RESUMO
FKBP12.6, a binding protein to the immunosuppressant FK506, which also binds the ryanodine receptor (RyR2) in the heart, has been proposed to regulate RyR2 function and to have antiarrhythmic properties. However, the level of FKBP12.6 expression in normal hearts remains elusive and some controversies still persist regarding its effects, both in basal conditions and during ß-adrenergic stimulation. We quantified FKBP12.6 in the left ventricles (LV) of WT (wild-type) mice and in two novel transgenic models expressing distinct levels of FKBP12.6, using a custom-made specific anti-FKBP12.6 antibody and a recombinant protein. FKBP12.6 level in WT LV was very low (0.16 ± 0.02 nmol/g of LV), indicating that <15% RyR2 monomers are bound to the protein. Mice with 14.1 ± 0.2 nmol of FKBP12.6 per g of LV (TG1) had mild cardiac hypertrophy and normal function and were protected against epinephrine/caffeine-evoked arrhythmias. The ventricular myocytes showed higher [Ca2+]i transient amplitudes than WT myocytes and normal SR-Ca2+ load, while fewer myocytes showed Ca2+ sparks. TG1 cardiomyocytes responded to 50 nM Isoproterenol increasing these [Ca2+]i parameters and producing RyR2-Ser2808 phosphorylation. Mice with more than twice the TG1 FKBP12.6 value (TG2) showed marked cardiac hypertrophy with calcineurin activation and more arrhythmias than WT mice during ß-adrenergic stimulation, challenging the protective potential of high FKBP12.6. RyR2R420Q CPVT mice overexpressing FKBP12.6 showed fewer proarrhythmic events and decreased incidence and duration of stress-induced bidirectional ventricular tachycardia. Our study, therefore, quantifies for the first time endogenous FKBP12.6 in the mouse heart, questioning its physiological relevance, at least at rest due its low level. By contrast, our work demonstrates that with caution FKBP12.6 remains an interesting target for the development of new antiarrhythmic therapies.
Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina , Taquicardia Ventricular , Proteínas de Ligação a Tacrolimo , Animais , Camundongos , Adrenérgicos , Antiarrítmicos/farmacologia , Cardiomegalia , Incidência , Miócitos Cardíacos , Taquicardia Ventricular/genéticaRESUMO
AIM: Left ventricular remodeling (LVR) after myocardial infarction (MI) can lead to heart failure, arrhythmia, and death. We aim to describe adverse LVR patterns at 6 months post-MI and their relationships with subsequent outcomes and to determine baseline. METHODS AND RESULTS: A multicenter cohort of 410 patients (median age 57 years, 87% male) with reperfused MI and at least 3 akinetic LV segments on admission was analyzed. All patients had transthoracic echocardiography performed 4 days and 6 months post-MI, and 214 also had cardiac magnetic resonance imaging performed on day 4. To predict LVR, machine learning methods were employed in order to handle many variables, some of which may have complex interactions. Six months post-MI, echocardiographic increases in LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), and LV ejection fraction (LVEF) were 14.1% [interquartile range 0.0, 32.0], 5.0% [- 14.0, 25.8], and 8.7% [0.0, 19.4], respectively. At 6 months, ≥ 15% or 20% increases in LVEDV were observed in 49% and 42% of patients, respectively, and 37% had an LVEF < 50%. The rate of death or new-onset HF at the end of 5-year follow-up was 8.8%. Baseline variables associated with adverse LVR were determined best by random forest analysis and included stroke volume, stroke work, necrosis size, LVEDV, LVEF, and LV afterload, the latter assessed by Ea or Ea/Ees. In contrast, baseline clinical and biological characteristics were poorly predictive of LVR. After adjustment for predictive baseline variables, LV dilation > 20% and 6-month LVEF < 50% were significantly associated with the risk of death and/or heart failure: hazard ratio (HR) 2.12 (95% confidence interval (CI) 1.05-4.43; p = 0.04) and HR 2.68 (95% CI 1.20-6.00; p = 0.016) respectively. CONCLUSION: Despite early reperfusion and cardioprotective therapy, adverse LVR remains frequent after acute MI and is associated with a risk of death and HF. A machine learning approach identified and prioritized early variables that are associated with adverse LVR and which were mainly hemodynamic, combining LV volumes, estimates of systolic function, and afterload.
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BACKGROUND: Myocardial infarction (MI) induces a repair response that ultimately generates a stable fibrotic scar. Although the scar prevents cardiac rupture, an excessive profibrotic response impairs optimal recovery by promoting the development of noncontractile fibrotic areas. The mechanisms that lead to cardiac fibrosis are diverse and incompletely characterized. We explored whether the expansion of cardiac fibroblasts after MI can be regulated through a paracrine action of cardiac stromal cells. METHODS: We performed a bioinformatic secretome analysis of cardiac stromal PW1+ cells isolated from normal and post-MI mouse hearts to identify novel secreted proteins. Functional assays were used to screen secreted proteins that promote fibroblast proliferation. The expressions of candidates were subsequently analyzed in mouse and human hearts and plasmas. The relationship between levels of circulating protein candidates and adverse post-MI cardiac remodeling was examined in a cohort of 80 patients with a first ST-segment-elevation MI and serial cardiac magnetic resonance imaging evaluations. RESULTS: Cardiac stromal PW1+ cells undergo a change in paracrine behavior after MI, and the conditioned media from these cells induced a significant increase in the proliferation of fibroblasts. We identified a total of 12 candidates as secreted proteins overexpressed by cardiac PW1+ cells after MI. Among these factors, GDF3 (growth differentiation factor 3), a member of the TGF-ß (transforming growth factor-ß) family, was markedly upregulated in the ischemic hearts. Conditioned media specifically enriched with GDF3 induced fibroblast proliferation at a high level by stimulation of activin-receptor-like kinases. In line with the secretory nature of this protein, we next found that GDF3 can be detected in mice and human plasma samples, with a significant increase in the days after MI. In humans, higher GDF3 circulating levels (measured in the plasma at day 4 after MI) were significantly associated with an increased risk of adverse remodeling 6 months after MI (adjusted odds ratio, 1.76 [1.03-3.00]; P=0.037), including lower left ventricular ejection fraction and a higher proportion of akinetic segments. CONCLUSIONS: Our findings define a mechanism for the profibrotic action of cardiac stromal cells through secreted cardiokines, such as GDF3, a candidate marker of adverse fibrotic remodeling after MI. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT01113268.
Assuntos
Infarto do Miocárdio , Miocárdio , Animais , Humanos , Camundongos , Cicatriz/patologia , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Fibrose , Fator 3 de Diferenciação de Crescimento/metabolismo , Miocárdio/metabolismo , Volume Sistólico , Fator de Crescimento Transformador beta/metabolismo , Função Ventricular Esquerda , Remodelação VentricularRESUMO
Heart Failure (HF) is defined as the inability of the heart to efficiently pump out enough blood to maintain the body's needs, first at exercise and then also at rest. Alterations in Ca2+ handling contributes to the diminished contraction and relaxation of the failing heart. While most Ca2+ handling protein expression and/or function has been shown to be altered in many models of experimental HF, in this review, we focus in the sarcoplasmic reticulum (SR) Ca2+ release channel, the type 2 ryanodine receptor (RyR2). Various modifications of this channel inducing alterations in its function have been reported. The first was the fact that RyR2 is less responsive to activation by Ca2+ entry through the L-Type calcium channel, which is the functional result of an ultrastructural remodeling of the ventricular cardiomyocyte, with fewer and disorganized transverse (T) tubules. HF is associated with an elevated sympathetic tone and in an oxidant environment. In this line, enhanced RyR2 phosphorylation and oxidation have been shown in human and experimental HF. After several controversies, it is now generally accepted that phosphorylation of RyR2 at the Calmodulin Kinase II site (S2814) is involved in both the depressed contractile function and the enhanced arrhythmic susceptibility of the failing heart. Diminished expression of the FK506 binding protein, FKBP12.6, may also contribute. While these alterations have been mostly studied in the left ventricle of HF with reduced ejection fraction, recent studies are looking at HF with preserved ejection fraction. Moreover, alterations in the RyR2 in HF may also contribute to supraventricular defects associated with HF such as sinus node dysfunction and atrial fibrillation.
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BACKGROUND: Microvascular obstruction (MVO) is associated with poor outcome after ST-segment elevation myocardial infarction (STEMI). Vascular endothelial growth factor-A (VEGF-A) is a vascular permeability inducer playing a key role in MVO pathogenesis. We aimed to assess whether VEGF-A levels are associated with MVO, when evaluated by magnetic resonance imaging (MRI) in STEMI patients. METHODS: The multicenter prospective PREGICA study included a CMR substudy with all consecutive patients with a first STEMI who had undergone cardiac MRI at baseline and at 6-month follow-up. Patients with initial TIMI flow >1 were excluded. VEGF-A levels were measured in blood samples drawn at inclusion. RESULTS: Between 2010 and 2017, 147 patients (mean age 57⯱â¯10â¯years; 84% males) were included. MVO was present in 65 (44%) patients. After multivariate analysis, higher troponin peak (OR 1.005; 95% CI 1.001-1.008; pâ¯=â¯0.007) and VEGF-A levels (OR 1.003; 95% CI 1.001-1.005; pâ¯=â¯0.015) were independently associated with MVO. When considering only patients with successful percutaneous coronary intervention (final TIMI flow 3, nâ¯=â¯130), higher troponin peak (pâ¯=â¯0.004) and VEGF-A levels (pâ¯=â¯0.03) remained independently predictive of MVO. Moreover, MVO was associated with adverse left ventricular (LV) remodeling and VEGF-A levels were significantly and inversely correlated with LV ejection fraction (EF) at 6-month follow-up. CONCLUSION: Our results show that VEGF-A levels were independently associated with MVO during STEMI and correlated with mid-term LVEF alteration. VEGF-A could therefore be considered as a biomarker of MVO in STEMI patients and be used to stratify patient prognosis.
Assuntos
Oclusão Coronária/sangue , Oclusão Coronária/diagnóstico por imagem , Microcirculação/fisiologia , Infarto do Miocárdio com Supradesnível do Segmento ST/sangue , Infarto do Miocárdio com Supradesnível do Segmento ST/diagnóstico por imagem , Fator A de Crescimento do Endotélio Vascular/sangue , Idoso , Biomarcadores/sangue , Oclusão Coronária/cirurgia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Intervenção Coronária Percutânea/métodos , Estudos Prospectivos , Infarto do Miocárdio com Supradesnível do Segmento ST/cirurgiaAssuntos
Infarto Miocárdico de Parede Anterior/diagnóstico por imagem , Imageamento por Ressonância Magnética , Infarto do Miocárdio com Supradesnível do Segmento ST/diagnóstico por imagem , Trombose/diagnóstico por imagem , Idoso , Infarto Miocárdico de Parede Anterior/complicações , Feminino , França , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Fatores de Risco , Infarto do Miocárdio com Supradesnível do Segmento ST/complicações , Trombose/etiologia , Fatores de TempoRESUMO
OBJECTIVES: Current methods for infarct size and microvascular obstruction (MVO) quantification by cardiac magnetic resonance (CMR) imaging rely on planimetry. This method is time-consuming. We sought to evaluate a direct assessment of MVO severity based on visual evaluation and to compare it to a reference method. METHODS: CMR was performed in 112 consecutive patients after reperfused myocardial infarction. MVO was estimated by direct visual assessment based on a three-grade severity scale (MVO 1, mild; MVO 2, moderate; MVO 3, severe) on late gadolinium-enhancement (LGE). RESULTS: MVO was present in 69 patients (61.6 %). Quantitative MVO extent significantly increased accordingly to visual MVO grading (p < 0.01). Correlation between visual grading and quantitative assessment was excellent (r = 0.92, IQR 0.88-0.95, p < 0.001). CMR inter- and intraobserver variability for visual MVO evaluation was low (κ = 0.93 and κ = 0.96, respectively), whereas quantitative MVO assessment suffered from moderate agreement (interobserver, bias = -0.81 ± 1.8 g LV; intraobserver, -0.83 ± 2.1 g LV). Visual evaluation was significantly faster than reference method (0.65 ± 0.37 vs. 10.2 ± 2.9 min, p < 0.0001). CONCLUSIONS: MVO severity based on direct visual assessment on LGE images is feasible, rapid, reproducible and agrees very well with quantitative methods, with a very low inter- and intraobserver variability. Our approach could be used for routine evaluation in patients undergoing CMR after acute myocardial infarction. KEY POINTS: ⢠Microvascular obstruction direct visual evaluation is feasible, rapid and highly reproducible. ⢠Microvascular obstruction direct visual evaluation correlates well with quantification by planimetry. ⢠Microvascular obstruction or no-reflow phenomenon is determined on late gadolinium-enhanced images. ⢠Cardiac MRI is useful for myocardial damage assessment after myocardial infarction.
Assuntos
Doença da Artéria Coronariana/diagnóstico por imagem , Microvasos/diagnóstico por imagem , Infarto do Miocárdio/diagnóstico por imagem , Reperfusão Miocárdica , Infarto do Miocárdio com Supradesnível do Segmento ST/diagnóstico por imagem , Arteriopatias Oclusivas/diagnóstico por imagem , Meios de Contraste , Angiografia Coronária , Feminino , Gadolínio , Humanos , Imageamento por Ressonância Magnética , Imagem Cinética por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/terapia , Reperfusão Miocárdica/métodos , Intervenção Coronária Percutânea , Estudos Prospectivos , Infarto do Miocárdio com Supradesnível do Segmento ST/terapia , Trombectomia , Terapia TrombolíticaRESUMO
Diabetes mellitus is one of the most common medical conditions. It is associated to medical complications in numerous organs and tissues, of which the heart is one of the most important and most prevalent organs affected by this disease. In fact, cardiovascular complications are the most common cause of death among diabetic patients. At the end of the 19th century, the weakness of the heart in diabetes was noted as part of the general muscular weakness that exists in that disease. However, it was only in the eighties that diabetic cardiomyopathy was recognized, which comprises structural and functional abnormalities in the myocardium in diabetic patients even in the absence of coronary artery disease or hypertension. This disorder has been associated with both type 1 and type 2 diabetes, and is characterized by early-onset diastolic dysfunction and late-onset systolic dysfunction, in which alteration in Ca(2+) signaling is of major importance, since it controls not only contraction, but also excitability (and therefore is involved in rhythmic disorder), enzymatic activity, and gene transcription. Here we attempt to give a brief overview of Ca(2+) fluxes alteration reported on diabetes, and provide some new data on differential modulation of Ca(2+) handling alteration in males and females type 2 diabetic mice to promote further research. Due to space limitations, we apologize for those authors whose important work is not cited.
Assuntos
Sinalização do Cálcio/genética , Diabetes Mellitus Tipo 2/genética , Cardiomiopatias Diabéticas/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/fisiopatologia , Feminino , Humanos , Masculino , Camundongos , Miócitos Cardíacos/patologia , Estresse Oxidativo/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Caracteres SexuaisRESUMO
BACKGROUND: Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling. METHODOLOGY AND PRINCIPAL FINDINGS: Five week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity. CONCLUSIONS/SIGNIFICANCE: Gender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases.
Assuntos
Cardiomegalia/enzimologia , Hipertensão/enzimologia , Fosfoproteínas Fosfatases/fisiologia , Animais , Feminino , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Miocárdio/enzimologia , Caracteres Sexuais , Remodelação VentricularRESUMO
Chronic pressure overload (PO) induces pathological left ventricular hypertrophy (LVH) leading to congestive heart failure (HF). Overexpression of FKBP12.6 (FK506-binding protein [K]) in mice should prevent Ca2+-leak during diastole and may improve overall cardiac function. In order to decipher molecular mechanisms involved in thoracic aortic constriction (TAC)-induced cardiac remodeling and the influence of gender and genotype, we performed a proteomic analysis using two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and bioinformatics techniques to identify alterations in characteristic biological networks. Wild-type (W) and K mice of both genders underwent TAC. Thirty days post-TAC, the altered cardiac remodeling was accompanied with systolic and diastolic dysfunction in all experimental groups. A gender difference in inflammatory protein expression (fibrinogen, α-1-antitrypsin isoforms) and in calreticulin occurred (males > females). Detoxification enzymes and cytoskeletal proteins were noticeably increased in K mice. Both non- and congestive failing mouse heart exhibited down- and upregulation of proteins related to mitochondrial function and purine metabolism, respectively. HF was characterized by a decrease in enzymes related to iron homeostasis, and altered mitochondrial protein expression related to fatty acid metabolism, glycolysis, and redox balance. Moreover, two distinct differential protein profiles characterized TAC-induced pathological LVH and congestive HF in all TAC mice. FKBP12.6 overexpression did not influence TAC-induced deleterious effects. Huntingtin was revealed as a potential mediator for HF. A broad dysregulation of signaling proteins associated with congestive HF suggested that different sets of proteins could be selected as useful biomarkers for HF progression and might predict outcome in PO-induced pathological LVH.
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Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca(2+) handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca(2+)](i) transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca(2+) content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca(2+) content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca(2+)](i) transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca(2+) content and Na(+)- Ca(2+) exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 µm)(-1) s(-1) in Tg MI versus 1.6 ± 0.2 sparks (100 µm)(-1) s(-1) in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted.
Assuntos
Infarto do Miocárdio/fisiopatologia , Proteínas de Ligação a Tacrolimo/biossíntese , Remodelação Ventricular/efeitos dos fármacos , Animais , Cálcio/metabolismo , Camundongos , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
Alterations in RyR2 function have been proposed as a major pathophysiological mechanism of arrhythmias and heart failure (HF). Cardiac FKBP12.6 overexpression protects against myocardial infarction-induced HF and catecholamine-promoted ventricular arrhythmias. We tested the hypothesis that FKBP12.6 overexpression protects against maladaptive LVH and triggered ventricular arrhythmias following transverse aorta constriction (TAC) in the mouse. The TAC-associated mortality rate was significantly lower in male transgenic (DT) than in Ctr mice (p < 0.05). TAC-associated maladaptive hypertrophy was blunted in DT mice especially 1 month post-TAC and their SERCA2a/PLB ratio remained unchanged 1 and 2 months post-TAC. Two months after TAC, trains of 30 stimuli (burst pacing) performed following isoproterenol injection (0.2 mg/kg, ip), induced VT in 50% of the TAC-Ctr and in none of the TAC-DT mice (p = 0.022). The increase in myocyte shortening and Ca(2+) spark frequency observed in sham-operated Ctr mice in response to 50 nM isoproterenol was reduced in DT mice, and abolished in TAC-DT mice. NCX1 function was reduced in Sham-DT and TAC-DT compared with Sham-Ctr and TAC-Ctr mice, respectively (p < 0.05 for the 2 comparisons). In mice killed after isoproterenol injection and burst pacing, RyR2 S2814 phosphorylation was decreased by 50% in TAC-DT versus TAC-Ctr mice (p < 0.05), with no change in RyR2 S2808 and PLB S16 and T17 phosphorylation. Cardiac FKBP12.6 overexpression in the mouse blunts pressure overload-induced maladaptive LV remodelling and protects against catecholamine-promoted burst pacing-induced ventricular tachycardia by decreasing cardiac sensitivity to adrenergic stress and RyR2 S2814 phosphorylation, and decreasing NCX1 activity.
Assuntos
Miocárdio/metabolismo , Taquicardia Ventricular/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Remodelação Ventricular/genética , Animais , Eletrocardiografia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Immunoblotting , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatologia , Proteínas de Ligação a Tacrolimo/genética , Regulação para CimaRESUMO
Preventing Ca(2+)-leak during diastole may provide a means to improve overall cardiac function. The immunosuppressant FK506-binding protein 12.6 (FKBP12.6) regulates ryanodine receptor-2 (RyR2) gating and binds to and inhibits calcineurin (Cn). It is also involved in the pathophysiology of heart failure (HF). Here, we investigated the effects of FKBP12.6 over-expression and gender on Ca(2+)-handling proteins (RyR2, SERCA2a/PLB, and NCX), and on pro-(CaMKII, Cn/NFAT) and anti-hypertrophic (GSK3ß) signalling pathways in a thoracic aortic constriction (TAC) mouse model. Wild type mice (WT) and mice over-expressing FKBP12.6 of both genders underwent TAC or sham-operation (Sham). FKBP12.6 over-expression ameliorated post-TAC survival rates in both genders. Over time, FKBP12.6 over-expression reduced the molecular signature of left ventricular hypertrophy (LVH) and the transition to HF (BNP and ß-MHC mRNAs) and attenuated Cn/NFAT activation in TAC-males only. The gender difference in pro- and anti-hypertrophic LVH signals was time-dependent: TAC-females exhibited earlier pathological LVH associated with concomitant SERCA2a down-regulation, CaMKII activation, and GSK3ß inactivation. Both genotypes showed systolic dysfunction, possibly related to down-regulated RyR2, but only FK-TAC-males exhibited preserved diastolic LV function. Although FKBP12.6 over-expression did not impact the vicious cycle of TAC-induced HF, this study reveals some subtle sequential and temporal gender differences in Ca(2+)-signalling pathways of pathological LVH.
Assuntos
Sinalização do Cálcio/genética , Perfilação da Expressão Gênica , Hipertrofia Ventricular Esquerda/fisiopatologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Proteínas de Ligação a Tacrolimo/fisiologia , Pressão Ventricular/fisiologia , Animais , Aorta Torácica/patologia , Inibidores de Calcineurina , Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Quinase 3 da Glicogênio Sintase , Glicogênio Sintase Quinase 3 beta , Coração/anatomia & histologia , Coração/fisiopatologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/cirurgia , Proteínas de Homeodomínio/antagonistas & inibidores , Humanos , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Complexo Principal de Histocompatibilidade , Masculino , Camundongos , Camundongos Transgênicos , Peptídeo Natriurético Encefálico/análise , Peptídeo Natriurético Encefálico/metabolismo , Fenótipo , Distribuição Aleatória , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Fatores Sexuais , Transdução de Sinais/fisiologiaRESUMO
Left ventricular hypertrophy (LVH) is an adaptive response to chronic biomechanical stress that generally progresses to maladaptive hypertrophy and heart failure (HF). We studied the activation of protein kinase B (Akt/PKB), glycogen synthase kinase 3 beta (GSK3ß), and calcineurin (Cn) at 3, 7, 15, 30, and 60 days following transverse aortic constriction (TAC) in 4-week-old mice. Following TAC, GSK3ß inactivation at day 3 was associated with Akt activation, whereas at days 15 and 30, it appeared to be controlled by other kinases. Moderate nonsignificant Cn activation occurred at the early stages, and peak activation at day 30, concomitant with GSK3ß inactivation and overt LVH and HF. At the latest stage (day 60), despite further progression of LVH and HF, Cn activation appeared attenuated. Early stages of LVH were associated with Ca2+-handling protein upregulation, whereas major Cn activation, associated with GSK3ß inactivation, appeared to engage maladaptive hypertrophy and progression to HF associated with Ca2+-handling protein downregulation.
Assuntos
Aorta Torácica/metabolismo , Calcineurina/fisiologia , Quinase 3 da Glicogênio Sintase/fisiologia , Insuficiência Cardíaca/etiologia , Hipertrofia Ventricular Esquerda/etiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/fisiologia , Animais , Cálcio/metabolismo , Glicogênio Sintase Quinase 3 beta , Masculino , Camundongos , Fatores de Transcrição NFATC/metabolismoRESUMO
Heart failure is one of the most frequent reasons for hospitalization due to a cardiac event. In most instances, the main difficulty is how to accurately evaluate left ventricular filling pressure. It can be evaluated clinically, biologically and invasively. Although historically, invasive management has been the reference, it is being used less and less frequently and expertise in the technique is being lost. This paper discusses the strength and weaknesses of the different techniques for evaluating filling pressure in these patients, and the importance of this parameter for their optimal treatment.
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Insuficiência Cardíaca/diagnóstico , Função Ventricular Esquerda , Pressão Ventricular , Doença Aguda , Biomarcadores/sangue , Cateterismo Cardíaco , Ecocardiografia Doppler , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/terapia , Humanos , Peptídeos Natriuréticos/sangue , Seleção de Pacientes , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Resultado do TratamentoRESUMO
BACKGROUND: Defects in cardiomyocyte Ca(2+) cycling are a signature feature of heart failure (HF) that occurs in response to sustained hemodynamic overload, and they largely account for contractile dysfunction. Neuronal nitric oxide synthase (NOS1) influences myocyte excitation-contraction coupling through modulation of Ca(2+) cycling, but the potential relevance of this in HF is unknown. METHODS AND RESULTS: We generated a transgenic mouse with conditional, cardiomyocyte-specific NOS1 overexpression (double-transgenic [DT]) and studied cardiac remodeling, myocardial Ca(2+) handling, and contractility in DT and control mice subjected to transverse aortic constriction (TAC). After TAC, control mice developed eccentric hypertrophy with evolution toward HF as revealed by a significantly reduced fractional shortening. In contrast, DT mice developed a greater increase in wall thickness (P<0.0001 versus control+TAC) and less left ventricular dilatation than control+TAC mice (P<0.0001 for both end-systolic and end-diastolic dimensions). Thus, DT mice displayed concentric hypertrophy with fully preserved fractional shortening (43.7+/-0.6% versus 30.3+/-2.6% in control+TAC mice, P<0.05). Isolated cardiomyocytes from DT+TAC mice had greater shortening, intracellular Ca(2+) transients, and sarcoplasmic reticulum Ca(2+) load (P<0.05 versus control+TAC for all parameters). These effects could be explained, at least in part, through modulation of phospholamban phosphorylation status. CONCLUSIONS: Cardiomyocyte NOS1 may be a useful target against cardiac deterioration during chronic pressure-overload-induced HF through modulation of calcium cycling.
Assuntos
Cálcio/metabolismo , Insuficiência Cardíaca/fisiopatologia , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo I/biossíntese , Animais , Aorta/fisiopatologia , Pressão Sanguínea , Separação Celular , Modelos Animais de Doenças , Progressão da Doença , Ativação Enzimática/genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Camundongos , Camundongos Transgênicos , Contração Miocárdica/genética , Miócitos Cardíacos/enzimologia , Óxido Nítrico Sintase Tipo I/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Função Ventricular EsquerdaRESUMO
Block of Na/Ca exchange (NCX) has potential therapeutic applications, in particular, if a mode-selective block could be achieved, but also carries serious risks for disturbing the normal Ca2+ balance maintained by NCX. We have examined the effects of partial inhibition of NCX by SEA-0400 (1 or 0.3 micromol/L) in left ventricular myocytes from healthy pigs or mice and from mice with heart failure (MLP-/-). During voltage clamp ramps with [Ca2+](i) buffering, block of reverse mode block was slightly larger than of forward mode (by 25+/-5%, P<0.05). In the absence of [Ca2+](i) buffering and with sarcoplasmic reticulum (SR) fluxes blocked, rate constants for Ca2+ influx and Ca2+ efflux were reduced to the same extent (to 36+/-6% and 32+/-4%, respectively). With normal SR function the reduction of inward NCX current (I(NCX)) was 57+/-10% (n=10); during large caffeine-induced Ca2+ transients, it was larger (82+/-3%). [Ca2+](i) transients evoked during depolarizing steps increased (from 424+/-27 to 994+/-127 nmol/L at +10 mV, P<0.05), despite a reduction of I(CaL) by 27%. Resting [Ca2+](i) increased; there was a small decrease in the rate of decline of [Ca2+](i). SR Ca2+) content increased more than 2-fold. Contraction amplitude of field-stimulated myocytes increased in healthy myocytes but not in myocytes from MLP-/- mice, in which SR Ca2+ content remained unchanged. These data provide proof-of-principle that even partial inhibition of NCX results in a net gain of Ca2+. Further development of NCX blockers, in particular, for heart failure, must balance potential benefits of I(NCX) reduction against effects on Ca2+ handling by refining mode dependence and/or including additional targets.
Assuntos
Compostos de Anilina/farmacologia , Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Éteres Fenílicos/farmacologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Cardiotônicos/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Feminino , Masculino , Camundongos , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , SuínosRESUMO
Tetracycline is a powerful tool for controlling the expression of specific transgenes (TGs) in various tissues, including heart. In these mouse systems, TG expression is repressed/enhanced by adding doxycycline (Dox) to the diet. However, Dox has been shown to attenuate matrix metalloproteinase (MMP) expression and activity in various tissues, and MMP inactivation mitigates left ventricular (LV) remodeling in animal models of heart failure. Therefore, we examined the influence of Dox on LV remodeling and MMP expression in mice after transverse aortic constriction (TAC). One month after TAC, cardiac hypertrophy (99% vs. 67%) and the proportion of mice exhibiting congestive heart failure (CHF, 74% vs. 32%) were higher in the TAC + Dox group than in the TAC group (P < 0.05). These differences were no longer seen 2 mo after TAC, although LV was more severely dilated in TAC + Dox mice than in TAC mice (P < 0.05). One month after TAC, the increase in brain natriuretic peptide and beta-myosin heavy chain mRNA levels was 1.6 and 1.7 times higher, respectively, in TAC + Dox mice than in TAC mice (P < 0.01). MMP-2 gelatin zymographic activity increased 1.9- and 2.4-fold in TAC and TAC + Dox mice, respectively (P < 0.01 and P < 0.05 relative to respective sham-operated animals), but the difference between TAC + Dox and TAC mice did not reach statistical significance. Dox did not significantly alter TAC-associated perivascular and interstitial myocardial fibrosis. These findings demonstrate that Dox accelerates the onset of cardiac hypertrophy and the progression to CHF following TAC in mice. Accordingly, care should be taken when designing and interpreting studies based on TG mouse models of LV hypertrophy using the tetracycline-regulated (tet)-on/tet-off system.
Assuntos
Doxiciclina/toxicidade , Insuficiência Cardíaca/etiologia , Hipertrofia Ventricular Esquerda/induzido quimicamente , Miocárdio/enzimologia , Inibidores de Proteases/toxicidade , Remodelação Ventricular/efeitos dos fármacos , Animais , Aorta Torácica/cirurgia , Proteínas de Ligação ao Cálcio/metabolismo , Constrição , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Camundongos , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Peptídeo Natriurético Encefálico/genética , Peptídeo Natriurético Encefálico/metabolismo , Reação em Cadeia da Polimerase , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores de Tempo , UltrassonografiaRESUMO
BACKGROUND: Ca(2+) release from the sarcoplasmic reticulum via the ryanodine receptor (RyR2) activates cardiac myocyte contraction. An important regulator of RyR2 function is FKBP12.6, which stabilizes RyR2 in the closed state during diastole. Beta-adrenergic stimulation has been suggested to dissociate FKBP12.6 from RyR2, leading to diastolic sarcoplasmic reticulum Ca(2+) leakage and ventricular tachycardia (VT). We tested the hypothesis that FKBP12.6 overexpression in cardiac myocytes can reduce susceptibility to VT in stress conditions. METHODS AND RESULTS: We developed a mouse model with conditional cardiac-specific overexpression of FKBP12.6. Transgenic mouse hearts showed a marked increase in FKBP12.6 binding to RyR2 compared with controls both at baseline and on isoproterenol stimulation (0.2 mg/kg i.p.). After pretreatment with isoproterenol, burst pacing induced VT in 10 of 23 control mice but in only 1 of 14 transgenic mice (P<0.05). In isolated transgenic myocytes, Ca(2+) spark frequency was reduced by 50% (P<0.01), a reduction that persisted under isoproterenol stimulation, whereas the sarcoplasmic reticulum Ca(2+) load remained unchanged. In parallel, peak I(Ca,L) density decreased by 15% (P<0.01), and the Ca(2+) transient peak amplitude decreased by 30% (P<0.001). A 33.5% prolongation of the caffeine-evoked Ca(2+) transient decay was associated with an 18% reduction in the Na(+)-Ca(2+) exchanger protein level (P<0.05). CONCLUSIONS: Increased FKBP12.6 binding to RyR2 prevents triggered VT in normal hearts in stress conditions, probably by reducing diastolic sarcoplasmic reticulum Ca(2+) leak. This indicates that the FKBP12.6-RyR2 complex is an important candidate target for pharmacological prevention of VT.
Assuntos
Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Taquicardia Ventricular/prevenção & controle , Proteínas de Ligação a Tacrolimo/fisiologia , Potenciais de Ação , Agonistas Adrenérgicos beta/toxicidade , Animais , Sinalização do Cálcio , Estimulação Cardíaca Artificial , Catecolaminas/fisiologia , Doxiciclina/farmacologia , Isoproterenol/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Contração Miocárdica , Fosforilação , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Retículo Sarcoplasmático/metabolismo , Proteínas de Ligação a Tacrolimo/biossíntese , Proteínas de Ligação a Tacrolimo/genética , Regulação para Cima/efeitos dos fármacosRESUMO
AIMS: A decrease in L-type calcium current (ICaL) is an important mechanism favouring atrial fibrillation (AF). Here, we aimed to identify pathogenic factors associated with ICaL downregulation. METHODS AND RESULTS: Atrial myocytes were isolated from right atrial appendages obtained from 86 adult patients in sinus rhythm with coronary artery disease, aortic valve disease, or mitral valve disease (MVD). Current was recorded in isolated myocytes using the whole-cell patch-clamp technique. The ICaL recorded in the 172 myocytes studied showed a marked variability of peak density ranging from 0.1 to 9.0 pA/pF. The ICaL peak density did not correlate with membrane capacitance or changes in current biophysical properties. The ICaL peak density was homogeneous for a given sample. Small ICaL values were recorded in patients with MVD or with a low left ventricular ejection fraction (<45%). Small ICaL values were more sensitive to the beta-adrenergic agonist, isoproterenol (1 microM), and to the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (10 microM). CONCLUSION: In human atrial myocytes, the variability of ICaL is related to the clinical history of the donors. The downregulation of ICaL is already observed in patients in sinus rhythm with a high risk of AF and is associated with the greatest response to beta-adrenergic agonist.