysiological properties of these two VGSCs and how their differences contribute to fine-tuning of neuronal excitability. However, due to lack of reliable labeling and imaging methods, the sub-cellular localization and dynamics of these homologous Nav1.2 and Nav1.6 channels remain understudied. To overcome this challenge, we combined genome editing, super-resolution and live-cell single molecule imaging to probe subcellular composition, relative abundances and trafficking dynamics of Nav1.2 and Nav1.6 in cultured mouse and rat neurons and in male and female mouse brain. We discovered a previously uncharacterized trafficking pathway that targets Nav1.2 to the distal axon of unmyelinated neurons. This pathway utilizes distinct signals residing in the intracellular loop 1 (ICL1) between transmembrane domain I and II to suppress the retention of Nav1.2 in the axon initial segment (AIS) and facilitate its membrane loading at the distal axon. As mouse pyramidal neurons undergo myelination, Nav1.2 is gradually excluded from the distal axon as Nav1.6 becomes the dominant VGSC in the axon initial segment and nodes of Ranvier. In addition, we revealed exquisite developmental regulation of Nav1.2 and Nav1.6 localizations in the axon initial segment and dendrites, clarifying the molecular identity of sodium channels in these subcellular compartments. Together, these results unveiled compartment-specific localizations and trafficking mechanisms for VGSCs, which could be regulated separately to modulate membrane excitability in the brain.SIGNIFICANCE STATEMENTDirect observation of endogenous voltage-gated sodium channels reveals a previously uncharacterized distal axon targeting mechanism and the molecular identity of sodium channels in distinct subcellular compartments.

8.
Circ Res ; 128(1): 76-88, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33086983

RESUMO

RATIONALE: Changing activity of cardiac CaV1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic. OBJECTIVE: The primary goal of this study was to determine how various factors converging at the CaV1.2 I-II loop interact to regulate channel activity under basal conditions, during ß-adrenergic stimulation, and in heart failure. METHODS AND RESULTS: We generated transgenic mice with expression of CaV1.2 α1C subunits with (1) mutations ablating interaction between α1C and ß-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of CaVß to α1C I-II loop and eliminated ß-adrenergic agonist stimulation of CaV1.2 current. In contrast, introduction of the exon 9* splice variant in the α1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to ß-adrenergic agonists when reconstituted heterologously with ß2B and Rad or transgenically expressed in cardiomyocytes. CONCLUSIONS: Ca2+ channel activity is dynamically modulated under basal conditions, during ß-adrenergic stimulation, and in heart failure by mechanisms converging at the α1C I-II loop. CaVß binding to α1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the ß-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca2+ channel activity by ß-adrenergic agonists/PKA also requires this rigid linker and ß-binding to α1C.


Assuntos
Agonistas Adrenérgicos beta/farmacologia , Canais de Cálcio Tipo L/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Proteínas ras/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Células HEK293 , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Potenciais da Membrana , Camundongos Transgênicos , Mutação , Miócitos Cardíacos/metabolismo , Fosforilação , Conformação Proteica , Coelhos , Relação Estrutura-Atividade , Proteínas ras/genética
10.
FASEB J ; 33(10): 11579-11594, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31339804

RESUMO

Fibroblast growth factor (FGF)13, a nonsecreted, X-linked, FGF homologous factor, is differentially expressed in adipocytes in response to diet, yet Fgf13's role in metabolism has not been explored. Heterozygous Fgf13 knockouts fed normal chow and housed at 22°C showed hyperactivity accompanying reduced core temperature and obesity when housed at 30°C. Those heterozygous knockouts showed defects in thermogenesis even at 30°C and an inability to protect core temperature. Surprisingly, we detected trivial FGF13 in adipose of wild-type mice fed normal chow and no obesity in adipose-specific heterozygous knockouts housed at 30°C, and we detected an intact brown fat response through exogenous ß3 agonist stimulation, suggesting a defect in sympathetic drive to brown adipose tissue. In contrast, hypothalamic-specific ablation of Fgf13 recapitulated weight gain at 30°C. Norepinephrine turnover in brown fat was reduced at both housing temperatures. Thus, our data suggest that impaired CNS regulation of sympathetic activation of brown fat underlies obesity and thermogenesis in Fgf13 heterozygous knockouts fed normal chow.-Sinden, D. S., Holman, C. D., Bare, C. J., Sun, X., Gade, A. R., Cohen, D. E., Pitt, G. S. Knockout of the X-linked Fgf13 in the hypothalamic paraventricular nucleus impairs sympathetic output to brown fat and causes obesity.


Assuntos
Tecido Adiposo Marrom/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Obesidade/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Adipócitos/metabolismo , Tecido Adiposo Branco/metabolismo , Adiposidade/fisiologia , Animais , Dieta Hiperlipídica/métodos , Metabolismo Energético/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Termogênese/fisiologia , Aumento de Peso/fisiologia
11.
Proc Natl Acad Sci U S A ; 114(1): 154-159, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-27994149

RESUMO

KCNQ2/3 (Kv7.2/7.3) channels and voltage-gated sodium channels (VGSCs) are enriched in the axon initial segment (AIS) where they bind to ankyrin-G and coregulate membrane potential in central nervous system neurons. The molecular mechanisms supporting coordinated regulation of KCNQ and VGSCs and the cellular mechanisms governing KCNQ trafficking to the AIS are incompletely understood. Here, we show that fibroblast growth factor 14 (FGF14), previously described as a VGSC regulator, also affects KCNQ function and localization. FGF14 knockdown leads to a reduction of KCNQ2 in the AIS and a reduction in whole-cell KCNQ currents. FGF14 positively regulates KCNQ2/3 channels in a simplified expression system. FGF14 interacts with KCNQ2 at a site distinct from the FGF14-VGSC interaction surface, thus enabling the bridging of NaV1.6 and KCNQ2. These data implicate FGF14 as an organizer of channel localization in the AIS and provide insight into the coordination of KCNQ and VGSC conductances in the regulation of membrane potential.


Assuntos
Segmento Inicial do Axônio/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Potenciais da Membrana/fisiologia , Anquirinas/metabolismo , Células Cultivadas , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Neurônios/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Canais de Sódio Disparados por Voltagem/metabolismo
12.
Proc Natl Acad Sci U S A ; 114(20): E4010-E4019, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28461495

RESUMO

The fibroblast growth factor (FGF) homologous factor FGF13, a noncanonical FGF, has been best characterized as a voltage-gated Na+ channel auxiliary subunit. Other cellular functions have been suggested, but not explored. In inducible, cardiac-specific Fgf13 knockout mice, we found-even in the context of the expected reduction in Na+ channel current-an unanticipated protection from the maladaptive hypertrophic response to pressure overload. To uncover the underlying mechanisms, we searched for components of the FGF13 interactome in cardiomyocytes and discovered the complete set of the cavin family of caveolar coat proteins. Detailed biochemical investigations showed that FGF13 acts as a negative regulator of caveolae abundance in cardiomyocytes by controlling the relative distribution of cavin 1 between the sarcolemma and cytosol. In cardiac-specific Fgf13 knockout mice, cavin 1 redistribution to the sarcolemma stabilized the caveolar structural protein caveolin 3. The consequent increase in caveolae density afforded protection against pressure overload-induced cardiac dysfunction by two mechanisms: (i) enhancing cardioprotective signaling pathways enriched in caveolae, and (ii) increasing the caveolar membrane reserve available to buffer membrane tension. Thus, our results uncover unexpected roles for a FGF homologous factor and establish FGF13 as a regulator of caveolae-mediated mechanoprotection and adaptive hypertrophic signaling.


Assuntos
Cardiomegalia/metabolismo , Cavéolas/fisiologia , Caveolinas/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Miócitos Cardíacos/fisiologia , Animais , Cardiomegalia/etiologia , Cardiomegalia/patologia , Modelos Animais de Doenças , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fibrose , Masculino , Microdomínios da Membrana/metabolismo , Camundongos Knockout , Miocárdio/patologia , Miócitos Cardíacos/ultraestrutura , Pressão , Sarcolema/fisiologia , Sarcolema/ultraestrutura
13.
Proc Natl Acad Sci U S A ; 114(34): 9194-9199, 2017 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-28784807

RESUMO

Calcium influx through the voltage-dependent L-type calcium channel (CaV1.2) rapidly increases in the heart during "fight or flight" through activation of the ß-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of ß-adrenergic activation of cardiac CaV1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α1C and C-terminal proteolytic cleavage of the α1C subunit. We generated transgenic mice expressing an α1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for ß-adrenergic regulation of CaV1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute ß-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α1C Prevention of C-terminal cleavage did not alter ß-adrenergic stimulation of CaV1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α1C in ß-adrenergic stimulation of CaV1.2, and show that phosphoregulatory sites on α1C are not redundant and do not each fractionally contribute to the net stimulatory effect of ß-adrenergic stimulation. Further, proteolytic cleavage of α1C is not required for ß-adrenergic stimulation of CaV1.2.


Assuntos
Adrenérgicos/metabolismo , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miocárdio/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Cobaias , Humanos , Camundongos , Camundongos Transgênicos , Fosforilação , Domínios Proteicos , Proteólise , Coelhos , Ratos
14.
Proc Natl Acad Sci U S A ; 113(19): E2665-74, 2016 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-27044086

RESUMO

Clustering of voltage-gated sodium channels (VGSCs) within the neuronal axon initial segment (AIS) is critical for efficient action potential initiation. Although initially inserted into both somatodendritic and axonal membranes, VGSCs are concentrated within the axon through mechanisms that include preferential axonal targeting and selective somatodendritic endocytosis. How the endocytic machinery specifically targets somatic VGSCs is unknown. Here, using knockdown strategies, we show that noncanonical FGF13 binds directly to VGSCs in hippocampal neurons to limit their somatodendritic surface expression, although exerting little effect on VGSCs within the AIS. In contrast, homologous FGF14, which is highly concentrated in the proximal axon, binds directly to VGSCs to promote their axonal localization. Single-point mutations in FGF13 or FGF14 abrogating VGSC interaction in vitro cannot support these specific functions in neurons. Thus, our data show how the concerted actions of FGF13 and FGF14 regulate the polarized localization of VGSCs that supports efficient action potential initiation.


Assuntos
Potenciais de Ação , Canais de Sódio Disparados por Voltagem/metabolismo , Axônios/metabolismo , Humanos , Neurônios/metabolismo , Sódio/metabolismo , Canais de Sódio/genética
15.
Proc Natl Acad Sci U S A ; 112(41): E5618-27, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26424448

RESUMO

Cardiac pacemaking is governed by specialized cardiomyocytes located in the sinoatrial node (SAN). SAN cells (SANCs) integrate voltage-gated currents from channels on the membrane surface (membrane clock) with rhythmic Ca(2+) release from internal Ca(2+) stores (Ca(2+) clock) to adjust heart rate to meet hemodynamic demand. Here, we report that stromal interaction molecule 1 (STIM1) and Orai1 channels, key components of store-operated Ca(2+) entry, are selectively expressed in SANCs. Cardiac-specific deletion of STIM1 in mice resulted in depletion of sarcoplasmic reticulum (SR) Ca(2+) stores of SANCs and led to SAN dysfunction, as was evident by a reduction in heart rate, sinus arrest, and an exaggerated autonomic response to cholinergic signaling. Moreover, STIM1 influenced SAN function by regulating ionic fluxes in SANCs, including activation of a store-operated Ca(2+) current, a reduction in L-type Ca(2+) current, and enhancing the activities of Na(+)/Ca(2+) exchanger. In conclusion, these studies reveal that STIM1 is a multifunctional regulator of Ca(2+) dynamics in SANCs that links SR Ca(2+) store content with electrical events occurring in the plasma membrane, thereby contributing to automaticity of the SAN.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Nó Sinoatrial/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Proteína ORAI1 , Retículo Sarcoplasmático/genética , Nó Sinoatrial/citologia , Molécula 1 de Interação Estromal
16.
Proc Natl Acad Sci U S A ; 112(40): 12528-33, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26392562

RESUMO

Nav channels are essential for metazoan membrane depolarization, and Nav channel dysfunction is directly linked with epilepsy, ataxia, pain, arrhythmia, myotonia, and irritable bowel syndrome. Human Nav channelopathies are primarily caused by variants that directly affect Nav channel permeability or gating. However, a new class of human Nav channelopathies has emerged based on channel variants that alter regulation by intracellular signaling or cytoskeletal proteins. Fibroblast growth factor homologous factors (FHFs) are a family of intracellular signaling proteins linked with Nav channel regulation in neurons and myocytes. However, to date, there is surprisingly little evidence linking Nav channel gene variants with FHFs and human disease. Here, we provide, to our knowledge, the first evidence that mutations in SCN5A (encodes primary cardiac Nav channel Nav1.5) that alter FHF binding result in human cardiovascular disease. We describe a five*generation kindred with a history of atrial and ventricular arrhythmias, cardiac arrest, and sudden cardiac death. Affected family members harbor a novel SCN5A variant resulting in p.H1849R. p.H1849R is localized in the central binding core on Nav1.5 for FHFs. Consistent with these data, Nav1.5 p.H1849R affected interaction with FHFs. Further, electrophysiological analysis identified Nav1.5 p.H1849R as a gain-of-function for INa by altering steady-state inactivation and slowing the rate of Nav1.5 inactivation. In line with these data and consistent with human cardiac phenotypes, myocytes expressing Nav1.5 p.H1849R displayed prolonged action potential duration and arrhythmogenic afterdepolarizations. Together, these findings identify a previously unexplored mechanism for human Nav channelopathy based on altered Nav1.5 association with FHF proteins.


Assuntos
Arritmias Cardíacas/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Células Cultivadas , Canalopatias/genética , Canalopatias/metabolismo , Canalopatias/fisiopatologia , Saúde da Família , Feminino , Predisposição Genética para Doença/genética , Células HEK293 , Humanos , Immunoblotting , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Linhagem , Ligação Proteica
17.
J Mol Cell Cardiol ; 104: 63-74, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28119060

RESUMO

The intracellular fibroblast growth factors (iFGF/FHFs) bind directly to cardiac voltage gated Na+ channels, and modulate their function. Mutations that affect iFGF/FHF-Na+ channel interaction are associated with arrhythmia syndromes. Although suspected to modulate other ionic currents, such as Ca2+ channels based on acute knockdown experiments in isolated cardiomyocytes, the in vivo consequences of iFGF/FHF gene ablation on cardiac electrical activity are still unknown. We generated inducible, cardiomyocyte-restricted Fgf13 knockout mice to determine the resultant effects of Fgf13 gene ablation. Patch clamp recordings from ventricular myocytes isolated from Fgf13 knockout mice showed a ~25% reduction in peak Na+ channel current density and a hyperpolarizing shift in steady-state inactivation. Electrocardiograms on Fgf13 knockout mice showed a prolonged QRS duration. The Na+ channel blocker flecainide further prolonged QRS duration and triggered ventricular tachyarrhythmias only in Fgf13 knockout mice, suggesting that arrhythmia vulnerability resulted, at least in part, from a loss of functioning Na+ channels. Consistent with these effects on Na+ channels, action potentials in Fgf13 knockout mice, compared to Cre control mice, exhibited slower upstrokes and reduced amplitude, but unexpectedly had longer durations. We investigated candidate sources of the prolonged action potential durations in myocytes from Fgf13 knockout mice and found a reduction of the transient outward K+ current (Ito). Fgf13 knockout did not alter whole-cell protein levels of Kv4.2 and Kv4.3, the Ito pore-forming subunits, but did decrease Kv4.2 and Kv4.3 at the sarcolemma. No changes were seen in the sustained outward K+ current or voltage-gated Ca2+ current, other candidate contributors to the increased action potential duration. These results implicate that FGF13 is a critical cardiac Na+ channel modulator and Fgf13 knockout mice have increased arrhythmia susceptibility in the setting of Na+ channel blockade. The unanticipated effect on Ito revealed new FGF13 properties and the unexpected lack of an effect on voltage-gated Ca2+ channels highlight potential compensatory changes in vivo not readily revealed with acute Fgf13 knockdown in cultured cardiomyocytes.


Assuntos
Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Fatores de Crescimento de Fibroblastos/deficiência , Predisposição Genética para Doença , Canais Iônicos/metabolismo , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Eletrocardiografia , Feminino , Técnicas de Inativação de Genes , Marcação de Genes , Loci Gênicos , Masculino , Camundongos , Camundongos Knockout , Canais de Sódio/metabolismo , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia
18.
Proc Natl Acad Sci U S A ; 116(52): 26150-26151, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31796599
19.
J Mol Cell Cardiol ; 92: 52-62, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26801742

RESUMO

BACKGROUND: Inherited autosomal dominant mutations in cardiac sodium channels (NaV1.5) cause various arrhythmias, such as long QT syndrome and Brugada syndrome. Although dozens of mutations throughout the protein have been reported, there are few reported mutations within a voltage sensor S4 transmembrane segment and few that are homozygous. Here we report analysis of a novel lidocaine-sensitive recessive mutation, p.R1309H, in the NaV1.5 DIII/S4 voltage sensor in a patient with a complex arrhythmia syndrome. METHODS AND RESULTS: We expressed the wild type or mutant NaV1.5 heterologously for analysis with the patch-clamp and voltage clamp fluorometry (VCF) techniques. p.R1309H depolarized the voltage-dependence of activation, hyperpolarized the voltage-dependence of inactivation, and slowed recovery from inactivation, thereby reducing the channel availability at physiologic membrane potentials. Additionally, p.R1309H increased the "late" Na(+) current. The location of the mutation in DIIIS4 prompted testing for a gating pore current. We observed an inward current at hyperpolarizing voltages that likely exacerbates the loss-of-function defects at resting membrane potentials. Lidocaine reduced the gating pore current. CONCLUSIONS: The p.R1309H homozygous NaV1.5 mutation conferred both gain-of-function and loss-of-function effects on NaV1.5 channel activity. Reduction of a mutation-induced gating pore current by lidocaine suggested a therapeutic mechanism.


Assuntos
Arritmias Cardíacas/genética , Síndrome de Brugada/genética , Sistema de Condução Cardíaco/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/fisiopatologia , Síndrome de Brugada/tratamento farmacológico , Síndrome de Brugada/fisiopatologia , Doença do Sistema de Condução Cardíaco , Humanos , Lactente , Lidocaína/administração & dosagem , Masculino , Potenciais da Membrana/genética , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp
20.
J Biol Chem ; 290(4): 2166-74, 2015 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-25505241

RESUMO

Voltage-gated Ca(2+) channels play a key role in initiating muscle excitation-contraction coupling, neurotransmitter release, gene expression, and hormone secretion. The association of CaV1.2 with a supramolecular complex impacts trafficking, localization, turnover, and, most importantly, multifaceted regulation of its function in the heart. Several studies hint at an important role for the C terminus of the α1C subunit as a hub for multidimensional regulation of CaV1.2 channel trafficking and function. Recent studies have demonstrated an important role for the four-residue PDZ binding motif at the C terminus of α1C in interacting with scaffold proteins containing PDZ domains, in the subcellular localization of CaV1.2 in neurons, and in the efficient signaling to cAMP-response element-binding protein in neurons. However, the role of the α1C PDZ ligand domain in the heart is not known. To determine whether the α1C PDZ motif is critical for CaV1.2 trafficking and function in cardiomyocytes, we generated transgenic mice with inducible expression of an N-terminal FLAG epitope-tagged dihydropyridine-resistant α1C with the PDZ motif deleted (ΔPDZ). These mice were crossed with α-myosin heavy chain reverse transcriptional transactivator transgenic mice, and the double-transgenic mice were fed doxycycline. The ΔPDZ channels expressed, trafficked to the membrane, and supported robust excitation-contraction coupling in the presence of nisoldipine, a dihydropyridine Ca(2+) channel blocker, providing functional evidence that they appropriately target to dyads. The ΔPDZ Ca(2+) channels were appropriately regulated by isoproterenol and forskolin. These data indicate that the α1C PDZ motif is not required for surface trafficking, localization to the dyad, or adrenergic stimulation of CaV1.2 in adult cardiomyocytes.


Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/fisiologia , Coração/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Motivos de Aminoácidos , Animais , Bloqueadores dos Canais de Cálcio/química , Colforsina/química , Epitopos/química , Deleção de Genes , Humanos , Ligantes , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Nisoldipino/química , Estrutura Terciária de Proteína , Coelhos , Propriedades de Superfície
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