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1.
Life Sci ; 237: 116944, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31604108

RESUMO

AIMS: Endoplasmic reticulum stress (ERS) is an evolutionarily conserved cell stress response. Recently, it was found that ERS induces not only apoptosis but also endoplasmic reticulophagy (ER-phagy). A previous study demonstrated that inhibition of ER-phagy alleviates cell injury. The purpose of this study was to investigate the involvement of the protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in ERS-induced ER-phagy in H9c2 cardiomyoblasts. To address this aim, cells were treated with ERS inhibitors and a Nrf2 inhibitor before establishment of thapsigargin (TG)- or tunicamycin (TM)-induced ERS models in H9c2 cardiomyoblasts. MAIN METHODS: Transmission electron microscopy and immunofluorescence staining were used to detect ER-phagy. Western blotting was employed to detect the levels of calreticulin (CRT), total and phosphorylated PERK, nuclear Nrf2, activated transcription factor 4 (ATF4), light chain 3B (LC3B)-II and Beclin 1. Immunofluorescence staining was used to assess subcellular location of Nrf2. KEY FINDING: TG or TM induced H9c2 cell injury and ER-phagy and upregulated CRT expression, PERK phosphorylation, Nrf2 nuclear translocation, and expression of ATF4, Beclin 1, and LC3B-II compared with control cells. Treatment with ERS inhibitors decreased TG- or TM-induced ER-phagy, downregulated CRT expression, PERK phosphorylation, Nrf2 nuclear translocation and the expression of ATF4, Beclin 1 and LC3B-II. Moreover, a Nrf2 inhibitor downregulated the expression of ATF4, Beclin 1 and LC3B-II and alleviated TG- or TM-induced ER-phagy and H9c2 cell injury. SIGNIFICANCE: These findings suggest that the PERK/Nrf2 pathway mediates upregulation of ER-phagy, thereby inducing cell injury in H9c2 cardiomyoblasts.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/patologia , Miócitos Cardíacos/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteína Beclina-1/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos , Transdução de Sinais
2.
Chem Biol Interact ; 314: 108848, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610156

RESUMO

Cardiomyocyte injury induced by acute myocardial infarction contributes to myocardial dysfunction. Accumulating evidence has demonstrated that pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) is a cytoprotective protein that protects against various adverse injuries. However, whether PHLPP2 participates in regulating myocardial-infarction-induced cardiomyocyte injury remains unknown. In the present study, we aimed to investigate the biological role and molecular mechanism of PHLPP2 in regulating hypoxia-induced cardiomyocyte injury. Cardiomyocytes were cultured in an anaerobic chamber for 24 h to induce hypoxic injury in vitro. The expression of PHLPP2 was determined by real-time quantitative PCR and Western blot analysis. Cell viability was measured by MTT assay. Cell apoptosis was assessed by TUNEL and caspase-3 activity assays. Intracellular reactive oxygen species (ROS) levels were measured by DCFH-DA probe. PHLPP2 expression was highly upregulated in hypoxia-injured cardiomyocytes. Inhibition of PHLPP2 by small interfering RNA (siRNA)-mediated gene silencing significantly improved the viability of hypoxia-injured cardiomyocytes and attenuated hypoxia-induced apoptosis and ROS production. In contrast, PHLPP2 overexpression exacerbated hypoxia-induced apoptosis and ROS production in cardiomyocytes. Mechanism research revealed that PHLPP2 silencing increased the phosphorylation of glycogen synthase kinase (GSK)-3ß and promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In addition, PHLPP2 inhibition promoted Nrf2/antioxidant response element (ARE) transcriptional activity. However, Nrf2 silencing markedly reversed PHLPP2-inhibition-mediated cardioprotection, while GSK-3ß inhibition partially blocked the PHLPP2-overexpression-induced adverse effect. Taken together, these findings demonstrate that PHLPP2 inhibition alleviates hypoxia-induced cardiomyocyte injury by reinforcing Nrf2/ARE antioxidant signaling via inactivating GSK-3ß, a pathway that highlights the importance of the PHLPP2/GSK-3ß/Nrf2/ARE signaling axis in regulation of cardiomyocyte injury. Our study suggests a potential relevance for PHLPP2 in acute myocardial infarction, and this protein may serve as a promising target for cardioprotection.


Assuntos
Elementos de Resposta Antioxidante/genética , Hipóxia Celular , Fator 2 Relacionado a NF-E2/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular , Regulação para Baixo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação , Pirimidinas/farmacologia , Pirróis/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
Sheng Li Xue Bao ; 71(5): 783-791, 2019 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-31646332

RESUMO

Circadian rhythms widely exist in living organisms, and they are regulated by the biological clock. Growing evidence has shown that circadian rhythms are tightly related to the physiological function of the cardiovascular system, including blood pressure, heart rate, metabolism of cardiomyocytes, function of endothelial cells, and vasoconstriction and vasodilation. In addition, disruption of circadian rhythms has been considered as one of the important risk factors for cardiovascular diseases, such as myocardial infarction. This review summarizes the recent research advances in the relationship between circadian clock and cardiovascular diseases, hoping to improve treatment strategies for patients with cardiovascular diseases according to the theory of biological clock.


Assuntos
Doenças Cardiovasculares/fisiopatologia , Relógios Circadianos , Ritmo Circadiano , Pressão Sanguínea , Células Endoteliais/citologia , Frequência Cardíaca , Humanos , Miócitos Cardíacos/metabolismo , Vasoconstrição , Vasodilatação
4.
Nat Cell Biol ; 21(9): 1152-1163, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481791

RESUMO

Ca2+/calmodulin-dependent kinase II (CaMKII) is a multifunctional serine/threonine kinase family, and its δ isoform is predominant in the heart. Excessive CaMKII activation plays a pivotal role in the pathogenesis of severe heart conditions, including myocardial infarction, cardiomyopathy and heart failure. However, the identity of CaMKII splice variants and the mechanism(s) underlying CaMKII-mediated cardiac pathology remain elusive. Here, we show that CaMKII-δ9, the most abundant CaMKII-δ splice variant in human heart, potently promotes cardiomyocyte death, cardiomyopathy and heart failure by disrupting cardiomyocyte genome stability. Mechanistically, CaMKII-δ9, but not the previously well-studied CaMKII-δ2 and CaMKII-δ3, targets the ubiquitin-conjugating enzyme E2T (UBE2T) for phosphorylation and degradation, disrupting UBE2T-dependent DNA repair and leading to the accumulation of DNA damage and genome instability. These findings not only reveal a crucial role of CaMKII in the regulation of DNA repair, but also mark the CaMKII-δ9-UBE2T-DNA damage pathway as an important therapeutic target for cardiomyopathy and heart failure.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Reparo do DNA/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Cálcio/metabolismo , Cardiomiopatias/metabolismo , Insuficiência Cardíaca/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Fosforilação , Isoformas de Proteínas/metabolismo , Enzimas de Conjugação de Ubiquitina/genética
5.
Life Sci ; 235: 116842, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31494170

RESUMO

MicroRNAs plays important role in the development of myocardial infarction (MI). The aim of this study was to analyze whether miR-429 has effect on the process of autophagy in myocardial anoxia/reoxygenation (AR) or ischemia/reperfusion (IR) injury and explore the underlying mechanism. The results showed that miR-429 was significantly decreased in MI mouse hearts and AR treated cardiomyocytes. Dual luciferase activity assay proved that MO25 was the direct target of miR-429. MO25 was dramatically decreased in AR treated cardiomyocytes. Overexpression of miR-429 dramatically decreased the expression of MO25, whereas inhibition of miR-429 noticeably increased the expression of MO25. In addition, overexpression of miR-429 reduced GFP-LC3B labelled cells, decreased the number of vesicle and autophagosome in each cardiomyocyte, and induced cell apoptosis in AR treated cardiomyocytes. In contrast, inhibition of miR-429 had the opposite effect. The further in vivo study showed that when mouse in IR group were injected with antagomiR-429, the weight of left ventricular was increased and infarct size was significantly decreased. Finally, both the in vitro and in vivo study showed that the expression of MO25, LKB1, pAMPKa, ATG13, p62 and LC3BI/II was noticeably increased by antagomiR-429. In conclusion, our results suggested that antagonism of miR-429 ameliorates anoxia/reoxygenation injury in cardiomyocytes by enhancing MO25/LKB1/AMPK mediated autophagy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Hipóxia/metabolismo , MicroRNAs/antagonistas & inibidores , Miócitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/efeitos dos fármacos , Contagem de Células , Vesículas Citoplasmáticas/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Traumatismo por Reperfusão Miocárdica , Miocárdio/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo
6.
Life Sci ; 235: 116863, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31513817

RESUMO

AIMS: To determine whether dimethyl fumarate (DMF) can protect against lipopolysaccharide (LPS) -induced myocardial injury. MAIN METHODS: H9c2 cells pretreated with or without DMF were stimulated with LPS. Cell viability and apoptosis were evaluated. Nrf2 and HO-1 expression were detected using Western blotting. Mitochondrial morphology, mitochondrial superoxide production were observed using confocal microscope. Mitochondrial respiration function was measured using Seahorse bioanalyzer. KEY FINDINGS: (1) The cell viability decreased, LDH release and apoptosis increased in LPS- challenged H9c2 cells. DMF pretreatment brought a higher cell viability, and a lower LDH leakage and apoptosis than those of LPS group (P < 0.01). (2) DMF pretreatment resulted in an increased Nrf2 and HO-1 expression, and enhanced nuclear Nrf2 level in LPS-challenged cells (P < 0.01). (3) Nrf2-siRNA could inhibit DMF-induced enhancement of HO-1 expression and cell viability, and partly abolish DMF-induced reduction of LDH leakage and apoptosis. (4) ERK1/2 inhibitor PD98059 could not only prevent the DMF-induced enhancement of nuclear Nrf2 and HO-1, but also inhibit DMF-induced increase in cell viability. (5) Compared with LPS-challenged cells, DMF pretreatment caused a lower production of mitochondrial superoxide and a higher mitochondrial membrane potential, which could be abolished by Nrf2-siRNA. (6) DMF could attenuate LPS-induced mitochondrial fragmentation and improve mitochondrial respiration function by enhancement of the oxygen consumption rate of basal respiration and ATP production in LPS-challenged cells (P < 0.01). SIGNIFICANCE: DMF protects cardiomyocytes against LPS-induced damage. ERK1/2-dependent activation of Nrf2/HO-1 pathway is responsible for DMF-induced cardioprotection via reduction of oxidative stress, improvement of mitochondrial morphology and energy metabolism.


Assuntos
Fumarato de Dimetilo/farmacologia , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/biossíntese , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fumarato de Dimetilo/antagonistas & inibidores , Flavonoides/farmacologia , Heme Oxigenase-1/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Lipopolissacarídeos/efeitos adversos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Consumo de Oxigênio/efeitos dos fármacos , Substâncias Protetoras/farmacologia , RNA Interferente Pequeno/farmacologia , Superóxidos/metabolismo
7.
Life Sci ; 235: 116802, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31472150

RESUMO

Substrate stiffness is essential for cell functions, but the mechanisms by which cell sense mechanical cues are still unclear. Here we show that the frequency and the amplitude of spontaneous Ca2+ oscillations were greater in chick cardiomyocytes cultured on the stiff substrates than that on the soft substrates. The spontaneous Ca2+ oscillations were increased on stiff substrates. However, an eliminated dependence of the Ca2+ oscillations on substrate stiffness was observed after applying blocker of the large-conductance Ca2+-activated K+ (BK) channels. In addition, the activity of BK channels in cardiomyocytes cultured on the stiff substrates was decreased. These results provide compelling evidences to show that BK channels are crucial in substrate stiffness-dependent regulation of the Ca2+ oscillation in cardiomyocytes.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Embrião de Galinha , Galinhas , Miócitos Cardíacos/citologia , Especificidade por Substrato
8.
Int J Nanomedicine ; 14: 5595-5609, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31413565

RESUMO

Background: Platinum nanoparticles (PtNPs) have been considered a nontoxic nanomaterial and been clinically used in cancer chemotherapy. PtNPs can also be vehicle exhausts and environmental pollutants. These situations increase the possibility of human exposure to PtNPs. However, the potential biotoxicities of PtNPs including that on cardiac electrophysiology have been poorly understood. Methods: Ion channel currents of cardiomyocytes were recorded by patch clamp. Heart rhythm was monitored by electrocardiogram recording. Morphology and characteristics of PtNPs were examined by transmission electron microscopy, dynamic light scattering and electrophoretic light scattering analyses. Results: In cultured neonatal mice ventricular cardiomyocytes, PtNPs with diameters 5 nm (PtNP-5) and 70 nm (PtNP-70) concentration-dependently (10-9 - 10-5 g/mL) depolarized the resting potentials, suppressed the depolarization of action potentials and delayed the repolarization of action potentials. At the ion channel level, PtNPs decreased the current densities of INa, IK1 and Ito channels, but did not affect the channel activity kinetics. In vivo, PtNP-5 and PtNP-70 dose-dependently (3-10 mg/kg, i.v.) decreased the heart rate and induced complete atrioventricular conduction block (AVB) at higher doses. Both PtNP-5 and PtNP-70 (10-9 - 10-5 g/mL) did not significantly increase the generation of ROS and leak of lactate dehydrogenase (LDH) from cardiomyocytes within 5 mins after exposure except that only very high PtNP-5 (10-5 g/mL) slightly increased LDH leak. The internalization of PtNP-5 and PtNP-70 did not occur within 5 mins but occurred 1 hr after exposure. Conclusion: PtNP-5 and PtNP-70 have similar acute toxic effects on cardiac electrophysiology and can induce threatening cardiac conduction block. These acute electrophysiological toxicities of PtNPs are most likely caused by a nanoscale interference of PtNPs on ion channels at the extracellular side, rather than by oxidative damage or other slower biological processes.


Assuntos
Frequência Cardíaca/efeitos dos fármacos , Canais Iônicos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Miócitos Cardíacos/metabolismo , Platina/toxicidade , Testes de Toxicidade Aguda , Animais , Animais Recém-Nascidos , Células Cultivadas , Eletrocardiografia , Endocitose/efeitos dos fármacos , Sistema de Condução Cardíaco/efeitos dos fármacos , Ventrículos do Coração/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Platina/administração & dosagem
9.
Hypertension ; 74(4): 833-842, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31446800

RESUMO

Heart failure (HF) is a major cause of morbidity and mortality in patients with various cardiovascular diseases. Restoration of cardiac function is critical in improving the clinical outcomes of patients with HF. Long noncoding RNAs are widely involved in the development of multiple cardiac diseases, whereas their role in regulating cardiac function remains unclear. In this study, we found that the expression of long noncoding RNA-DACH1 (dachshund homolog 1) was upregulated in the failing hearts of mice and human. We tested the hypothesis that the intronic long noncoding RNA of DACH1 (LncDACH1) can participate in the regulation of cardiac function and HF. Transgenic overexpression of LncDACH1 in the cardiac myocytes of mice led to impaired cardiac function, reduced calcium transient and cell shortening, and decreased SERCA2a (sarcoplasmic reticulum calcium ATPase 2a) protein expression. In contrast, conditional knockout of LncDACH1 in cardiac myocytes resulted in increased calcium transient, cell shortening, SERCA2a protein expression, and improved cardiac function of transverse aortic constriction induced HF mice. The same qualitative data were obtained by overexpression or knockdown of LncDACH1 with adenovirus carrying LncDACH1 or its siRNA. Moreover, therapeutic administration of adenovirus carrying LncDACH1 siRNA to transverse aortic constriction mice abolished the development of HF. Mechanistically, LncDACH1 directly binds to SERCA2a. Overexpression of LncDACH1 augments the ubiquitination of SERCA2a. LncDACH1 upregulation impairs cardiac function by promoting ubiquitination-related degradation of SERCA2a.


Assuntos
Proteínas do Olho/metabolismo , Insuficiência Cardíaca/metabolismo , Coração/fisiologia , RNA Longo não Codificante/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Proteínas do Olho/genética , Feminino , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Humanos , Masculino , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/genética , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Fatores de Transcrição/genética
10.
Cell Physiol Biochem ; 53(2): 337-354, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31373783

RESUMO

BACKGROUND/AIMS: The availability of truly maturated cardiomyocytic subtypes is a major prerequisite for cardiovascular cell replacement therapies. Pluripotent stem cells provide a suitable source for the development of new strategies to overcome enormous hurdles such as yield, purity and safety of in vitro generated cells. METHODS: To address these issues, we have refined existing forward programming protocols by combining forced exogenous overexpression of the early cardiovascular transcription factor Nkx2.5 with a αMHC-promoter-based antibiotic selection step. Additionally, we applied small molecules such as ascorbic acid to enhance cardiomyogenic differentiation efficiency. Subsequently, we evaluated the cell fate of the resulting cardiomyocytes on the mRNA as well as protein levels. The latter was performed using high-resolution confocal microscopy. Furthermore, we examined the response of the cells` beating activities to pharmacological substance administration. RESULTS: Our results reveal an apparent influence of Nkx2.5 on the cell fate of ESC-derived cardiomyocytes. Resulting single cells exhibit characteristics of early ventricular cardiomyocytes, such as sarcomeric marker expression, spontaneous beating frequency, and distinct L-type calcium channel occurrence. CONCLUSION: Therefore, we demonstrate cardiovascular subtype forward programming of ESCs using a combination of transcription factors along with small molecule administration. However, our findings also underline current assumptions, that a terminal maturation of PSC derived cardiomyocytes in vitro is still an unsolved problem which urgently needs to be addressed in the field.


Assuntos
Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Ácido Ascórbico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Proteína Homeobox Nkx-2.5/antagonistas & inibidores , Proteína Homeobox Nkx-2.5/genética , Camundongos , Microscopia Confocal , Miócitos Cardíacos/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Verapamil/farmacologia
11.
Int Heart J ; 60(4): 944-957, 2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31257341

RESUMO

Cardiac fibrosis plays an important role in cardiac remodeling after myocardial infarction (MI). The molecular mechanisms that promote cardiac fibrosis after MI are well studied; however, the mechanisms by which the progression of cardiac fibrosis becomes attenuated after MI remain poorly understood. Recent reports show the role of cellular senescence in limiting tissue fibrosis. In the present study, we tested whether cellular senescence of cardiac fibroblasts (CFs) plays a role in attenuating the progression of cardiac fibrosis after MI. We found that the number of γH2AX-positive CFs increased up to day 7, whereas the number of proliferating CFs peaked at day 4 after MI. Senescent CFs were also observed at day 7, suggesting that attenuation of CF proliferation occurred simultaneously with the activation of the DNA damage response (DDR) system and the appearance of senescent CFs. We next cultured senescent CFs with non-senescent CFs and showed that senescent CFs suppressed proliferation of the surrounding non-senescent CFs in a juxtacrine manner. We also found that the blockade of DDR by Atm gene deletion sustained the proliferation of CFs and exacerbated the cardiac fibrosis at the early stage after MI. Our results indicate the role of DDR activation and cellular senescence in limiting cardiac fibrosis after MI. Regulation of cellular senescence in CFs may become one of the therapeutic strategies for preventing cardiac remodeling after MI.


Assuntos
Senescência Celular/genética , Dano ao DNA/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Remodelação Ventricular/genética , Animais , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/patologia
12.
Life Sci ; 232: 116620, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31291594

RESUMO

AIMS: Cell-based biological pacemakers aim to overcome limitations and side effects of electronic pacemaker devices. We here developed and tested different approaches to achieve nodal-type differentiation using human adipose- and bone marrow-derived mesenchymal stem cells (haMSC, hbMSC). MAIN METHODS: haMSC and hbMSC were differentiated using customized protocols. Quantitative RT-PCR was applied for transcriptional pacemaker-gene profiling. Protein membrane expression was analyzed by immunocytochemistry. Pacemaker current (If) was studied in haMSC with and without lentiviral HCN4-transduction using patch clamp recordings. Functional characteristics were evaluated by co-culturing with neonatal rat ventricular myocytes (NRVM). KEY FINDINGS: Culture media-based differentiation for two weeks generated cells with abundant transcription of ion channel genes (Cav1.2, NCX1), transcription factors (TBX3, TBX18, SHOX2) and connexins (Cx31.9 and Cx45) characteristic for cardiac pacemaker tissue, but lack adequate HCN transcription. haMSC-derived cells revealed transcript levels, which were closer related to sinoatrial nodal cells than hbMSC-derived cells. To substitute for the lack of If, we performed lentiviral HCN4-transduction of haMSC resulting in stable If. Co-culturing with NRVM demonstrated that differentiated haMSC expressing HCN4 showed earlier onset of spontaneous contractions and higher beating regularity, synchrony and rate compared to co-cultures with non-HCN4-transduced haMSC or HCN4-transduced, non-differentiated haMSC. Confocal imaging indicated increased membrane expression of cardiac gap junctional proteins in differentiated haMSC. SIGNIFICANCE: By differentiation haMSC, rather than hbMSC attain properties favorable for cardiac pacemaking. In combination with lentiviral HCN4-transduction, a cellular phenotype was generated that sustainably controls and stabilizes rate in co-culture with NRVM.


Assuntos
Relógios Biológicos/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas Musculares/metabolismo , Canais de Potássio/metabolismo , Tecido Adiposo/fisiologia , Animais , Células da Medula Óssea/fisiologia , Diferenciação Celular/fisiologia , Técnicas de Cocultura , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Células-Tronco Mesenquimais/metabolismo , Células Musculares/metabolismo , Proteínas Musculares/fisiologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Ratos , Nó Sinoatrial
13.
Life Sci ; 232: 116623, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31279781

RESUMO

AIMS: Doxorubicin, an antibiotic belonging to anthracycline family, has been used for treatment of malignancies. Cardiotoxicity is the main adverse effect of doxorubicin. Apigenin, as a flavonoid, has antioxidant, anti-inflammatory and anti-tumoral properties. The aim of this study was the assessment of any protective effect of apigenin on cardiotoxicity induced by doxorubicin. MAIN METHODS: 40 male Wistar rats were randomly divided into 4 groups: control, cardiotoxicity (DOX), apigenin treated group (DOX + Api 25) and apigenin group (Api 25). At the end of the experiment, the markers of cardiac function (%EF, %FS, LVIDs, LVIDd), cardiac and liver injury (LDH, CK-MB, cTn-I, ALT, and AST), cardiac apoptosis (Bax, Bcl-2 and Caspase3), cardiac oxidative stress (SOD, GSH, MDA) and cardiac fibrosis were measured. KEY FINDINGS: Apigenin improved cardiac functional parameters. The levels of cardiac and liver injury markers were significantly decreased in DOX + Api 25 compared to DOX. Treatment with apigenin caused significant decrease in percentage of cardiac fibrosis in comparison with DOX. Apigenin in DOX + Api 25 group led to significant decrease in apoptotic proteins (Casp3, Bax) and a significant increase in anti-apoptotic proteins (Bcl2). In apigenin treatment groups, SOD levels significantly increased while a significant decrease was observed in MDA. The amount of GSH in DOX + Api 25 had no significant change in comparison to control and Api 25 groups. SIGNIFICANCE: Apigenin reduced cardiac injuries induced by DOX through anti-fibrotic, antioxidant and anti-apoptotic properties. It seems that apigenin prevents cardiac injuries and improves cardiac function.


Assuntos
Apigenina/farmacologia , Cardiotoxicidade/tratamento farmacológico , Animais , Antioxidantes/farmacologia , Apigenina/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Doxorrubicina/efeitos adversos , Doxorrubicina/metabolismo , Flavonoides/farmacologia , Testes de Função Cardíaca , Inflamação/patologia , Masculino , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar
14.
Braz J Med Biol Res ; 52(7): e8732, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31314855

RESUMO

Inflammation plays an important role in the development of cardiovascular diseases (CVDs), suggesting that the immune system is a target of therapeutic interventions used for treating CVDs. This study evaluated mechanisms underlying inflammatory response and cardiomyocyte hypertrophy associated with bacterial lipopolysaccharide (LPS)- or heat shock protein 60 (HSP60)-induced Toll-like receptor (TLR) stimulation and the effect of a small interfering RNA (siRNA) against Ca2+/calmodulin-dependent kinase II delta B (CaMKIIδB) on these outcomes. Our results showed that treatment with HSP60 or LPS (TLR agonists) induced cardiomyocyte hypertrophy and complement system C3 and factor B gene expression. In vitro silencing of CaMKIIδB prevented complement gene transcription and cardiomyocyte hypertrophy associated with TLR 2/4 activation but did not prevent the increase in interleukin-6 and tumor necrosis factor-alfa gene expression in primary cultured cardiomyocytes. Moreover, CaMKIIδB silencing attenuated nuclear factor-kappa B expression. These findings supported the hypothesis that CaMKIIδB acts as a link between inflammation and cardiac hypertrophy. Furthermore, the present study is the first to show that extracellular HSP60 activated complement gene expression through CaMKIIδB. Our results indicated that a stress stimulus induced by LPS or HSP60 treatment promoted cardiomyocyte hypertrophy and initiated an inflammatory response through the complement system. However, CaMKIIδB silencing prevented the cardiomyocyte hypertrophy independent of inflammatory response induced by LPS or HSP60 treatment.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/patologia , Receptores Toll-Like/metabolismo , Animais , Chaperonina 60/farmacologia , Expressão Gênica , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , RNA Interferente Pequeno , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia
15.
Life Sci ; 232: 116635, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31283925

RESUMO

AIMS: The pathological cardiac hypertrophy will develop into heart failure, which has no effective treatment currently. Previous studies have proved that microRNAs (miRNAs) participate in the development of cardiac hypertrophy and regulate the pathological progress. In this study, we want to investigate the role of microRNA-92b-3p (miR-92b-3p) in cardiomyocyte hypertrophy and the mechanisms involved. MATERIALS AND METHODS: Neonatal mouse ventricular cells (NMVCs) were isolated from the hearts of 1-3-d-old newborn C57BL6 mice. The isolated NMVCs were induced hypertrophic phenotype by Angiotensin-II (Ang-II) and the cell size was examined by FITC-phalloidin staining assay. The expression of miR-92b-3p was determined by quantitative real-time PCR (qRT-qPCR). MRNA and protein level of ß-MHC, ACTA1 and HAND2 in NMVCs transfected with miR-92b-3p mimic and inhibition were assessed by RT-qPCR assay and western blot assay, respectively. Dual luciferase assay was used to verify the interaction between miR-92b-3p and the 3'-untranslated region (UTR) of HAND2 gene. KEY FINDINGS: MiR-92b-3p and HAND2 were significantly increased in Ang-II-induced NMVCs. Overexpression of miR-92b-3p can ameliorate Ang-II-induced cardiomyocyte hypertrophy. MiR-92b-3p negatively regulated HAND2 expression at the transcriptional level. Both miR-92b-3p mimic and HAND2 siRNA could efficiently inhibit Ang-II-induced hypertrophy in mouse cardiomyocytes. SIGNIFICANCE: MiR-92b-3p inhibits Ang-II-induced cardiomyocyte hypertrophy via targeting HAND2.


Assuntos
Angiotensina II/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/patologia , Regiões 3' não Traduzidas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Modelos Animais de Doenças , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima
16.
Int Heart J ; 60(4): 958-963, 2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31308330

RESUMO

Myocardial infarction (MI) occurs when the heart muscle is severely damaged due to a decrease in blood flow from the coronary arteries. During recovery from an MI, cardiac fibroblasts become activated and produce extracellular matrices, contributing to the wound healing process in the damaged heart. Inappropriate activation of the fibroblasts leads to excessive fibrosis in the heart. However, the molecular pathways by which cardiac fibroblasts are activated have not yet been fully elucidated.Here we show that serum deprivation, which recapitulates the cellular microenvironment of the MI area, strikingly induces collagen production in C3H/10T1/2 cells. Based on transcriptomic and pharmacological studies, we found that cell cycle perturbation is directly linked to collagen production in fibroblasts. Importantly, collagen synthesis is increased independently of the transcriptional levels of type I collagen genes. These results reveal a novel mode of fibroblast activation in the ischemic area, which will allow us to gain insights into the molecular mechanisms underlying cardiac fibrosis and establish a basis for anti-fibrotic therapy.


Assuntos
Colágeno/biossíntese , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Ciclo Celular , Células Cultivadas , Fibroblastos/metabolismo , Camundongos , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Transdução de Sinais
17.
Life Sci ; 232: 116619, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31265855

RESUMO

AIMS: Clinical treatment strategies for patients with myocardial ischemia typically include coronary artery recanalization to restore myocardial blood supply. However, myocardial reperfusion insult often induces oxidative stress and inflammation, which further leads to apoptosis and necrosis of myocardial cells. Increasing evidence suggests that microRNAs (miRNAs) participate in the pathological and physiological processes associated with myocardial ischemia reperfusion. MAIN METHODS: In this study, we established a myocardial H/R H9C2 cell model and a mouse I/R model to detect molecules implicated in myocardial I/R regulation and to determine the underlying signal transduction pathways. KEY FINDINGS: Herein, we showed that the expression of miR-374a-5p decreased in a myocardial cell model (H9C2 cells) of hypoxia/reoxygenation (H/R) and mouse model of ischemia/reperfusion (I/R). Alternatively, overexpression of miR-374a-5p was found to ameliorate myocardial cell damage within both in vivo and in vitro models of ischemia. Further, mitogen-activated protein kinase 6 (MAPK6) was identified as a direct target of miR-374a-5p. Thus, by targeting MAPK6, miR-374a-5p was found to negatively regulate MAPK6 expression. However, up-regulation of MAPK6 functioned to inhibit the previously observed protective effect of miR-374a-5p in the H9C2 H/R model. SIGNIFICANCE: Taken together, our study suggests that miR-374a-5p may have protective effects against cardiac I/R injury in vivo, and H/R injury in vitro, thereby providing novel insights into the molecular mechanisms associated with ischemia/reperfusion injury and a potential novel therapeutic target.


Assuntos
MicroRNAs/biossíntese , Traumatismo por Reperfusão/metabolismo , Animais , Apoptose/efeitos dos fármacos , Hipóxia Celular/fisiologia , Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Proteína Quinase 6 Ativada por Mitógeno/genética , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/fisiologia , Ratos , Traumatismo por Reperfusão/genética , Transdução de Sinais
18.
Phytochemistry ; 166: 112065, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31362147

RESUMO

Ten undescribed neo-clerodane diterpenoids, named hispanins A-J, together with six known ones, were isolated from the aerial parts of Salvia hispanica L. Their structures were established by extensive spectroscopic analysis. The absolute configurations of the undescribed compounds were determined by the ECD data and single crystal X-ray diffraction analysis. Hispanins B and C represented the first neo-clerodane diterpenoids with a unique oxygen bridge between C-19 and C-20. All isolated compounds were evaluated for their protective effects against H2O2-induced cardiomyocyte injury. Five of these compounds showed significant cardioprotective effects.


Assuntos
Cardiotônicos/química , Cardiotônicos/farmacologia , Diterpenos Clerodânicos/química , Diterpenos Clerodânicos/farmacologia , Componentes Aéreos da Planta/química , Salvia/química , Animais , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos
19.
Chem Biol Interact ; 309: 108723, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31228469

RESUMO

Ischemic preconditioning and pharmacological preconditioning are common strategies to prevent lethal myocardial injury, especially nutritional preconditioning (NPC). In this study, we investigated the effects of astragaloside IV (Ast), as an NPC agent, on myocardium suffered anoxia/reoxygenation (A/R) injury. Rats received 5 mg/kg Ast daily for 3 weeks by intragastric administration. Then, hearts were harvested and underwent A/R treatment using a Langendorff apparatus. Ast- pretreatment significantly promoted functional recovery of the myocardium, reduced infarct size, and oxidative stress, and decreased the apoptotic index. Similar findings were demonstrated in H9c2 cardiomyocytes that were pretreated with Ast for 24 h. Moreover, Ast-pretreatment significantly upregulated Bcl-2 expression, especially in mitochondria. The effects of Ast treatment against A/R injury were also reflected by increased antioxidant potential, inhibited reactive oxygen species (ROS) burst, increased oxygen consumption rate, maintained mitochondrial membrane potential (MMP), inhibited mitochondrial permeability transition pore (mPTP) opening, and prevented apoptosis. Selective inhibition of Bcl-2 by ABT-737 decreased myocardial injury protection of Ast. Ast-pretreatment resulted in NPC- related effects against A/R, and mitochondria may be the target of a cascade of events elicited by upregulating Bcl-2 expression, promoting translocation of Bcl-2 into mitochondria, maintaining MMP, inhibiting ROS bursts, thereby leading to recovery of mitochondrial respiration, preventing mPTP opening, decreasing cytochrome C release, preventing apoptosis, and ultimately alleviating myocardial injury.


Assuntos
Mitocôndrias/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Nitrofenóis/farmacologia , Nitrofenóis/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Superóxido Dismutase/metabolismo
20.
Toxicol Lett ; 313: 77-90, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31220554

RESUMO

Quetiapine is a common atypical antipsychotic used to treat mental disorders such as schizophrenia, bipolar disorder, and major depressive disorder. There has been increasing number of reports describing its cardiotoxicity. However, the molecular mechanisms underlying quetiapine-induced myocardial injury remain largely unknown. Herein, we reported a novel cell death type, quetiapine-induced necroptosis, which accounted for quetiapine cardiotoxicity in mice and proposed novel therapeutic strategies. Quetiapine-treated hearts showed inflammatory infiltration and evident fibrosis after 21-day continuous injection. The specific increases of protein levels of RIP3, MLKL and the phosphorylation of MLKL showed that quetiapine induced necroptotic cell death both in vivo and in vitro. Pharmacologic blockade of necroptosis using its specific inhibitor Necrostatin-1 attenuated quetiapine-induced myocardial injury in mice. In addition, quetiapine imbalanced the endocannabinoid system and caused opposing effects on two cannabinoid receptors (CB1R and CB2R). Specific antagonists of CB1R (AM 281, Rimonabant), but not its agonist ACEA significantly ameliorated the heart histopathology induced by chronic quetiapine exposure. By contrast, specific agonists of CB2R (JWH-133, AM 1241), but not its antagonist AM 630 exerted beneficial roles against quetiapine cardiotoxicity. The protective agents (AM 281, Rimonabant, AM 1241, and JWH-133) consistently inactivated the quetiapine-induced necroptosis signaling. Quetiapine bidirectionally regulates cannabinoid receptors and induces myocardial necroptosis, leading to cardiac toxic effects. Therefore, pharmacologic inhibition of CB1R or activation of CB2R represents promising therapeutic strategies against quetiapine-induced cardiotoxicity.


Assuntos
Antipsicóticos/toxicidade , Apoptose/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/toxicidade , Antagonistas de Receptores de Canabinoides/toxicidade , Cardiomiopatias/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Fumarato de Quetiapina/toxicidade , Receptor CB1 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Animais , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiotoxicidade , Linhagem Celular , Endocanabinoides/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Necrose , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Transdução de Sinais/efeitos dos fármacos
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