Ad5-spike COVID-19 vaccine does not aggravate heart damage after ischemic injury

Abstract Hopes for a COVID-19 vaccine are now a reality. The spike protein of SARS-CoV-2, which majorly binds to the host receptor ACE2 for cell entry, is used by most of the COVID-19 vaccine candidates as a choice of antigen. ACE2 is highly expressed in the heart and is known to be protective in multiple organs. Interaction of spike with ACE2 has been reported to reduce ACE2 expression and affect ACE2-mediated signal transduction in the heart. However, whether a spike-encoding vaccine will aggravate myocardial damage after a heart attack via affecting ACE2 remains unclear. Therefore, for patients with or at risk of heart diseases, questions arise around the safety of the spike-based vaccines. Here, we demonstrate that ACE2 is up-regulated and protective in the injured mouse heart after myocardial ischemia/reperfusion (I/R). Infecting human cardiomyocyte, smooth muscle cells, endothelial cells, and cardiac fibroblasts with a recombinant adenovirus type-5 vectored COVID-19 vaccine expressing the spike protein (AdSpike) does not affect cell survival and cardiomyocyte function, whether the cells are subjected to hypoxia-reoxygenation injury or not. This observation is further confirmed in human engineered heart tissues. Furthermore, AdSpike vaccination does not aggravate heart damage in wild-type or humanized ACE2 mice after I/R injury, even at a dose that is ten-fold higher as used in human. This study represents the first systematic evaluation of the safety of a leading COVID-19 vaccine under a disease context and may provide important information to ensure maximal protection from COVID-19 in patients with or at risk of heart diseases.

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Abstract 103: Mechanistic Interplay Between Cardioprotection And Heart Regeneration Mediated By The ER-bound Transcription Factor Nrf1

Circulation Research ◽

10.1161/res.129.suppl_1.103 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Miao Cui ◽

Atmanli Ayhan ◽

Ning Liu ◽

Rhonda S Bassel-duby ◽

Eric N Olson

Keyword(s):

Transcription Factor ◽

Ischemia Reperfusion Injury ◽

Heart Diseases ◽

Ischemia Reperfusion ◽

Induced Pluripotent Stem Cell ◽

Redox Balance ◽

Mouse Heart ◽

Heart Regeneration ◽

Neonatal Cardiomyocytes ◽

Underlying Mechanisms

Cardiomyocyte loss is the underlying basis for a majority of heart diseases. Preventing cardiomyocytes from death (cardioprotection) and replenishing the lost myocardium (regeneration) are the central goals for heart repair. Although cardioprotection and heart regeneration have been traditionally thought to involve separate mechanisms, protection of cardiomyocytes from injury or disease stimuli is a prerequisite to any meaningful regenerative response. In our study, we sought to understand how neonatal cardiomyocytes cope with injury-induced stress to regenerate damaged myocardium and whether the underlying mechanisms could be leveraged to promote heart regeneration and repair in adults. Using spatial transcriptomic profiling, we visualized regenerative cardiomyocytes reconstituting damaged myocardium after ischemia, and found that they are marked by expression of Nrf1, an ER-bound stress responsive transcription factor. Single-nucleus RNA sequencing revealed that genetic deletion of Nrf1 prevented neonatal cardiomyocytes from activating a transcriptional program required for heart regeneration. Conversely, overexpression of Nrf1 protected the adult mouse heart from ischemia/reperfusion injury. Nrf1 also protected human induced pluripotent stem cell-derived cardiomyocytes from cardiotoxicity induced by the chemotherapeutic drug doxorubicin. The cardioprotective function of Nrf1 is mediated by a dual stress response mechanism involving activation of the proteasome and maintenance of redox balance. Taken together, our study uncovers a unique adaptive mechanism activated in response to injury that maintains the tissue homeostatic balance required for heart regeneration. Reactivating these mechanisms in the adult heart represents a potential therapeutic approach for cardiac repair.

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Influence of Low Concentrations of Carbon Monoxide on Metabolism of Isolated Heart under Conditions of Ischemia-Reperfusion

Ukraïnsʹkij žurnal medicini bìologìï ta sportu ◽

10.26693/jmbs06.06.230 ◽

2021 ◽

Vol 6 (6) ◽

pp. 230-238

Author(s):

S. P. Beschasnyi ◽

◽

Ye. M. Lysenko

Keyword(s):

Carbon Monoxide ◽

Isolated Heart ◽

Ischemia Reperfusion ◽

Volumetric Flow Rate ◽

Myocardial Damage ◽

Mouse Heart ◽

Triphenyltetrazolium Chloride ◽

Isolated Hearts ◽

Low Concentrations ◽

Vasoconstrictor Effect

The purpose of the study was to determine the effect of different concentrations of carbon monoxide on the metabolism of isolated mice hearts. Materials and methods. To elucidate the effect of low concentrations of carbon monoxide on the myocardium, we performed retrograde perfusion of isolated hearts of laboratory mice with Krebs-Henseleit solution, which was saturated with carbon monoxide for 5, 10, and 30 minutes. We then determined how different concentrations of carbon monoxide affected coronary volumetric flow rate, myocardial glucose and calcium uptake, creatinine release, and aspartate aminotransferase release. During perfusion, R-wave amplitude and R-R interval were measured using an electrocardiograph. To determine the effect of ischemia on the heart muscle during perfusion with solutions of different concentrations, we measured the area of the affected myocardium after staining with 2,3,5-triphenyltetrazolium chloride. Results and discussion. After these studies, it was found that different concentrations of carbon monoxide had a dose-dependent effect on the isolated mouse heart. However, the dependence of the effects does not follow the pattern «lowest concentration – lowest effect». At the same time, an increase in concentration did not mean an increase in adverse effects on the myocardium. Even on the contrary, the smallest concentration led to increased signs of ischemic myocardial damage. In particular, the use of the solution, through which carbon monoxide was passed for 5 minutes, caused vasoconstrictor effect during perfusion. At the end of reperfusion, vasoconstrictor effect was observed after using a solution through which carbon monoxide was passed for 10 minutes. Increased glucose uptake was observed in the group with 30-minute carbon monoxide permeation against the background of the minimal myocardial creatinine release. In this group there was also a decrease in Ca2+ loss at the beginning of reperfusion (immediately after ischemia). The above phenomenon explains the least degree of ischemic myocardial damage in the isolated mouse heart. The obtained data should be expanded. Since it is difficult to accurately determine the dose of carbon monoxide, then the use of donor compounds is promising. Such compounds include CORM-2 and CORM-3. Under physiological conditions, they decompose in a controlled manner, releasing a specific amount of carbon monoxide. Conclusion. The obtained results indicate that at different concentrations of carbon monoxide can differently influence different structures of cardiomyocyte: at one concentration it binds to calcium channels, other concentrations influence ion channels of plasma membrane, which can explain all these dependencies

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Abstract 112: Microrna-532-5p Protects the Mouse Heart Against Myocardial Infarction by Repressing a Positive Regulator of Endothelial-to-mesenchymal Transition, Prss23

Circulation Research ◽

10.1161/res.119.suppl_1.112 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Ahmed S Bayoumi ◽

Jian P Teoh ◽

Il-man Kim

Keyword(s):

Myocardial Infarction ◽

Endothelial Cell ◽

Heart Diseases ◽

Ischemia Reperfusion ◽

Locked Nucleic Acid ◽

Mouse Heart ◽

Intramyocardial Injection ◽

Mesenchymal Transition ◽

Endothelial To Mesenchymal Transition

Myocardial infarction (MI) leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after MI has been identified as a critical event in this process. MicroRNAs (miRs), negative regulators of gene expression, have emerged as important players in heart diseases including MI. We previously showed that miR-532-5p (miR-532) is up-regulated by the β-arrestin-biased β-blocker carvedilol (Carv), which activates protective pathways in the heart independent of G protein-mediated second messenger signaling (Figure A-B). Here, we test whether a β2-adrenergic receptor (β2AR)/β-arrestin-responsive miR, miR-532 functions as a cardioprotective miR against MI. Using cultured cardiac endothelial cell (CEC) and in vivo approaches in mice, we demonstrate that knockdown of miR-532 induces CEC apoptosis when subjected to simulated ischemia-reperfusion. In vivo blocking of miR-532 via intramyocardial injection of its locked nucleic acid-anti-miR results in post-MI abnormalities in cardiac function and remodeling. Mechanistically, miR-532 promotes cardiac vascularization after MI in part by repressing a predicted target, Prss23 and subsequently activating endothelial cell markers such as CD-31 (Figure C). Prss23, which is a vascular protease and modulates Snail transcription to promote endothelial-to-mesenchymal transition (EndoMT), is also down-regulated by Carv in mouse hearts. In conclusion, our novel findings indicate that miR-532-Prss23 axis acts as an important regulator of CEC function during MI and can be suitable for therapeutic intervention in the setting of ischemic heart disease.

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Reperfusion injury is not affected by blockade of P-selectin in the diabetic mouse heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.277.2.h763 ◽

1999 ◽

Vol 277 (2) ◽

pp. H763-H769 ◽

Cited By ~ 27

Author(s):

Steven P. Jones ◽

Wesley G. Girod ◽

D. Neil Granger ◽

Anthony J. Palazzo ◽

David J. Lefer

Keyword(s):

At Risk ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Myocardial Necrosis ◽

Coronary Artery Occlusion ◽

Diabetic Mouse ◽

Type Ii Diabetes Mellitus ◽

Mouse Heart ◽

Area At Risk

We examined the mechanisms responsible for myocardial ischemia-reperfusion (MI-R) injury in a well-characterized animal model of type II diabetes mellitus. Diabetic ( db/db) mice and their littermate nondiabetic controls were subjected to 30 min of left anterior descending coronary artery occlusion and 2 h of reperfusion. Diabetic and nondiabetic mice experienced similar-sized areas at risk per left ventricle: 50.4 ± 2.0 and 53.4 ± 4.1%, respectively. However, myocardial necrosis (percentage of area at risk) was significantly greater ( P < 0.001) in diabetic than in nondiabetic animals: 56.3 ± 2.8 and 27.2 ± 3.1%, respectively. Histological examination revealed significantly ( P < 0.05) more neutrophils (PMNs) in the diabetic than in the nondiabetic hearts. Coronary endothelial expression of P-selectin was determined using radiolabeled monoclonal antibodies (MAbs). MI-R elicited a more intense ( P < 0.05) upregulation of P-selectin in the ischemic zone of diabetic than of nondiabetic myocardium: 0.310 ± 0.034 and 0.161 ± 0.042 μg MAb/g tissue. Immunoneutralization of P-selectin (RB40.34) reduced PMN accumulation in the diabetic myocardium but failed to reduce the extent of myocardial necrosis. Conversely, administration of an MAb directed against CD18 (GAME46) reduced PMN infiltration and attenuated the infarct size in the diabetic hearts. These results suggest that the diabetic heart is more susceptible to ischemia-reperfusion injury than normal myocardium. Furthermore, the mechanism of this injury may not be critically dependent on P-selectin in diabetic hearts.

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Remifentanil preconditioning alleviates myocardial ischemia/reperfusion injury in rats via activating Jagged-1/Notch signaling pathway

Bioscience Reports ◽

10.1042/bsr20210534 ◽

2021 ◽

Author(s):

Dejun Cao ◽

Shaoxing Liu ◽

Mengchang Yang ◽

Keyu Xie ◽

Zhuo Zheng ◽

...

Keyword(s):

Notch Signaling ◽

Reperfusion Injury ◽

Signaling Pathway ◽

Ischemia Reperfusion Injury ◽

Heart Diseases ◽

Ischemia Reperfusion ◽

Myocardial Damage ◽

Notch Signaling Pathway ◽

Protective Effects ◽

Myocardial Ischemia Reperfusion

Ischemic heart diseases have emerged as great threats to human health. Nowadays, restoration of cardiac blood flow supply is widely regarded as a feasible treatment choice for ischemic heart diseases; however, this intervention would contradictorily elicit reperfusion injury. Recently, myocardial ischemia/reperfusion injury (MI/RI) has aroused widespread public concerns. Remifentanil, an ultra-short acting opioid analgesic, is frequently used for surgical anesthesia. Previous studies have demonstrated the cardioprotective effects of remifentanil preconditioning in clinical practice and in vitro experimental models; however, its exact mechanisms remain largely unclear. This study aimed to further evaluate the protective effects of remifentanil preconditioning against MI/RI and elucidate the potential molecular mechanisms. Rat models of MI/RI were successfully established via ligation of left anterior descending coronary artery for 30 minutes and restoration of blood flow for 2 hours. Herein, animal experiments displayed that remifentanil preconditioning could alleviate myocardial damage in rat models of MI/RI. Consistently, cell model experiments implied that remifentanil preconditioning attenuated hypoxia/reoxygenation exposure-induced injury in rat cardiomyocytes. Moreover, our findings verified the involvement of Notch signaling pathway in the protective effects of remifentanil preconditioning. In addition, mechanistic studies revealed that remifentanil preconditioning could up-regulate Jagged-1 expression and that Jagged-1 mediated the cardioprotective effects of remifentanil preconditioning through activating Notch signaling pathway. Taken together, our data indicate that remifentanil preconditioning ameliorates myocardial damage in rat MI/RI models via Jagged-1-mediated Notch signaling pathway activation. Thus, this study may offer some novel clues for understanding the cardioprotective mechanisms of remifentanil preconditioning against MI/RI.

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Abstract 350: Toll-like Receptor 2 Activation, Especially In Circulating Leukocytes, Mediates Myocardial Ischemia/Reperfusion Injury In Mice

Circulation ◽

10.1161/circ.118.suppl_18.s_288 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Fatih Arslan ◽

Gerard Pasterkamp ◽

Leo Timmers ◽

Ben van Middelaar ◽

Pieter A Doevendans ◽

...

Keyword(s):

At Risk ◽

Myocardial Ischemia ◽

Infarct Size ◽

Ischemia Reperfusion ◽

Myocardial Damage ◽

Blue Dye ◽

Tlr2 Expression ◽

Area At Risk ◽

Saline Treatment ◽

Myocardial Ischemia Reperfusion

OBJECTIVES. Myocardial ischemia/reperfusion (MI/R) injury is characterized by an inflammatory response through NF-κB, increase of infarct size and worsening of cardiac function. Toll-like receptors (TLRs) are part of innate immunity and initiate the same inflammatory reaction. Here we studied in vivo to what extent TLR2 is involved in myocardial damage and what the relative contribution is of TLR2 expression in parenchymal cells and leukocytes to myocardial damage during MI/R in mice. METHODS. C57Bl6J mice underwent 30 minutes ischemia - 24 hours of reperfusion. Four experimental groups were studied: TLR2 knock-out (TLR2 KO) mice (n=10), saline treated wild-type (WT) mice (n=10), generated chimeric WT mice with TLR2 KO bone marrow (BLOOD KO; n=7) and chimeric TLR2 KO mice with WT hematopoietic cells (ORGAN KO; n=7). Saline was administered via the tail vein 5 minutes prior to reperfusion. After 24 hours, the LCA was ligated again at the level marked by the suture left in place. Mice were terminated and infarct size (IS) was measured as a percentage of the area at risk (AAR) using 4% Evans’ blue dye injection in the aortic root and triphenyltetrazolium chloride (TTC) staining (fig. 1). Data are presented as Mean±SEM. RESULTS. The AAR as percentage of the left ventricle was similar between groups: TLR2 KO 41%, saline 41%, Blood KO 41%, Organ KO 42%. Saline treatment resulted in 34.5%±3.3 of infarction, whereas in TLR2 KO mice infarct size decreased to 23.0%±2.9 (p=0.029 vs. saline). Infarct size in BLOOD KO mice was 22.9%±2.7 (p=0.024 vs. saline), while ORGAN KO mice had 33.9%±3.2 (p=0.998 vs. saline) of infarction within the area at risk (fig. 2). CONCLUSION. TLR2 deficiency significantly reduces infarct size with ~33% compared to saline treatment in mice after 30 minutes of ischemia and 24 hours of reperfusion. We show for the first time that TLR2 expression in circulating leukocytes plays an important role in infarction after MI/R injury. Systemic inhibition of TLR2 may be a potential therapeutic target in the treatment of patients with acute myocardial infarction.

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Expressing a truncated SARS-COV-2 spike protein in mouse heart induces cardiac hypertrophy

European Heart Journal ◽

10.1093/eurheartj/ehab724.3287 ◽

2021 ◽

Vol 42 (Supplement_1) ◽

Author(s):

S Negron ◽

C Kessinger ◽

Z Lin

Keyword(s):

Immune Responses ◽

Cardiac Hypertrophy ◽

Systolic Function ◽

Heart Diseases ◽

Cardiac Injury ◽

Innate Immune ◽

Host Cells ◽

Spike Protein ◽

Mouse Heart ◽

Innate Immune Responses

Abstract Cardiac injury is common in hospitalized and non-hospitalized COVID-19 patients, for which systemic inflammation stress is one of the causes (Topol, 2020). Although rare, COVID-19 cases that SARS-CoV-2 infecting cardiomyocytes (CMs) have been reported. In vitro, SARS-CoV-2 infection of human induced-pluripotent-cells derived CMs triggered innate immune responses and induced apoptosis (Bojkova et al., 2020; Chen et al., 2020). Therefore, the current literature indicates that the heart is attacked by SARS-CoV-2 directly or indirectly; however, the underlying mechanism remains largely unknown. Involved in the pathogenesis of heart diseases, Toll-like receptors (TLR) are a family of pattern recognition receptors that sense the pathogenic stimuli and signal the cardiac residential cells to cope with harsh conditions (Yu and Feng, 2018). Among the best characterized TLR signaling pathways is TLR4/NF-kB axis (Lu et al., 2008), in which TLR4 convey the danger signals through its down stream kinases, such as TAK1 and TBK1, to activate NF-kB. SARS-CoV-2 Spike protein is well known for its role of mediating virus entry into host cells, but its immunogenic role has not been clearly defined. Recently, we have found that SARS-CoV-2 Spike protein directly interacts with TLR4 and activates NF-kB transcriptional activity. Pharmaceutically blocking either TBK1 or TAK1 attenuates Spike protein's immunogenic activity. To pinpoint Spike protein's role in the heart, we generated an AAV to specifically express a truncated Spike protein (S1-TM) in the CMs. Our data show that expressing S1-TM in CMs induces cardiac hypertrophy and decreases heart systolic function in mice. On the molecular level, Spike protein increases RelA (p65 subunit of the NF-kB complex) and activates the expression of pro-inflammatory cytokine genes. In summary, our study suggests that Spike protein directly interacts with TLR4 to trigger innate immune signaling, and that Spike protein induced CM innate immune responses might be one of the underlying mechanisms of cardiac injury in COVID-19. FUNDunding Acknowledgement Type of funding sources: Other. Main funding source(s): Masonic Medical Research Institute

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The neuro-cardiac junction defines an extracellular microdomain required for neurotrophic signaling

European Heart Journal ◽

10.1093/ehjci/ehaa946.3589 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

M Mongillo ◽

M Franzoso ◽

V Prando ◽

L Dokshokova ◽

A Di Bona ◽

...

Keyword(s):

Culture Medium ◽

Heart Diseases ◽

Sympathetic Neurons ◽

Confocal Imaging ◽

Public Institution ◽

Mdx Mice ◽

Mouse Heart ◽

Funding Source ◽

Neurotrophic Signaling

Abstract Background Sympathetic neurons (SNs) innervate the myocardium with a defined topology that allows physiological modulation of cardiac activity. Neurotrophins released by cardiac cells control SN viability and myocardial distribution, which are impaired in heart diseases with reduced (e.g. heart failure) or heterogenous sympathetic stimulation (e.g. arrhythmias). We previously demonstrated that SNs interact directly with cardiomyocytes (CMs) at neuro-cardiac junctions (NCJ), and such structured contact sites allow neurons to efficiently activate β-adrenoceptors on the myocyte membrane. Aims We here asked whether NCJs are functional for retrograde (myocyte to neuron) neurotrophic signaling. Methods and results Electron microscopy and immunofluorescence on mouse heart slices and SN/CM co-cultures showed that the NGF receptor, TrkA, is preferentially found in correspondence of the NCJ. Consistently, neurons taking structured contact with CMs showed fast TrkA activation and its retrograde transport to the soma, which was monitored using live confocal imaging in cells expressing TrkA-RFP. In accord with NGF dependent effects, CM-contacted SN showed larger synaptic varicosities and did not require NGF supplementation in the culture medium. In support that NGF locally released at NCJs sustains SN viability, the neurotrophin concentration in the culture medium was 1.61 pg/mL, and did not suffice to maintain neuronal viability, which was also perturbed (66% decrease of neuronal density) by silencing NGF expression in CMs. These results support that the NCJ is essential for intercellular neurotrophin signaling. Consistently, by applying competitive inhibition of TrkA with increasing doses of K252a, we estimated NGF concentration at the contact site to be about 1000-fold higher than that released by CM in the culture medium. To seek for the structural determinants of the NCJ, we focused on dystrophin, based on the finding that the protein accumulates on the CM membrane portion contacted by SNs, as observed in mouse heart slices, and co-cultured CMs. In support of a role of CM-expressed dystrophin in neurotrophic signaling, hearts from dystrophin-KO (mdx) mice showed 74.36% decrease of innervation, with no significant changes of NGF expression. In line with the purported role of NCJs, in co-cultures between wild type SNs and mdx CMs, TrkA activation (TrkA movements toward SN soma (%): WTCM-WTSN=18±4; MDXCM-WTSN= 12±3; p<0,05) and neuronal survival were reduced. Conclusions Taken together, our results suggest that NGF-dependent signaling to SNs requires a direct and specialized interaction with myocytes, and that loss of dystrophin at the CM membrane impairs retrograde signaling to the neurons leading to cardiac sympathetic dys-innervation. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): University of Padova

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Overexpression of miR-431 attenuates hypoxia/reoxygenation-induced myocardial damage via autophagy-related 3

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa154 ◽

2020 ◽

Author(s):

Kang Zhou ◽

Yan Xu ◽

Qiong Wang ◽

Lini Dong

Keyword(s):

Cell Viability ◽

Cell Apoptosis ◽

Myocardial Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Myocardial Damage ◽

Protective Role ◽

Human Cardiomyocytes ◽

Hypoxia Reoxygenation

Abstract Myocardial injury is still a serious condition damaging the public health. Clinically, myocardial injury often leads to cardiac dysfunction and, in severe cases, death. Reperfusion of the ischemic myocardial tissues can minimize acute myocardial infarction (AMI)-induced damage. MicroRNAs are commonly recognized in diverse diseases and are often involved in the development of myocardial ischemia/reperfusion injury. However, the role of miR-431 remains unclear in myocardial injury. In this study, we investigated the underlying mechanisms of miR-431 in the cell apoptosis and autophagy of human cardiomyocytes in hypoxia/reoxygenation (H/R). H/R treatment reduced cell viability, promoted cell apoptotic rate, and down-regulated the expression of miR-431 in human cardiomyocytes. The down-regulation of miR-431 by its inhibitor reduced cell viability and induced cell apoptosis in the human cardiomyocytes. Moreover, miR-431 down-regulated the expression of autophagy-related 3 (ATG3) via targeting the 3ʹ-untranslated region of ATG3. Up-regulated expression of ATG3 by pcDNA3.1-ATG3 reversed the protective role of the overexpression of miR-431 on cell viability and cell apoptosis in H/R-treated human cardiomyocytes. More importantly, H/R treatments promoted autophagy in the human cardiomyocytes, and this effect was greatly alleviated via miR-431-mimic transfection. Our results suggested that miR-431 overexpression attenuated the H/R-induced myocardial damage at least partly through regulating the expression of ATG3.

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