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1.
Circ Res ; 107(9): 1140-9, 2010 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-20814022

RESUMO

RATIONALE: Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein-coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function. OBJECTIVE: A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac ß-adrenergic receptor (ßAR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the ßARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model. METHODS AND RESULTS: We demonstrate, using cardiac-specific GRK2 and ßARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with ßARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2-overexpressing mice (45.0±2.8% versus 31.3±2.3% in controls) and significantly smaller in ßARKct mice (16.8±1.3%, P<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via ßARKct, and these antiapoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of ß(2)ARs abolished ßARKct-mediated cardioprotection, suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival. CONCLUSION: The novel effect of reducing acute ischemic myocardial injury via increased Akt activity and NO production adds significantly to the therapeutic potential of GRK2 inhibition with the ßARKct not only in chronic HF but also potentially in acute ischemic injury conditions.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/fisiologia , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Animais , Células Cultivadas , Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Camundongos , Camundongos Transgênicos , Ratos
2.
Circ Res ; 107(12): 1445-53, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-20966393

RESUMO

RATIONALE: coronary artery ligation to induce myocardial infarction (MI) in mice is typically performed by an invasive and time-consuming approach that requires ventilation and chest opening (classic method), often resulting in extensive tissue damage and high mortality. We developed a novel and rapid surgical method to induce MI that does not require ventilation. OBJECTIVE: the purpose of this study was to develop and comprehensively describe this method and directly compare it to the classic method. METHODS AND RESULTS: male C57/B6 mice were grouped into 4 groups: new method MI (MI-N) or sham (S-N) and classic method MI (MI-C) or sham (S-C). In the new method, heart was manually exposed without intubation through a small incision and MI was induced. In the classic method, MI was induced through a ventilated thoracotomy. Similar groups were used in an ischemia/reperfusion injury model. This novel MI procedure is rapid, with an average procedure time of 1.22 ± 0.05 minutes, whereas the classic method requires 23.2 ± 0.6 minutes per procedure. Surgical mortality was 3% in MI-N and 15.9% in MI-C. The rate of arrhythmia was significantly lower in MI-N. The postsurgical levels of tumor necrosis factor-α and myeloperoxidase were lower in new method, indicating less inflammation. Overall, 28-day post-MI survival rate was 68% with MI-N and 48% with MI-C. Importantly, there was no difference in infarct size or post-MI cardiac function between the methods. CONCLUSIONS: this new rapid method of MI in mice represents a more efficient and less damaging model of myocardial ischemic injury compared with the classic method.


Assuntos
Vasos Coronários/cirurgia , Modelos Animais de Doenças , Infarto do Miocárdio/etiologia , Projetos de Pesquisa/normas , Animais , Ligadura/efeitos adversos , Ligadura/métodos , Masculino , Métodos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo
3.
J Mol Cell Cardiol ; 47(1): 121-32, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19376127

RESUMO

A novel Cl(-) inward rectifier channel (Cl,ir) encoded by ClC-2, a member of the ClC voltage-gated Cl(-) channel gene superfamily, has been recently discovered in cardiac myocytes of several species. However, the physiological role of Cl,ir channels in the heart remains unknown. In this study we tested the hypothesis that Cl,ir channels may play an important role in cardiac pacemaker activity. In isolated guinea-pig sinoatrial node (SAN) cells, Cl,ir current was activated by hyperpolarization and hypotonic cell swelling. RT-PCR and immunohistological analyses confirmed the molecular expression of ClC-2 in guinea-pig SAN cells. Hypotonic stress increased the diastolic depolarization slope and decreased the maximum diastolic potential, action potential amplitude, APD(50), APD(90), and the cycle-length of the SAN cells. These effects were largely reversed by intracellular dialysis of anti-ClC-2 antibody, which significantly inhibited Cl,ir current but not other pacemaker currents, including the hyperpolarization-activated non-selective cationic "funny" current (I(f)), the L-type Ca(2+) currents (I(Ca,L)), the slowly-activating delayed rectifier I(Ks) and the volume-regulated outwardly-rectifying Cl(-) current (I(Cl,vol)). Telemetry electrocardiograph studies in conscious ClC-2 knockout (Clcn2(-/-)) mice revealed a decreased chronotropic response to acute exercise stress when compared to their age-matched Clcn2(+/+) and Clcn2(+/-) littermates. Targeted inactivation of ClC-2 does not alter intrinsic heart rate but prevented the positive chronotropic effect of acute exercise stress through a sympathetic regulation of ClC-2 channels. These results provide compelling evidence that ClC-2-encoded endogenous Cl,ir channels may play an important role in the regulation of cardiac pacemaker activity, which may become more prominent under stressed or pathological conditions.


Assuntos
Canais de Cloreto/fisiologia , Nó Sinoatrial/citologia , Nó Sinoatrial/metabolismo , Potenciais de Ação/fisiologia , Animais , Canais de Cloro CLC-2 , Eletrofisiologia Cardíaca , Células Cultivadas , Canais de Cloreto/genética , Eletrocardiografia , Cobaias , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nó Sinoatrial/fisiologia
4.
Sci Signal ; 6(299): ra95, 2013 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-24170934

RESUMO

Heart failure caused by ischemic heart disease is a leading cause of death in the developed world. Treatment is currently centered on regimens involving G protein-coupled receptors (GPCRs) or nitric oxide (NO). These regimens are thought to target distinct molecular pathways. We showed that these pathways were interdependent and converged on the effector GRK2 (GPCR kinase 2) to regulate myocyte survival and function. Ischemic injury coupled to GPCR activation, including GPCR desensitization and myocyte loss, required GRK2 activation, and we found that cardioprotection mediated by inhibition of GRK2 depended on endothelial nitric oxide synthase (eNOS) and was associated with S-nitrosylation of GRK2. Conversely, the cardioprotective effects of NO bioactivity were absent in a knock-in mouse with a form of GRK2 that cannot be S-nitrosylated. Because GRK2 and eNOS inhibit each other, the balance of the activities of these enzymes in the myocardium determined the outcome to ischemic injury. Our findings suggest new insights into the mechanism of action of classic drugs used to treat heart failure and new therapeutic approaches to ischemic heart disease.


Assuntos
Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Western Blotting , Células Cultivadas , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Coração/efeitos dos fármacos , Coração/fisiopatologia , Isoproterenol/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , S-Nitrosoglutationa/farmacologia , S-Nitrosotióis/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
5.
J Thorac Cardiovasc Surg ; 143(3): 720-726.e3, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22143102

RESUMO

OBJECTIVE: Genetic modulation of heart function is a novel therapeutic strategy. We investigated the effect of molecular cardiac surgery with recirculating delivery (MCARD)-mediated carboxyl-terminus of the ß-adrenergic receptor kinase (ßARKct) gene transfer on cardiac mechanoenergetics and ß-adrenoreceptor (ßAR) signaling. METHODS: After baseline measurements, sheep underwent MCARD-mediated delivery of 10(14) genome copies of self-complimentary adeno-associated virus (scAAV6)-ßARKct. Four and 8 weeks after MCARD, mechanoenergetic studies using magnetic resonance imaging were performed. Tissues were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. ßAR density, cyclic adenosine monophosphate levels, and physiologic parameters were evaluated. RESULTS: There was a significant increase in dP/dt(max) at 4 weeks: 1384 ± 76 versus 1772 ± 182 mm Hg/s; and the increase persisted at 8 weeks in response to isoproterenol (P < .05). Similarly, the magnitude of dP/dt(min) increased at both 4 weeks and 8 weeks with isoproterenol stimulation (P < .05). At 8 weeks, potential energy was conserved, whereas in controls there was a decrease in potential energy (P < .05) in response to isoproterenol. RT-qPCR confirmed robustness of ßARKct expression throughout the left ventricle and undetectable expression in extracardiac tissues. Quantitative Western blot data confirmed higher expression of ßARKct in the left ventricle: 0.46 ± 0.05 versus 0.00 in lung and liver (P < .05). Survival was 100% and laboratory parameters of major organ function were within normal limits. CONCLUSIONS: MCARD-mediated ßARKct delivery is safe, results in robust cardiac-specific gene expression, enhances cardiac contractility and lusitropy, increases adrenergic reserve, and improves energy utilization efficiency in a preclinical large animal model.


Assuntos
Procedimentos Cirúrgicos Cardíacos , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Ventrículos do Coração/enzimologia , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Quinases de Receptores Adrenérgicos beta/biossíntese , Agonistas Adrenérgicos beta/farmacologia , Animais , Western Blotting , AMP Cíclico/metabolismo , Ecocardiografia Doppler , Regulação da Expressão Gênica , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/efeitos dos fármacos , Hemodinâmica , Isoproterenol/farmacologia , Imageamento por Ressonância Magnética , Masculino , Contração Miocárdica , Reação em Cadeia da Polimerase em Tempo Real , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/genética , Ovinos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Tempo , Pressão Ventricular , Quinases de Receptores Adrenérgicos beta/genética
6.
ACS Chem Biol ; 7(11): 1830-9, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-22882301

RESUMO

G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. Herein we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine-Gßγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.


Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Paroxetina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Animais , Domínio Catalítico/efeitos dos fármacos , Células Cultivadas , Quinase 2 de Receptor Acoplado a Proteína G/química , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Coração/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Fosforilação/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Hormônio Liberador de Tireotropina/metabolismo
7.
J Biol Chem ; 280(27): 25871-80, 2005 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-15883157

RESUMO

A novel volume-regulated hyperpolarization-activated chloride inward rectifier channel (Cl.ir) was identified in mammalian heart. To investigate whether ClC-2 is the gene encoding Cl.ir channels in heart, ClC-2 cDNAs cloned from rat (rClC-2) and guinea pig (gpClC-2) hearts were functionally characterized. When expressed in NIH/3T3 cells, full-length rClC-2 yielded inwardly rectifying whole-cell currents with very slow activation kinetics (time constants > 1.7 s) upon hyperpolarization under hypotonic condition. The single-channel rClC-2 currents had a unitary slope conductance of 3.9 +/- 0.2 picosiemens. A novel variant with an in-frame deletion at the beginning of exon 15 that leads to a deletion of 45 bp (corresponding to 15 amino acids in alpha-helices O and P, rClC-2(Delta509-523)) was identified in rat heart. The relative transcriptional expression levels of full-length rClC-2 and rClC-2(Delta509-523) in rat heart were 0.018 +/- 0.003 and 0.028 +/- 0.006 arbitrary units, respectively, relative to glyceraldehyde-3-phosphate dehydrogenase (n = 5, p = nonsignificant). A similar partial exon 15 skipping with a deletion of 105 bp (35 amino acids in alpha-helices O-Q, gpClC-2(Delta509-543)) was also identified in guinea pig heart. Expression of both rClC-2(Delta509-523) and gpClC-2(Delta509-543) resulted in functional channels with phenotypic activation kinetics and many properties identical to those of endogenous Cl.ir channels in native rat and guinea pig cardiac myocytes, respectively. Intracellular dialysis of anti-ClC-2 antibody inhibited expressed ClC-2 channels and endogenous Cl.ir currents in native rat and guinea pig cardiac myocytes. These results demonstrate that novel deletion variants of ClC-2 due to partial exon 15 skipping may be expressed normally in heart and contribute to the formation of endogenous Cl.ir channels in native cardiac cells.


Assuntos
Processamento Alternativo/fisiologia , Canais de Cloreto/genética , Ativação do Canal Iônico/fisiologia , Miócitos Cardíacos/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Canais de Cloro CLC-2 , Canais de Cloreto/química , Canais de Cloreto/imunologia , Clonagem Molecular , Cobaias , Ventrículos do Coração/citologia , Masculino , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley
8.
Acta Pharmacol Sin ; 26(3): 265-78, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15715921

RESUMO

In comparison to cation (K+, Na+, and Ca2+) channels, much less is currently known about the functional role of anion (Cl-) channels in cardiovascular physiology and pathophysiology. Over the past 15 years, various types of Cl- currents have been recorded in cardiac cells from different species including humans. All cardiac Cl- channels described to date may be encoded by five different Cl- channel genes: the PKA- and PKC-activated cystic fibrosis tansmembrane conductance regulator (CFTR), the volume-regulated ClC-2 and ClC-3, and the Ca2+-activated CLCA or Bestrophin. Recent studies using multiple approaches to examine the functional role of Cl- channels in the context of health and disease have demonstrated that Cl- channels might contribute to: 1) arrhythmogenesis in myocardial injury; 2) cardiac ischemic preconditioning; and 3) the adaptive remodeling of the heart during myocardial hypertrophy and heart failure. Therefore, anion channels represent very attractive novel targets for therapeutic approaches to the treatment of heart diseases. Recent evidence suggests that Cl- channels, like cation channels, might function as a multiprotein complex or functional module. In the post-genome era, the emergence of functional proteomics has necessitated a new paradigm shift to the structural and functional assessment of integrated Cl- channel multiprotein complexes in the heart, which could provide new insight into our understanding of the underlying mechanisms responsible for heart disease and protection.


Assuntos
Arritmias Cardíacas/fisiopatologia , Cardiomegalia/fisiopatologia , Canais de Cloreto/fisiologia , Precondicionamento Isquêmico , Animais , Arritmias Cardíacas/genética , Cardiomegalia/genética , Cardiomegalia/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos
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