The Role of the Immune System on the Cardiac Complications Observed in SARS-CoV-2

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.

Cenário Disfuncional dos Principais Componentes Responsáveis pelo Equilíbrio do Trânsito de Cálcio Miocárdico na Insuficiência Cardíaca Induzida por Estenose Aórtica / The Dysfunctional Scenario of the Major Components Responsible for Myocardial Calcium Balance in Heart Failure Induced by Aortic Stenosis

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.

Oral administration of TRPV4 inhibitor improves atrial calcium handling abnormalities in sterile pericarditis rats / 生理学报

Atrial Ca2+ handling abnormalities, mainly involving the dysfunction of ryanodine receptor (RyR) and sarcoplasmic reticulum Ca2+-ATPase (SERCA), play a role in the pathogenesis of atrial fibrillation (AF). Previously, we found that the expression and function of transient receptor potential vanilloid subtype 4 (TRPV4) are upregulated in a sterile pericarditis (SP) rat model of AF, and oral administration of TRPV4 inhibitor GSK2193874 alleviates AF in this animal model. The aim of this study was to investigate whether oral administration of GSK2193874 could alleviate atrial Ca2+ handling abnormalities in SP rats. A SP rat model of AF was established by daubing sterile talcum powder on both atria of Sprague-Dawley (SD) rats after a pericardiotomy, to simulate the pathogenesis of postoperative atrial fibrillation (POAF). On the 3rd postoperative day, Ca2+ signals of atria were collected in isolated perfused hearts by optical mapping. Ca2+ transient duration (CaD), alternan, and the recovery properties of Ca2+ transient (CaT) were quantified and analyzed. GSK2193874 treatment reversed the abnormal prolongation of time to peak (determined mainly by RyR activity) and CaD (determined mainly by SERCA activity), as well as the regional heterogeneity of CaD in SP rats. Furthermore, GSK2193874 treatment relieved alternan in SP rats, and reduced its incidence of discordant alternan (DIS-ALT). More importantly, GSK2193874 treatment prevented the reduction of the S2/S1 CaT ratio (determined mainly by RyR refractoriness) in SP rats, and decreased its regional heterogeneity. Taken together, oral administration of TRPV4 inhibitor alleviates Ca2+ handling abnormalities in SP rats primarily by blocking the TRPV4-Ca2+-RyR pathway, and thus exerts therapeutic effect on POAF.

Construction of an adenovirus vector expressing engineered splicing factor for regulating alternative splicing of YAP1 in neonatal rat cardiomyocytes / 南方医科大学学报

OBJECTIVE@#To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes.@*METHODS@#The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag.@*RESULTS@#The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05).@*CONCLUSION@#We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.

Sacubitril/valsartan attenuates left ventricular remodeling and improve cardiac function by upregulating apelin/APJ pathway in rats with heart failure / 中华心血管病杂志

Objective: To investigate the effect and mechanism of sacubitril/valsartan on left ventricular remodeling and cardiac function in rats with heart failure. Methods: A total of 46 SPF-grade male Wistar rats weighed 300-350 g were acclimatized to the laboratory for 7 days. Rats were then divided into 4 groups: the heart failure group (n=12, intraperitoneal injection of adriamycin hydrochloride 2.5 mg/kg once a week for 6 consecutive weeks, establishing a model of heart failure); heart failure+sacubitril/valsartan group (treatment group, n=12, intragastric administration with sacubitril/valsartan 1 week before the first injection of adriamycin, at a dose of 60 mg·kg-1·d-1 for 7 weeks); heart failure+sacubitril/valsartan+APJ antagonist F13A group (F13A group, n=12, adriamycin and sacubitril/valsartan, intraperitoneal injection of 100 μg·kg-1·d-1 APJ antagonist F13A for 7 weeks) and control group (n=10, intraperitoneal injection of equal volume of normal saline). One week after the last injection of adriamycin or saline, transthoracic echocardiography was performed to detect the cardiac structure and function, and then the rats were executed, blood and left ventricular specimens were obtained for further analysis. Hematoxylin-eosin staining and Masson trichrome staining were performed to analyze the left ventricular pathological change and myocardial fibrosis. TUNEL staining was performed to detect cardiomyocyte apoptosis. mRNA expression of left ventricular myocardial apelin and APJ was detected by RT-qRCR. ELISA was performed to detect plasma apelin-12 concentration. The protein expression of left ventricular myocardial apelin and APJ was detected by Western blot. Results: Seven rats survived in the heart failure group, 10 in the treatment group, and 8 in the F13A group. Echocardiography showed that the left ventricular end-diastolic diameter (LVEDD) and the left ventricular end-systolic diameter (LVESD) were higher (both P<0.05), while the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were lower in the heart failure group than in the control group (both P<0.05). Compared with the heart failure group, rats in the treatment group were featured with lower LVEDD and LVESD (both P<0.05), higher LVEF and LVFS (both P<0.05), these beneficial effects were reversed in rats assigned to F13A group (all P<0.05 vs. treatment group). The results of HE staining showed that the cardiomyocytes of rats in the control group were arranged neatly and densely structured, the cardiomyocytes in the heart failure group were arranged in disorder, distorted and the gap between cells was increased, the cardiomyocytes in the treatment group were slightly neat and dense, and cardiomyocytes in the F13A group were featured similarly as the heart failure group. Masson staining showed that there were small amount of collagen fibers in the left ventricular myocardial interstitium of the control group, while left ventricular myocardial fibrosis was significantly increased, and collagen volume fraction (CVF) was significantly higher in the heart failure group than that of the control group (P<0.05). Compared with the heart failure group, the left ventricular myocardial fibrosis and the CVF were reduced in the treatment group (both P<0.05), these effects were reversed in the F13A group (all P<0.05 vs. treatment group). TUNEL staining showed that the apoptosis index (AI) of cardiomyocytes in rats was higher in the heart failure group compared with the control group (P<0.05), which was reduced in the treatment group (P<0.05 vs. heart failure group), this effect again was reversed in the F13A group (P<0.05 vs. treatment group). The results of RT-qPCR and Western blot showed that the mRNA and protein levels of apelin and APJ in left ventricular myocardial tissue of rats were downregulated in heart failure group (all P<0.05) compared with the control group. Compared with the heart failure group, the mRNA and protein levels of apelin and APJ were upregulated in the treatment group (all P<0.05), these effects were reversed in the F13A group (all P<0.05 vs. treatment group). ELISA test showed that the plasma apelin concentration of rats was lower in the heart failure group compared with the control group (P<0.05); compared with the heart failure group, the plasma apelin concentration of rats was higher in the treatment group (P<0.05), this effect was reversed in the F13A group (P<0.05 vs. treatment group). Conclusion: Sacubitril/valsartan can partially reverse left ventricular remodeling and improve cardiac function in rats with heart failure through modulating Apelin/APJ pathways.

Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro / 中西医结合学报

OBJECTIVE@#Qili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.@*METHODS@#The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia-reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.@*RESULTS@#Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.@*CONCLUSION@#QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

Role of PNPT1 in cardiomyocyte apoptosis induced by oxygen-glucose deprivation / 南方医科大学学报

OBJECTIVE@#To explore the effect of inhibiting polyribonucleotide nucleotidyl-transferase 1 (PNPT1) on oxygen-glucose deprivation (OGD)-induced apoptosis of mouse atrial myocytes.@*METHODS@#Cultured mouse atrial myocytes (HL-1 cells) with or without OGD were transfected with PNPT1-siRNA or a negative control siRNA (NC-siRNA group), and the cell survival rate was detected using CCK-8 assay. The expression levels of ACTB and TUBA mRNA were detected with qPCR, and the protein expression of PNPT1 was detected with Western blotting. The apoptosis rate of the treated cells was determined with flow cytometry, the mitochondrial membrane potential was detected using JC-1 kit, and the mitochondrial morphology was observed using transmission electron microscope.@*RESULTS@#With the extension of OGD time, the protein expression levels of PNPT1 increased progressively in the cytoplasm of HL-1 cells (P < 0.05). Transfection with PNPT1-siRNA significantly reduced PNPT1 expression in HL-1 cells (P < 0.05). Exposure to OGD significantly enhanced degradation of ACTB and TUBA mRNA (P < 0.05) and markedly increased the apoptosis rate of HL-1 cells (P < 0.05), and these changes were significantly inhibited by transfection with PNPT1-siRNA (P < 0.05), which obviously increased mitochondrial membrane potential and improved mitochondrial morphology of HL-1 cells exposed to OGD.@*CONCLUSION@#Inhibition of PNPT1 improves mitochondrial damage and reduces degradation of apoptotic-associated mRNAs to alleviate OGD-induced apoptosis of mouse atrial myocyte.

Palmitic acid suppresses autophagy in neonatal rat cardiomyocytes via the cGAS-STING-IRF3 pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of palmitic acid (PA) on autophagy in neonatal rat cardiomyocytes (NRCMs) and explore the underlying mechanism.@*METHODS@#NRCMs were isolated and cultured for 24 h before exposure to 10% BSA and 0.1, 0.3, 0.5, or 0.7 mmol/L PA for 24 h. After the treatments, the expressions of Parkin, PINK1, p62, LC3Ⅱ/ LC3Ⅰ, cGAS, STING and p-IRF3/IRF3 were detected using Western blotting and the cell viability was assessed with CCK8 assay, based on which 0.7 mmol/L was selected as the optimal concentration in subsequent experiments. The effects of cGAS knockdown mediated by cGAS siRNA in the presence of PA on autophagy-related proteins in the NRCMs were determined using Western blotting, and the expressions of P62 and LC3 in the treated cells were examined using immunofluorescence assay.@*RESULTS@#PA at different concentrations significantly lowered the expressions of Parkin, PINK1, LC3 Ⅱ/LC3 Ⅰ and LC3 Ⅱ/LC3 Ⅰ+Ⅱ (P < 0.05), increased the expression of p62 (P < 0.05), and inhibited the viability of NRCMs (P < 0.05). Knockdown of cGAS obviously blocked the autophagy-suppressing effect of PA and improved the viability of NRCMs (P < 0.05).@*CONCLUSION@#PA inhibits autophagy by activating the cGAS-STING-IRF3 pathway to reduce the viability of NRCMs.

Effects of Qilong Capsules on myocardial fibrosis and insufficient blood circulation in ischemic cardiomyopathy with Qi deficiency and blood stasis / 中国中药杂志

Protective effect of Qilong Capsules(QL) on the myocardial fibrosis and blood circulation of rats with coronary heart disease of Qi deficiency and blood stasis type was investigated. Sleep deprivation and coronary artery ligation were used to construct a disease-symptom combination model, and 60 SD rats were divided into sham operation(sham) group, syndrome(S) group, disease and syndrome(M) group and QL group randomly. The treatment group received administration of QL 0.4 g·kg~(-1)·d~(-1). Other groups were given the same amount of normal saline. The disease indexes of each group [left ventricular end diastolic diameter(LVESD), left ventricular end systolic diameter(LVEDD), left ventricular ejection fraction(LVEF), left ventricular axis shortening rate(LVFS), myocardial histopathology, platelet morphology, peripheral blood flow] and syndrome indexes(tongue color, pulse, grip power) were detected. In sham group, cardiomyocytes and myocardial fibers were arranged neatly and densely with clear structures. The tongues' color in sham were light red, and the pulse shape were regular. RGB is a parameter reflected the brightness of the image of the tongue. In the S group, the amplitude and frequency of the animal's pulse increased accompanied by decreasing R,G,B, however, the decreased R,G,B was accompanied by reduced pulse amplitude in M group. And in M group, we observed fuzzy cell morphology, hypertrophied myocytes, disordered arrangement of cardiomyocytes and myocardial fibers, reduced peripheral blood flow and increased collagen volume fraction(CVF). Increased LVESD and LVEDD, and decreased LVEF and LVFS represented cardiac function in S group was significantly lower than that in sham. In QL group, the tongue's color was red and the pulse was smooth. The myocardial fibers of the QL group were arranged neatly and secreted less collagen. It improved the blood circulation in the sole and tail, and reversed the increasing of LVEDD, LVESD and the decreasing of LVEF and LVFS of M group. Platelets in M and S group showed high reactivity, and QL could decrease aggregation risk. In conclusion, Qilong Capsules has an obvious myocardial protective effect on ischemic cardiomyopathy, which may inhibit the degree of myocardial fibrosis and reduce platelet reactivity.

Progress in the Role of Mechanical Stimulus in Cardiac Development / 中国医学科学院学报

Mechanical stimulus is critical to cardiovascular development during embryogenesis period.The mechanoreceptors of endocardial cells and cardiac myocytes may sense mechanical signals and initiate signal transduction that induce gene expression at a cellular level,and then translate molecular-level events into tissue-level deformations,thus guiding embryo development.This review summarizes the regulatory roles of mechanical signals in the early cardiac development including the formation of heart tube,looping,valve and septal morphogenesis,ventricular development and maturation.Further,we discuss the potential mechanical transduction mechanisms of platelet endothelial cell adhesion molecule 1-vascular endothelial-cadherin-vascular endothelial growth factor receptor 2 complex,primary cilia,ion channels,and other mechanical sensors that affect some cardiac malformations.

Efeito Cardioprotetor da Suplementação Materna com Resveratrol sobre a Toxicidade Induzida por Doxorrubicina em Cardiomiócitos de Neonatos / Cardioprotective Effect of Maternal Supplementation with Resveratrol on Toxicity Induced by Doxorubicin in Offspring Cardiomyocytes

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.

VEGF121 como Mediador de Efeitos Cardioprotetores Pós-Hipóxia via CaSR e via da Protease Dependente de Mitocôndria / VEGF121 Mediates Post-Hypoxia Cardioprotective Effects Via CaSR and Mitochondria-Dependent Protease Pathway

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.

A Inibição do Metabolismo da Glicose por miR-34a e miR-125b Protege contra a Morte Celular de Cardiomiócitos Causada por Hiperglicemia / Inhibiting Glucose Metabolism By miR-34a and miR-125b Protects Against Hyperglycemia-Induced Cardiomyocyte Cell Death

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.

MicroRNA-369 attenuates hypoxia-induced cardiomyocyte apoptosis and inflammation via targeting TRPV3

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.

Silencing LncRNA SNHG7 alleviates hypoxia/reoxygenation-induced cardiomyocyte damage by regulating the expression of miR-181b-5p / 中华医学遗传学杂志

OBJECTIVE@#To study the effect of silencing LncRNA SNHG7 on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its targeted regulation on miR-181b-5p.@*METHODS@#Rat cardiomyocytes H9c2 were cultured in vitro and randomly divided into control group, H/R group, H/R + si-NC group, H/R + si-SNHG7 group, H/R + si-SNHG7 + anti-miR-NC group and H/R + si-SNHG7 + anti-miR-181b-5p group. The content of lactate dehydrogenase (LDH), malondialedhyde (MDA) and the activity of superoxide dismutase (SOD) were detected. Flow cytometry was carried out to detect the rate of apoptosis. qRT-PCR was used to detect the expression of SNHG7 and miR-181b-5p. Dual luciferase report experiment was used to verify the targeting relationship between SNHG7 and miR-181b-5p. Western blotting was used to detect the expression of Bax and Bcl-2.@*RESULTS@#Compared with the control group, the H/R group showed significantly increased SNHG7 expression in cardiomyocytes, reduced miR-181b-5p expression, higher levels of LDH and MDA, reduced activity of SOD, increased cell apoptosis rate, higher level of Bax protein, and reduced level of Bcl-2 protein (all P< 0.05). Compared with the H/R and H/R + si-NC groups, the H/R + si-SNHG7 group had significantly reduced level of LDH and MDA, increased activity of SOD, reduced apoptosis rate, reduced level of Bax protein, increased level of Bcl-2 protein (all P< 0.05). The dual luciferase report experiment confirmed that SNHG7 could target miR-181b-5p. Interference with the expression of miR-181b-5p could reduce the effect of silencing SNHG7 on H/R-induced cardiomyocyte oxidative stress and apoptosis.@*CONCLUSION@#Silencing SNHG7 may inhibit H/R-induced cardiomyocyte oxidative stress and apoptosis by up-regulating the expression of miR-181b-5p, thereby exerting a protective effect on cardiomyocytes.

Biocompatibility of extracellular matrix hydrogel with human iPSCs differentiated cardiomyocytes / 中华心血管病杂志

Objective: To observe the biocompatibility of porcine omental derived extracellular matrix (ECM) hydrogel with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and the feasibility of ECM hydrogel as a delivery vector of cell transplantation. Methods: A series of chemical, physical and enzymatic methods were applied to acellularize the porcine omentum. Subsequently, the extracted ECM was prepared into thermosensitive hydrogel. The biochemical composition of the hydrogel was identified by histological staining. The microstructure was observed by scanning electron microscopy. The hydrogel was then injected into the myocardium of mice to observe its in situ gelation ability. Differentiation of human induced pluripotent stem cells into cardiomyocytes was achieved by small molecule induction, and then the obtained hiPSC-CMs were cultured. hiPSC-CMs cultured onto the prepared hydrogel were defined as the hydrogel group, while conventionally cultured hiPSC-CMs were defined as the control group. Cardiomyocyte viability and growth patterns were detected using live/dead staining, CCK-8 and phalloidin staining. Immunofluorescence staining and Western blot of cardiomyocytes were used to determine the survival and phenotypic maintenance markers of cardiomyocytes in materials. Results: The results of HE staining, oil red O staining and DAPI fluorescence staining showed that there was no significant cell debris, nucleus and lipid residue in the prepared ECM hydrogel. The Sirius red staining and Alcian blue staining showed that the hydrogel retained collagen and glycolaminoglycan, which were the main components of ECM. The prepared hydrogel behaves as a viscous liquid at 4 ℃ and as a gel state at 37 ℃. Scanning electron microscope results showed that the microstructure of the hydrogel was composed of irregular fibers and pores of different sizes. Under the guidance of ultrasound, the prepared ECM hydrogel could be successfully injected into the myocardium of mice. Immediately after the injection, the hyperechoic signal could be observed under ultrasound, suggesting that the hydrogel remained in the myocardium. HE staining of myocardial tissue evidenced that there was lump of gel in the injection area. The differentiated hiPSC-CMs were co-cultured with the prepared ECM hydrogel, and the results of live/dead staining showed that most of the hiPSC-CMs in the hydrogel group and the control group were alive, dead cells were scanty. The results of CCK-8 test showed that the absorbance values of the two groups were similar (P>0.05). The results of phalloidin staining showed that hiPSC-CMs could extend normally when co-cultured with ECM hydrogel. The cell morphology of the hydrogel group was similar with that of the control group, and there was no statistically significant difference in the F-actin coverage area per cell between the two groups (P>0.05). Immunofluorescence staining of cardiomyocyte markers showed that there was no significant difference in the coverage area of α-actinin and connexin-43 (Cx-43) per field between the hydrogel group and the control group (both P>0.05), the quantitative results of DAPI staining showed that there was no statistically significant difference in the number of cells between the two groups (P>0.05). Meanwhile, the results of Western blot showed that the expression levels of α-actinin and Cx-43 in cardiomyocytes in the hydrogel group were similar as those in the control group (both P>0.05). Conclusions: These results show that preparation of the ECM hydrogel from porcine omentum is successful. The hydrogel has good biocompatibility and no obvious cytotoxicity. Besides, the hydrogel can support the survival of hiPSC-CMs in vitro and maintain its phenotype. These properties make it a promising injectable cardiac tissue engineering material.

Change of gene expression profiles in human cardiomyocytes and macrophages infected with SARS / 中南大学学报(医学版)

OBJECTIVES@#Coronavirus disease 2019 (COVID-19) is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 can damage the myocardium directly, or activate the immune system, trigger a cytokine storm, and cause inflammatory cells to infiltrate the myocardial tissue and damage the myocardium. This study is based on the sequencing data to analyze the changes in gene expression of cardiomyocytes and macrophages after SARS-CoV-2 infection, and explore the potential effects of SARS-CoV-2 on the heart and immune system.@*METHODS@#The public data set GSE151879 was retrieved. The online software Network Analyst was used to preprocess the data, and the differentially expressed genes (DEGs) [log@*RESULTS@#After data standardization, the data quality was excellent and it can ensure reliable results. Myocardial cell infection with SARS-CoV-2 and gene expression spectrum were changed significantly, including a total of 484 DEGs in adult cardiomyoblasts, a total of 667 DEGs in macrophages, and a total of 1 483 DEGs in human embryo source of cardiomyopathy. The Stum, mechanosensory transduction mediator homolog (STUM), dehydrogenase/reductase 9 (DHRS9), calcium/calmodulin dependent protein kinase II beta (CAMK2B), claudin 1(CLDN1), C-C motif chemokine ligand 2 (CCL2), TNFAIP3 interacting protein 3 (TNIP3), G protein-coupled receptor 84 (GPR84), and C-X-C motif chemokine ligand 1 (CXCL1) were identical in expression patterns in 3 types of cells. The protein-protein interaction suggested that CAMK2B proteins may play a key role in the antiviral process in 3 types of cells; and silicon dioxide (SiO@*CONCLUSIONS@#CAMK2B, CLDN1, CCL2, and DHRS9 genes play important roles in the immune response of cardiomyocytes against SARS-CoV-2. SiO

Down-regulation of miR-488 targeting to promote Jag1 expression inhibits hypoxia-reoxygenation myocardial H9c2 cell damage / 中华医学遗传学杂志

OBJECTIVE@#To study the effect of down-regulating miR-488 targeting Jag1 on the injury of hypoxia-reoxygenation myocardial H9c2 cells.@*METHODS@#A hypoxic-reoxygenated myocardial H9c2 cell injury model was constructed. miR-488 inhibitor was used to transfect the cells. CCK-8 method and flow cytometry were used to detect cell proliferation and apoptosis in each group. Lactate dehydrogenase (LDH), superoxide dismutase (SOD), malonaldehyde (MDA), catalase (CAT) levels were detected. Western blotting was used to detect the expression of Bcl-2 associated X Protein (Bax) and B cell lymphoma/lewkmia-2 (Bcl-2). Target genes of miR-488 were predicted, and a luciferase reporter system was used to verify the targeting relationship between the two. Myocardial H9c2 cells were co-transfected with miR-488 inhibitor and Jag1 siRNA, and treated with hypoxia and reoxygenation, cell proliferation, apoptosis, LDH, SOD, MDA, CAT levels, and Bax, Bcl-2 protein expression were detected.@*RESULTS@#The expression of miR-488 in the hypoxia-reoxygenated myocardial H9c2 cells was increased, along with reduced cell proliferation, increased apoptosis, increased Bax protein expression, decreased Bcl-2 protein expression, increased MDA, decreased CAT and SOD, and increased LDH level in the supernatant of cell culture. When myocardial H9c2 cells were transfected with miR-488 inhibitor and treated with hypoxia and reoxygenation, the expression of miR-488 was decreased, along with increased cell proliferation, decreased apoptosis, decreased Bax protein expression, increased Bcl-2 protein expression, decreased MDA, increased CAT and SOD, and decreased LDH level in the supernatant of cell culture. Down-regulation of miR-488 could target and down-regulate Jag1 expression. And Jag1 siRNA could reverse the effect of miR-488 inhibitor on the proliferation, apoptosis, LDH, SOD, MDA, CAT levels and the expression of Bax and Bcl-2 of hypoxic-reoxygenated myocardial H9c2 cells.@*CONCLUSION@#Down-regulating miR-488 targeted Jag1 can attenuate hypoxia-reoxygenation induced myocardial H9c2 cell injury.

Molecular mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion injury in H9c2 cardiomyocytes based on molecular docking / 中国中药杂志

The aim of this study was to investigate the mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion(OGD/R) injury in H9 c2 cardiomyocytes. First, Discovery Studio 2019 was used for the molecular docking of luteolin with three key enzymes including lipoxygenase 5(ALOX5), lipoxygenase 12(ALOX12), and lipoxygenase 15(ALOX15) in lipoxygenase pathway. The docking results showed that luteolin had high docking score and similar functional groups with the original ligand. From this, H9 c2 cardiomyocytes were cultured in vitro, and then the injury model of H9 c2 cardiomyocytes was induced by deprivation of oxygen-glucose for 8 h, and rehabilitation of oxygen-glucose for 12 h. Cell viability was detected by tetrazolium(MTT) colorimetry. H9 c2 cardiomyocytes were observed with a fluorescence inverted microscope, and colorimetry was used to detect the level of lactate dehydrogenase(LDH) in cell supernatant. The results showed that luteolin could significantly protect the morphology of H9 c2 cells, significantly improve the survival rate of H9 c2 cardiomyocytes in OGD/R injury model, reduce the level of LDH in cell supernatant, inhibit cytotoxicity, and maintain the integrity of cell membrane. The inflammatory cytokines interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with the model group, luteolin can significantly reduce the release of IL-6 and TNF-α. Western blot was employed to detect the protein levels of ALOX5, ALOX12, and ALOX15 in lipoxygenase pathway. After luteolin intervention, the protein levels of ALOX5, ALOX12, and ALOX15 were significantly down-regulated compared with those in model group. These results indicate that luteolin can inhibit the release of IL-6 and TNF-α by restraining the activation of lipoxygenase pathway, thereby playing a protective role in the cardiomyocyte injury model induced by OGD/R.