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1.
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
2.
Annu Rev Physiol ; 84: 285-306, 2022 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-34752709

RESUMO

Each heartbeat is initiated by the action potential, an electrical signal that depolarizes the plasma membrane and activates a cycle of calcium influx via voltage-gated calcium channels, calcium release via ryanodine receptors, and calcium reuptake and efflux via calcium-ATPase pumps and sodium-calcium exchangers. Agonists of the sympathetic nervous system bind to adrenergic receptors in cardiomyocytes, which, via cascading signal transduction pathways and protein kinase A (PKA), increase the heart rate (chronotropy), the strength of myocardial contraction (inotropy), and the rate of myocardial relaxation (lusitropy). These effects correlate with increased intracellular concentration of calcium, which is required for the augmentation of cardiomyocyte contraction. Despite extensive investigations, the molecular mechanisms underlying sympathetic nervous system regulation of calcium influx in cardiomyocytes have remained elusive over the last 40 years. Recent studies have uncovered the mechanisms underlying this fundamental biologic process, namely that PKA phosphorylates a calcium channel inhibitor, Rad, thereby releasing inhibition and increasing calcium influx. Here, we describe an updated model for how signals from adrenergic agonists are transduced to stimulate calcium influx and contractility in the heart.


Assuntos
Adrenérgicos , Canais de Cálcio Tipo L , Adrenérgicos/metabolismo , Adrenérgicos/farmacologia , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/farmacologia , Humanos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/farmacologia
3.
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
4.
J Clin Invest ; 134(5)2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38227371

RESUMO

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on ß-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavß subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVß, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVß, is sufficient for sympathetic upregulation of Ca2+ currents.


Assuntos
Adrenérgicos , Proteínas Monoméricas de Ligação ao GTP , Humanos , Adrenérgicos/metabolismo , Adrenérgicos/farmacologia , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Arritmias Cardíacas/metabolismo
5.
Nat Cardiovasc Res ; 1(11): 1022-1038, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36424916

RESUMO

Fight-or-flight responses involve ß-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart's innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for ß-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of ß-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel ß-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility.

6.
JACC Basic Transl Sci ; 6(7): 598-609, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34368510

RESUMO

Protein-protein interactions are of paramount importance in regulating normal cardiac physiology. Methodologies to elucidate these interactions in vivo have been limited. Recently, proximity-dependent biotinylation, with the use of BioID, TurboID, and ascorbate peroxidase, has been developed to uncover cellular neighborhoods and novel protein-protein interactions. These cutting-edge techniques have enabled the identification of subcellular localizations of specific proteins and the neighbors or interacting proteins within these subcellular regions. In contrast to classic methods such as affinity purification and subcellular fractionation, these techniques add covalently bound tags in living cells, such that spatial relationships and interaction networks are not disrupted. Recently, these methodologies have been used to identify novel protein-protein interactions relevant to the cardiovascular system. In this review, we discuss the development and current use of proximity biotin-labeling for cardiovascular research.

7.
J Clin Invest ; 129(2): 647-658, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30422117

RESUMO

Ca2+ channel ß-subunit interactions with pore-forming α-subunits are long-thought to be obligatory for channel trafficking to the cell surface and for tuning of basal biophysical properties in many tissues. Unexpectedly, we demonstrate that transgenic expression of mutant α1C subunits lacking capacity to bind CaVß can traffic to the sarcolemma in adult cardiomyocytes in vivo and sustain normal excitation-contraction coupling. However, these ß-less Ca2+ channels cannot be stimulated by ß-adrenergic pathway agonists, and thus adrenergic augmentation of contractility is markedly impaired in isolated cardiomyocytes and in hearts. Similarly, viral-mediated expression of a ß-subunit-sequestering peptide sharply curtailed ß-adrenergic stimulation of WT Ca2+ channels, identifying an approach to specifically modulate ß-adrenergic regulation of cardiac contractility. Our data demonstrate that ß subunits are required for ß-adrenergic regulation of CaV1.2 channels and positive inotropy in the heart, but are dispensable for CaV1.2 trafficking to the adult cardiomyocyte cell surface, and for basal function and excitation-contraction coupling.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Cobaias , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Transporte Proteico , Sarcolema/genética
8.
Biomaterials ; 102: 107-19, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27328431

RESUMO

Stem cell-based therapy is emerging as a promising approach for chronic diabetic wounds, but strategies for optimizing both cellular differentiation and delivery remain as major obstacles. Here, we study bioengineered vascularized constructs as a therapeutic modality for diabetic wound healing. We developed a wound model in immunodeficient rodent and treated it with engineered vascularized constructs from endothelial progenitors or early vascular cells-derived from human induced pluripotent stem cells (hiPSCs) reprogrammed either from healthy donor or type-1 diabetic patient. We found that all vascularized constructs expedited wound closure and reperfusion, with endothelial progenitor constructs having the earliest maximum closure rate followed closely by healthy and diabetic hiPSC-derivative constructs. This was accompanied by rapid granulation layer formation and regression in all vascularized construct groups. Macrophage infiltration into the hydrogel matrix occurred during early stages of healing, seeming to facilitate rapid neovascularization of the wound that could then better persist in the vascularized constructs. Blood perfusion of the human vasculature could be detected after three days, indicating rapid integration with the host vasculature. Overall, we propose a potential therapeutic strategy using allograft or autologous vascularized constructs to treat type-1 diabetic wounds. This approach highlights the unprecedented prospects of designing patient-specific stem cell therapy.


Assuntos
Complicações do Diabetes/terapia , Diabetes Mellitus Experimental/complicações , Células Endoteliais/transplante , Células-Tronco Pluripotentes Induzidas/transplante , Neovascularização Fisiológica , Alicerces Teciduais/química , Cicatrização , Animais , Linhagem Celular , Diabetes Mellitus Tipo 1/complicações , Modelos Animais de Doenças , Células Endoteliais/citologia , Feminino , Humanos , Ácido Hialurônico/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos Nus , Transplante de Células-Tronco/métodos
9.
J Invest Dermatol ; 135(10): 2519-2529, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26358387

RESUMO

Currently available skin grafts and skin substitutes for healing following third-degree burn injuries are fraught with complications, often resulting in long-term physical and psychological sequelae. Synthetic treatment that can promote wound healing in a regenerative manner would provide an off-the-shelf, non-immunogenic strategy to improve clinical care of severe burn wounds. Here, we demonstrate the vulnerary efficacy and accelerated healing mechanism of a dextran-based hydrogel in a third-degree porcine burn model. The model was optimized to allow examination of the hydrogel treatment for clinical translation and its regenerative response mechanisms. Hydrogel treatment accelerated third-degree burn wound healing by rapid wound closure, improved re-epithelialization, enhanced extracellular matrix remodeling, and greater nerve reinnervation, compared with the dressing-treated group. These effects appear to be mediated through the ability of the hydrogel to facilitate a rapid but brief initial inflammatory response that coherently stimulates neovascularization within the granulation tissue during the first week of treatment, followed by an efficient vascular regression to promote a regenerative healing process. Our results suggest that the dextran-based hydrogels may substantially improve healing quality and reduce skin grafting incidents and thus pave the way for clinical studies to improve the care of severe burn injury patients.


Assuntos
Queimaduras/terapia , Hidrogéis/farmacologia , Pele Artificial , Cicatrização/efeitos dos fármacos , Animais , Biópsia por Agulha , Queimaduras/patologia , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Distribuição Aleatória , Regeneração/efeitos dos fármacos , Sensibilidade e Especificidade , Higiene da Pele/métodos , Transplante de Pele/métodos , Sus scrofa , Suínos
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