RESUMO
Cardiac hypertrophy resulting from sympathetic nervous system activation triggers the development of heart failure. The transcription factor Y-box binding protein 1 (YB-1) can interact with transcription factors involved in cardiac hypertrophy and may thereby interfere with the hypertrophy growth process. Therefore, the question arises as to whether YB-1 influences cardiomyocyte hypertrophy and might thereby influence the development of heart failure. YB-1 expression is downregulated in human heart biopsies of patients with ischemic cardiomyopathy (n = 8), leading to heart failure. To study the impact of reduced YB-1 in cardiac cells, we performed small interfering RNA (siRNA) experiments in H9C2 cells as well as in adult cardiomyocytes (CMs) of rats. The specificity of YB-1 siRNA was analyzed by a miRNA-like off-target prediction assay identifying potential genes. Testing three high-scoring genes by transfecting cardiac cells with YB-1 siRNA did not result in downregulation of these genes in contrast to YB-1, whose downregulation increased hypertrophic growth. Hypertrophic growth was mediated by PI3K under PE stimulation, as well by downregulation with YB-1 siRNA. On the other hand, overexpression of YB-1 in CMs, caused by infection with an adenovirus encoding YB-1 (AdYB-1), prevented hypertrophic growth under α-adrenergic stimulation with phenylephrine (PE), but not under stimulation with growth differentiation factor 15 (GDF15; n = 10-16). An adenovirus encoding the green fluorescent protein (AdGFP) served as the control. YB-1 overexpression enhanced the mRNA expression of the Gq inhibitor regulator of G-protein signaling 2 (RGS2) under PE stimulation (n = 6), potentially explaining its inhibitory effect on PE-induced hypertrophic growth. This study shows that YB-1 protects cardiomyocytes against PE-induced hypertrophic growth. Like in human end-stage heart failure, YB-1 downregulation may cause the heart to lose its protection against hypertrophic stimuli and progress to heart failure. Therefore, the transcription factor YB-1 is a pivotal signaling molecule, providing perspectives for therapeutic approaches.
Assuntos
Adrenérgicos , Insuficiência Cardíaca , Adulto , Humanos , Animais , Ratos , Fenilefrina , Insuficiência Cardíaca/genética , Miócitos Cardíacos , RNA Interferente Pequeno/genética , Adenoviridae , Cardiomegalia/genética , Fatores de TranscriçãoRESUMO
Fractional flow reserve (FFR) measurement was compared to dobutamine stress echocardiography (DSE) instable angina (SA) with stable coronary lesion (s) (SCL (s) ) in a few trials; however, similar comparisons in patients with acute coronary syndrome (ACS) with non-culprit lesion (s) (NCL (s) ) are lacking. Our objectives were to prospectively evaluate the diagnostic performance of FFR with two different cutoff values (< 0.80 and < 0.75) relative to DSE in moderate (30%-70% diameter stenosis) NCLs (ACS group) and to compare these observations with those measured in SCLs (SA group). One hundred seventy-five consecutive patients with SA (n = 86) and ACS (n = 89) with 225 coronary lesions (109 SCLs and 116 NCLs) were enrolled. In contrast to the ACS cohort in SA patients, normal DSE was associated with higher FFR values compared to those with abnormal DSE (P = 0.051 versus P = 0.006). In addition, in the SA group, a significant correlation was observed between DSE (regional wall motion score index at peak stress) and FFR (r = -0.290; P = 0.002), whereas a similar association was absent (r = -0.029; P = 0.760) among ACS patients. In the SA group, decreasing the FFR cutoff value (< 0.80 versus < 0.75) improved the concordance of FFR with DSE (70.6% versus 81.7%) without altering its discriminatory power (area under the curve; 0.68 versus 0.63; P = 0.369), whereas in the ACS group, concordance remained similar (69.0% versus 71.6%) and discriminatory power decreased (0.62 versus 0.51; P = 0.049), respectively. In conclusion, lesion-specific FFR assessment may have different relevance in patients with moderate NCLs than in patients with SCLs.
Assuntos
Síndrome Coronariana Aguda/diagnóstico , Angina Estável/diagnóstico , Ecocardiografia sob Estresse/métodos , Reserva Fracionada de Fluxo Miocárdico/fisiologia , Síndrome Coronariana Aguda/fisiopatologia , Síndrome Coronariana Aguda/cirurgia , Idoso , Angina Estável/classificação , Angina Estável/fisiopatologia , Estudos de Casos e Controles , Angiografia Coronária/métodos , Estenose Coronária/diagnóstico , Estenose Coronária/patologia , Ecocardiografia sob Estresse/estatística & dados numéricos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ensaios Clínicos Controlados não Aleatórios como Assunto/métodos , Intervenção Coronária Percutânea/instrumentação , Estudos ProspectivosRESUMO
Although the application of cardioprotective ischaemia/reperfusion (I/R) stimuli after myocardial infarction (MI) is a promising concept for salvaging the myocardium, translation to a clinical scenario has not fulfilled expectations. We have previously shown that in pigs, ischaemic postconditioning (IPostC) reduces myocardial oedema and microvascular obstruction (MVO), without influencing myocardial infarct size. In the present study, we analyzed the mechanisms underlying the IPostC-induced microvascular protection by transcriptomic analysis, followed by pathway analysis. Closed-chest reperfused MI was induced by 90 min percutaneous balloon occlusion of the left anterior descending coronary artery, followed by balloon deflation in anaesthetised pigs. Animals were randomised to IPostC (n = 8), MI (non-conditioned, n = 8), or Control (sham-operated, n = 4) groups. After three hours or three days follow-up, myocardial tissue samples were harvested and subjected to RNA-seq analysis. Although the transcriptome analysis revealed similar expression between IPostC and MI in transcripts involved in cardioprotective pathways, we identified gene expression changes responding to IPostC at the three days follow-up. Focal adhesion signaling, downregulated genes participating in cardiomyopathy and activation of blood cells may have critical consequences for microvascular protection. Specific analyses of the gene subsets enriched in the endothelium of the infarcted area, revealed strong deregulation of transcriptional functional clusters, DNA processing, replication and repair, cell proliferation, and focal adhesion, suggesting sustentative function in the endothelial cell layer protection and integrity. The spatial and time-dependent transcriptome analysis of porcine myocardium supports a protective effect of IPostC on coronary microvasculature post-MI.
Assuntos
Pós-Condicionamento Isquêmico , Microvasos/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Transcrição Gênica , Animais , Tamanho Celular , Análise por Conglomerados , Modelos Animais de Doenças , Adesões Focais/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Análise de Sobrevida , SuínosRESUMO
Feeding rats with high-fat diet (HFD) with a single streptozotocin (STZ) injection induced obesity, slightly elevated fasting blood glucose and impaired glucose and insulin tolerance, and caused cardiac hypertrophy and mild diastolic dysfunction as published before by Koncsos et al. in 2016. Here we aimed to explore the renal consequences in the same groups of rats. Male Long-Evans rats were fed normal chow (CON; n = 9) or HFD containing 40% lard and were administered STZ at 20 mg/kg (i.p.) at week four (prediabetic rats, PRED, n = 9). At week 21 blood and urine samples were taken and kidney and liver samples were collected for histology, immunohistochemistry and for analysis of gene expression. HFD and STZ increased body weight and visceral adiposity and plasma leptin concentration. Despite hyperleptinemia, plasma C-reactive protein concentration decreased in PRED rats. Immunohistochemistry revealed elevated collagen IV protein expression in the glomeruli, and Lcn2 mRNA expression increased, while Il-1ß mRNA expression decreased in both the renal cortex and medulla in PRED vs. CON rats. Kidney histology, urinary protein excretion, plasma creatinine, glomerular Feret diameter, desmin protein expression, and cortical and medullary mRNA expression of TGF-ß1, Nrf2, and PPARγ were similar in CON and PRED rats. Reduced AMPKα phosphorylation of the autophagy regulator Akt was the first sign of liver damage, while plasma lipid and liver enzyme concentrations were similar. In conclusion, glomerular collagen deposition and increased lipocalin-2 expression were the early signs of kidney injury, while most biomarkers of inflammation, oxidative stress and fibrosis were negative in the kidneys of obese, prediabetic rats with mild heart and liver injury.
Assuntos
Colágeno/metabolismo , Glomérulos Renais/lesões , Glomérulos Renais/metabolismo , Lipocalina-2/metabolismo , Obesidade/metabolismo , Estado Pré-Diabético/metabolismo , Tecido Adiposo/metabolismo , Animais , Biomarcadores/metabolismo , Peso Corporal , Dieta Hiperlipídica , Fibrose , Regulação da Expressão Gênica , Inflamação/genética , Inflamação/patologia , Glomérulos Renais/patologia , Lipídeos/sangue , Fígado/enzimologia , Fígado/patologia , Fígado/fisiopatologia , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Obesidade/sangue , Estresse Oxidativo/genética , Fosforilação , Fosfosserina/metabolismo , Estado Pré-Diabético/sangue , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Long-Evans , EstreptozocinaRESUMO
BACKGROUND: Cardioprotective value of ischemic post- (IPostC), remote (RIC) conditioning in acute myocardial infarction (AMI) is unclear in clinical trials. To evaluate cardioprotection, most translational animal studies and clinical trials utilize necrotic tissue referred to the area at risk (AAR) by magnetic resonance imaging (MRI). However, determination of AAR by MRI' may not be accurate, since MRI-indices of microvascular damage, i.e., myocardial edema and microvascular obstruction (MVO), may be affected by cardioprotection independently from myocardial necrosis. Therefore, we assessed the effect of IPostC, RIC conditioning and ischemic preconditioning (IPreC; positive control) on myocardial necrosis, edema and MVO in a clinically relevant, closed-chest pig model of AMI. METHODS AND RESULTS: Acute myocardial infarction was induced by a 90-min balloon occlusion of the left anterior descending coronary artery (LAD) in domestic juvenile female pigs. IPostC (6 × 30 s ischemia/reperfusion after 90-min occlusion) and RIC (4 × 5 min hind limb ischemia/reperfusion during 90-min LAD occlusion) did not reduce myocardial necrosis as assessed by late gadolinium enhancement 3 days after reperfusion and by ex vivo triphenyltetrazolium chloride staining 3 h after reperfusion, however, the positive control, IPreC (3 × 5 min ischemia/reperfusion before 90-min LAD occlusion) did. IPostC and RIC attenuated myocardial edema as measured by cardiac T2-weighted MRI 3 days after reperfusion, however, AAR measured by Evans blue staining was not different among groups, which confirms that myocardial edema is not a measure of AAR, IPostC and IPreC but not RIC decreased MVO. CONCLUSION: We conclude that IPostC and RIC interventions may protect the coronary microvasculature even without reducing myocardial necrosis.
Assuntos
Cardiotônicos/metabolismo , Pós-Condicionamento Isquêmico , Precondicionamento Isquêmico Miocárdico , Imageamento por Ressonância Magnética/métodos , Microvasos/patologia , Traumatismo por Reperfusão Miocárdica/diagnóstico , Animais , Edema/patologia , Eletrocardiografia , Feminino , Testes de Função Cardíaca , Hemodinâmica , Traumatismo por Reperfusão Miocárdica/diagnóstico por imagem , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Necrose , Coloração e Rotulagem , Sus scrofaRESUMO
BACKGROUND: We have previously shown that efficiency of ischemic conditioning is diminished in hypercholesterolemia and that autophagy is necessary for cardioprotection. However, it is unknown whether isolated hypercholesterolemia disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways. Therefore, we investigated whether isolated hypercholesterolemia modulates cardiac autophagy-related pathways or programmed cell death mechanisms such as apoptosis and necroptosis in rat heart. METHODS: Male Wistar rats were fed either normal chow (NORM; n = 9) or with 2% cholesterol and 0.25% cholic acid-enriched diet (CHOL; n = 9) for 12 weeks. CHOL rats exhibited a 41% increase in plasma total cholesterol level over that of NORM rats (4.09 mmol/L vs. 2.89 mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out. Left ventricles were snap-frozen for determination of markers of autophagy, mTOR pathway, apoptosis, and necroptosis by Western blot. RESULTS: Isolated hypercholesterolemia was associated with a significant reduction in expression of cardiac autophagy markers such as LC3-II, Beclin-1, Rubicon and RAB7 as compared to controls. Phosphorylation of ribosomal S6, a surrogate marker for mTOR activity, was increased in CHOL samples. Cleaved caspase-3, a marker of apoptosis, increased in CHOL hearts, while no difference in the expression of necroptotic marker RIP1, RIP3 and MLKL was detected between treatments. CONCLUSIONS: This is the first comprehensive analysis of autophagy and programmed cell death pathways of apoptosis and necroptosis in hearts of hypercholesterolemic rats. Our data show that isolated hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an activated mTOR pathway. Reduced autophagy was accompanied by increased apoptosis, while cardiac necroptosis was not modulated by isolated hypercholesterolemia. Decreased basal autophagy and elevated apoptosis may be responsible for the loss of cardioprotection reported in hypercholesterolemic animals.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Colesterol/efeitos adversos , Ácido Cólico/efeitos adversos , Hipercolesterolemia/metabolismo , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Biomarcadores/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Colesterol/administração & dosagem , Ácido Cólico/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Hipercolesterolemia/etiologia , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Necrose/etiologia , Necrose/genética , Necrose/metabolismo , Necrose/patologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Wistar , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
BACKGROUND: Lipid accumulation in the liver and pancreas is primarily caused by combined hyperlipidemia. However, the effect of isolated hypercholesterolemia without hypertriglyceridemia is not fully described. Therefore, our aim was to investigate whether hypercholesterolemia alone leads to alterations both in hepatic and pancreatic lipid panel and histology in rats. METHODS: Male Wistar rats were fed with 2% cholesterol +0.25% cholate-supplemented diet or standard chow for 12 weeks. Blood was collected at weeks 0, 4, 8 and 12 to measure serum cholesterol and triglyceride levels. At week 12, both the pancreas and the liver were isolated for further histological and biochemical analysis. Hepatic and plasma fatty acid composition was assessed by gas chromatography. Expression of mRNA of major enzymes involved in saturated/unsaturated fatty acid synthesis was analyzed by qPCR. In separate experiments serum enzyme activities and insulin levels were measured at week 9. RESULTS: At week 12, rats fed with 2% cholesterol +0.25% cholate-supplemented diet were characterized by elevated serum cholesterol (4.09 ± 0.20 vs. 2.89 ± 0.22 mmol/L, *p < 0.05) while triglyceride (2.27 ± 0.05 vs. 2.03 ± 0.03 mmol/L) and glucose levels (5.32 ± 0.14 vs. 5.23 ± 0.10 mmol/L) remained unchanged. Isolated hypercholesterolemia increased hepatic lipid accumulation, hepatic cholesterol (5.86 ± 0.22 vs. 1.60 ± 0.15 ng/g tissue, *p < 0.05) and triglyceride contents (19.28 ± 1.42 vs. 6.78 ± 0.71 ng/g tissue, *p < 0.05), and hepatic nitrotyrosine level (4.07 ± 0.52 vs. 2.59 ± 0.31 ng/mg protein, *p < 0.05). The histology and tissue lipid content of the pancreas was not affected. Serum total protein level, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities remained unchanged in response to isolated hypercholesterolemia while serum alkaline phosphatase activity (ALP) significantly increased. Plasma insulin levels did not change in response to isolated hypercholesterolemia suggesting an intact endocrine function of the pancreas. Isolated hypercholesterolemia caused a significantly increased hepatic and serum fatty acid level associated with a marked alteration of fatty acid composition. Hepatic expression of Δ9-desaturase (SCD1) was increased 4.92×, while expression of Δ5-desaturase and Δ6-desaturase were decreased (0.447× and 0.577×, respectively) due to isolated hypercholesterolemia. CONCLUSIONS: Isolated hypercholesterolemia leads to hepatic steatosis and marked alterations in the hepatic lipid profile without affecting the pancreas. Altered fatty acid profile might mediate harmful effects of cholesterol in the liver.
Assuntos
Fígado Gorduroso/etiologia , Hipercolesterolemia/complicações , Fígado/patologia , Pâncreas/patologia , Animais , Glicemia/metabolismo , Peso Corporal , Colesterol/sangue , Enzimas/sangue , Enzimas/genética , Ácidos Graxos/biossíntese , Fígado Gorduroso/sangue , Hipercolesterolemia/sangue , Hipercolesterolemia/enzimologia , Insulina/sangue , Masculino , Estresse Nitrosativo , Tamanho do Órgão , Estresse Oxidativo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Triglicerídeos/sangue , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Although incidence and prevalence of prediabetes are increasing, little is known about its cardiac effects. Therefore, our aim was to investigate the effect of prediabetes on cardiac function and to characterize parameters and pathways associated with deteriorated cardiac performance. Long-Evans rats were fed with either control or high-fat chow for 21 wk and treated with a single low dose (20 mg/kg) of streptozotocin at week 4 High-fat and streptozotocin treatment induced prediabetes as characterized by slightly elevated fasting blood glucose, impaired glucose and insulin tolerance, increased visceral adipose tissue and plasma leptin levels, as well as sensory neuropathy. In prediabetic animals, a mild diastolic dysfunction was observed, the number of myocardial lipid droplets increased, and left ventricular mass and wall thickness were elevated; however, no molecular sign of fibrosis or cardiac hypertrophy was shown. In prediabetes, production of reactive oxygen species was elevated in subsarcolemmal mitochondria. Expression of mitofusin-2 was increased, while the phosphorylation of phospholamban and expression of Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3, a marker of mitophagy) decreased. However, expression of other markers of cardiac auto- and mitophagy, mitochondrial dynamics, inflammation, heat shock proteins, Ca2+/calmodulin-dependent protein kinase II, mammalian target of rapamycin, or apoptotic pathways were unchanged in prediabetes. This is the first comprehensive analysis of cardiac effects of prediabetes indicating that mild diastolic dysfunction and cardiac hypertrophy are multifactorial phenomena that are associated with early changes in mitophagy, cardiac lipid accumulation, and elevated oxidative stress and that prediabetes-induced oxidative stress originates from the subsarcolemmal mitochondria.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Mitocôndrias Cardíacas/metabolismo , Estresse Oxidativo , Estado Pré-Diabético/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Adipocinas/metabolismo , Tecido Adiposo , Animais , Apoptose , Autofagia , Composição Corporal , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Neuropatias Diabéticas , Diástole , Dieta Hiperlipídica , Ecocardiografia , GTP Fosfo-Hidrolases , Proteínas de Choque Térmico/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Mitocôndrias Cardíacas/ultraestrutura , Proteínas Mitocondriais/metabolismo , Mitofagia , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Fosforilação , Estado Pré-Diabético/fisiopatologia , Ratos , Ratos Long-Evans , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sarcolema , Serina-Treonina Quinases TOR/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Pressão VentricularRESUMO
BACKGROUND: Myostatin (Mstn) is a key regulator of heart metabolism and cardiomyocyte growth interacting tightly with insulin-like growth factor I (IGF-I) under physiological conditions. The pathological role of Mstn has also been suggested since Mstn protein was shown to be upregulated in the myocardium of end-stage heart failure. However, no data are available about the regulation of gene expression of Mstn and IGF-I in different regions of healthy or pathologic human hearts, although they both might play a crucial role in the pathomechanism of heart failure. METHODS: In the present study, heart samples were collected from left ventricles, septum and right ventricles of control healthy individuals as well as from failing hearts of dilated (DCM) or ischemic cardiomyopathic (ICM) patients. A comprehensive qRT-PCR analysis of Mstn and IGF-I signaling was carried out by measuring expression of Mstn, its receptor Activin receptor IIB (ActRIIB), IGF-I, IGF-I receptor (IGF-IR), and the negative regulator of Mstn miR-208, respectively. Moreover, we combined the measured transcript levels and created complex parameters characterizing either Mstn- or IGF-I signaling in the different regions of healthy or failing hearts. RESULTS: We have found that in healthy control hearts, the ratio of Mstn/IGF-I signaling was significantly higher in the left ventricle/septum than in the right ventricle. Moreover, Mstn transcript levels were significantly upregulated in all heart regions of DCM but not ICM patients. However, the ratio of Mstn/IGF-I signaling remained increased in the left ventricle/septum compared to the right ventricle of DCM patients (similarly to the healthy hearts). In contrast, in ICM hearts significant transcript changes were detected mainly in IGF-I signaling. In parallel with these results miR-208 showed mild upregulation in the left ventricle of both DCM and ICM hearts. CONCLUSIONS: This is the first demonstration of a spatial asymmetry in the expression pattern of Mstn/IGF-I in healthy hearts, which is likely to play a role in the different growth regulation of left vs. right ventricle. Moreover, we identified Mstn as a massively regulated gene in DCM but not in ICM as part of possible compensatory mechanisms in the failing heart.
Assuntos
Insuficiência Cardíaca/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Miostatina/metabolismo , Reação em Cadeia da Polimerase/métodos , Transdução de Sinais , Adulto , Idoso , Sequência de Bases , Estudos de Casos e Controles , Primers do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Remote ischemic perconditioning (RIPerC) has a promising therapeutic insight to improve the prognosis of acute myocardial infarction. Chronic comorbidities such as diabetes are known to interfere with conditioning interventions by modulating cardioprotective signaling pathways, such as e.g., mTOR pathway and autophagy. However, the effect of acute hyperglycemia on RIPerC has not been studied so far. Therefore, here we investigated the effect of acute hyperglycemia on cardioprotection by RIPerC. METHODS: Wistar rats were divided into normoglycemic (NG) and acute hyperglycemic (AHG) groups. Acute hyperglycemia was induced by glucose infusion to maintain a serum glucose concentration of 15-20 mM throughout the experimental protocol. NG rats received mannitol infusion of an equal osmolarity. Both groups were subdivided into an ischemic (Isch) and a RIPerC group. Each group underwent reversible occlusion of the left anterior descending coronary artery (LAD) for 40 min in the presence or absence of acute hyperglycemia. After the 10-min LAD occlusion, RIPerC was induced by 3 cycles of 5-min unilateral femoral artery and vein occlusion and 5-min reperfusion. After 120 min of reperfusion, infarct size was measured by triphenyltetrazolium chloride staining. To study underlying signaling mechanisms, hearts were harvested for immunoblotting after 35 min in both the NG and AHG groups. RESULTS: Infarct size was significantly reduced by RIPerC in NG, but not in the AHG group (NG + Isch: 46.27 ± 5.31 % vs. NG + RIPerC: 24.65 ± 7.45 %, p < 0.05; AHG + Isch: 54.19 ± 4.07 % vs. 52.76 ± 3.80 %). Acute hyperglycemia per se did not influence infarct size, but significantly increased the incidence and duration of arrhythmias. Acute hyperglycemia activated mechanistic target of rapamycine (mTOR) pathway, as it significantly increased the phosphorylation of mTOR and S6 proteins and the phosphorylation of AKT. In spite of a decreased LC3II/LC3I ratio, other markers of autophagy, such as ATG7, ULK1 phopsphorylation, Beclin 1 and SQSTM1/p62, were not modulated by acute hyperglycemia. Furthermore, acute hyperglycemia significantly elevated nitrative stress in the heart (0.87 ± 0.01 vs. 0.50 ± 0.04 µg 3-nitrotyrosine/mg protein, p < 0.05). CONCLUSIONS: This is the first demonstration that acute hypreglycemia deteriorates cardioprotection by RIPerC. The mechanism of this phenomenon may involve an acute hyperglycemia-induced increase in nitrative stress and activation of the mTOR pathway.
Assuntos
Arritmias Cardíacas/fisiopatologia , Autofagia , Hiperglicemia/metabolismo , Precondicionamento Isquêmico Miocárdico , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Estresse Fisiológico , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Arritmias Cardíacas/etiologia , Proteína 7 Relacionada à Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteína Beclina-1 , Proteínas de Choque Térmico/metabolismo , Hiperglicemia/complicações , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Infarto do Miocárdio/complicações , Traumatismo por Reperfusão Miocárdica/complicações , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Proteína Sequestossoma-1 , Índice de Gravidade de Doença , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
Remote ischemic preconditioning (RIPC) of the heart is exerted by brief ischemic insults affected on a remote organ or a remote area of the heart before a sustained cardiac ischemia. To date, little is known about the inter-organ transfer mechanisms of cardioprotection by RIPC. Exosomes and microvesicles/microparticles are vesicles of 30-100 nm and 100-1000 nm in diameter, respectively (collectively termed extracellular vesicles [EVs]). Their content of proteins, mRNAs and microRNAs, renders EV ideal conveyors of inter-organ communication. However, whether EVs are involved in RIPC, is unknown. Therefore, here we investigated whether (1) IPC induces release of EVs from the heart, and (2) EVs are necessary for cardioprotection by RIPC. Hearts of male Wistar rats were isolated and perfused in Langendorff mode. A group of donor hearts was exposed to 3 × 5-5 min global ischemia and reperfusion (IPC) or 30 min aerobic perfusion, while coronary perfusates were collected. Coronary perfusates of these hearts were given to another set of recipient isolated hearts. A group of recipient hearts received IPC effluent depleted of EVs by differential ultracentrifugation. Infarct size was determined after 30 min global ischemia and 120 min reperfusion. The presence or absence of EVs in perfusates was confirmed by dynamic light scattering, the EV marker HSP60 Western blot, and electron microscopy. IPC markedly increased EV release from the heart as assessed by HSP60. Administration of coronary perfusate from IPC donor hearts attenuated infarct size in non-preconditioned recipient hearts (12.9 ± 1.6% vs. 25.0 ± 2.7%), similarly to cardioprotection afforded by IPC (7.3 ± 2.7% vs. 22.1 ± 2.9%) on the donor hearts. Perfusates of IPC hearts depleted of EVs failed to exert cardioprotection in recipient hearts (22.0 ± 2.3%). This is the first demonstration that EVs released from the heart after IPC are necessary for cardioprotection by RIPC, evidencing the importance of vesicular transfer mechanisms in remote cardioprotection.
Assuntos
Micropartículas Derivadas de Células/fisiologia , Precondicionamento Isquêmico Miocárdico , Miocárdio/patologia , Animais , Micropartículas Derivadas de Células/química , Exossomos/fisiologia , Técnicas In Vitro , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Tamanho da Partícula , Ratos , Ratos WistarRESUMO
The role of coronary computed tomography angiography (CCTA) derived fractional flow reserve (CT-FFR) in the assessment of non-culprit lesions (NCL) in patients with acute coronary syndrome (ACS) is debated. In this prospective clinical study, a total of 68 ACS patients with 89 moderate (30−70% diameter stenosis) NCLs were enrolled to evaluate the diagnostic accuracy of on-site CT-FFR compared to invasive fractional flow reserve (FFRi) and dobutamine stress echocardiography (DSE) as reference standards. CT-FFR and FFRi values ≤0.80, as well as new or worsening wall motion abnormality in ≥2 contiguous segments on the supplying area of an NCL on DSE, were considered positive for ischemia. Sensitivity, specificity, positive, and negative predictive value of CT-FFR relative to FFRi and DSE were 51%, 89%, 75%, and 74% and 37%, 77%, 42%, and 74%, respectively. CT-FFR value (ß = 0.334, p < 0.001) and CT-FFR drop from proximal to distal measuring point [(CT-FFR drop), ß = −0.289, p = 0.002)] were independent predictors of FFRi value in multivariate linear regression analysis. Based on comparing their receiver operating characteristics area under the curve (AUC) values, CT-FFR value and CT-FFR drop provided better discriminatory power than CCTA-based minimal lumen diameter stenosis to distinguish between an NCL with positive and negative FFRi [0.77 (95% Confidence Intervals, CI: 0.67−0.86) and 0.77 (CI: 0.67−0.86) vs. 0.63 (CI: 0.52−0.73), p = 0.029 and p = 0.043, respectively]. Neither CT-FFR value nor CT-FFR drop was predictive of regional wall motion score index at peak stress (ß = −0.440, p = 0.441 and ß = 0.403, p = 0.494) or was able to confirm ischemia on the territory of an NCL revealed by DSE (AUC = 0.54, CI: 0.43−0.64 and AUC = 0.55, CI: 0.44−0.65, respectively). In conclusion, on-site CT-FFR is superior to conventional CCTA-based anatomical analysis in the assessment of moderate NCLs; however, its diagnostic capacity is not sufficient to make it a gatekeeper to invasive functional evaluation. Moreover, based on its comparison with DSE, CT-FFR might not yield any information on the microvascular dysfunction in the territory of an NCL.
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Little is known about the role of the neuropeptide somatostatin (SST) in myocardial ischemia/reperfusion injury and cardioprotection. Here, we investigated the direct cardiocytoprotective effect of SST on ischemia/reperfusion injury in cardiomyocyte cultures, as well as the expression of SST and its receptors in pig and human heart tissues. SST induced a bell-shaped, concentration-dependent cardiocytoprotection in both adult rat primary cardiomyocytes and H9C2 cells subjected to simulated ischemia/reperfusion injury. Furthermore, in a translational porcine closed-chest acute myocardial infarction model, ischemic preconditioning increased plasma SST-like immunoreactivity. Interestingly, SST expression was detectable at the protein, but not at the mRNA level in the pig left ventricles. SSTR1 and SSTR2, but not the other SST receptors, were detectable at the mRNA level by PCR and sequencing in the pig left ventricle. Moreover, remote ischemic conditioning upregulated SSTR1 mRNA. Similarly, SST expression was also detectable in healthy human interventricular septum samples at the protein level. Furthermore, SST-like immunoreactivity decreased in interventricular septum samples of patients with ischemic cardiomyopathy. SSTR1, SSTR2, and SSTR5 but not SST and the other SST receptors were detectable at the mRNA level by sequencing in healthy human left ventricles. In addition, in healthy human left ventricle samples, SSTR1 and SSTR2 mRNAs were expressed especially in vascular endothelial and some other cell types as detected by RNA Scope® in situ hybridization. This is the first demonstration that SST exerts a direct cardiocytoprotective effect against simulated ischemia/reperfusion injury. Moreover, SST is expressed in the heart tissue at the peptide level; however, it is likely to be of sensory neural origin since its mRNA is not detectable. SSTR1 and SSTR2 might be involved in the cardioprotective action of SST, but other mechanisms cannot be excluded.
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The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an unmet clinical need for prevention and therapy of post-myocardial infarction heart failure (post-MI HF). Clinically relevant pig models of post-MI HF are prerequisites for final proof-of-concept studies before entering into clinical trials in drug and medical device development. Here we aimed to characterize a closed-chest porcine model of post-MI HF in adult Göttingen minipigs with long-term follow-up including serial cardiac magnetic resonance imaging (CMRI) and to compare it with the commonly used Landrace pig model. MI was induced by intraluminal balloon occlusion of the left anterior descending coronary artery for 120 min in Göttingen minipigs and for 90 min in Landrace pigs, followed by reperfusion. CMRI was performed to assess cardiac morphology and function at baseline in both breeds and at 3 and 6 months in Göttingen minipigs and at 2 months in Landrace pigs, respectively. Scar sizes were comparable in the two breeds, but MI resulted in a significant decrease of left ventricular ejection fraction (LVEF) only in Göttingen minipigs, while Landrace pigs did not show a reduction of LVEF. Right ventricular (RV) ejection fraction increased in both breeds despite the negligible RV scar sizes. In contrast to the significant increase of left ventricular end-diastolic (LVED) mass in Landrace pigs at 2 months, Göttingen minipigs showed a slight increase in LVED mass only at 6 months. In summary, this is the first characterization of post-MI HF in Göttingen minipigs in comparison to Landrace pigs, showing that the Göttingen minipig model reflects post-MI HF parameters comparable to the human pathology. We conclude that the Göttingen minipig model is superior to the Landrace pig model to study the development of post-MI HF.
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Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Infarto do Miocárdio/complicações , Animais , Oclusão Coronária/diagnóstico por imagem , Oclusão Coronária/fisiopatologia , Feminino , Coração/diagnóstico por imagem , Coração/fisiopatologia , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/fisiopatologia , Imageamento por Ressonância Magnética , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/fisiopatologia , Reperfusão Miocárdica , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Suínos , Porco Miniatura , Função Ventricular EsquerdaRESUMO
AIMS: Interleukin-1ß (IL-1ß) is an important pathogenic factor in cardiovascular diseases including chronic heart failure (HF). The CANTOS trial highlighted that inflammasomes as primary sources of IL-1 ß are promising new therapeutic targets in cardiovascular diseases. Therefore, we aimed to assess inflammasome activation in failing hearts to identify activation patterns of inflammasome subtypes as sources of IL-1ß. METHODS AND RESULTS: Out of the four major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human HF regardless of the aetiology (ischaemic or dilated cardiomyopathy), while the NLRP1/NALP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change in HF samples. AIM2 expression was primarily detected in monocytes/macrophages of failing hearts. Translational animal models of HF (pressure or volume overload, and permanent coronary artery ligation in rat, as well as ischaemia/reperfusion-induced HF in pigs) demonstrated activation pattern of AIM2 similar to that of observed in end-stages of human HF. In vitro AIM2 inflammasome activation in human Tohoku Hospital Pediatrics-1 (THP-1) monocytic cells and human AC16 cells was significantly reduced by pharmacological blockade of pannexin-1 channels by the clinically used uricosuric drug probenecid. Probenecid was also able to reduce pressure overload-induced mortality and restore indices of disease severity in a rat chronic HF model in vivo. CONCLUSIONS: This is the first report showing that AIM2 and NLRC4 inflammasome activation contribute to chronic inflammation in HF and that probenecid alleviates chronic HF by reducing inflammasome activation. The present translational study suggests the possibility of repositioning probenecid for HF indications.
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Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Insuficiência Cardíaca/metabolismo , Inflamassomos/metabolismo , Miócitos Cardíacos/metabolismo , Receptores de Superfície Celular/metabolismo , Adolescente , Adulto , Idoso , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Estudos de Casos e Controles , Conexinas/antagonistas & inibidores , Conexinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Modelos Animais de Doenças , Feminino , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/imunologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Inflamassomos/imunologia , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/imunologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Probenecid/farmacologia , Ratos Wistar , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Transdução de Sinais , Sus scrofa , Células THP-1 , Função Ventricular Esquerda , Adulto JovemRESUMO
UNLABELLED: Hypertriglyceridemia is reported to cause 1-7% of the cases of acute pancreatitis. AIM: The aim of the present study was to assess the clinical features and management of pancreatitis with hypertriglyceridemia in our tertiary center. METHODS: Between 1 January 2007 and 31 December 2009, patients with a diagnosis of hypertriglyceridemia-induced acute pancreatitis were reviewed. Patients with pancreatitis and serum triglyceride levels greater than 11.3 mmol/l (≈1000 mg/dl) were included. Acute pancreatitis with other etiologies was excluded. RESULTS: 26 patients (2 women, 24 men; median age at presentation 42 years; range: 22-70) were diagnosed with hypertriglyceridemiainduced acute pancreatitis; 3 patients had altogether 7 relapses. The total number of cases was 33, which accounted for 4.71% of the total number of acute pancreatitis cases in the examined period. There was a failure in diet in 30.3% and a history of regular alcohol abuse in 57.6% of the cases. A history of diabetes mellitus was present in 38.1%, and gallstones in 9.1% of the cases. Lactescent serum was described on admission in 27.3%. Mean triglyceride level was 47.24 mmol/l (≈4181 mg/dl; 12.4-103.8 mmol/l). Amylase level was elevated to three times the normal in 54.5%, and that of lipase to three times the normal in 58.8%. Necrotizing acute pancreatitis was diagnosed in 7 patients (26.9%), and pseudocyst in 8 patients (30.7%). Administration of insulin, heparin, plasmapheresis and fibrates lowered the triglyceride to 3.71 mmol/l (≈328 mg/dl). CONCLUSION: The clinical course of acute pancreatitis with hypertriglyceridemia does not differ from acute pancreatitis of other causes. Interestingly, levels of serum pancreatic enzymes may be normal or only minimally elevated. Insulin, heparin, plasmapheresis and fibrates effectively reduce lipid levels and relieve symptoms. A low triglyceride level is necessary to prevent relapses.
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Complicações do Diabetes/diagnóstico , Hipertrigliceridemia/complicações , Pancreatite/etiologia , Pancreatite/terapia , Triglicerídeos/sangue , Doença Aguda , Adulto , Idoso , Alcoolismo/complicações , Amilases/sangue , Anticoagulantes/administração & dosagem , Complicações do Diabetes/sangue , Complicações do Diabetes/tratamento farmacológico , Comportamento Alimentar , Feminino , Ácidos Fíbricos/administração & dosagem , Heparina/administração & dosagem , Humanos , Hipertrigliceridemia/sangue , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Lipase/sangue , Masculino , Pessoa de Meia-Idade , Pancreatite/sangue , Pancreatite/diagnóstico , Pancreatite/tratamento farmacológico , Pancreatite Necrosante Aguda/diagnóstico , Pancreatite Necrosante Aguda/etiologia , Plasmaferese , Estudos RetrospectivosRESUMO
Cardiac adverse effects are among the leading causes of the discontinuation of clinical trials and the withdrawal of drugs from the market. The novel concept of 'hidden cardiotoxicity' is defined as cardiotoxicity of a drug that manifests in the diseased (e.g. ischemic/reperfused), but not in the healthy heart or as a drug-induced deterioration of cardiac stress adaptation (e.g. ischemic conditioning). Here, we aimed to test if the cardiotoxicity of a selective COX-2 inhibitor rofecoxib that was revealed during its clinical use, i.e., increased occurrence of proarrhythmic and thrombotic events, could have been revealed in early phases of drug development by using preclinical models of ischemia/reperfusion (I/R) injury. Rats that were treated with rofecoxib or vehicle for four weeks were subjected to 30 min. coronary artery occlusion and 120 min. reperfusion with or without cardioprotection that is induced by ischemic preconditioning (IPC). Rofecoxib increased overall the arrhythmias including ventricular fibrillation (VF) during I/R. The proarrhythmic effect of rofecoxib during I/R was not observed in the IPC group. Rofecoxib prolonged the action potential duration (APD) in isolated papillary muscles, which was not seen in the simulated IPC group. Interestingly, while showing hidden cardiotoxicity manifested as a proarrhythmic effect during I/R, rofecoxib decreased the infarct size and increased the survival of adult rat cardiac myocytes that were subjected to simulated I/R injury. This is the first demonstration that rofecoxib increased acute mortality due to its proarrhythmic effect via increased APD during I/R. Rofecoxib did not interfere with the cardiprotective effect of IPC; moreover, IPC was able to protect against rofecoxib-induced hidden cardiotoxicity. These results show that cardiac safety testing with simple preclinical models of I/R injury uncovers hidden cardiotoxicity of rofecoxib and might reveal the hidden cardiotoxicity of other drugs.
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Cardiotoxicidade/complicações , Lactonas/efeitos adversos , Traumatismo por Reperfusão/complicações , Sulfonas/efeitos adversos , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/complicações , Cardiotônicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Precondicionamento Isquêmico , Masculino , Infarto do Miocárdio/complicações , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Ratos WistarRESUMO
INTRODUCTION: In the heart, subsarcolemmal (SSM), interfibrillar (IFM) and perinuclear mitochondria represent three subtypes of mitochondria. The most commonly used protease during IFM isolation is the nagarse, however, its effect on the detection of mitochondrial proteins is still unclear. Therefore, we investigated whether nagarse treatment influences the quantification of mitochondrial proteins. METHODS: SSM and IFM were isolated from hearts of mice and rats. During IFM isolation, nagarse activity was either stopped by centrifugation (common protocol, IFM+N) or inhibited by phenylmethylsulfonyl fluoride (PMSF, IFM+N+I). The amounts of proteins located in different mitochondrial compartments (outer membrane: mitofusin 1 (MFN1) and 2 (MFN2); intermembrane space: p66shc; inner membrane (connexin 43 (Cx43)), and of protein deglycase DJ-1 were determined by Western blot. RESULTS: MFN2 and Cx43 were found predominantly in SSM isolated from mouse and rat hearts. MFN1 and p66shc were present in similar amounts in SSM and IFM+N, whereas the level of DJ-1 was higher in IFM+N compared to SSM. In IFM+N+I samples from mice, the amount of MFN2, but not that of Cx43 increased. Nagarse or nagarse inhibition by PMSF had no effect on oxygen consumption of SSM or IFM. DISCUSSION: Whereas the use of the common protocol indicates the localization of MFN2 predominantly in SSM, the inhibition of nagarse by PMSF increases the signal of MFN2 in IFM to that of in SSM, indicating an underestimation of MFN2 in IFM. Therefore, protease sensitivity should be considered when assessing distribution of mitochondrial proteins using nagarse-based isolation.
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Mitocôndrias/metabolismo , Proteínas Mitocondriais/análise , Subtilisinas/metabolismo , Animais , Fracionamento Celular/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Modelos Animais , Miocárdio/citologia , Consumo de Oxigênio/efeitos dos fármacos , Fluoreto de Fenilmetilsulfonil/farmacologia , Projetos Piloto , Proteômica/métodos , Ratos , Ratos Long-Evans , Ratos WistarRESUMO
Little is known about the molecular mechanism including microRNAs (miRNA) in hypercholesterolemia-induced cardiac dysfunction. We aimed to explore novel hypercholesterolemia-induced pathway alterations in the heart by an unbiased approach based on miRNA omics, target prediction and validation. With miRNA microarray we identified forty-seven upregulated and ten downregulated miRNAs in hypercholesterolemic rat hearts compared to the normocholesterolemic group. Eleven mRNAs with at least 4 interacting upregulated miRNAs were selected by a network theoretical approach, out of which 3 mRNAs (beta-2 adrenergic receptor [Adrb2], calcineurin B type 1 [Ppp3r1] and calcium/calmodulin-dependent serine protein kinase [Cask]) were validated with qRT-PCR and Western blot. In hypercholesterolemic hearts, the expression of Adrb2 mRNA was significantly decreased. ADRB2 and PPP3R1 protein were significantly downregulated in hypercholesterolemic hearts. The direct interaction of Adrb2 with upregulated miRNAs was demonstrated by luciferase reporter assay. Gene ontology analysis revealed that the majority of the predicted mRNA changes may contribute to the hypercholesterolemia-induced cardiac dysfunction. In summary, the present unbiased target prediction approach based on global cardiac miRNA expression profiling revealed for the first time in the literature that both the mRNA and protein product of Adrb2 and PPP3R1 protein are decreased in the hypercholesterolemic heart.