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Objective:To investigate the efficacy of phenolamine in the treatment of sepsis-induced myocardial dysfunction and its effect on cardiac function, myocardial injury index, and hemodynamics in patients.Methods:The clinical data of 79 patients with sepsis-induced myocardial dysfunction who received treatment in Huangshi Central Hospital, Edong Healthcare Group from February 2017 to February 2020 were retrospectively analyzed. These patients were divided into a control group (without phenolamine treatment, n = 41) and an observation group (with phenolamine treatment, n = 38) according to whether they received phenolamine treatment or not. Clinical efficacy, cardiac function, myocardial injury index, and hemodynamic index pre- and post-treatment were compared between the two groups. Results:There was no significant difference in 28-day mortality rate between the two groups ( P > 0.05). Intensive care unit length of stay and mechanical ventilation duration in the observation group were (9.33 ± 3.52) days and 83.00 (28.50, 138.00) hours, which were significantly shorter than (12.17 ± 4.15) days and 111.00 (47.50, 169.00) hours in the control group ( t = 3.26, Z = -2.27, both P < 0.05). The response rate in the observation group was significantly higher than that in the control group [81.58% (31/38) vs. 60.98% (25/41), χ2 = 4.05, P < 0.05]. After 7 days of treatment, the left ventricular ejection fraction in each group was significantly increased, and the left ventricular end-diastolic diameter and left ventricular end-systolic diameter in each group were significantly decreased compared with before treatment (all P < 0.05). After 7 days of treatment, the left ventricular ejection fraction in the observation group was significantly higher than that in the control group ( t = 3.29, P < 0.05), and left ventricular end-diastolic diameter and left ventricular end-systolic diameter were significantly lower than those in the control group ( t = 5.94, 11.21, both P < 0.05). N-terminal pro-brain natriuretic peptide and cardiac troponin I levels in each group were significantly decreased with time (both P < 0.05). At 24 and 72 hours and 7 days after treatment, N-terminal pro-brain natriuretic peptide and cardiac troponin I levels in the observation group were significantly lower than those in the control group (both P < 0.05). After 7 days of treatment, heart rate in each group decreased significantly compared with that before treatment (both P < 0.05), mean arterial pressure, cardiac index, and stroke output index in each group increased significantly compared with those before treatment (all P < 0.05). After 7 days of treatment, heart rate in the observation group was significantly lower than that in the control group ( t = 4.90, P < 0.05), and mean arterial pressure, cardiac index, and stroke output index in the observation group were significantly higher than those in the control group ( t = 4.37, 3.23, 6.01, all P < 0.05). Conclusion:Phentolamine can improve hemodynamics, reduce myocardial injury and improve cardiac function in patients with sepsis-induced myocardial dysfunction.
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Resumo Fundamento O período neonatal é marcado por muitas alterações importantes no sistema cardiovascular, principalmente na primeira semana de vida. Diferentemente da população adulta, estudos sobre dados de eletrocardiograma (ECG) no período neonatal são escassos. Este é o primeiro estudo a descrever alterações eletrocardiográficas em uma coorte de recém-nascidos com ecocardiogramas normais. Objetivos Analisar padrões eletrocardiográficos de uma população de recém-nascidos a termo, sem anomalias morfológicas ou funcionais cardíacas, e comparar os resultados com a literatura. Métodos Neste estudo observacional, ecocardiogramas e resultados de ECG de 94 neonatos divididos em três grupos etários (até 24 horas, entre 25 e 72 horas, e entre 73 e 168 horas de vida) foram avaliados e comparados com aqueles descritos por Davignon et al. Um valor de p < 0,05 foi considerado estatisticamente significativo. Resultados Diferenças significativas na direção da onda T foram detectadas nas derivações V1 (p= 0,04), V2 (p= 0,02), V3 (p= 0,008) e V4 (p= 0,005). Houve diferenças entre nossos resultados e a literatura atual na maioria dos parâmetros. Conclusão Recém-nascidos a termo com menos de 24 horas de vida apresentaram significativamente mais ondas T positivas que aqueles com mais horas de vida. Encontramos muitas diferenças nos parâmetros de ECG em comparação aos descritos por Davignon et al., particularmente nas amplitudes de P, Q, R, S, duração do QRS, R/S e R+S. Esses achados indicam a necessidade de mais estudos para uma interpretação definitiva do ECG em recém-nascidos.
Abstract Background The neonatal period is marked by major changes in the cardiovascular system, especially in the first week of life. Unlike the adult population, studies on electrocardiogram (ECG) data in the neonatal period are scarce. This is the first study to describe electrocardiographic changes in a cohort of newborns with normal echocardiograms. Objectives To analyze the electrocardiographic patterns of a population of full-term NB, without any cardiac morphological or functional anomalies, and compare the results with the literature. Methods In this observational study, echocardiograms and ECG results from 94 newborns divided in three age groups (up to 24 hours, between 25 and 72 hours, and between 73 and 168 hours of life) were evaluated and compared with those reported by Davignon et al. A p-value <0.05 was considered statistically significant. Results There were significant differences in T-wave direction in leads V1 (p= 0.04), V2 (p= 0.02), V3 (p= 0.008) and V4 (p= 0.005) between the three age groups. There were differences between our findings and the current literature in most of the parameters. Conclusion Term newborns within 24 hours of life showed significantly more positive T waves than older ones. Many differences from the Davignon's ECG parameters were found, particularly in the P, Q, R, S amplitudes, QRS duration, R/S and R+S. These findings indicate that more studies are needed for a definitive interpretation of the ECG in newborns.
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Resumo Fundamento A hipertrofia e a dilatação do ventrículo direito observadas na hipertensão arterial pulmonar (HAP) prejudicam a dinâmica do ventrículo esquerdo (VE) achatando o septo interventricular. Objetivo Investigar se o treinamento físico resistido (TFR) de intensidade baixa a moderada é benéfico para funções contráteis do VE e de cardiomiócitos em ratos durante o desenvolvimento de HAP induzida por monocrotalina (MCT). Métodos Foram usados ratos Wistar machos (Peso corporal: ~ 200 g). Para avaliar o tempo até o possível surgimento de insuficiência cardíaca (ou seja, ponto de desfecho), os ratos foram divididos em dois grupos, hipertensão com sedentarismo até a insuficiência (HSI, n=6) e hipertensão com treinamento até a insuficiência (HTI, n=6). Para testar os efeitos do TFR, os ratos foram divididos entre grupos de controle sedentários (CS, n=7), hipertensão com sedentarismo (HS, n=7) e hipertensão com treinamento (HT, n=7). A HAP foi induzida por duas injeções de MCT (20 mg/kg, com um intervalo de 7 dias). Os grupos com treinamento foram submetidos a um protocolo de TFR (subir escadas; 55-65% da máxima carga carregada), 5 dias por semana. A significância estatística foi definida em p <0,05. Resultados O TFR prolongou o ponto de desfecho (~25%), melhorou a tolerância ao esforço físico (~55%) e atenuou as disfunções de contratilidade de VE e de cardiomiócitos promovidas pela MCT preservando a fração de ejeção e o encurtamento fracional, a amplitude do encurtamento, e as velocidades de contração e relaxamento nos cardiomiócitos. O TFR também preveniu os aumentos de fibrose e colágeno tipo I no ventrículo esquerdo causados pela MCT, além de manter as dimensões de miócitos e colágeno tipo III reduzidas por MCT. Conclusão O TFR de intensidade baixa a moderada é benéfico para funções contráteis de VE e cardiomiócitos em ratos durante o desenvolvimento de HAP induzida por MCT.
Abstract Background The right ventricular hypertrophy and dilation observed in pulmonary artery hypertension (PAH) damages the left ventricle (LV) dynamics by flattening the interventricular septum. Objective To investigate whether low- to moderate-intensity resistance exercise training (RT) is beneficial to LV and cardiomyocyte contractile functions in rats during the development of monocrotaline (MCT)-induced PAH. Methods Male Wistar rats (Body weight: ~ 200 g) were used. To assess the time to potential heart failure onset (i.e., end point), rats were divided into sedentary hypertension until failure (SHF, n=6) and exercise hypertension until failure (EHF, n=6) groups. To test RT effects, rats were divided into sedentary control (SC, n = 7), sedentary hypertension (SH, n=7), and exercise hypertension (EH, n=7) groups. PAH was induced by two MCT injections (20 mg/kg, with 7 days interval). Exercise groups were submitted to an RT protocol (Ladder climbing; 55-65% of carrying maximal load), 5 times/week. Statistical significance was assumed at P < 0.05. Results RT prolonged the end point (~25 %), enhanced the physical effort tolerance (~ 55%), and mitigated the LV and cardiomyocyte contractility dysfunctions promoted by MCT by preserving the ejection fraction and fractional shortening, the amplitude of shortening, and the velocities of contraction and relaxation in cardiomyocytes. RT also prevented increases in left ventricle fibrosis and type I collagen caused by MCT, and maintained the type III collagen and myocyte dimensions reduced by MCT. Conclusion Low- to moderate-intensity RT benefits LV and cardiomyocyte contractile functions in rats during the development of MCT-induced PAH.
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Objective:To evaluate the effects of different densities of rat cardiac fibroblasts (RCF) subjected to hypothermic hypoxia-reoxygenation on cardiomyocyte injury and intercellular coupling.Methods:RCF was cultured in vitro and divided into 3 groups ( n=12 each) using a random number table method: RCF density 0.5×10 5 cells/ml group (T 0.5 group), RCF density 1.0×10 5 cells/ml group (T 1.0 group), and RCF density 2.0×10 5 cells/ml group (T 2.0 group). The three groups were placed in an anoxic device, into which 95% N 2 + 5% CO 2 was continuously blown at the speed of 5 L/min for 15 min, and then placed in a 4 ℃ refrigerator for 1 h for low temperature treatment.After completion of culture, cells were placed in a incubator containing 95% air + 5% CO 2 at 37 ℃ for 4 h of reoxygenation.After the end of culture, RCF in three groups were indirectly co-cultured with cardiomyocytes of the same density (1.0×10 5 cells/ml) in a Transwell chamber for 16 h, cardiomyocytes were seeded in the lower chamber of Transwell, and RCF were seeded in the upper chamber of Transwell.After the end of co-culture, cardiomyocytes were collected for determination of the cell viability (by CCK8 method), apoptosis rate (by flow cytometry), expression of connexin 43 (Cx43) mRNA (by real-time fluorescence quantitative polymerase chain reaction), and expression of Cx43 and phosphorylated Cx43 (p-Cx43) (by Western blot). Results:Compared with T 0.5 group, the cell viability, apoptosis rate and expression of Cx43, p-Cx43 and Cx43 mRNA were significantly decreased in T 1.0 and T 2.0 groups ( P<0.01). Compared with T 1.0 group, the cell viability, apoptosis rate and expression of Cx43 and p-Cx43 were significantly decreased ( P<0.01), and no significant change was found in expression of Cx43 mRNA in cardiomyocytes in T 2.0 group ( P>0.05). Conclusions:RCF subjected to hypothermic hypoxia-reoxygenation induces cardiomyocyte injury in a density-dependent manner in a certain range, and the mechanism may be related to down-regulation of the expression of Cx43 and reduction of the activity of Cx43.
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Objective:To evaluate the role of ferroptosis in hypoxia-reoxygenation (H/R) injury in cardiomyocytes cultured in high-fat high-glucose (HFHG) medium.Methods:Cardiomyocytes H9c2 cells were commonly cultured and divided into 3 groups ( n=20 each) using a random number table method: control group (C group), HFHG-H/R group and Ferrostatin-1 (Fer-1) plus HFHG-H/R group (Fer-1+ HFHG+ H/R group). H9c2 cells were cultured in a HFHG medium for 12 h and then exposed to 1%O 2-5%CO 2-94%N 2 for 4 h, followed by 2 h reoxygenation in a cell incubator.Fer-1 at a final concentration of 10 μmol/L was added while the cells were cultured in the HFHG medium in group Fer-1+ HFHG+ H/R.At 2 h of reoxygenation, the cell viability was measured using CCK-8 assay, the activity of lactate dehydrogenase (LDH) in the supernatant was measured using 2, 4-dinitrophenylhydrazine color method, the activity of reactive oxygen species (ROS) was measured by fluorescent probe DCFH-DA flow cytometry, and the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), nuclear receptor coactivator 4 (NCOA4), and glutathione peroxidase 4 (GPX4) was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the activities of LDH release and ROS were increased, and the expression of ACSL4 and NCOA4 was up-regulated ( P<0.05), and no significant change was found in the expression of GPX4 in group HFHG+ H/R ( P>0.05). Compared with group HFHG+ H/R, the cell activity was significantly increased, the activities of LDH and ROS were decreased, and the expression of ACSL4 and NCOA4 was down-regulated ( P<0.05), and no significant change was found in the expression of GPX4 in Fer-1+ HFHG+ H/R group ( P>0.05). Conclusions:Ferroptosis is involved in the process of H/R injury in cardiomyocytes cultured in HFHG medium.
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Objective:To evaluate the effect of rat cardiac fibroblasts (RCF) on the expression of connexin43 (Cx43) in H9c2 cells during hypothermic hypoxia/reoxygenation.Methods:H9c2 cells cultured in vitro were divided into 4 groups ( n=12 each) using the random number table method: control group (group C), hypothermic hypoxia/reoxygenation group (group HHR), RCF co-culture group (group Co) and RCF co-culture plus hypothermic hypoxia/reoxygenation group (group Co+ HHR). Group C was incubated at 37℃ in 5% CO 2 + 95% air for 5 h. Group HHR was incubated at 4 ℃ in 5% CO 2 + 95% N 2 for 1 h and then at 37 ℃ in 5% CO 2 + 95% air for 4 h. In group Co and group Co+ HHR, H9c2 cells 0.3×10 5 cells/well were inoculated in the lower chamber and RCF 0.6×10 5 cells/well in the the upper chamber of a transwell ? culture dish.Group Co was incubated at 37 ℃ in 5% CO 2 + 95% air for 5 h. Group Co+ HHR was incubated at 4℃ in 95% N 2 + 5% CO 2 for 1 h, and then incubated at 37 ℃ in 5% CO 2 + 95% air for 4 h. The mortality rate of H9c2 cells was measured by trypan blue staining, the expression of Cx43 and extracellular signal-regulated protein kinases 1/2 (ERK1/2) by immunofluorescence, and the expression of Cx43, phosphorylated Cx43, ERK1/2 and phosphorylated ERK1/2 by Western blot. Results:Compared with group C, the mortality rate of H9c2 cells was significantly increased, the expression and phosphorylation of Cx43 were decreased, and the expression and phosphorylation of ERK1/2 were increased in group HHR ( P<0.05), and no significant change was found in the mortality rate of H9c2 cells or expression and phosphorylation of Cx43 and ERK1/2 in group Co ( P>0.05). Compared with group Co, the mortality rate of H9c2 cells was significantly increased, and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+ HHR ( P<0.05). Compared with group HHR, the mortality rate of H9c2 cells was significantly increased, and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+ HHR ( P<0.05). Conclusions:RCFs can decrease the expression and activity of Cx43 in H9c2 cells during hypothermic hypoxia/reoxygenation, and the mechanism may be related to the down-regulation of ERK1/2 expression and inhibition of ERK1/2 activity.
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Objective:To evaluate the role of reactive oxygen species (ROS) in hypoxia postconditioning-induced activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway in rat cardiomyocytes.Methods:Primary cardiomyocytes of adult rats were isolated and cultured and divided into 4 groups ( n=20 each) using a random number table method: normal group (group N), hypoxia-reoxygenation group (group HR), hypoxia postconditioning group (group HPO) and hypoxia postconditioning plus an ROS scavenger N-(2-Amidinopropionyl)-glycine (MPG) group (group HPO+ MPG). Cells were exposed to hypoxia for 45 min followed by 60 min reoxygenation to develop the cardiomyocyte hypoxia-reoxygenation injury model.In HPO group, cells were subjected to 3 cycles of 5-min hypoxia/5-min reoxygenation after 45 min hypoxia, followed by reoxygenation for 60 min.In HPO+ MPG group, MPG (final concentration 2 mmol/L) was added at 35 min of hypoxia, cells were subjected to hypoxia for 10 min, and the other treatments were similar to those previously described in group HPO.At the end of reoxygenation, the intracellular calcium level and Nrf2 activity were measured, the ultrastructure of cardiomyocytes was observed, and the Flameng score of mitochondria was assessed, and the expression of Nrf2, NAD(P)H quinine oxidoreductase 1 (NQO1), superoxide dismutase 1 (SOD1) and heme oxygenase-1 (HO-1) protein and mRNA was detected using real-time polymerase chain reaction and Western blot. Results:Compared with group N, the intracellular free Ca 2+ level, Nrf2 activity and Flameng score were significantly increased, and the expression of Nrf2, NQO1, SOD1 and HO-1 protein and mRNA was down-regulated in group HR ( P<0.05). Compared with group HR, the intracellular free Ca 2+ level and Flameng score were significantly decreased, the Nrf2 activity was increased, and the expression of Nrf2, NQO1, SOD1 and HO-1 protein and mRNA was up-regulated in group HPO ( P<0.05). Compared with group HPO, the intracellular free Ca 2+ level and Flameng score were significantly increased, the Nrf2 activity was decreased, and the expression of Nrf2, NQO1, SOD1 and HO-1 protein and mRNA was down-regulated in group HPO+ MPG ( P<0.05). Conclusions:The mechanism by which hypoxia postconditioning activates the Nrf2/ARE signaling pathway in rat cardiomyocytes may be related to ROS.
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ABSTRACT Objective Analyze the effects of aerobic and anaerobic training on different ergometers on muscle and cardiac hypertrophy in rats. Methods The animals were separated into the following groups: Control (C), Aerobic Training in Water (ATW), Resistance Training in Water (RTW), Aerobic Training on Treadmill (ATT), and Resistance Training in Climbing (RTC). All training protocols were carried out for 4 weeks, 3 times/week. The cross-sectional area (CSA) of the gastrocnemius muscle cells and the areas of the cardiomyocytes were measured. Results In the fast-twitch fibers, there was an increase in CSA in the RTW and RTC groups compared to the ATW (p<0.01 and p<0.01) and ATT groups (p<0.01 and p<0.01). In the slow-twitch fibers, the ATW and ATT groups demonstrated a lower CSA compared to the RTW (p=0.03 and p<0.00) and RTC groups (p<0.01 and p<0.01). In the cardiomyocytes, there was an increase in the area of the RTW and RTC groups compared to groups C (p<0.01; p<0.01), ATW (p=0.02; p<0.01), and ATT (p<0.01; p<0.01). Conclusion The anaerobic training effectively promotes hypertrophy in the fast-twitch fibers and the cardiomyocytes. Level of Evidence V; Animal experimental study.
RESUMO Objetivo Analisar os efeitos dos treinamentos aeróbios e anaeróbios em diferentes ergômetros na hipertrofia muscular e cardíaca de ratos. Métodos Os animais foram separados nos grupos controle (C), treinamento aeróbio em natação (ATW), treinamento resistido em meio aquático (RTW), treinamento aeróbio em esteira rolante (ATT) e treinamento resistido em escalada (RTC). Os protocolos de treinamento foram realizados por 4 semanas, 3 x/semana. Foram mensurados a área de secção transversa (CSA) das células do músculo gastrocnêmio e as áreas dos cardiomiócitos. Resultados Nas fibras de contração rápida houve aumento da CSA dos grupos RTW e RTC em relação aos grupos ATW (p<0,01 e p<0,01) e ATT (p<0,01 e p<0,01). Nas fibras de contração lenta os grupos ATW e ATT demonstraram menor CSA comparado aos grupos RTW (p=0,03 e p<0,00) e RTC (p<0,01 e p<0,01). Nos cardiomiócitos houve aumento da área dos grupos RTW e RTC em comparação com os grupos C (p<0,01 e p<0,01), ATW (p=0,02 e p<0,01) e ATT (p<0,01 e p<0,01). Conclusão Os treinamentos anaeróbios promoveram hipertrofia nas fibras de contração rápida e nos cardiomiócitos. Nível de Evidência V; Estudo experimental em animais.
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Resumo Fundamento: É sabido que a resistência à insulina e a hiperglicemia são causas patológicas importantes no desenvolvimento de cardiomiopatia diabética (CMD). Entretanto, seus mecanismos moleculares precisos na patogênese da CMD ainda não estão claros. Objetivos: Estudos recentes revelam que os microRNAs (miRNAs) desempenham papéis essenciais na patogênese da CMD. Este projeto tem o objetivo de determinar os papéis de miR-34a e miR-125b na morte celular de cardiomiócitos causada por hiperglicemia. Métodos: Cardiomiócitos primários de ratos foram isolados e expostos a concentrações de glicose normais e altas. A viabilidade das células foi medida utilizando-se o ensaio MTT. As expressões de miR-34a e miR-125b foram detectadas por qRT-PCR. Alvos potenciais de miR-34a e miR-125b foram previstos pelo www.Targetscan.org, e validados a partir de tecidos cardíacos humanos. Um p<0,05 foi considerado significância estatística. Resultados: Demonstra-se neste estudo que o miR-34a e o miR-125b têm resposta celular reduzida no coração humano diabético. Além disso, os dados in vitro de cardiomiócitos primários de ratos demonstraram que o tratamento com glicose alta em curto prazo estimula a expressão de miR-34a e miR-125b. Demonstrou-se que, em condições de glicose alta, os cardiomiócitos de ratos apresentaram metabolismo de glicose intracelular, e a captação de glicose e a produção de lactato aumentaram significativamente. Foi identificado que as principais enzimas metabólicas da glicose, hexoquinase 2 (HK2) e lactato desidrogenase-A (LDHA) eram alvos diretos de miR-125b e miR-34a, respectivamente. A superexpressão de miR-125b e miR-34a poderia evitar a morte de celular de cardiomiócitos causada por hiperglicemia. Por fim, a recuperação de HK2 e LDHA em cardiomiócitos com superexpressão de miR-125b e miR-34a restaurou a sensibilidade de cardiomiócitos à hiperglicemia. Conclusões: Nossos resultados propõem um mecanismo molecular para proteção cardiovascular diabética mediada por microRNA e contribuirão para o desenvolvimento de estratégias de tratamento de disfunção cardiovascular associada a diabetes.
Abstract Background: It is well-known that insulin resistance and hyperglycemia are important pathological causes for the development of diabetic cardiomyopathy (DCM). However, its precise molecular mechanisms in the pathogenesis of DCM remain unclear. Objectives: Recent studies reveal that microRNAs (miRNA) play essential roles in the pathogenesis of DCM. This project aimed to determine the roles of miR-34a and miR-125b in hyperglycemia-induced cardiomyocyte cell death. Methods: Rat primary cardiomyocytes were isolated and exposed to normal and high concentrations of glucose. Cell viability was measured using MTT assay. Expressions of miR-34a and miR-125b were detected by qRT-PCR. Potential targets of miR-34a and miR-125b were predicted from www.Targetscan.org and validated from human heart tissues. A statistical significance of p<0.05 was considered. Results: The present study shows that miR-34a and miR-125b are downregulated in a human diabetic heart. Moreover, in vitro data from rat primary cardiomyocytes showed that short-term high glucose treatment stimulates miR-34a and miR-125b expressions. Under high glucose, it was found that rat cardiomyocytes displayed increased intracellular glucose metabolism, and glucose uptake and lactate production were significantly increased. It was also found that the key glucose metabolic enzymes, Hexokinase 2 (HK2) and Lactate dehydrogenase-A (LDHA), were direct targets of miR-125b and miR-34a, respectively. Overexpression of miR-125b and miR-34a could prevent hyperglycemia-induced cardiomyocyte cell death. Finally, the restoration of HK2 and LDHA in miR-125b and miR-34a overexpressed cardiomyocytes recovered the cardiomyocytes' sensitivity to hyperglycemia. Conclusion: Our results proposed a molecular mechanism for the microRNA-mediated diabetic cardiovascular protection and will contribute to developing treatment strategies for diabetes-associated cardiovascular dysfunction.
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Animals , Rats , MicroRNAs/genetics , Hyperglycemia , Cell Death , Myocytes, Cardiac , GlucoseABSTRACT
Objective:To evaluate the role of histone deacetylase 3 (HDAC3) in high glucose hypoxia/reoxygenation (H/R) injury to primary rat cardiomyocytes and the relationship with autophagy.Methods:The primary cardiomyocytes extracted from newborn Sprague-Dawley rats, aged about 1-3 days, were divided into 5 groups ( n=24 each) according to the random number table method: control group (C group, glucose concentration 5.5 mmol/L), H/R group, high glucose group (H group, glucose concentration 30 mmol/L), high glucose H/R group (HH/R group), and high glucose H/R + HDAC3 inhibitor RGFP966 group (HH/R+ RG group). Fifty percent glucose injection was used to prepare high-glucose medium (final concentration 30 mmol/L). Cells were cultured in a hypoxic environment (5% CO 2-0.9% O 2-94.1% N 2) for 6 h, followed by reoxygenation in a normoxic environment for 2 h to establish the cardiomyocyte H/R model in H/R group.RGFP966 at a final concentration of 10 μmol/L was added at 24 h before H/R in HH/R+ RG group.At 2 h of reoxygenation, the cell viability was measured using CCK-8 kit, the activity of lactic dehydrogenase (LDH) in the cell supernatant was determined using enzyme-linked immunosorbent assay, the level of autophagy was detected with a confocal microscope after cells were transfected with autophagy double-labeled adenovirus (mRFP-GFP-LC3), and the expression of HDAC3, p62, LC3 Ⅱ and LC3 Ⅰ was detected using Western blot.LC3Ⅱ/LC3Ⅰ ratio was calculated. Results:Compared with group C, the cell viability was significantly decreased, and the activity of LDH in supernatant was increased in H/R and H groups, the number of autophagosomes was significantly increased, the expression of HDAC3 in cardiomyocytes was up-regulated, the expression of p62 was down-regulated, and the LC3 Ⅱ/I ratio was increased in group H/R, and the number of autophagosomes was significantly decreased, the expression of HDAC3 and p62 in cardiomyocytes was up-regulated, and the LC3 Ⅱ/I ratio was decreased in group H ( P<0.05). Compared with group H/R, the cell viability was significantly decreased, the activity of LDH in supernatant was increased, the number of autophagosomes was decreased, the expression of HDAC3 and p62 in cardiomyocytes was up-regulated, and the LC3 Ⅱ/I ratio was decreased in group HH/R ( P<0.05). Compared with group H, the cell viability was significantly decreased, the activity of LDH in supernatant was increased, the number of autophagosomes was increased, the expression of HDAC3 and p62 in cardiomyocytes was up-regulated, and the LC3 Ⅱ/I ratio was increased in group HH/R ( P<0.05). Compared with group HH/R, the cell viability was significantly increased, the activity of LDH in supernatant was decreased, the number of autophagosomes was increased, the expression of HDAC3 and p62 in cardiomyocytes was down-regulated, and the LC3 Ⅱ/I ratio was increased in group HH/R+ RG ( P<0.05). Conclusion:Up-regulation of HDAC3 expression is involved in high glucose H/R injury to primary rat cardiomyocytes, which is related to decreasing the level of autophagy.
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Objective:To evaluate the effect of TBK1 overexpression on hypoxia-reoxygenation (H/R) injury in isolated mouse cardiomyocytes subjected to high glucose and the relationship with mitochondrial autophagy.Methods:Normally cultured log-phase HL-1 mouse cardiomyocytes were inoculated in a 6-well plate at a density of 1×10 6 cells/ml and were divided into 4 groups ( n=10 each) using a random number table method: control group (group C), high glucose group (group HG), high glucose and H/R group (group HG+ H/R), and TBK1 overexpression group (group TBK1). The cells were incubated in culture medium with 1% fetal bovine serum and 1% double antibody for 24 h when the cell density reached 50%.When the cell density reached 80%, pcDNA3.1 (+ ) was used as a vector to achieve TBK1 overexpression.The cells were cultured with high glucose medium (33 mmol/L) for 24 h, exposed to 94% N 2+ 5% CO 2+ 1% O 2 for 24 h in an incubator at 37℃ followed by 12 h reoxygenation in an incubator containing 5% CO 2 at 37°C to establish the model of H/R injury to cardiomyocytes subjected to high glucose.After reoxygenation, CCK-8 assay was used to detect cell viability, the activity of lactic dehydrogenase (LDH) in supernatant was detected using LDH kit, mitochondrial contents were determined using Mito-Tracter green fluorescent probe, and the expression of TBK1 and mitophagy-related proteins PINK1, Parkin, LC3B and P62 was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the activity of LDH in supernatant was increased, mitochondrial contents were decreased, the expression of TBK1, PINK1, Parkin and LC3B was down-regulated, and the expression of P62 was up-regulated in HG group and HG+ H/R group ( P<0.05). Compared with group HG, the cell viability was significantly decreased, the activity of LDH in supernatant was increased, mitochondrial contents were decreased, the expression of TBK1, PINK1, Parkin and LC3B was down-regulated, and the expression of P62 was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG+ H/R, the the cell viability was significantly increased, the activity of LDH in supernatant was decreased, mitochondrial contents were increased, the expression of TBK1, PINK1, Parkin and LC3B was up-regulated, and the expression of P62 was down-regulated in group TBK1 ( P<0.05). Conclusion:The mechanism by which TBK1 overexpression reduces the H/R injury is related to restoring mitophagy in isolated mouse cardiomyocytes subjected to high glucose.
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Objective:To evaluate the relationship between phosphorylation of glycogen synthase kinase-3β (GSK-3β) and high glucose-caused abolition of cardioprotection induced by sevoflurane postconditioning.Methods:H9c2 cells were incubated in normal glucose (5.56 mmol/L) DMEM culture medium or high glucose (33 mmol/L) DMEM culture medium.The cells were divided into 8 groups ( n=24 each) using a random number table method: normal control group (group NC), normal glucose-cultured hypoxia/reoxygenation (H/R) group (group NH/R), normal glucose-cultured sevoflurane postconditioning group (group NS), normal glucose-cultured GSK-3β inhibitor SB216763 group (group NSB), high glucose-cultured group (group HC), high glucose-cultured H/R group (group HH/R), high glucose-cultured sevoflurane postconditioning group (group HS) and high glucose-cultured GSK-3β inhibitor SB216763 group (group HSB). The model of cardiomyocyte H/R was established by subjecting cardiomyocytes to 3 h of hypoxia followed by reoxygenation.Immediately after onset of reoxygenation, cardiomyocytes were exposed to 2.4% sevoflurane for 30 min in Ns and HS groups.Before the beginning of reoxygenation, GSK-3β inhibitor SB216763 was added to the culture medium with the final concentration of 10 μmol/L in NSB and HSB groups.At 3 h of reoxygenation, the apoptosis rate was determined by Anexin V-PI flow cytometry, the expression of GSK-3β and phosphorylated GSK-3β (p-GSK-3β) was detected by Western blot, superoxide dismutase (SOD) activity was measured using xanthineoxidase method, and lactic dehydrogenase (LDH) activity and malondialdehyde (MDA) content were determined by colorimetric assay. Results:Compared with group NC, apoptosis rate, LDH activity and MDA content were significantly increased, and SOD activity was decreased in group NH/R and group HC, expression of GSK-3β was up-regulated, and expression of p-GSK-3β was down-regulated in group NH/R, expression of p-GSK-3β was up-regulated in group NS, and expression of p-GSK-3β was down-regulated in group HC ( P<0.05). Compared with group NH/R, apoptosis rate, LDH activity and MDA content were significantly decreased, and SOD activity was increased in group NS and NSB groups, and expression of GSK-3β was down-regulated, and expression of p-GSK-3β was up-regulated in group NS ( P<0.05). Compared with group HC, apoptosis rate, LDH activity and MDA content were significantly increased, SOD activity was decreased, expression of GSK-3β was up-regulated, and expression of p-GSK-3β was down-regulated in group HH/R ( P<0.05). Compared with group HH/R, apoptosis rate, LDH activity and MDA content were significantly decreased, and SOD activity was increased in group HSB ( P<0.05). Conclusion:The mechanism by which high glucose abolishes cardioprotection induced by sevoflurane postconditioning is related to inhibiting phosphorylation of GSK-3β.
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Objective:To evaluate the effects of different density rat fibroblasts on the expression of conjunctin 43 (Cx43) in cardiomyocytes and cell viability.Methods:Cardiomyocytes and fibroblasts were co-cultured using Transwell, cardiomyocytes were inoculated into the lower chamber of Transwell and fibroblasts into the upper chamber of Transwell.The cells were divided into 3 groups ( n=12 each) by a random number table method: fibroblast density 0.5×10 5 cells/ml group (group C 0.5), fibroblast density 1×10 5 cells/ml group (group C 1), and fibroblast density 2×10 5 cells/ml group (group C 2), with the density of cardiomyocytes 1×10 5 cells/ml in three groups.Cardiomyocytes and fibroblasts were co-cultured for 20 h in three groups.Cardiomyocytes were collected after co-culture for determination of cell viability (by CCK8 method), apoptosis rate (by flow cytometry), and expression of Cx43 mRNA (by quantitative real-time polymerase chain reaction) and expression of Cx43 and phosphorylated Cx43 (p-Cx43) (by Western blot). Results:There was no significant difference in the apoptosis rate of cardiomyocytes among the three groups ( P>0.05). Compared with group C 0.5, the expression of Cx43 protein and mRNA and p-Cx43 was significantly up-regulated in group C 1, the cardiomyocyte viability was significantly increased, and the expression of Cx43 protein and mRNA and p-Cx43 was up-regulated in group C 2 ( P<0.05). Compared with group C 1, the cardiomyocyte viability was significantly increased, and the expression of Cx43 protein and mRNA and p-Cx43 was up-regulated in group C 2 ( P<0.05). Conclusion:Rat fibroblasts up-regulate the expression of Cx43 and enhance the activity of Cx43 in cardiomyocytes and enhance cell viability in a density-dependent manner in a certain range.
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Abstract Objective: To investigate the effect of Shenfu (SF) injection on donor heart preservation. Methods: Twelve pigs were randomly divided into SF group (n=6) and control group (n=6). After eight hours of perfusion, the differences in hemoglobin, the expression of Bcl-2 and BAX, and changes in the myocardial ultrastructure were compared to illustrate the effects of SF injection in heart preservation. Results: The differences in free hemoglobin between the SF group and the control group were statistically significant (P=0.001), and there was significant interaction of groups with times (P=0.019), but the perfusion time may not be associated with the hemoglobin concentration (P=0.616). According to Western blotting analysis, the expression of Bcl-2 was higher in the SF group than in the control group, while the expression of BAX was not different between the two groups. As to ultrastructural changes, both groups exhibited mitochondrial swelling and myofilament lysis, but the degree of damage in the SF group was smaller. Conclusion: Our study suggests that the application of SF injection for heart preservation may protect against cardiomyocytes and erythrocytes apoptosis, and Bcl-2 protein may play a role in these physiological processes.
Subject(s)
Animals , Male , Drugs, Chinese Herbal , Heart Transplantation , Swine , Swine, Miniature , Tissue DonorsABSTRACT
Objective: To explore the role and related mechanism of mammalian sterile 20-like kinase 1(Mst-1)in regulating hypoxia reoxygenation (HR) induced myocardial cell autophagy and apoptosis. Methods: Enzyme digestion method combined with differential adherent method was used to culture neonatal mouse myocardial cells. HR model was established by hypoxia for 24 hours and reoxygenation for 6 hours. The experimental groups including control group (normal cultured cardiomyocytes), Mst-1 empty virus group (cardiomyocytes transfected with recombinant lentiviral empty vector for 48 hours), Mst-1 knockdown group (recombinant lentivirus carrying Mst-1small interfering RNA (siRNA) was transfected into cardiomyocytes for 48 hours), Mst-1 overexpression group (cardiomyocytes were transfected with recombinant lentivirus carrying Mst-1 gene for 48 hours), HR group (cardiomyocytes exposed to HR), Mst-1 knockdown+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1siRNA) and Mst-1 overexpression+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1 gene). Real-time fluorescence quantitative RCR (qPCR) and Western blot were used to detect the relative expression of Mst-1 mRNA and protein in the cells, immunofluorescence staining was used to detect cardiomyocyte troponin T (cTnT), and autophagosomes and autophagy enzyme changes. TUNEL method was used to detect myocardial cell apoptosis, Western blot was adopted to detect autophagy-related protein microtubule-related protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, P62 and apoptosis-related protein cleaved-caspase 9, pro-caspase 9, cleaved-caspase-3, pro-caspase-3, and myeloid leukemia 1 (MCL-1) expression. MCL-1 inhibitor A1210477 was used to validate the signaling pathway of Mst-1 on regulating cardiomyocyte apoptosis and autophagy. Results: Immunofluorescence detection revealed that the cultured cells expressed cardiomyocyte-specific marker cTnT. The expression of Mst-1 in cardiomyocytes increased in HR model. Lentiviral transfection could effectively inhibit or overexpress Mst-1 in treated cells. The levels of autophagosomes and autophagolysosomes in cardiomyocytes undergoing HR and in Mst-1 overexpression+HR group were lower than those of control group, while autophagosomes and autophagolysosomes in cardiomyocytes of Mst-1 knockdown+HR group was significantly higher than in the HR group (all P<0.05). The TUNEL results showed that the proportion of TUNEL positive cells was significantly increased in the HR group and Mst-1 overexpression+HR group than in the control group, while the proportion of TUNEL positive cells was significantly decreased in the Mst-1 knockdown group+HR group as compared to the HR group (all P<0.05). Western blot results showed that the LC3 Ⅱ/LC3 Ⅰ levels were significantly lower, while the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly higher in the HR group and Mst-1 overexpression+HR group than in control group (all P<0.05). The LC3 Ⅱ/LC3 Ⅰ value was significantly higher, and the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly lower in the Mst-1 knockdown+HR group than in the HR group (P both<0.05). The expression level of P-MCL-1 protein was significantly lower in cardiomyocytes of HR and Mst-1 overexpression+HR group than in control group, and the expression level of P-MCL-1 protein was higher in Mst-1 knockdown+HR group than in HR group (P both<0.05). The recovery experiment showed that inhibiting MCL-1 in cells can block the regulatory effect of Mst-1 siRNA on cell autophagy and apoptosis. Conclusion: Inhibiting Mst-1 expression in cardiomyocytes can promote the autophagy of cardiomyocytes induced by hypoxic reoxygenation and reduce the apoptosis of cardiomyocytes via activating McL-1.
Subject(s)
Animals , Mice , Apoptosis , Autophagy , Hypoxia , Myocytes, Cardiac , Signal TransductionABSTRACT
Objective: To explore the effects of 3-phosphate dependent protein kinase 1-protein kinase B (PDK1-Akt) signaling pathway on the transcription, expression and function of cardiac hyperpolarized activated cyclic nucleotide gated 4 (HCN4) ion channels. Methods: Atrial myocytes were obtained from healthy male wild-type C57 mice and heart-specific PDK1 knockout mice (PDK1-KO) by enzymolysis. Then the atrial myocytes were divided into blank control group and PDK1-KO group. In further studies, the isolated atrial myocytes were cultured and further divided into drug control group (treated with dimethyl sulfoxide (DMSO)) and PDK1 knockdown group (treated with 1 μg/ml PDK1 short hairpin RNA (shRNA) interference plasmid), SC79 group (treated with 8 μmol/ml SC79), GSK2334470 group (treated with 10 nmol/L GSK2334470) and PDK1 knockdown+SC79 group (8 μmol/ml SC79 and 1 μg/ml PDK1 shRNA interference plasmid). Real time quantitative PCR (qRT-PCR) was used to detect the mRNA expression levels of PDK1 and HCN4, Western blot was used to detect the protein expression levels of PDK1, Akt and HCN4, the whole cell patch clamp was used to detecte the current density of HCN, and immunofluorescence was used to detecte the expression of HCN4 protein on atrial cells. Results: (1) the expression levels of HCN4 mRNA (1.46±0.03 vs. 0.99±0.01, P<0.001) and protein (1.14±0.02 vs. 1.00±0.06, P=0.017) in PDK1-KO group were higher than those in blank control group. The HCN current density in PDK1-KO group was higher than that in blank control group((-17.47±2.00) pA/pF vs. (-12.15±2.25) pA/pF, P=0.038). (2) The functions of PDK1 shRNA and specific Akt agonist SC79 were verified by comparing the PDK1 knockdown group and SC79 group with the drug control group. The results showed that the expression levels of PDK1 mRNA and protein in PDK1 knockdown group were lower than those in drug control group, and the expression level of phosphorylated Akt (Thr 308) protein in SC79 group was higher than that in drug control group. (3) The expression levels of HCN4 mRNA (3.61±0.46 vs. 1.00±0.08, P<0.001) and protein (2.33±0.11 vs. 1.00±0.05, P<0.001) in GSK2334470 group were higher than those in drug control group. (4) To reduce the effect of drug-miss target, the cultured atrial myocytes were transfected with shRNA plasmid of PDK1 and intervened with SC79. The results showed that the expression of HCN4 mRNA in PDK1 knockdown group was higher than that in the drug control group (1.76±0.11 vs. 1.00±0.06, P<0.001), and PDK1 knockdown+SC79 group (1.76±0.11 vs. 1.33±0.07, P=0.003). In PDK1 knockdown+SC79 group, the mRNA expression level was also higher than that in the drug control group (1.33±0.07 vs. 1.00±0.06, P<0.001). The expression level of HCN4 protein in PDK1 knockdown group was higher than that in drug control group (1.15±0.04 vs. 1.00±0.05, P=0.003). As for the The expression level of HCN4 protein, there was no significantly statistical difference between the PDK1 knockdown+SC79 group and the drug control group (P>0.05), but PDK1 knockdown+SC79 group was lower than PDK1 knockdown group (0.95±0.01 vs. 1.15±0.04, P<0.001). In patch clamp experiments, the results showed that the HCN current density was (-13.27±1.28) pA/pF in the drug control group, (-18.76±2.03) pA/pF in the PDK1 knockdown group, (-13.50±2.58) pA/pF in the PDK1 knockdown+SC79 group; the HCN current density of PDK1 knockdown group was higher than that of drug control group (P<0.001), but there was no significant difference between PDK1 knockdown+SC79 group and drug control group (P>0.05). (5) The results of immunofluorescence showed that the brightness of green fluorescence of PDK1 knockdown group was higher than that of drug control group, indicating that the expression of HCN4 localized on cell membrane was increased. However, the green fluorescence of PDK1 knockdown+SC79 group was lighter than that of PDK1 knockdown group, suggesting that the expression of HCN4 in PDK1-knockdown cell membrane decreased after further activating Akt. Conclusion: PDK1-Akt signaling pathway is involved in the regulation of HCN4 ion channel transcription, expression and function.
Subject(s)
Animals , Male , Mice , Cyclic Nucleotide-Gated Cation Channels , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Myocytes, Cardiac/metabolism , Potassium Channels/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal TransductionABSTRACT
Objective: To investigate if microRNA (miR) -23a knockdown could attenuate angiotensin Ⅱ(AngⅡ) induced cardiac hypertrophy by activating phosphatase and tensin homolog deleted on chromosome ten(PTEN) and AMP-activated protein kinase(AMPK) pathway. Methods: Rat H9c2 cells were cultured in DMEM high glucose medium and put in 5% CO(2) incubator at 37 ℃(normal group). After 48 hours of culture, H9c2 cells were stimulated with 10 nmol/L AngⅡ to establish cell hypertrophy model (AngⅡgroup). The H9c2 cells were inoculated in a 6-well cell culture plate and cultured in an incubator at 37 ℃. When the confluence degree of cell growth was about 70%, the cells were transfected with different reagents, and 24 hours after transfection, 10 nmol/L AngⅡ was used to interfere with the cells. The H9c2 cells were divided into different groups according to the reagents, namely AngⅡ+anti-miR group(transfected with miR-23a inhibitor), Ang Ⅱ+NC group(transfected with miR-23a inhibitor negative control), Ang Ⅱ+anti-miR+si-PTEN group(cotransfected with miR-23a inhibitor and PTEN small interference RNA(siRNA)), and AngⅡ+anti-miR+si-NC group(cotransfected with miR-23a inhibitor and PTEN siRNA negative control). The surface area of single cell was measured by Image J software.The mRNA expression levels of α-actin 1 (ACTA1) and β-myosin heavy chain (β-MHC) and miR-23a were detected by quantitative real-time PCR(qRT-PCR). The expression levels of PTEN and AMPK signal pathway related proteins were detected by Western blot. In order to verify whether miR-23a targets PTEN gene, double luciferase reporter gene experiment was performed. The luciferase reporter gene vector recombinant plasmids of wild type pGL-WT-PTEN and mutant pGL-MUT-PTEN were constructed and prepared after normal sequencing. H9c2 cells was inoculated into 24-well cell culture plate and cultured overnight in 37 ℃ incubator. The cells were co-transfected with miR-23a mimic or miR-23a mimic negative control and wild type or mutant reporter gene recombinant plasmid. Forty-eight hours after transfection, firefly luciferase activity and sea kidney luciferase activity were measured, and the ratio of them was recorded as relative luciferase activity. Results: Compared with the normal group, the cell surface area, the mRNA expression levels of ACTA1, β-MHC and miR-23a were significantly higher, while the protein expression levels of PTEN and p-AMPK were significantly lower in the Ang Ⅱ group(all P<0.05). The results of double luciferase reporter gene assay showed that the relative luciferase activity of cells co-transfected with miR-23a mimic and wild-type reporter gene recombinant plasmid was lower than that of miR-23a mimic negative control (P<0.05), and PTEN served as the target gene of miR-23a. In AngⅡ+anti-miR group the mRNA expression levels of miR-23a, ACTA1 and β-MHC were lower, and the cell surface area was smaller, while the protein expression levels of PTEN and p-AMPK were higher than that in AngⅡ group and AngⅡ+NC group(all P<0.05). Compared with AngⅡ+anti-miR group, the cell surface area was bigger, the expression of ACTA1 and β-MHC mRNA was up-regulated, and the protein expression levels of PTEN and p-AMPK were down-regulated in Ang Ⅱ+anti-miR+si-PTEN group(all P<0.05). Conclusion: Inhibition of miR-23a can attenuate Ang Ⅱ-induced hypertrophy in H9c2 cells through targeting PTEN and activating AMPK signaling pathway.
Subject(s)
Animals , Rats , AMP-Activated Protein Kinases , Angiotensin II , Cardiomegaly , Cell Line , Cell Proliferation , MicroRNAs/genetics , PTEN Phosphohydrolase , Signal TransductionABSTRACT
Abstract Objective: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). Methods: The DACT1 expression and its associations with the degree of fibrosis and β-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of β-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. Results: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and β-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in β-catenin and subsequent partial colocalization of DACT1 and β-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. Conclusion: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by β-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.