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1.
Cardiovasc Diabetol ; 20(1): 7, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33413413

RESUMO

BACKGROUND: Sodium-glucose linked transporter type 2 (SGLT-2) inhibition has been shown to reduce cardiovascular mortality in heart failure independently of glycemic control and prevents the onset of atrial arrhythmias, a common co-morbidity in heart failure with preserved ejection fraction (HFpEF). The mechanism behind these effects is not fully understood, and it remains unclear if they could be further enhanced by additional SGLT-1 inhibition. We investigated the effects of chronic treatment with the dual SGLT-1&2 inhibitor sotagliflozin on left atrial (LA) remodeling and cellular arrhythmogenesis (i.e. atrial cardiomyopathy) in a metabolic syndrome-related rat model of HFpEF. METHODS: 17 week-old ZSF-1 obese rats, a metabolic syndrome-related model of HFpEF, and wild type rats (Wistar Kyoto), were fed 30 mg/kg/d sotagliflozin for 6 weeks. At 23 weeks, LA were imaged in-vivo by echocardiography. In-vitro, Ca2+ transients (CaT; electrically stimulated, caffeine-induced) and spontaneous Ca2+ release were recorded by ratiometric microscopy using Ca2+-sensitive fluorescent dyes (Fura-2) during various experimental protocols. Mitochondrial structure (dye: Mitotracker), Ca2+ buffer capacity (dye: Rhod-2), mitochondrial depolarization (dye: TMRE) and production of reactive oxygen species (dye: H2DCF) were visualized by confocal microscopy. Statistical analysis was performed with 2-way analysis of variance followed by post-hoc Bonferroni and student's t-test, as applicable. RESULTS: Sotagliflozin ameliorated LA enlargement in HFpEF in-vivo. In-vitro, LA cardiomyocytes in HFpEF showed an increased incidence and amplitude of arrhythmic spontaneous Ca2+ release events (SCaEs). Sotagliflozin significantly reduced the magnitude of SCaEs, while their frequency was unaffected. Sotagliflozin lowered diastolic [Ca2+] of CaT at baseline and in response to glucose influx, possibly related to a ~ 50% increase of sodium sodium-calcium exchanger (NCX) forward-mode activity. Sotagliflozin prevented mitochondrial swelling and enhanced mitochondrial Ca2+ buffer capacity in HFpEF. Sotagliflozin improved mitochondrial fission and reactive oxygen species (ROS) production during glucose starvation and averted Ca2+ accumulation upon glycolytic inhibition. CONCLUSION: The SGLT-1&2 inhibitor sotagliflozin ameliorated LA remodeling in metabolic HFpEF. It also improved distinct features of Ca2+-mediated cellular arrhythmogenesis in-vitro (i.e. magnitude of SCaEs, mitochondrial Ca2+ buffer capacity, diastolic Ca2+ accumulation, NCX activity). The safety and efficacy of combined SGLT-1&2 inhibition for the treatment and/or prevention of atrial cardiomyopathy associated arrhythmias should be further evaluated in clinical trials.


Assuntos
Arritmias Cardíacas/prevenção & controle , Função do Átrio Esquerdo/efeitos dos fármacos , Remodelamento Atrial/efeitos dos fármacos , Glicosídeos/farmacologia , Átrios do Coração/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Transportador 1 de Glucose-Sódio/antagonistas & inibidores , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Transportador 2 de Glucose-Sódio/metabolismo , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Modelos Animais de Doenças , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Síndrome Metabólica/complicações , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Ratos Endogâmicos WKY , Ratos Zucker , Espécies Reativas de Oxigênio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Transportador 1 de Glucose-Sódio/metabolismo
2.
Cell Mol Life Sci ; 75(23): 4403-4416, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30062428

RESUMO

Heart failure (HF) manifestation and progression are driven by systemic activation of neuroendocrine signaling cascades, such as the renin-angiotensin aldosterone system (RAAS). Fibroblast growth factor 23 (FGF23), an endocrine hormone, is linked to HF and cardiovascular mortality. It is also a mediator of left-ventricular hypertrophy (LVH). In vivo, high circulating levels of FGF23 are associated with an altered systemic RAAS response. FGF23 is proposed to trigger pathological signaling mediated by Ca2+-regulated transcriptional pathways. In the present study, we investigated Ca2+-dependent signaling of FGF23 in ventricular cardiomyocytes and its association with angiotensin II (ATII). In neonatal rat ventricular myocytes (NRVMs), both ATII and FGF23 induced hypertrophy as observed by an increase in cell area and hypertrophic gene expression. Furthermore, FGF23 activates nuclear Ca2+-regulated CaMKII-HDAC4 pathway, similar to ATII. In addition to a global increase in cytoplasmic Ca2+, FGF23, like ATII, induced inositol 1, 4, 5-triphosphate (IP3)-induced Ca2+ release from the nucleoplasmic Ca2+ store, associated with cellular hypertrophy. Interestingly, ATII receptor antagonist, losartan, significantly attenuated FGF23-induced changes in Ca2+ homeostasis and cellular hypertrophy suggesting an involvement of ATII receptor-mediated signaling. In addition, application of FGF23 increased intracellular expression of ATII peptide and its secretion in NRVMs, confirming the participation of ATII. In conclusion, FGF23 and ATII share a common mechanism of IP3-nuclear Ca2+-dependent cardiomyocyte hypertrophy. FGF23-mediated cellular hypertrophy is associated with increased production and secretion of ATII by cardiomyocytes. These findings indicate a pathophysiological role of the cellular angiotensin system in FGF23-induced hypertrophy in ventricular cardiomyocytes.


Assuntos
Angiotensina II/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Cardiomegalia/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Angiotensina II/metabolismo , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/genética , Células Cultivadas , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Receptor Tipo 1 de Angiotensina/metabolismo
3.
J Mol Cell Cardiol ; 115: 10-19, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29289652

RESUMO

Heart failure (HF) with preserved ejection fraction (HFpEF) is present in about 50% of HF patients. Atrial remodeling is common in HFpEF and associated with increased mortality. We postulate that atrial remodeling is associated with atrial dysfunction in vivo related to alterations in cardiomyocyte Calcium (Ca) signaling and remodeling. We examined atrial function in vivo and Ca transients (CaT) (Fluo4-AM, field stim) in atrial cardiomyocytes of ZSF-1 rats without (Ln; lean hypertensive) and with metabolic syndrome (Ob; obese, hypertensive, diabetic) and HFpEF. RESULTS: At 21weeks Ln showed an increased left ventricular (LV) mass and left ventricular end-diastolic pressure (LVEDP), but unchanged left atrial (LA) size and preserved atrial ejection fraction vs. wild-type (WT). CaT amplitude in atrial cardiomyocytes was increased in Ln (2.9±0.2 vs. 2.3±0.2F/F0 in WT; n=22 cells/group; p<0.05). Studying subcellular Ca release in more detail, we found that local central cytosolic CaT amplitude was increased, while subsarcolemmal CaT amplitudes remained unchanged. Moreover, Sarcoplasmic reticulum (SR) Ca content (caffeine) was preserved while Ca spark frequency and tetracaine-dependent SR Ca leak were significantly increased in Ln. Ob mice developed a HFpEF phenotype in vivo, LA area was significantly increased and atrial in vivo function was impaired, despite increased atrial CaT amplitudes in vitro (2.8±0.2; p<0.05 vs. WT). Ob cells showed alterations of the tubular network possibly contributing to the observed phenotype. CaT kinetics as well as SR Ca in Ob were not significantly different from WT, but SR Ca leak remained increased. Angiotensin II (Ang II) reduced in vitro cytosolic CaT amplitudes and let to active nuclear Ca release in Ob but not in Ln or WT. SUMMARY: In hypertensive ZSF-1 rats, a possibly compensatory increase of cytosolic CaT amplitude and increased SR Ca leak precede atrial remodeling and HFpEF. Atrial remodeling in ZSF-1 HFpEF is associated with an altered tubular network in-vitro and atrial contractile dysfunction in vivo, indicating insufficient compensation. Atrial cardiomyocyte dysfunction in vitro is induced by the addition of angiotensin II.


Assuntos
Átrios do Coração/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Síndrome Metabólica/fisiopatologia , Volume Sistólico , Angiotensina II , Animais , Remodelamento Atrial , Cálcio/metabolismo , Sinalização do Cálcio , Núcleo Celular/metabolismo , Citosol/metabolismo , Modelos Animais de Doenças , Acoplamento Excitação-Contração , Insuficiência Cardíaca/complicações , Ventrículos do Coração/fisiopatologia , Hipertensão/complicações , Hipertensão/fisiopatologia , Síndrome Metabólica/complicações , Miócitos Cardíacos/metabolismo , Ratos , Retículo Sarcoplasmático/metabolismo
4.
Europace ; 19(4): 544-551, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28431065

RESUMO

AIMS: Paroxysmal atrial fibrillation (PAF) is often asymptomatic but nonetheless harmful. We evaluated the performance of disease-related blood biomarkers and CHA2DS2-VASc score to discriminate for PAF in patients with continuous rhythm monitoring. METHODS AND RESULTS: Clinical data and blood samples were obtained from patients with dual-chamber pacemakers selected according to the absence (no_AHRE) or presence of Atrial High-Rate Episodes (AHRE) >6 min in recent device history (case-control approach). We included 93 patients (n = 49 AHRE, n = 44 no_AHRE). In a subgroup with high AHRE burden and confirmed PAF 15 biomarkers were evaluated (n = 19 AHRE-AF vs. n = 20 no_AHRE). Significantly regulated biomarkers were then tested in all patients to distinguish no_AHRE from AHRE (receiver operating characteristics analysis). Hsp27, TGFß1, cystatin C, matrix metalloproteinases MMP-2,-3,-9, albumin, and serum uric acid were not altered in the subgroup. Tissue inhibitors of metalloproteinases (TIMP) -1,-2,-4; NT-proANP, NT-proBNP, IL-6 and serum amyloid protein A were significantly different in AHRE vs. no_AHRE (subgroup and whole cohort), with best discriminatory performance for TIMP-4. Biomarkers performed better than CHADS2-VASc for AHRE discrimination. Intracardial electrograms and medical history from seven AHRE patients suggested atrial tachycardia and not AF (AHRE-AT). Four of the most relevant regulated biomarkers (TIMP-4, TIMP-2, SAA, NT-proBNP) behaved similarly in AHRE-AT and AHRE-AF. NT-proBNP >150 pg/mL indicated an odds ratio of 12.9 for AHRE. Combining two biomarkers significantly improved discrimination of AHRE. CONCLUSION: TIMP-4, NT-proANP, NT-proBNP were strongest associated with PAF and AHRE. The discriminatory performance of CHADS2-VASc for PAF was increased by addition of selected biomarkers.


Assuntos
Fibrilação Atrial/diagnóstico , Fibrilação Atrial/epidemiologia , Fator Natriurético Atrial/sangue , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/epidemiologia , Inibidores Teciduais de Metaloproteinases/sangue , Idoso , Idoso de 80 Anos ou mais , Fibrilação Atrial/sangue , Biomarcadores/sangue , Causalidade , Comorbidade , Eletrocardiografia/estatística & dados numéricos , Feminino , Alemanha/epidemiologia , Humanos , Incidência , Interleucina-6/sangue , Masculino , Reprodutibilidade dos Testes , Medição de Risco/métodos , Sensibilidade e Especificidade , Proteína Amiloide A Sérica/análise , Acidente Vascular Cerebral/sangue , Inibidor Tecidual 4 de Metaloproteinase
5.
J Mol Cell Cardiol ; 89(Pt B): 365-75, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26432951

RESUMO

Glucagon-like peptide-1 receptor (GLP-1R) agonists are a rapidly growing class of drugs developed for treating type-2 diabetes mellitus. Patients with diabetes carry an up to 5-fold greater mortality risk compared to non-diabetic patients, mainly as a result of cardiovascular diseases. Although beneficial cardiovascular effects have been reported, exact mechanisms of GLP-1R-agonist action in the heart, especially in human myocardium, are poorly understood. The effects of GLP-1R-agonists (exenatide, GLP-1(7-36)NH2, PF-06446009, PF-06446667) on cardiac contractility were tested in non-failing atrial and ventricular trabeculae from 72 patients. The GLP-1(7-36)NH2 metabolite, GLP-1(9-36)NH2, was also examined. In electrically stimulated trabeculae, the effects of compounds on isometric force were measured in the absence and presence of pharmacological inhibitors of signal transduction pathways. The role of ß-arrestin signaling was examined using a ß-arrestin partial agonist, PF-06446667. Expression levels were tested by immunoblots. Translocation of GLP-1R downstream molecular targets, Epac2, GLUT-1 and GLUT-4, were assessed by fluorescence microscopy. All tested GLP-1R-agonists significantly increased developed force in human atrial trabeculae, whereas GLP-1(9-36)NH2 had no effect. Exendin(9-39)NH2, a GLP-1R-antagonist, and H-89 blunted the inotropic effect of exenatide. In addition, exenatide increased PKA-dependent phosphorylation of phospholamban (PLB), GLUT-1 and Epac2 translocation, but not GLUT-4 translocation. Exenatide failed to enhance contractility in ventricular myocardium. Quantitative real-time PCR (qRT-PCR) revealed a significant higher GLP-1R expression in the atrium compared to ventricle. Exenatide increased contractility in a dose-dependent manner via GLP-1R/cAMP/PKA pathway and induced GLUT-1 and Epac2 translocation in human atrial myocardium, but had no effect in ventricular myocardium. Therapeutic use of GLP-1R-agonists may therefore impart beneficial effects on myocardial function and remodelling.


Assuntos
Cardiotônicos/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Átrios do Coração/metabolismo , Miocárdio/metabolismo , Peptídeos/farmacologia , Peçonhas/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Exenatida , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Átrios do Coração/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Humanos , Contração Miocárdica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
6.
Circulation ; 130(3): 244-55, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24928680

RESUMO

BACKGROUND: A hallmark of heart failure is impaired cytoplasmic Ca(2+) handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca(2+) handling via altered excitation-transcription coupling contribute to the development and progression of heart failure. METHODS AND RESULTS: Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca(2+) handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca(2+) pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca(2+) transporters. These changes were associated with altered nucleoplasmic Ca(2+) handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca(2+) levels with diminished and prolonged nuclear Ca(2+) transients and slowed intranuclear Ca(2+) diffusion. Altered nucleoplasmic Ca(2+) levels were translated to higher activation of nuclear Ca(2+)/calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca(2+) alterations occurred early during hypertrophy and preceded the cytoplasmic Ca(2+) changes that are typical of heart failure. CONCLUSIONS: During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca(2+) signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca(2+) regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Cardiomegalia/fisiopatologia , Núcleo Celular/metabolismo , Insuficiência Cardíaca/fisiopatologia , Remodelação Ventricular/fisiologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Modelos Animais de Doenças , Estimulação Elétrica , Feminino , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Histona Desacetilases/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Coelhos
7.
Circ Res ; 113(5): 527-38, 2013 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-23825358

RESUMO

RATIONALE: Synchronized release of Ca²âº into the cytosol during each cardiac cycle determines cardiomyocyte contraction. OBJECTIVE: We investigated synchrony of cytosolic [Ca²âº] decay during diastole and the impact of cardiac remodeling. METHODS AND RESULTS: Local cytosolic [Ca²âº] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca²âº] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca²âº release. Stimulation of sarcoplasmic reticulum Ca²âº ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca²âº] decay (TAUglobal). Na⁺/Ca²âº exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca²âº uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. CONCLUSIONS: In cardiomyocytes, cytosolic [Ca²âº] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca²âº] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.


Assuntos
Sinalização do Cálcio/fisiologia , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/citologia , Miócitos Cardíacos/fisiologia , Remodelação Ventricular/fisiologia , Compostos de Anilina/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Colforsina/farmacologia , Citosol/metabolismo , Diástole , Estimulação Elétrica , Etiocolanolona/análogos & derivados , Etiocolanolona/farmacologia , Humanos , Hipertrofia , Hipertrofia Ventricular Esquerda/fisiopatologia , Indóis/farmacologia , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Isquemia Miocárdica/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Éteres Fenílicos/farmacologia , Compostos de Rutênio/farmacologia , Sarcômeros/ultraestrutura , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/genética , Sus scrofa , Suínos
8.
Am J Physiol Heart Circ Physiol ; 307(5): H689-700, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25015964

RESUMO

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling.


Assuntos
Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Urocortinas/metabolismo , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Ventrículos do Coração/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Coelhos , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Serina/metabolismo , Transdução de Sinais
9.
J Cardiovasc Pharmacol ; 64(4): 293-305, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25286359

RESUMO

Cardiac hypertrophy is commonly observed in conditions of increased hemodynamic or metabolic stress. This hypertrophy is not compensatory but rather reflects activation of maladaptive cellular processes that promote disease progression. Myocardial hypertrophy serves as a diagnostic and prognostic marker of cardiac remodeling, and underlying regulatory processes have provided effective therapeutic targets to slow disease progression and improve outcome. We review hypertrophic signaling pathways in cardiomyocytes and discuss established and novel targets for pharmacological intervention. New drugs in the pipeline include the third generation aldosterone antagonists (PF-03882845 and BAY94-8862) and biased angiotensin II receptor agonists. Furthermore, different approaches to stimulate cGMP-dependent protective signaling are currently evaluated in clinical trials, including the combination of the vasopeptidase neprilysin inhibitor and an angiotensin receptor blocker (ARNi). In an overview on cardiomyocyte hypertrophic signaling, we also highlight emerging experimental treatment concepts such as inhibition of Ca-mediated transcriptional regulation, adeno-associated viruses for sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), PI3 kinase gene transfer and microRNA-based therapy. We conclude that antihypertrophic therapy extends beyond blocking the classical ß-adrenergic and renin-angiotensin-aldosterone system-dependent signaling cascades, although new therapies require clinical validation regarding outcome.


Assuntos
Anti-Hipertensivos/uso terapêutico , Cardiomegalia/tratamento farmacológico , Cardiomegalia/metabolismo , Insuficiência Cardíaca/prevenção & controle , Terapia de Alvo Molecular , Anti-Hipertensivos/administração & dosagem , Cardiomegalia/complicações , Cardiomegalia/patologia , Ensaios Clínicos como Assunto , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Remodelação Ventricular/efeitos dos fármacos
10.
J Biol Chem ; 287(13): 9827-9834, 2012 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-22228770

RESUMO

Insulin-like growth factor 1 (IGF1) promotes a physiological type of cardiac hypertrophy and has therapeutic effects in heart disease. Here, we report the relationship of IGF1 to GATA4, an essential transcription factor in cardiac hypertrophy and cell survival. In cultured neonatal rat ventricular myocytes, we compared the responses to IGF1 (10 nmol/liter) and phenylephrine (PE, 20 µmol/liter), a known GATA4 activator, in concentrations promoting a similar extent of hypertrophy. IGF1 and PE both increased nuclear accumulation of GATA4 and phosphorylation at Ser(105) (PE, 2.4-fold; IGF1, 1.8-fold; both, p < 0.05) and increased GATA4 DNA binding activity as indicated by ELISA and by chromatin IP of selected promoters. Although IGF1 and PE each activated GATA4 to the same degree, GATA4 knockdown by RNA interference only blocked hypertrophy by PE but not by IGF1. PE induction of a panel of GATA4 target genes (Nppa, Nppb, Tnni3, Myl1, and Acta1) was inhibited by GATA4 knockdown. In contrast, IGF1 regulated only Acta1 in a GATA4-dependent fashion. Consistent with the in vitro findings, Gata4 haploinsufficiency in mice did not alter cardiac structure, hyperdynamic function, or antifibrotic effects induced by myocardial overexpression of the IGF1 receptor. Our data indicate that GATA4 is activated by the IGF1 pathway, but although it is required for responses to pathological stimuli, it is not necessary for the effects of IGF1 on cardiac structure and function.


Assuntos
Cardiomegalia/metabolismo , Fator de Transcrição GATA4/metabolismo , Fator de Crescimento Insulin-Like I/efeitos adversos , Miócitos Cardíacos/metabolismo , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Cardiomegalia/patologia , Células Cultivadas , Fator de Transcrição GATA4/genética , Técnicas de Silenciamento de Genes , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Camundongos Knockout , Miócitos Cardíacos/patologia , Regiões Promotoras Genéticas/genética , Ratos , Ratos Wistar
12.
Front Cardiovasc Med ; 9: 859014, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35865376

RESUMO

Background: Although the angiotensin receptor-neprilysin inhibitor (ARNI) sacubitril/valsartan started a new era in heart failure (HF) treatment, less is known about the tissue-level effects of the drug on the atrial myocardial functional reserve and arrhythmogenesis. Methods and Results: Right atrial (RA) biopsies were retrieved from patients (n = 42) undergoing open-heart surgery, and functional experiments were conducted in muscle strips (n = 101). B-type natriuretic peptide (BNP) did not modulate systolic developed force in human myocardium during ß-adrenergic stimulation, but it significantly reduced diastolic tension (p < 0.01) and the probability of arrhythmias (p < 0.01). In addition, patient's plasma NTproBNP positively correlated with isoproterenol-induced contractile reserve in atrial tissue in vitro (r = 0.65; p < 0.01). Sacubitrilat+valsartan (Sac/Val) did not show positive inotropic effects on atrial trabeculae function but reduced arrhythmogeneity. Atrial and ventricular biopsies from patients with end-stage HF (n = 10) confirmed that neprilysin (NEP) is equally expressed in human atrial and ventricular myocardium. RA NEP expression correlates positively with RA ejection fraction (EF) (r = 0.806; p < 0.05) and left ventricle (LV) NEP correlates inversely with left atrial (LA) volume (r = -0.691; p < 0.05). Conclusion: BNP ameliorates diastolic tension during adrenergic stress in human atrial myocardium and may have positive long-term effects on the inotropic reserve. BNP and Sac/Val reduce atrial arrhythmogeneity during adrenergic stress in vitro. Myocardial NEP expression is downregulated with declining myocardial function, suggesting a compensatory mechanism in HF.

13.
ESC Heart Fail ; 8(3): 1806-1818, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33768692

RESUMO

AIMS: Heart failure with preserved ejection fraction (HFpEF) is an increasingly prevalent disease. Physical exercise has been shown to alter disease progression in HFpEF. We examined cardiomyocyte Ca2+ homeostasis and left ventricular function in a metabolic HFpEF model in sedentary and trained rats following 8 weeks of moderate-intensity continuous training (MICT) or high-intensity interval training (HIIT). METHODS AND RESULTS: Left ventricular in vivo function (echocardiography) and cardiomyocyte Ca2+ transients (CaTs) (Fluo-4, confocal) were compared in ZSF-1 obese (metabolic syndrome, HFpEF) and ZSF-1 lean (control) 21- and 28-week-old rats. At 21 weeks, cardiomyocytes from HFpEF rats showed prolonged Ca2+ reuptake in cytosolic and nuclear CaTs and impaired Ca2+ release kinetics in nuclear CaTs. At 28 weeks, HFpEF cardiomyocytes had depressed CaT amplitudes, decreased sarcoplasmic reticulum (SR) Ca2+ content, increased SR Ca2+ leak, and elevated diastolic [Ca2+ ] following increased pacing rate (5 Hz). In trained HFpEF rats (HIIT or MICT), cardiomyocyte SR Ca2+ leak was significantly reduced. While HIIT had no effects on the CaTs (1-5 Hz), MICT accelerated early Ca2+ release, reduced the amplitude, and prolonged the CaT without increasing diastolic [Ca2+ ] or cytosolic Ca2+ load at basal or increased pacing rate (1-5 Hz). MICT lowered pro-arrhythmogenic Ca2+ sparks and attenuated Ca2+ -wave propagation in cardiomyocytes. MICT was associated with increased stroke volume in HFpEF. CONCLUSIONS: In this metabolic rat model of HFpEF at an advanced stage, Ca2+ release was impaired under baseline conditions. HIIT and MICT differentially affected Ca2+ homeostasis with positive effects of MICT on stroke volume, end-diastolic volume, and cellular arrhythmogenicity.


Assuntos
Insuficiência Cardíaca , Animais , Ecocardiografia , Miócitos Cardíacos , Ratos , Retículo Sarcoplasmático , Volume Sistólico
14.
ESC Heart Fail ; 8(4): 3130-3144, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34002482

RESUMO

AIMS: Heart failure with preserved ejection fraction (HFpEF) is frequently (30%) associated with right ventricular (RV) dysfunction, which increases morbidity and mortality in these patients. Yet cellular mechanisms of RV remodelling and RV dysfunction in HFpEF are not well understood. Here, we evaluated RV cardiomyocyte function in a rat model of metabolically induced HFpEF. METHODS AND RESULTS: Heart failure with preserved ejection fraction-prone animals (ZSF-1 obese) and control rats (Wistar Kyoto) were fed a high-caloric diet for 13 weeks. Haemodynamic characterization by echocardiography and invasive catheterization was performed at 22 and 23 weeks of age, respectively. After sacrifice, organ morphometry, RV histology, isolated RV cardiomyocyte function, and calcium (Ca2+ ) transients were assessed. ZSF-1 obese rats showed a HFpEF phenotype with left ventricular (LV) hypertrophy, LV diastolic dysfunction (including increased LV end-diastolic pressures and E/e' ratio), and preserved LV ejection fraction. ZSF-1 obese animals developed RV dilatation (50% increased end-diastolic area) and mildly impaired RV ejection fraction (42%) with evidence of RV hypertrophy. In isolated RV cardiomyocytes from ZSF-1 obese rats, cell shortening amplitude was preserved, but cytosolic Ca2+ transient amplitude was reduced. In addition, augmentation of cytosolic Ca2+ release with increased stimulation frequency was lost in ZSF-1 obese rats. Myofilament sensitivity was increased, while contractile kinetics were largely unaffected in intact isolated RV cardiomyocytes from ZSF-1 obese rats. Western blot analysis revealed significantly increased phosphorylation of cardiac myosin-binding protein C (Ser282 cMyBP-C) but no change in phosphorylation of troponin I (Ser23, 24 TnI) in RV myocardium from ZSF-1 obese rats. CONCLUSIONS: Right ventricular dysfunction in obese ZSF-1 rats with HFpEF is associated with intrinsic RV cardiomyocyte remodelling including reduced cytosolic Ca2+ amplitudes, loss of frequency-dependent augmentation of Ca2+ release, and increased myofilament Ca2+ sensitivity.


Assuntos
Insuficiência Cardíaca , Disfunção Ventricular Direita , Animais , Insuficiência Cardíaca/etiologia , Homeostase , Humanos , Miócitos Cardíacos , Miofibrilas , Ratos , Volume Sistólico , Disfunção Ventricular Direita/etiologia
15.
ESC Heart Fail ; 8(1): 151-161, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33251761

RESUMO

AIMS: Atrial contractile dysfunction contributes to worse prognosis in hypertensive heart disease (HHD), but the role of cardiomyocyte dysfunction in atrial remodelling in HHD is not well understood. We investigated and compared cellular mechanisms of left (LA) and right atrial (RA) contractile dysfunction in pigs with HHD. METHODS AND RESULTS: In vivo electrophysiological and magnetic resonance imaging studies were performed in control and pigs treated with 11-deoxycorticosterone acetate (DOCA)/high-salt/glucose diet (12 weeks) to induce HHD. HHD leads to significant atrial remodelling and loss of contractile function in LA and a similar trend in RA (magnetic resonance imaging). Atrial remodelling was associated with a higher inducibility of atrial fibrillation but unrelated to changes in atrial refractory period or fibrosis (histology). Reduced atrial function in DOCA pigs was related to reduced contraction amplitude of isolated LA (already at baseline) and RA myocytes (at higher frequencies) due to reduced intracellular Ca release (Fura 2-AM, field stimulation). However, Ca regulation differed in LA and RA cardiomyocytes: LA cardiomyocytes showed reduced sarcoplasmic reticulum (SR) [Ca], whereas in RA, SR [Ca] was unchanged and SR Ca2+ -ATPase activity was increased. Sodium-calcium exchanger (NCX) activity was not significantly altered. We used ORM-10103 (3 µM), a specific NCX inhibitor to improve Ca availability in LA and RA cardiomyocytes from DOCA pigs. Partial inhibition of NCX increased Ca2+ transient amplitude and SR Ca in LA, but not RA cells. CONCLUSIONS: In this large animal model of HHD, atrial remodelling in sinus rhythm in vivo was related to differential LA and RA cardiomyocyte dysfunction and Ca signalling. Selective acute inhibition of NCX improved Ca release in diseased LA cardiomyocytes, suggesting a potential therapeutic approach to improve atrial inotropy in HHD.


Assuntos
Cálcio , Hipertensão , Animais , Cálcio/metabolismo , Átrios do Coração/diagnóstico por imagem , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio , Suínos
16.
Biochem J ; 400(2): 377-83, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16987104

RESUMO

It is reasonably well understood how the initiation of translation is controlled by reversible phosphorylation of the eukaryotic translation initiation factors eIF2alpha, eIF2Bepsilon and eIF4E. Other initiation factors, including eIF2beta, are also established phosphoproteins but the physiological impact of their phosphorylation is not known. Using a sequence homology search we found that the central region of eIF2beta contains a putative PP1-(protein phosphatase-1) binding RVxF-motif. The predicted eIF2beta-PP1 interaction was confirmed by PP1 binding and co-immunoprecipitation assays on cell lysates as well as with the purified components. Site-directed mutagenesis showed that eIF2beta contains, in addition to an RVxF-motif, at least one other PP1-binding site in its C-terminal half. eIF2beta functioned as an inhibitor for the dephosphorylation of glycogen phosphorylase and Ser51 of eIF2alpha by PP1, but did not affect the dephosphorylation of Ser464 of eIF2Bepsilon by this phosphatase. Strikingly, eIF2beta emerged as an activator of its own dephosphorylation (Ser2, Ser67, Ser218) by associated PP1, since the substrate quality of eIF2beta was decreased by the mere mutation of its RVxF-motif. These results make eIF2beta an attractive candidate substrate for associated PP1 in vivo. The overexpression of wild-type eIF2beta or eIF2beta with a mutated RVxF-motif did not differentially affect the rate of translation, indicating that the binding of PP1 is not rate-limiting for translation under basal conditions.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/genética , Humanos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosfoproteínas Fosfatases/genética , Biossíntese de Proteínas , Proteína Fosfatase 1 , Coelhos , Alinhamento de Sequência , Especificidade por Substrato , Suínos
17.
Eur J Heart Fail ; 18(8): 987-97, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27135883

RESUMO

AIMS: Heart failure with preserved ejection fraction (HFpEF) is increasingly common, but the underlying cellular mechanisms are not well understood. We investigated cardiomyocyte function and the role of SEA0400, an Na(+) /Ca(2+) exchanger (NCX) inhibitor in a rat model of chronic kidney disease (CKD) with HFpEF. METHODS AND RESULTS: Male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (Sham). After 8 and 24 weeks, in vivo (haemodynamics, echocardiography) and in vitro function (LV cardiomyocyte cell shortening (CS), and Ca(2+) transients (CaT)) were determined without and with SEA0400. In a subgroup of rats, SEA0400 or vehicle was given p.o. (1 mg/kg b.w.) between week 8 and 24. NXT resulted in stable compensated CKD and HFpEF [hypertrophied left ventricle, prolonged LV isovolumetric relaxation constant TAU (IVRc TAU), elevated end diastolic pressure (EDP), increased lung weight (pulmonary congestion), and preserved LV systolic function (EF, dP/dt)]. In NXT cardiomyocytes, the amplitude of CS and CaT were unchanged but relaxation and CaT decay were progressively prolonged at 8 and 24 weeks vs. Sham, individually correlating with diastolic dysfunction in vivo. NCX forward mode activity (caffeine response) was progressively reduced, while NCX protein expression was up-regulated, suggesting increased NCX reverse mode activity in NXT. SEA0400 acutely improved relaxation in NXT in vivo and in cardiomyocytes and improved cardiac remodelling and diastolic function when given chronically. CONCLUSIONS: This model of renal HFpEF is associated with slowed relaxation of LV cardiomyocytes. Treatment with SEA0400 improved cardiomyocyte function, remodelling, and HFpEF.


Assuntos
Compostos de Anilina/farmacologia , Insuficiência Cardíaca/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Éteres Fenílicos/farmacologia , Insuficiência Renal Crônica/fisiopatologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Volume Sistólico , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Ecocardiografia , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/complicações , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Ratos , Ratos Wistar , Insuficiência Renal Crônica/complicações
18.
J Pharmacol Toxicol Methods ; 63(2): 180-5, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-20883803

RESUMO

INTRODUCTION: Fundamental differences in Ca²+ homeostasis between mice and larger mammals require the validation of the mechanisms of arrhythmogenesis before translation into human pathophysiology. The purpose of this study was to create transgenic rabbits that express defective human cardiac ryanodine receptor (hRyR2) with a mutation (R4497C) causing a clinically relevant arrhythmogenic syndrome. METHODS: The construct pcDNA3-EGFP-hRyR2-R4497C with the CMV promoter was used to generate transgenic rabbits. The founder animals were created by microinjection and identified by PCR with specific primers for the EGFP sequence. The copy number of the transgene was quantified by real-time PCR using genomic DNA from blood cells. mRNA expression of EGFP-hRyR2-R4497C was quantified using RT-PCR with specific primers for the RyR2 and EGFP sequence. Protein expression of the transgene in heart and non-cardiac tissues was determined using immunoblots with antibodies directed against EGFP and RyR2. RESULTS: Real-time PCR in peripheral blood cells identified several rabbit lines with the construct integrated into their genome. Transcription levels of the transgene were low (Ct>30). On the protein level, neither EGFP nor hRyR2 R4497C was detected in either cardiac or non-cardiac tissue. A truncated gene product (3' end and central part of hRyR2 R4497C, but not EGFP) could be detected at the mRNA level in the heart. DISCUSSION: Lack of significant protein expression of the EGFP-RyR2 R4497C gene construct despite successful incorporation into the genomic DNA is due to combination of at least two factors: low mRNA expression, and truncation of the transgene on the mRNA level. Our results suggest that the CMV promoter may not be well suited for creating transgenic rabbits.


Assuntos
Animais Geneticamente Modificados/genética , Citomegalovirus/genética , Mutação , Regiões Promotoras Genéticas , Coelhos/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/biossíntese , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Expressão Gênica , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Miocárdio/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Coelhos/sangue , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Transgenes
19.
Cardiovasc Res ; 87(1): 50-9, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20080988

RESUMO

AIMS: Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content--in the absence of protein kinase A activation--affects RyR2 function and arrhythmogenesis in CPVT remains elusive. METHODS AND RESULTS: Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release, and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events. CONCLUSION: The elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.


Assuntos
Cálcio/metabolismo , Catecolaminas/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Sódio/metabolismo , Taquicardia Ventricular/metabolismo , Potenciais de Ação , Animais , Sinalização do Cálcio , Inibidores Enzimáticos/farmacologia , Feminino , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Miócitos Cardíacos/efeitos dos fármacos , Ouabaína/farmacologia , Técnicas de Patch-Clamp , Fosforilação , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/prevenção & controle , Tiazepinas/farmacologia , Fatores de Tempo
20.
J Biol Chem ; 278(21): 18817-23, 2003 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-12657641

RESUMO

Most interactors of protein phosphatase-1 (PP1) contain a variant of a so-called "RVXF" sequence that binds to a hydrophobic groove of the catalytic subunit. A combination of sequence alignments and site-directed mutagenesis has enabled us to further define the consensus sequence for this degenerate motif as [RK]-X(0-1)-[VI]-[P]-[FW], where X denotes any residue and [P] any residue except Pro. Naturally occurring RVXF sequences differ in their affinity for PP1, and we show by swapping experiments that this binding affinity is an important determinant of the inhibitory potency of the regulators NIPP1 and inhibitor-1. Also, inhibition by NIPP1-(143-224) was retained when the RVXF motif (plus the preceding Ser) was swapped for either of two unrelated PP1-binding sequences from human inhibitor-2, i.e. KGILK or RKLHY. Conversely, the KGILK motif of inhibitor-2 could be functionally replaced by the RVXF motif of NIPP1. Our data provide additional evidence for the view that the RVXF and KGILK motifs function as anchors for PP1 and thereby promote the interaction of secondary binding sites that determine the activity and substrate specificity of the enzyme.


Assuntos
Sequência Consenso , Endorribonucleases , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Ligação a RNA , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Células COS , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Glutationa Transferase/genética , Humanos , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosforilação , Proteína Fosfatase 1 , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Coelhos , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Relação Estrutura-Atividade
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