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1.
Int. j. cardiovasc. sci. (Impr.) ; 35(3): 410-418, May-June 2022. graf
Artigo em Inglês | LILACS | ID: biblio-1375650

RESUMO

Abstract An acute respiratory syndrome caused by SARS-CoV2 was declared a pandemic by the World Health Organization. Current data in the world and in Brazil show that approximately 40% of patients who died have some type of cardiac comorbidity. There are also robust reports showing an increase in IL-6 / IL-1B / TNF-alpha and the presence of lymphopenia in patients with COVID-19. Our team and others have shown that increased cytokines are the link between arrhythmias/Left ventricular dysfunction and the immune system in different diseases. In addition, it has been well demonstrated that lymphopenia can not only be a good marker, but also a factor that causes heart failure. Thus, the present review focused on the role of the immune system upon the cardiac alterations observed in the SARS-CoV2 infection. Additionally, it was well described that SARS-CoV-2 is able to infect cardiac cells. Therefore, here it will be reviewed in deep.


Assuntos
Arritmias Cardíacas/complicações , SARS-CoV-2/patogenicidade , COVID-19/complicações , Insuficiência Cardíaca/etiologia , Miocárdio/imunologia , Arritmias Cardíacas/fisiopatologia , Citocinas , Citocinas/imunologia , Coronavirus/patogenicidade , Disfunção Ventricular Esquerda/fisiopatologia , Miócitos Cardíacos/patologia , Síndrome Respiratória Aguda Grave , Insuficiência Cardíaca/complicações , Linfopenia/complicações
2.
Arq. bras. cardiol ; 118(2): 463-475, 2022. tab, graf
Artigo em Inglês, Português | LILACS | ID: biblio-1364328

RESUMO

Resumo Fundamento O remodelamento cardíaco patológico se caracteriza por disfunção diastólica e sistólica, levando à insuficiência cardíaca. Neste contexto, o cenário disfuncional do trânsito de cálcio miocárdico (Ca2+) tem sido pouco estudado. Um modelo experimental de estenose aórtica tem sido extensamente utilizado para aprimorar os conhecimentos sobre os principais mecanismos do remodelamento patológico cardíaco. Objetivo Entender o processo disfuncional dos principais componentes responsáveis pelo equilíbrio do cálcio miocárdico e sua influência sobre a função cardíaca na insuficiência cardíaca induzida pela estenose aórtica. Métodos Ratos Wistar de 21 dias de idade foram distribuídos em dois grupos: controle (placebo; n=28) e estenose aórtica (EaO; n=18). A função cardíaca foi analisada com o ecocardiograma, músculo papilar isolado e cardiomiócitos isolados. No ensaio do músculo papilar, SERCA2a e a atividade do canal de Ca2+ do tipo L foram avaliados. O ensaio de cardiomiócitos isolados avaliou o trânsito de cálcio. A expressão proteica da proteínas do trânsito de cálcio foi analisada com o western blot. Os resultados foram estatisticamente significativos quando p <0,05. Resultados Os músculos papilares e cardiomiócitos dos corações no grupo EaO demonstraram falhas mecânicas. Os ratos com EaO apresentaram menor tempo de pico do Ca2+, menor sensibilidade das miofibrilas do Ca2+, prejuízos nos processos de entrada e recaptura de cálcio pelo retículo sarcoplasmático, bem como disfunção no canal de cálcio do tipo L (CCTL). Além disso, os animais com EaO apresentaram maior expressão de SERCA2a, CCTL e trocador de Na+/Ca2+. Conclusão Insuficiência cardíaca sistólica e diastólica devido à estenose aórtica supravalvular acarretou comprometimento da entrada de Ca2+ celular e inibição da recaptura de cálcio pelo retículo sarcoplasmático devido à disfunção no CCTL e SERCA2a, assim como mudanças no trânsito de cálcio e na expressão das principais proteínas responsáveis pela homeostase de Ca2+ celular.


Abstract Background Maladaptive cardiac remodelling is characterized by diastolic and systolic dysfunction, culminating in heart failure. In this context, the dysfunctional scenario of cardiac calcium (Ca2+) handling has been poorly studied. An experimental model of aortic stenosis has been extensively used to improve knowledge about the key mechanisms of cardiac pathologic remodelling. Objective To understand the dysfunctional process of the major components responsible for Ca2+ balance and its influence on cardiac function in heart failure induced by aortic stenosis. Methods Male 21-day-old Wistar rats were distributed into two groups: control (sham; n= 28) and aortic stenosis (AoS; n= 18). Cardiac function was analysed by echocardiogram, isolated papillary muscle, and isolated cardiomyocytes. In the papillary muscle assay, SERCA2a and L-type Ca2+ channel activity was evaluated. The isolated cardiomyocyte assay evaluated Ca2+ handling. Ca2+ handling protein expression was analysed by western blot. Statistical significance was set at p <0.05. Results Papillary muscles and cardiomyocytes from AoS hearts displayed mechanical malfunction. AoS rats presented a slower time to the Ca2+ peak, reduced Ca2+ myofilament sensitivity, impaired sarcoplasmic reticulum Ca2+ influx and reuptake ability, and SERCA2a and L-type calcium channel (LTCC) dysfunction. Moreover, AoS animals presented increased expression of SERCA2a, LTCCs, and the Na+/Ca2+ exchanger. Conclusion Systolic and diastolic heart failure due to supravalvular aortic stenosis was paralleled by impairment of cellular Ca2+ influx and inhibition of sarcoplasmic reticulum Ca2+ reuptake due to LTCC and SERCA2a dysfunction, as well as changes in Ca2+ handling and expression of the major proteins responsible for cellular Ca2+ homeostasis.


Assuntos
Animais , Masculino , Ratos , Estenose da Valva Aórtica/patologia , Insuficiência Cardíaca/patologia , Músculos Papilares , Cálcio/metabolismo , Ratos Wistar , Miócitos Cardíacos/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Contração Miocárdica/fisiologia
3.
Arq. bras. cardiol ; 117(6): 1147-1158, dez. 2021. tab, graf
Artigo em Português | LILACS | ID: biblio-1350045

RESUMO

Resumo Fundamento A doxorrubicina (DOX) é frequentemente usada para tratar muitos tipos de cânceres, apesar da cardiotoxicidade dose-dependente. Como alternativa, o resveratrol é um polifenol que tem demonstrado efeitos cardioprotetores em vários modelos de disfunção cardíaca. Objetivo Este estudo investigou se o tratamento com resveratrol em ratas gestantes protege contra toxicidade induzida por doxorrubicina em cardiomiócitos da ninhada. Métodos Ratas Wistar (n-8) receberam sresveratrol como suplemento alimentar durante a gestação. No nascimento da ninhada, os corações (9-11) foram usados para se obter a cultura primária de cardiomiócitos. A cardiotoxicidade induzida por DOX e os efeitos da suplementação com resveratrol foram avaliados por marcadores de stress oxidativo, tais como oxidação da diclorofluoresceína diacetato, diminuição da atividade de enzimas antioxidantes, e oxidação do teor total de grupos sulfidrila, além da avaliação da viabilidade celular, geração de danos ao DNA, bem como a resposta de reparo aos danos ao DNA. Um valor de p <0,05 foi considerado estatisticamente significativo. Resultados Os cardiomiócitos de neonatos de ratas que receberam suplemento resveratrol apresentaram um aumento (p <0,01) na viabilidade das células, e diminuição (p <0,0001) de células apoptóticas/necróticas após o tratamento com DOX, o que está correlacionado às atividades de enzimas antioxidantes e produção de diclorofluoresceína. Além disso, o resveratrol protegeu os cardiomiócitos de danos ao DNA induzidos por DOX, apresentando uma diminuição (p <0,05) nas quebras de DNA induzidas por stress oxidativo, avaliadas pela atividade de enzimas reparadoras do DNA endonuclease III e formamidopirimidina glicosilase. A suplementação com resveratrol aumentou (p <0,05) a expressão da proteína reparadora Sirt6 nos cardiomiócitos dos filhotes. Conclusão Essa pesquisa indica que a suplementação com resveratrol durante o período gestacional tem um efeito cardioprotetor no coração da ninhada contra a toxicidade induzida por DOX, o que pode se dever a sua função antioxidante, e o aumento na resposta de danos ao DNA.


Abstract Background Doxorubicin (DOX) is frequently used to treat many types of cancers, despite its dose-dependent cardiotoxicity. Alternatively, resveratrol is a polyphenol that has shown useful cardioprotective effects in many heart dysfunction models. Objective This study investigated whether resveratrol treatment in pregnant rats protects against doxorubicin-induced toxicity in offspring cardiomyocytes. Methods Wistar rats (n=8) were supplemented with dietary resveratrol during pregnancy. Upon the offspring's birth, hearts (9-11) were used to obtain the primary culture of cardiomyocytes. DOX-induced cardiotoxicity and the effects of resveratrol supplementation were evaluated by oxidative stress markers, such as dichlorofluorescein diacetate oxidation, decrease in the activity of antioxidant enzymes, and oxidation of total sulfhydryl content, in addition to cell viability evaluation, DNA damage generation, and DNA damage repair response. A value of p<0.05 was considered statistically significant. Results Neonatal cardiomyocytes from resveratrol supplemented rats exhibiting an increase (p<0.01) in cell viability and lower (p<0.0001) apoptotic/necrotic cells after DOX treatment, which correlates with the activities of antioxidant enzymes and dichlorofluorescein production. Moreover, resveratrol protected cardiomyocytes from DOX-induced DNA damage, showing a decrease (p<0.05) in DNA breaks induced by oxidative stress, evaluated by the activity of DNA-repair enzymes endonuclease III and formamidopyrimidine glycosylase. Supplementation with resveratrol increased (p<0.05) the expression of the repair protein Sirt6 in the cardiomyocytes of the pups. Conclusion This research indicates that supplementation with resveratrol during the gestational period has a notable cardioprotective effect on the offspring's heart against DOX-induced toxicity, which may well be due to its antioxidant function, and the increase in the DNA damage repair response.


Assuntos
Animais , Feminino , Gravidez , Ratos , Doxorrubicina/toxicidade , Miócitos Cardíacos , Ratos Wistar , Suplementos Nutricionais , Resveratrol/farmacologia
4.
Arq. bras. cardiol ; 117(3): 476-483, Sept. 2021. graf
Artigo em Inglês, Português | LILACS | ID: biblio-1339188

RESUMO

Resumo Fundamento: A doença cardiovascular é a principal causa de morte em todo o mundo. A apoptose mediada por hipóxia em cardiomiócitos é uma das principais causas de distúrbios cardiovasculares. O tratamento com a proteína do fator de crescimento endotelial vascular (VEGF, do inglês vascular endothelial growth factor) foi testado, mas as dificuldades operacionais limitaram seu uso. Entretanto, com os avanços da terapia gênica, aumentou o interesse na terapia gênica baseada no VEGF em doenças cardiovasculares. No entanto, o mecanismo preciso pelo qual a reposição de VEGF resgata os danos pós-hipóxia em cardiomiócitos não é conhecido. Objetivos: Investigar o efeito da expressão de VEGF121 pós-hipóxia utilizando cardiomiócitos de ratos neonatos. Métodos: Cardiomiócitos isolados de ratos neonatos foram utilizados para estabelecer um modelo in vitro de lesão cardíaca induzida por hipóxia. O efeito da superexpressão de VEGF, isolado ou em conjunto com inibidores de moléculas pequenas que têm como alvo os canais de cálcio, receptores sensíveis ao cálcio (CaSR, do inglês calcium-sensitive receptors) e calpaína, no crescimento e proliferação celular em lesão de cardiomiócitos induzidos por hipóxia, foram determinados com ensaio de MTT, coloração TUNEL, coloração com Anexina V/PI, lactato desidrogenase e atividade da caspase. Para análise estatística, um valor de p<0,05 foi considerado significativo. Resultados: Verificou-se que o efeito do VEGF121 foi mediado por CaSR e calpaína, mas não foi dependente dos canais de cálcio. Conclusões: Nossos resultados, mesmo em um ambiente in vitro, estabelecem as bases para uma validação futura e testes pré-clínicos da terapia gênica baseada em VEGF em doenças cardiovasculares.


Abstract Background: Cardiovascular disease is the major cause of death worldwide. Hypoxia-mediated apoptosis in cardiomyocytes is a major cause of cardiovascular disorders. Treatment with vascular endothelial growth factor (VEGF) protein has been tested but operational difficulties have limited its use. However, with the advancements of gene therapy, interest has risen in VEGF-based gene therapy in cardiovascular disorders. However, the precise mechanism by which VEGF replenishment rescues post-hypoxia damage in cardiomyocytes is not known. Objectives: To investigate the effect of post-hypoxia VEGF121 expression using neonatal rat cardiomyocytes. Methods: Cardiomyocytes isolated from neonatal rats were used to establish an in vitro model of hypoxia-induced cardiac injury. The effect of VEGF overexpression, alone or in combination with small-molecule inhibitors targeting calcium channel, calcium sensitive receptors (CaSR), and calpain on cell growth and proliferation on hypoxia-induced cardiomyocyte injury were determined using an MTT assay, TUNEL staining, Annexin V/PI staining, lactate dehydrogenase and caspase activity. For statistical analysis, a value of P<0.05 was considered to be significant. Results: The effect of VEGF121 was found to be mediated by CaSR and calpain but was not dependent on calcium channels. Conclusions: Our findings, even though using an in vitro setting, lay the foundation for future validation and pre-clinical testing of VEGF-based gene therapy in cardiovascular diseases.


Assuntos
Animais , Ratos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Peptídeo Hidrolases/metabolismo , Miócitos Cardíacos/metabolismo , Hipóxia , Mitocôndrias
5.
Arq. bras. cardiol ; 116(3): 415-422, Mar. 2021. graf
Artigo em Inglês, Português | LILACS | ID: biblio-1248864

RESUMO

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.


Assuntos
Animais , Ratos , MicroRNAs/genética , Hiperglicemia , Morte Celular , Miócitos Cardíacos , Glucose
7.
Biol. Res ; 54: 3-3, 2021. graf, ilus
Artigo em Inglês | LILACS | ID: biblio-1505792

RESUMO

BACKGROUND: Testosterone regulates nutrient and energy balance to maintain protein synthesis and metabolism in cardiomyocytes, but supraphysiological concentrations induce cardiac hypertrophy. Previously, we determined that testosterone increased glucose uptake­via AMP-activated protein kinase (AMPK)­after acute treatment in cardiomyocytes. However, whether elevated glucose uptake is involved in long-term changes of glucose metabolism or is required during cardiomyocyte growth remained unknown. In this study, we hypothesized that glucose uptake and glycolysis increase in testosterone-treated cardiomyocytes through AMPK and androgen receptor (AR). METHODS: Cultured cardiomyocytes were stimulated with 100 nM testosterone for 24 h, and hypertrophy was verified by increased cell size and mRNA levels of ß-myosin heavy chain (ß-mhc). Glucose uptake was assessed by 2-NBDG. Glycolysis and glycolytic capacity were determined by measuring extracellular acidification rate (ECAR). RESULTS: Testosterone induced cardiomyocyte hypertrophy that was accompanied by increased glucose uptake, glycolysis enhancement and upregulated mRNA expression of hexokinase 2. In addition, testosterone increased AMPK phosphorylation (Thr172), while inhibition of both AMPK and AR blocked glycolysis and cardiomyocyte hypertrophy induced by testosterone. Moreover, testosterone supplementation in adult male rats by 5 weeks induced cardiac hypertrophy and upregulated ß-mhc, Hk2 and Pfk2 mRNA levels. CONCLUSION: These results indicate that testosterone stimulates glucose metabolism by activation of AMPK and AR signaling which are critical to induce cardiomyocyte hypertrophy.


Assuntos
Animais , Masculino , Ratos , Testosterona/farmacologia , Receptores Androgênicos/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Transdução de Sinais , Células Cultivadas , Hipertrofia , Miocárdio/patologia
8.
Braz. j. med. biol. res ; 54(3): e10550, 2021. graf
Artigo em Inglês | LILACS | ID: biblio-1153516

RESUMO

Hypoxia-induced apoptosis and inflammation play an important role in cardiovascular diseases including myocardial infarction (MI). miR-369 has been suggested to be a key regulator of cardiac fibrosis. However, the role of miR-369 in regulating hypoxia-induced heart injury remains unknown. Our data indicated that miR-369 expression was significantly down-regulated and TRPV3 was significantly up-regulated in myocardial tissue after MI in rats and in hypoxic-treated neonatal rat cardiomyocytes (NRCMs). In addition, we observed that hypoxia significantly promoted apoptosis and the inflammatory response, accompanied by increased caspase-3 activity and the secretion of the cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α. miR-369 overexpression significantly suppressed cell apoptosis and inflammatory factor production triggered by hypoxia, whereas miR-369 inhibition had an opposite effect. Importantly, we identified TRPV3 as a direct target of miR-369-3p. TRPV3 inhibition with small interfering RNA (siRNA) significantly inhibited hypoxia-induced inflammation and apoptosis, which can reverse the injury effects of miR-369 inhibitors. Our findings indicated that miR-369 reduced hypoxia-induced apoptosis and inflammation by targeting TRPV3.


Assuntos
Animais , Ratos , Miócitos Cardíacos , Apoptose , MicroRNAs , Canais de Cátion TRPV , Inflamação , Hipóxia
9.
Braz. j. med. biol. res ; 53(2): e9106, 2020. graf
Artigo em Inglês | LILACS | ID: biblio-1055491

RESUMO

Reperfusion strategies in acute myocardial infarction (AMI) can cause a series of additional clinical damage, defined as myocardial ischemia/reperfusion (I/R) injury, and thus there is a need for effective therapeutic methods to attenuate I/R injury. miR-26a-5p has been proven to be an essential regulator for biological processes in different cell types. Nevertheless, the role of miR-26a-5p in myocardial I/R injury has not yet been reported. We established an I/R injury model in vitro and in vivo. In vitro, we used cardiomyocytes to simulate I/R injury using hypoxia/reoxygenation (H/R) assay. In vivo, we used C57BL/6 mice to construct I/R injury model. The infarct area was examined by TTC staining. The level of miR-26a-5p and PTEN was determined by bioinformatics methods, qRT-PCR, and western blot. In addition, the viability and apoptosis of cardiomyocytes were separately detected by MTT and flow cytometry. The targeting relationship between miR-26a-5p and PTEN was analyzed by the TargetScan website and luciferase reporter assay. I/R and H/R treatment induced myocardial tissue injury and cardiomyocyte apoptosis, respectively. The results showed that miR-26a-5p was down-regulated in myocardial I/R injury. PTEN was found to be a direct target of miR-26a-5p. Furthermore, miR-26a-5p effectively improved viability and inhibited apoptosis in cardiomyocytes upon I/R injury by inhibiting PTEN expression to activate the PI3K/AKT signaling pathway. miR-26a-5p could protect cardiomyocytes against I/R injury by regulating the PTEN/PI3K/AKT pathway, which offers a potential approach for myocardial I/R injury treatment.


Assuntos
Animais , Coelhos , Traumatismo por Reperfusão Miocárdica/metabolismo , Isquemia Miocárdica/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Miócitos Cardíacos/patologia , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Western Blotting , Modelos Animais de Doenças , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Citometria de Fluxo , Camundongos Endogâmicos C57BL
10.
Braz. j. med. biol. res ; 53(10): e9849, 2020. tab, graf
Artigo em Inglês | LILACS, Coleciona SUS | ID: biblio-1132481

RESUMO

Testosterone has been demonstrated to antagonize doxorubicin-induced cardiomyocyte senescence. However, whether testosterone prevents the paraquat-induced cardiomyocyte senescence is largely unknown. The detection of SA-β-gal activity was performed using senescence β-gal staining kit and the reactive oxygen species levels were determined by reactive oxygen species assay kit. The plasmids for insulin-like growth factor 1 shRNA (sh-mIGF-1), sirtuin-1 shRNA (sh-SIRT1), scramble shRNA (sh-NC), overexpressing mIGF-1 (mIGF-1), overexpressing SIRT1 (SIRT1), and negative controls (NC) were obtained for this study. The expression of target genes was detected using quantitative real-time PCR, immunolabeling, and western blot. We found that testosterone significantly delayed the paraquat-induced HL-1 cardiomyocyte senescence as evidenced by decreasing senescence-associated β-galactosidase activity and reactive oxygen species generation, which were accompanied by the up-regulated expression of mIGF-1 and SIRT1. RNA interference to reduce mIGF-1 and SIRT1 expression showed that testosterone prevented paraquat-induced HL-1 senescence via the mIGF-1/SIRT1 signaling pathway. Furthermore, myocardial contraction was evaluated by expression of genes of the contractile proteins/enzymes, such as α-myosin heavy chain 6 (MHC6), α-myosin heavy chain 7 (MHC7), α-skeletal actin (ACTA-1), and sarco/endoplasmic reticulum calcium ATPase-2 (SERCA2). Testosterone adjusted the above four gene expressions and the adjustment was blocked by mIGF-1 or SIRT1 inhibition. Our findings suggested that the mIGF-1/SIRT1 signaling pathway mediated the protective function of testosterone against the HL-1 cardiomyocyte senescence by paraquat, which provided new clues for the mechanisms underlying the anti-aging role of testosterone in cardiomyocytes.


Assuntos
Paraquat/toxicidade , Testosterona/fisiologia , Miócitos Cardíacos , Sirtuína 1 , Transdução de Sinais , Células Cultivadas
11.
Braz. j. med. biol. res ; 53(9): e9693, 2020. tab, graf
Artigo em Inglês | LILACS, Coleciona SUS | ID: biblio-1132556

RESUMO

Ischemic heart disease (IHD) is one of the leading causes of death worldwide. C-type lectin domain family 3 member B (CLEC3B) is a C-type lectin superfamily member and is reported to promote tissue remodeling. The serum levels of CLEC3B are downregulated in patients with cardiovascular disease. However, the molecular mechanisms of CLEC3B in IHD is not well-characterized. Therefore, we overexpressed CLEC3B and silenced CLEC3B in H9c2 rat cardiomyocytes for the first time. We then constructed a model of IHD in vitro through culturing H9c2 cardiomyocytes in serum-free medium under oxygen-deficit conditions. Then, Cell Counting Kit-8 (CCK-8), flow cytometry, qRT-PCR, and western blot assays were performed to investigate cell viability, apoptosis, and expression levels of CLEC3B, phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (Akt), phosphorylated Akt (p-Akt), and cleaved-caspase 3. We observed that the mRNA expression of CLEC3B was decreased in hypoxic H9c2 cardiomyocytes (P<0.05). Overexpression of CLEC3B increased cell viability (P<0.01), inhibited cell apoptosis (P<0.05), upregulated the levels of p-PI3K/PI3K and p-Akt/Akt (P<0.01 or P<0.05), and downregulated expression of cleaved-caspase 3 (P<0.001) in hypoxic H9c2 cardiomyocytes while silencing of CLEC3B caused the opposite results. Inhibition of the PI3K/Akt pathway reversed the protective effect of CLEC3B on hypoxic H9c2 cardiomyocytes. Our study demonstrated that CLEC3B alleviated the injury of hypoxic H9c2 cardiomyocytes via the PI3K/Akt pathway.


Assuntos
Humanos , Animais , Ratos , Apoptose/fisiologia , Lectinas Tipo C/metabolismo , Transdução de Sinais , Fosfatidilinositol 3-Quinases , Miócitos Cardíacos/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinase , Hipóxia
12.
Braz. j. med. biol. res ; 53(3): e8761, 2020. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1089339

RESUMO

Nitric oxide (NO) inhibition by high-dose NG-nitro-L-arginine methyl ester (L-NAME) is associated with several detrimental effects on the cardiovascular system. However, low-dose L-NAME increases NO synthesis, which in turn induces physiological cardiovascular benefits, probably by activating a protective negative feedback mechanism. Aerobic exercise, likewise, improves several cardiovascular functions in healthy hearts, but its effects are not known when chronically associated with low-dose L-NAME. Thus, we tested whether the association between low-dose L-NAME administration and chronic aerobic exercise promotes beneficial effects to the cardiovascular system, evaluating the cardiac remodeling process. Male Wistar rats were randomly assigned to control (C), L-NAME (L), chronic aerobic exercise (Ex), and chronic aerobic exercise associated to L-NAME (ExL). Aerobic training was performed with progressive intensity for 12 weeks; L-NAME (1.5 mg·kg-1·day-1) was administered by orogastric gavage. Low-dose L-NAME alone did not change systolic blood pressure (SBP), but ExL significantly increased SBP at week 8 with normalization after 12 weeks. Furthermore, ExL promoted the elevation of left ventricle (LV) end-diastolic pressure without the presence of cardiac hypertrophy and fibrosis. Time to 50% shortening and relaxation were reduced in ExL, suggesting a cardiomyocyte contractile improvement. In addition, the time to 50% Ca2+ peak was increased without alterations in Ca2+ amplitude and time to 50% Ca2+ decay. In conclusion, the association of chronic aerobic exercise and low-dose L-NAME prevented cardiac pathological remodeling and induced cardiomyocyte contractile function improvement; however, it did not alter myocyte affinity and sensitivity to intracellular Ca2+ handling.


Assuntos
Animais , Masculino , Condicionamento Físico Animal/fisiologia , Cálcio/análise , Óxido Nítrico Sintase/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Inibidores Enzimáticos/farmacologia , Contração Miocárdica/efeitos dos fármacos , Peso Corporal/fisiologia , Ratos Wistar , Pressão Ventricular/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , NG-Nitroarginina Metil Éster/administração & dosagem , Modelos Animais , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Inibidores Enzimáticos/administração & dosagem , Adiposidade , Hemodinâmica , Atividade Motora/fisiologia , Miocárdio/patologia
13.
J. venom. anim. toxins incl. trop. dis ; 26: e20200005, 2020. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1135147

RESUMO

Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. Methods: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. Results: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. Conclusion: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.(AU)


Assuntos
Animais , Charibdotoxina/isolamento & purificação , Miócitos Cardíacos , Proteínas Cardiotóxicas de Elapídeos , Venenos Elapídicos , Cardiotoxinas , Ophiophagus hannah , Supressão , Citotoxicidade Imunológica
14.
Rev. bras. cir. cardiovasc ; 34(6): 711-722, Nov.-Dec. 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1057503

RESUMO

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.


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Adulto Jovem , Fibrilação Atrial/metabolismo , Citoesqueleto/metabolismo , Proteínas Nucleares/metabolismo , Conexina 43/metabolismo , Miócitos Cardíacos/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fibrilação Atrial/fisiopatologia , Fibrilação Atrial/genética , Imuno-Histoquímica , Proteínas Nucleares/genética , Movimento Celular , Conexina 43/genética , Proteínas Adaptadoras de Transdução de Sinal/genética
15.
Rev. chil. cardiol ; 38(1): 29-36, abr. 2019. graf
Artigo em Espanhol | LILACS | ID: biblio-1003635

RESUMO

Resumen: Antecedentes: La muerte de los cardiomiocitos es determinante en el desarrollo de patologías cardiacas posteriores al infarto del miocardio y la insuficiencia cardiaca. Las variaciones en la expresión de la familia de proteínas BCL-2 regulan vías, tanto de muerte, como de sobrevida celular. Así, BCL-2 es una proteína anti- apoptótica y NIX una proteína que induce la necrosis y/o la apoptosis celular. La Policistina-1 (PC1) es un mecanosensor vital para la función contráctil cardiaca; sin embargo, se desconoce su papel en la sobrevida de los cardiomiocitos durante el estrés mecánico. Objetivo: Determinar si PC-1 previene la muerte de los cardiomiocitos inducida por estrés mecánico y las proteínas BCL-2 y NIX. Métodos: Se utilizó cultivo de cardiomiocitos de ratas neonatas controles o deficientes en la expresión de PC1, estimulados con solución hiposmótica (HS), como modelo de estrés mecánico. Se midió la muerte por necrosis y apoptosis y los niveles de BCL-2 y NIX. Resultados: La deficiencia de la PC1 en los cardiomiocitos induce un aumento de la necrosis y los niveles proteicos de NIX en las células estimuladas con HS. El estrés mecánico induce la apoptosis basal relacionada a una disminución de BCL- 2, independiente de la expresión de la PC1. Conclusiones: La PC1 protege a los cardiomiocitos de la necrosis por estrés mecánico, lo que podría deberse en parte a su papel en la regulación de los niveles de las proteínas NIX.


Abstracts: Background: Cardiomyocytes death is a determining factor in the development of cardiac dysfunction after myocardial infarction and heart failure. The change in BCL-2 family protein expression regulates both cell death and survival pathways, whereas BCL-2 is an anti-apoptotic protein and NIX induces necrosis and/or apoptosis. Polycystin-1 (PC1) is a crucial mechanosensor for cardiac contractile function. However, its role in cardiomyocyte survival during mechanical stress is unknown. Aim: To study the relationship of PC1 with mechanical stretch-death in cardiomyocytes and the BCL-2, and NIX proteins. Methods. Controls or deficient expression of PC1 neonatal rat ventricular myocytes were stimulated with hypoosmotic solution (HS) and used as a model of mechanical stress. Necrosis or apoptosis cell death, BCL-2 and NIX protein levels were measured. Results: Deficient expression of PC1 increases cardiomyocyte necrosis and NIX protein levels in cells stimulated with HS. Mechanical stress induces basal apoptosis related to a decrease in BCL-2, independent of PC1 expression. Conclusion: PC1 protects cardiomyocytes from mechanical stress necrosis, at least in part, by regulating NIX protein levels.


Assuntos
Animais , Masculino , Ratos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPP/metabolismo , Necrose/prevenção & controle , Estresse Mecânico , Western Blotting , Ratos Sprague-Dawley , Apoptose , Citometria de Fluxo , Proteínas de Membrana/metabolismo
16.
Braz. j. med. biol. res ; 52(4): e7626, 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1001516

RESUMO

Reactive oxygen species (ROS) are highly reactive chemical species that may cause irreversible tissue damage, and play a critical role in cardiovascular diseases. Hydrogen sulfide (H2S) is a gasotransmitter that acts as a ROS scavenger with cardio-protective effects. In this study, we investigated the cytoprotective effect of H2S against H2O2-induced apoptosis in cardiomyocytes. H9c2 rat cardiomyoblasts were treated with H2S (100 μM) 24 h before challenging with H2O2 (100 μM). Apoptosis was then assessed by annexin V and PI, and mitochondrial membrane potential was measured using a fluorescent probe, JC-1. Our results revealed that H2S improved cell viability, reduced the apoptotic rate, and preserved mitochondrial membrane potential. An increased Bcl-2 to Bax ratio was also seen in myocytes treated with H2S after H2O2-induced stress. Our findings indicated a therapeutic potential for H2S in preventing myocyte death following ischemia/reperfusion.


Assuntos
Animais , Ratos , Apoptose/efeitos dos fármacos , Mioblastos Cardíacos/efeitos dos fármacos , Peróxido de Hidrogênio , Antioxidantes/farmacologia , Valores de Referência , Sulfetos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Western Blotting , Reprodutibilidade dos Testes , Espécies Reativas de Oxigênio/metabolismo , Apoptose/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Mioblastos Cardíacos/metabolismo , Potencial da Membrana Mitocondrial , Citometria de Fluxo/métodos , Sulfeto de Hidrogênio/farmacologia
17.
Braz. j. med. biol. res ; 52(3): e7994, 2019. graf
Artigo em Inglês | LILACS | ID: biblio-984040

RESUMO

Myocardial infarction (MI) is a common presentation for ischemic heart disease, which is a leading cause of death. Emodin is a Chinese herbal anthraquinone used in several diseases. However, the effect of emodin in hypoxia-induced injury in cardiomyocytes has not been clearly elucidated. Our study aimed to clarify the functions of emodin in hypoxia-induced injury in rat cardiomyocytes H9c2 and explore the underlying mechanism. The effects of emodin on cell viability and apoptosis were analyzed by the Cell counting kit-8 assay and flow cytometry assay, respectively. The cell proliferation- and cell apoptosis-related proteins were detected by western blot. qRT-PCR was used to determine the relative expression of miR-138. Cell transfection was performed to alter miR-138 and MLK3 expression. miR-138 target was performed by dual luciferase activity assay. Sirt1/AKT and Wnt/β-catenin pathways-related factors phosphorylation were analyzed by western blot. Emodin inhibited hypoxia-induced injury in H9c2 cells by promoting cell viability and reducing cell apoptosis. miR-138 was down-regulated by hypoxia treatment but up-regulated by emodin. Up-regulation of miR-138 alleviated hypoxia-induced cell injury. Down-regulation of miR-138 attenuated the growth-promoting effect of emodin on hypoxia-induced injury, whereas up-regulation of miR-138 enhanced the growth-promoting effects of emodin. The underlying mechanism might be by inactivating Sirt1/AKT and Wnt/β-catenin pathways. MLK3 was negatively regulated by miR-138 expression and inactivated Sirt1/AKT and Wnt/β-catenin pathways. Emodin alleviated hypoxia-induced injury in H9c2 cells via up-regulation of miR-138 modulated by MLK3, as well as by activating Sirt1/AKT and Wnt/β-catenin pathways.


Assuntos
Animais , Ratos , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Emodina/uso terapêutico , Miócitos Cardíacos/patologia , Proliferação de Células/efeitos dos fármacos , Hipóxia/complicações , Transdução de Sinais , Regulação para Cima , Linhagem Celular , Miócitos Cardíacos/efeitos dos fármacos , MicroRNAs
18.
Mem. Inst. Oswaldo Cruz ; 114: e180593, 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1020079

RESUMO

BACKGROUND Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in β-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.


Assuntos
Animais , Camundongos , Trypanosoma cruzi/fisiologia , Cardiomiopatia Chagásica/metabolismo , Miócitos Cardíacos/parasitologia , Mecanotransdução Celular/genética , Western Blotting , Reação em Cadeia da Polimerase , Imunofluorescência , Paxilina/metabolismo
19.
Braz. j. med. biol. res ; 52(12): e8834, 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1055472

RESUMO

Polydatin (PD), a monocrystalline polyphenolic drug mainly found in the roots of Polygonum cuspidatum, has various pharmacological activities. Long non-coding RNAs (lncRNA) DiGeorge syndrome critical region gene 5 (DGCR5) was found to participate in the suppression of multiple cancers. Here, we proposed to study the effect of PD on myocardial infarction (MI) by inducing DGCR5. CCK-8 assay was performed to detect the viability of H9c2 cells. Flow cytometry was utilized to test apoptosis of H9c2 cells. These results determined the optimal concentration and effect time of hypoxia as well as PD. Si-DGCR5 was transfected into cells and the expression level was determined by qRT-PCR. Western blot was utilized to evaluate the expression of apoptosis-related proteins, Bcl-2, Bax, and cleaved-caspase-3, as well as autophagy-associated proteins including Beclin-1, p62, and LC3-II/LC3-I. As a result, PD efficiently attenuated hypoxia-induced apoptosis and autophagy in H9c2 cells. The expression of DGCR5 was down-regulated by hypoxia and up-regulated by PD. Besides, knocking-down the expression of DGCR5 inhibited the protection of PD in H9c2 cells. In addition, PD up-regulated the accumulation of DGCR5, DGCR5 decreased the expression of Bcl-2 and p62, raised the expression of Bax and cleaved-caspase-3, and the proportion of LC3-II/LC3-I. PD stimulated the PI3K/AKT/mTOR and MEK/ERK signaling pathways via up-regulating the expression of DGCR5. Our data demonstrated that PD reduced cell apoptosis and autophagy induced by hypoxia in cardiomyocytes. Moreover, PD activated PI3K/AKT/mTOR and MEK/ERK signaling pathways by up-regulating the expression of DGCR5.


Assuntos
Animais , Ratos , Estilbenos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , RNA Longo não Codificante/efeitos dos fármacos , Glucosídeos/farmacologia , Transdução de Sinais , Regulação para Cima/efeitos dos fármacos , Linhagem Celular , Citoproteção , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia
20.
Braz. j. med. biol. res ; 52(7): e8732, 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1011598

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
Animais , Ratos , Miócitos Cardíacos/patologia , Receptores Toll-Like/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Transdução de Sinais/fisiologia , Expressão Gênica , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Ratos Wistar , Chaperonina 60/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RNA Interferente Pequeno , Inflamação/metabolismo
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