RESUMO
BACKGROUND: Autoantibodies have been identified as major predisposing factors for dilated cardiomyopathy (DCM). Patients with DCM show elevated serum levels of vascular endothelial growth factor (VEGF) whose source is unknown. Besides its well-investigated effects on angiogenesis, evidence is present that VEGF signaling is additionally involved in fibroblast proliferation and cardiomyocyte hypertrophy, hence in cardiac remodeling. Whether autoimmune effects in DCM impact cardiac VEGF signaling needs to be elucidated. METHODS: Five DCM patients were treated by the immunoadsorption (IA) therapy on five consecutive days. The eluents from the IA columns were collected and prepared for cell culture. Cardiomyocytes from neonatal rats (NRCM) were incubated with increasing DCM-immunoglobulin-G (IgG) concentrations for 48 h. Polyclonal IgG (Venimmun N), which was used to restore IgG plasma levels in DCM patients after the IA therapy was additionally used for control cell culture purposes. RESULTS: Elevated serum levels of VEGF decreased significantly after IA (Serum VEGF (ng/ml); DCM pre-IA: 45 ± 9.1 vs. DCM post-IA: 29 ± 6.7; P < 0.05). In cell culture, pretreatment of NRCM by DCM-IgG induced VEGF expression in a time and dose dependent manner. Biologically active VEGF that was secreted by NRCM significantly increased BNP mRNA levels in control cardiomyocytes and induced cell-proliferation of cultured cardiac fibroblast (Fibroblast proliferation; NRCM medium/HC-IgG: 1 ± 0.0 vs. NRCM medium/DCM-IgG 100 ng/ml: 5.6 ± 0.9; P < 0.05). CONCLUSION: The present study extends the knowledge about the possible link between autoimmune signaling in DCM and VEGF induction. Whether this observation plays a considerable role in cardiac remodeling during DCM development needs to be further elucidated.
Assuntos
Autoanticorpos/farmacologia , Cardiomiopatia Dilatada/genética , Fibroblastos/efeitos dos fármacos , Imunoglobulina G/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Animais Recém-Nascidos , Autoanticorpos/sangue , Cardiomiopatia Dilatada/imunologia , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/terapia , Proliferação de Células/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Imunoglobulina G/sangue , Técnicas de Imunoadsorção , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
RATIONALE: Recently, we provided a technique of chronic high-frequency electric stimulation (HFES) of the right inferior ganglionated plexus for ventricular rate control during atrial fibrillation in dogs and humans. In these experiments, we observed a decrease of the intrinsic ventricular rate during the first 4 to 5 months when HFES was intermittently shut off. OBJECTIVE: We thus hypothesized that HFES might elicit trophic effects on cardiac neurons, which in turn increase baseline parasympathetic tone of the atrioventricular node. METHODS AND RESULTS: In mongrel dogs atrial fibrillation was induced by rapid atrial pacing. Endocardial HFES of the right inferior ganglionated plexus, which contains abundant fibers to the atrioventricular node, was performed for 2 years. Sham-operated nonstimulated dogs served as control. In chronic neurostimulated dogs, we found an increased neuronal cell size accompanied by an increase of choline acetyltransferase and unchanged tyrosine hydroxylase protein expression as compared with unstimulated dogs. Moreover, ß-nerve growth factor (NGF) and neurotrophin (NT)-3 were upregulated in chronically neurostimulated dogs. In vitro, HFES of cultured neurons of interatrial ganglionated plexus from adult rats increased neuronal growth accompanied by upregulation of NGF, NT-3, glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) expression. NGF was identified as the main growth-inducing factor, whereas NT-3 did not affect HFES-induced growth. However, NT-3 could be identified as an important acetylcholine-upregulating factor. CONCLUSIONS: HFES of cardiac neurons in vivo and in vitro causes neuronal cellular hypertrophy, which is mediated by NGF and boosters cellular function by NT-3-mediated acetylcholine upregulation. This knowledge may contribute to develop HFES techniques to augment cardiac parasympathetic tone.
Assuntos
Função do Átrio Direito/fisiologia , Fatores de Crescimento Neural/fisiologia , Neurônios/fisiologia , Neurotrofina 3/fisiologia , Fibras Parassimpáticas Pós-Ganglionares/fisiologia , Regulação para Cima/fisiologia , Animais , Células Cultivadas , Cães , Estimulação Elétrica/métodos , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: Recently, increased cardiac norepinephrine levels were observed in patients who were exposed to irregular stimulation during electrophysiological testing. The molecular mechanisms remain unclear. Intrinsic cardiac adrenergic (ICA) cells are present in mammalian hearts and contain catecholamine-synthesizing enzymes sufficient to produce biologically active norepinephrine levels. Thus, we aimed to investigate the expression of catecholamine-synthesizing enzymes by ICA cells exposed to irregular pacing. METHODS: Co-cultures of cardiomyocytes and ICA cells were exposed to irregular pacing for 48h (standard deviation (SD)=5%, 25% and 50% of mean cycle length) at a constant rate of 5Hz. The expression of catecholamine-synthesizing enzymes including tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) were analyzed on mRNA and protein levels. RESULTS: First, immunolabeling identified ICA cells presenting TH and DBH staining around the cell nucleus. Irregular pacing with 25% SD at a constant rate of 5Hz significantly increased the expression of TH and DBH enzyme synthesis. Pharmacological approaches have shown that both metoprolol and losartan reversed the irregular pacing induced DBH increase, whereas the expression of TH was only blocked by metoprolol in a significant manner. Blockade of the endothelin-A receptor by BQ123 or the calcineurin-NFAT pathway by cyclosporine-A, 11R-VIVIT or FK506 revealed a potential role of both cascades in irregular pacing induced catecholamine-synthesizing enzyme expression. CONCLUSIONS: ICA cells respond to irregular electrical activation with an increase in catecholamine-synthesizing enzymes. Drugs commonly used in clinical routine significantly influence the expression of TH and DBH by ICA cells via different signaling routes.
Assuntos
Dopamina beta-Hidroxilase/biossíntese , Epinefrina/fisiologia , Miocárdio/citologia , Miocárdio/enzimologia , Miócitos Cardíacos/fisiologia , Tirosina 3-Mono-Oxigenase/biossíntese , Animais , Inibidores de Calcineurina , Catecolaminas/biossíntese , Técnicas de Cocultura , Ciclosporina/farmacologia , Dopamina beta-Hidroxilase/genética , Estimulação Elétrica , Antagonistas do Receptor de Endotelina A , Losartan/farmacologia , Metoprolol/farmacologia , Miócitos Cardíacos/enzimologia , Fatores de Transcrição NFATC/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Ratos , Transdução de Sinais , Tacrolimo/farmacologia , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
Mechanical stretch has been shown to increase vascular endothelial growth factor (VEGF) expression in cultured myocytes. Sympathetic neurons (SN) also possess the ability to express and secrete VEGF, which is mediated by the NGF/TrkA signaling pathway. Recently, we demonstrated that SN respond to stretch with an upregulation of nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF). Whether stretch increases neuronal VEGF expression still remains to be clarified. Therefore, SN from the superior cervical ganglia of neonatal Sprangue Dawley rats were exposed to a gradual increase of stretch from 3% up to 13% within 3days (3%, 7% and 13%). Under these conditions, the expression and secretion of VEGF was analyzed. Mechanical stretch significantly increased VEGF mRNA and protein expression (mRNA: control=1 vs. stretch=3.1; n=3/protein: control=1 vs. stretch=2.7; n=3). ELISA experiments to asses VEGF content in the cell culture supernatant showed a time and dose dependency in VEGF increment due to stretch. NGF and CNTF neutralization decreased stretch-induced VEGF augmentation in a significant manner. This response was mediated in part by TrkA receptor activation. The stretch-induced VEGF upregulation was accompanied by an increase in HIF-1α expression. KDR levels remained unchanged under conditions of stretch, but showed a significant increase due to NGF neutralization. In summary, SN respond to stretch with an upregulation of VEGF, which is mediated by the NGF/CNTF and TrkA signaling pathway paralleled by HIF-1α expression. NGF signaling seems to play an important role in regulating neuronal KDR expression.
Assuntos
Fator Neurotrófico Ciliar/metabolismo , Mecanotransdução Celular , Fator de Crescimento Neural/metabolismo , Neurônios/metabolismo , Estresse Mecânico , Sistema Nervoso Simpático/citologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Células Cultivadas , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
Recently, we have shown that high frequency electrical field stimulation (HFES) of sympathetic neurons (SN) induces nerve sprouting by up-regulation of nerve growth factor (NGF) which targets the tyrosine kinase A receptor (TrkA) in an autocrine/paracrine manner. There is increasing evidence that matrix metalloproteinase-2 (MMP-2) is not only involved in extracellular matrix (ECM) turnover but may also exert beneficial effects during neuronal growth. Therefore, this study aimed to investigate the regulation and function of MMP-2 and its major activator membrane type 1-matrix metalloproteinase (MT1-MMP) as well its inhibitor TIMP-1 in SN under conditions of HFES. Moreover, we analyzed molecular mechanisms of the beneficial effect of losartan, an angiotensin II type I receptor (AT-1)blocker on HFES-induced nerve sprouting. Cell cultures of SN from the superior cervical ganglia (SCG) of neonatal rats were electrically stimulated for 48 h with a frequency of 5 or 50 Hz. HFES increased MMP-2 and MT1-MMP mRNA and protein expression, whereas TIMP-1 expression remained unchanged. Under conditions of HFES, we observed a shift from pro- to active-MMP-2 indicating an increase in MMP-2 enzyme activity. Specific pharmacological MMP-2 inhibition contributed to an increase in pro-NGF amount in the cell culture supernatant and significantly reduced HFES-induced neurite outgrowth. Losartan abolished HFES-induced nerve sprouting in a significant manner by preventing HFES-induced NGF, MMP-2, and MT1-MMP up-regulation. In summary, specific MMP-2 blockade prevents sympathetic nerve sprouting (SNS) by inhibition of pro-NGF conversion while losartan abolishes HFES-induced SNS by reducing total NGF, MMP-2 and MT1-MMP expression.
Assuntos
Metaloproteinase 14 da Matriz/fisiologia , Metaloproteinase 2 da Matriz/fisiologia , Fatores de Crescimento Neural/metabolismo , Neuritos/fisiologia , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Sistema Nervoso Simpático/metabolismo , Antagonistas de Receptores de Angiotensina/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Estimulação Elétrica/métodos , Losartan/farmacologia , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/fisiologiaRESUMO
Pressure overload is the major stimulus for cardiac hypertrophy. Accumulating evidence suggests an important role for calcium-induced activation of calcineurin in mediating hypertrophic signaling. Hypertrophy is an important risk factor for cardiovascular morbidity and mortality. We therefore employed an in vitro mechanical stretch model of cultured neonatal cardiomyocytes to evaluate proposed mechanisms of calcium-induced calcineurin activation in terms of inhibition of calcineurin activity and hypertrophy. The protein/DNA ratio and ANP gene expression were used as markers for stretch-induced hypertrophy. Stretch increased the calcineurin activity, MCIP1 gene expression and DNA binding of NFATc as well as the protein/DNA ratio and ANP mRNA in a significant manner. The specific inhibitor of calcineurin, cyclosporin A, inhibited the stretch-induced increase in calcineurin activity, MCIP1 gene expression and hypertrophy. The L-type Ca2+ channel blocker nifedipine and a blocker of the Na+/H+ exchanger (cariporide) both suppressed stretch-dependent enhanced calcineurin activity and hypertrophy. Also application of a blocker of the Na+/Ca2+ exchanger (KB-R7943) was effective in preventing calcineurin activation and increases in the protein/DNA ratio. Inhibition of capacitative Ca2+ entry with SKF 96365 was also sufficient to abrogate calcineurin activation and hypertrophy. The blocker of stretch-activated ion channels, streptomycin, was without effect on stretch-induced hypertrophy and calcineurin activity. The present work suggests that of the proposed mechanisms for the calcium-induced activation of calcineurin (L-type Ca2+ channels, capacitative Ca2+ entry, Na+/H+ exchanger, Na+/Ca2+ exchanger and stretch-activated channels) all but stretch-activated channels are possible targets for the inhibition of hypertrophy.
Assuntos
Calcineurina/fisiologia , Cálcio/metabolismo , Cardiomegalia/fisiopatologia , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Inibidores de Calcineurina , Cardiomegalia/metabolismo , Células Cultivadas , Modelos Cardiovasculares , Ratos , Ratos Wistar , Resistência à TraçãoRESUMO
Subthreshold electrical stimulation (SES) has been shown to induce an improvement of angiogenesis in ischemic and nonischemic skeletal muscles, mediated by increased VEGF expression. VEGF plays a key role in physiological and pathological angiogenesis. Cardiomyocytes possess the ability to synthesize and secrete VEGF. Thus, we thought to investigate the effect of SES on VEGF regulation in cultured neonatal rat ventricular myocytes (NRVMs), in the aim to reveal new techniques for therapeutic angiogenesis in ischemic heart disease. Cell cultures of NRVMs were electrically stimulated with field strengths below the myocyte depolarization threshold (0.5 V/cm with 1 ms bipolar impulse duration). Frequencies ranging from 5 Hz up to 25, 50, and 99 Hz were applied over a period of 48 h. The expression of VEGF and its receptor KDR was determined with Western blot and ELISA. To reveal the biological activity of the secreted VEGF amount, cultured human coronary artery endothelial cells (HCAECs) were treated with the cell culture supernatant of NRVMs exposed to SES. A dominant effect of SES was observed at 25 Hz. Within this particular frequency the VEGF protein amount in the cytoplasm as well as in the cell culture supernatant increased significantly. In parallel, the protein expression of the KDR receptor decreased in a significant manner. Moreover, cell culture supernatant of NRVMs exposed to SES augmented the growth of HCAECs. Cardiomyocytes respond to SES with an increase in biologically active VEGF expression that promotes cell proliferation of HCAECs. This mechanism may provide new approaches to develop therapeutic angiogenesis in the ischemic heart.
Assuntos
Estimulação Elétrica , Miócitos Cardíacos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/citologia , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Miócitos Cardíacos/citologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: To evaluate the effects of various methods of sterilization on ovarian function, in a rat model. STUDY DESIGN: Forty-eight female Whistar albino rats weighing 200-250g are divided equally into four groups. All rats underwent laparotomy, while no specific intervention was made to the first group. Bilateral tubal ligation by Pomeroy's technique, unipolar and bipolar cautery was done to the second, third and fourth groups, respectively. All rats were then individually caged and fed on demand for 6 months. Afterwards, the rats were sacrificed and underwent bilateral oophorectomy. A pathologist blinded to the groups made histological examination by counting number of healthy tertiary follicles and corpora lutea in each ovary. The results of the groups were statistically compared by one-way ANOVA using post-hoc Bonferroni correction. RESULTS: Rats in group 1 had significantly higher number of healthy tertiary follicles than every other group. Rats in group 1 also had significantly more corpora lutea than those in group 3. CONCLUSION: Tubal ligation may affect ovarian function, which in turn may reflect to ovarian histology in rats.
Assuntos
Ovário/fisiologia , Esterilização Tubária/efeitos adversos , Animais , Corpo Lúteo/anatomia & histologia , Feminino , Folículo Ovariano/anatomia & histologia , Ratos , Ratos Wistar , Esterilização Tubária/métodosRESUMO
OBJECTIVE: To determine the effects of intraperitoneal Lipiodol and methylene blue in prevention of postsurgical adhesion formation. STUDY DESIGN: Thirty female rats were divided into three equal groups and a standard damage to provoke adhesion formation was made by laparotomy to each of them. Methylene blue and Lipiodol was administered intraperitoneally to the first and second groups, respectively; while the third group was taken as control and no additional intervention was made. Adhesions were scored by a second look laparotomy which was made 21 days after the first operation. RESULTS: Adhesion scores of the second group (i.e. Lipiodol group) were significantly lower than that of controls. No significant differences were present between any other groups (i.e. methylene blue versus control and methylene blue versus Lipiodol). CONCLUSION: Intraperitoneal administration of Lipiodol inhibits postsurgical adhesion formation in rats.
Assuntos
Óleo Iodado/uso terapêutico , Azul de Metileno/uso terapêutico , Complicações Pós-Operatórias/prevenção & controle , Aderências Teciduais/prevenção & controle , Animais , Feminino , Peritônio/efeitos dos fármacos , Ratos , Ratos Wistar , Aderências Teciduais/patologiaRESUMO
BACKGROUND: Neurofilament light chain (NF-L) is the major intermediate filament specifically expressed in neurons and their axons. No data are available concerning serum levels of NF-L after global cerebral ischemia due to cardiac arrest. To find a specific neuronal marker of long-term neurological outcome, we examined serum levels of NF-L in patients after cardiac arrest. METHODS: A prospective observational cohort study was conducted. Blood samples for the measurement of NF-L were analyzed from 85 patients within 2h after admission, as well as on 2nd, 3rd, 5th, and 7th day. Neurological outcome was assessed 6 months after cardiac arrest by employing the Modified Glasgow Outcome Score (MGOS). RESULTS: The serum course of NF-L in patients with poor neurological outcome (MGOS 1+2) was significantly augmented compared to patients with good neurological outcome (MGOS 3+4+5) (on admission (pg/ml): good: 125 ± 11.7 vs. poor: 884.4 ± 86.2 pg/ml; 3rd day: good: 153.1 ± 13.2 vs. poor: 854.4 ± 119.1; 7th day: good: 112.5 ± 10.4 vs. poor: 1011.8 ± 100.8; P<0.001). Intermediate NF-L serum values were found in patients with MGOS 0, which represents a mixture of patients who died with and without certified brain damage (on admission (pg/dl): 433.7 ± 49.8; 3rd day: 598.3 ± 86.6; 7th day: 474 ± 77.4). A prediction power of 0.93 (c-statistic, 95%-CI 0.87-0.99) on 1st, 0.85 (0.81-0.95) on 2nd, 0.92 (0.85-0.99) on 3rd, 0.97 (0.92-1) on 5th and 0.99 (0.98-1) on 7th day was achieved for NF-L predicting poor neurological outcome. CONCLUSIONS: The present data suggest that within 7 days after cardiac arrest serum NF-L is a valuable marker of long-term neurological outcome.
Assuntos
Parada Cardíaca/sangue , Parada Cardíaca/diagnóstico , Doenças do Sistema Nervoso/sangue , Doenças do Sistema Nervoso/diagnóstico , Proteínas de Neurofilamentos/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Coortes , Diagnóstico Precoce , Feminino , Parada Cardíaca/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso/epidemiologia , Valor Preditivo dos Testes , Estudos Prospectivos , Fatores de Tempo , Resultado do TratamentoRESUMO
Age has been identified as an independent risk factor for cardiovascular diseases. A shift of the cardiac autonomic nervous system towards an increase in sympathetic tone has been reported in the elderly. Nerve growth factor (NGF) is the main neurotrophic factor that increases the sympathetic activity of the heart. If there is a shift of NGF expression in old compared to young cardiomyocytes and whether there are regional differences in the heart still remain unclear. Therefore, we chose a rat model of different-aged rats (3-4 days = neonatal, 6-8 weeks = young, 20-24 months = old), and isolated cardiomyocytes from the left and the right atrium (LA, RA), as well as from the left and the right ventricle (LV, RV), were used to determine NGF expression on mRNA and protein levels. In neonatal, young, and old rats, NGF amount in LA and RA was significantly lower as compared to LV and RV. In young and old rats, we found significant higher NGF protein levels in LA compared to RA. In addition, both atria showed an increase in NGF expression between age groups neonatal, young, and old. In both ventricles, we observed a significant decrease in NGF expression from neonatal to young rats and a significant increase from young to old rats. The highest NGF amount in LV and RV was observed in neonatal rats. Regarding tyrosine kinase A receptor (TrkA) expression, the main receptor for NGF signaling, both atria showed the largest expression in old rats; while in LV and RV, TrkA was expressed mainly in young rats. These results point to a contribution of nerve growth factors to the change of autonomic tone observed in elderly patients.
Assuntos
Envelhecimento/genética , Sistema Nervoso Autônomo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Coração/inervação , Fator de Crescimento Neural/genética , RNA/genética , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Sistema Nervoso Autônomo/citologia , Sistema Nervoso Autônomo/crescimento & desenvolvimento , Western Blotting , Células Cultivadas , Coração/crescimento & desenvolvimento , Masculino , Fator de Crescimento Neural/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo RealRESUMO
An irregular ventricular response during atrial fibrillation (AF) has been shown to mediate an increase in sympathetic nerve activity in human subjects. The molecular mechanisms remain unclear. This study aimed to investigate the impact of rate and irregularity on nerve growth factor (NGF) expression in cardiomyocytes, since NGF is known to be the main contributor to cardiac sympathetic innervation density. Cell cultures of neonatal rat ventricular myocytes were electrically stimulated for 48 h with increasing rates (0, 5 and 50 Hz) and irregularity (standard deviation (SD)=5%, 25% and 50% of mean cycle length). Furthermore, we analyzed the calcineurin-NFAT and the endothelin-1 signalling pathways as possible contributors to NGF regulation during arrhythmic stimulation. We found that the increase of NGF expression reached its maximum at the irregularity of 25% SD by 5 Hz (NGF: 5 Hz 0% SD=1 vs. 5Hz 25% SD=1.57, P<0.05). Specific blockade of the ET-A receptor by BQ123 could abolish this NGF increase (NGF: 5 Hz 25% SD+BQ123=0.66, P<0.05). High frequency electrical field stimulation (HFES) with 50 Hz decreased the NGF expression in a significant manner (NGF: 50Hz=0.55, P<0.05). Inhibition of calcineurin-NFAT signalling with cyclosporine-A or 11R-VIVIT abolished the HFES induced NGF down-regulation (NGF: 50 Hz+CsA=1.14, P<0.05). In summary, this study reveals different signalling routes of NGF expression in cardiomyocytes exposed to increasing rates and irregularity. Whether this translates into different degrees of NGF expression and possibly neural sympathetic growth in various forms of ventricular rate control during AF remains to be elucidated in further studies.
Assuntos
Fibrilação Atrial/fisiopatologia , Ventrículos do Coração/citologia , Miócitos Cardíacos/metabolismo , Fator de Crescimento Neural/genética , Transdução de Sinais , Animais , Fibrilação Atrial/metabolismo , Fator Natriurético Atrial/metabolismo , Calcineurina/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/química , Estimulação Elétrica , Antagonistas do Receptor de Endotelina A , Endotelina-1/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição NFATC/metabolismo , Fator de Crescimento Neural/metabolismo , Neuritos/fisiologia , Peptídeos Cíclicos/farmacologia , Cultura Primária de Células , Ratos , Transcrição GênicaRESUMO
The Glasgow-Pittsburgh cerebral performance categories (GP-CPC) and the Glasgow Outcome Score (GOS) have been used to categorize patients according to their neurological outcome for prognostic predictors in patients after cardiac arrest (CA). We postulated that inclusion of deaths without knowing the cerebral status into the group of patients with poor outcome after CA using the GP-CPC and GOS will lead to dilution of the prognostic power of the investigated biochemical marker. The present study was conducted to verify this issue by employing a modified outcome score, which we termed as Modified Glasgow Outcome Score (MGOS). In the present study, 97 patients were enrolled in a prospective manner. Serum NSE and S100B levels were measured daily for 7 days after admission to the intensive care unit. Neurological outcome was assessed by employing the GOS and MGOS after 6 months. By employing the GOS, 46 patients were categorized into the group of patients with poor outcome and 51 patients survived with good neurological outcome. Patients who died without certified brain damage or with unknown cerebral status after CA (n = 20) were separated from patients with poor outcome in the MGOS. The magnitude of NSE (S100B) elevation in patients with poor outcome categorized by the MGOS was approximately 1.7-fold (1.5) higher as compared with patients divided by the GOS. The mean calculated sensitivities and area under the curve values of NSE and S100B predicting poor outcome classified by the MGOS were significantly higher as compared with the GOS. Conclusively, inclusion of deaths without certified brain damage or with unknown cerebral status into the group of patients with poor outcome will lead to underestimation of the prognostic power of investigated biochemical markers such as NSE and S100B. The MGOS will help to avoid this bias.
Assuntos
Escala de Resultado de Glasgow , Parada Cardíaca/diagnóstico , Hipóxia Encefálica/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Biomarcadores/sangue , Reanimação Cardiopulmonar , Causas de Morte , Avaliação da Deficiência , Feminino , Alemanha , Parada Cardíaca/sangue , Parada Cardíaca/complicações , Parada Cardíaca/mortalidade , Parada Cardíaca/fisiopatologia , Parada Cardíaca/terapia , Humanos , Hipóxia Encefálica/sangue , Hipóxia Encefálica/etiologia , Hipóxia Encefálica/mortalidade , Hipóxia Encefálica/fisiopatologia , Hipóxia Encefálica/terapia , Masculino , Pessoa de Meia-Idade , Fatores de Crescimento Neural/sangue , Exame Neurológico , Fosfopiruvato Hidratase/sangue , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Curva ROC , Recuperação de Função Fisiológica , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/sangue , Fatores de TempoRESUMO
The biomechanical environment to which cells are exposed is important to their normal growth, development, interaction, and function. Accordingly, there has been much interest in studying the role of biomechanical forces in cell biology and pathophysiology. This has led to the introduction and even commercialization of many experimental devices. Many of the early devices were limited by the heterogeneity of deformation of cells cultivated in different locations of the culture plate membranes and were also attached with complicated technical/electronic efforts resulting in a restriction of the reproducibility of these devices. The objective of this study was to design and build a simple device to allow the application of dose-dependent homogeneous equibiaxial static stretch to cells cultured on flexible silicone membranes to investigate biological and biomedical questions. In addition, cultured neonatal rat atrial cardiomyocytes were stretched with the proposed device with different strain gradients. For the first time with this study we could demonstrate that stretch up to 21% caused dose-dependent changes in biological markers such as the calcineurin activity, modulatory calcineurin-interacting protein-1, voltage-gated potassium channel isoform 4.2, and voltage-gated K(+) channel-interacting proteins-2 gene expression and transient outward potassium current densities but not the protein-to-DNA ratio and atrial natriuretic peptide mRNA. With both markers mentioned last, dose-dependent stretch alterations could only be achieved with stretch up to 13%. The simple and low-cost device presented here might be applied to a wide range of experimental settings in different fields of research.
Assuntos
Técnicas de Cultura de Células/instrumentação , Membranas Artificiais , Miócitos Cardíacos/metabolismo , Silicones/química , Animais , Animais Recém-Nascidos , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Calcineurina/metabolismo , Tamanho Celular , Células Cultivadas , Desenho de Equipamento , Átrios do Coração/metabolismo , Hipertrofia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Teste de Materiais , Potenciais da Membrana , Miócitos Cardíacos/patologia , Maleabilidade , Potássio/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo , Estresse Mecânico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Atrial fibrillation (AF) is the most frequent arrhythmia found in clinical practice. In recent studies, a decrease in the development or recurrence of AF was found in hypertensive patients treated with angiotensin-converting enzyme inhibitors or angiotensin receptor-blocking agents. Hypertension is related to an increased wall tension in the atria, resulting in increased stretch of the individual myocyte, which is one of the major stimuli for the remodeling process. In the present study, we used a model of cultured atrial neonatal rat cardiomyocytes under conditions of stretch to provide insight into the mechanisms of the preventive effect of the angiotensin receptor-blocking agent losartan against AF on a molecular level. Stretch significantly increased protein-to-DNA ratio and atrial natriuretic factor mRNA expression, indicating hypertrophy. Expression of genes encoding for the inward rectifier K(+) current (I(K1)), Kir 2.1, and Kir 2.3, as well as the gene encoding for the ultrarapid delayed rectifier K(+) current (I(Kur)), Kv 1.5, was significantly increased. In contrast, mRNA expression of Kv 4.2 was significantly reduced in stretched myocytes. Alterations of gene expression correlated with the corresponding current densities: I(K1) and I(Kur) densities were significantly increased in stretched myocytes, whereas transient outward K(+) current (I(to)) density was reduced. These alterations resulted in a significant abbreviation of the action potential duration. Losartan (1 microM) prevented stretch-induced increases in the protein-to-DNA ratio and atrial natriuretic peptide mRNA expression in stretched myocytes. Concomitantly, losartan attenuated stretch-induced alterations in I(K1), I(Kur), and I(to) density and gene expression. This prevented the stretch-induced abbreviation of action potential duration. Prevention of stretch-induced electrical remodeling might contribute to the clinical effects of losartan against AF.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Anti-Hipertensivos/farmacologia , Expressão Gênica/efeitos dos fármacos , Losartan/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Animais Recém-Nascidos , Anti-Hipertensivos/uso terapêutico , Fibrilação Atrial/etiologia , Fibrilação Atrial/prevenção & controle , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Crescimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Átrios do Coração/citologia , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Hipertensão/complicações , Hipertensão/tratamento farmacológico , Cinética , Canal de Potássio Kv1.5/efeitos dos fármacos , Canal de Potássio Kv1.5/metabolismo , Losartan/uso terapêutico , Miócitos Cardíacos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , RNA Mensageiro/metabolismo , Ratos , Canais de Potássio Shal/efeitos dos fármacos , Canais de Potássio Shal/metabolismoRESUMO
OBJECTIVE: The purpose of this study was to investigate the insulin response to a 3-hour oral glucose tolerance test and to compare the insulin levels in the gestational diabetes mellitus and single abnormal test value groups with a nondiabetic control group. STUDY DESIGN: One hundred ten Turkish women with uncomplicated pregnancy participated in this prospective controlled study between 24 to 28 weeks of gestation. A 100-g 3-hour oral glucose tolerance test was given, and glucose and insulin plasma levels were assayed. The subjects were classified according to established criteria. Early-phase insulin secretion was assessed by the insulinogenic index. Total insulin secretion was assessed by mean insulin level during the oral glucose tolerance test; insulin resistance was assessed by fasting insulin concentration and by the use of the homeostasis model. Data were analyzed by the Student t test and 1-way analysis of variance, with posthoc Bonferroni correction. RESULTS: The fasting insulin levels of patients with normal oral glucose tolerance test results were significantly lower than those of patients with gestational diabetes mellitus and a single value abnormality (P <.001 and P <.005, respectively). The insulinogenic index as a marker of early-phase insulin secretion was significantly lower in gestational diabetes mellitus, compared with that of patients with normal oral glucose tolerance test results (P <.05). The worsening of glycemic profile from normal oral glucose tolerance test results to gestational diabetes mellitus was associated with an increase in the homeostasis model; no significant difference was found between gestational diabetes mellitus and a single value abnormality group in terms of both the homeostasis model and the insulinogenic index. Values for total insulin secretion were highest in gestational diabetes mellitus, followed by the single value abnormality group, both significantly differing from the values of patients with normal oral glucose tolerance test results (P <.001 and P <.005, respectively). CONCLUSION: In this prospective study of Turkish subjects, we found a striking similarity in terms of patient characteristics between the gestational diabetes mellitus group and the single value abnormality group. Additionally, when we used fasting insulin level and insulin resistance as 2 separate criteria of analysis, patients with single value abnormality were indistinguishable from patients with gestational diabetes mellitus; both groups were significantly different from the normal oral glucose tolerance test group. Our findings suggest that a single abnormal test value on an oral glucose tolerance test should be regarded as a pathologic finding and that the patient with a single abnormal test value may be treated similarly to the patient with gestational diabetes mellitus.