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The high mobility group box 1 (HMGB1) is a potential biomarker and therapeutic target in various human diseases. However, a systematic, comprehensive pan-cancer analysis of HMGB1 in human cancers remains to be reported. This study analysed the genetic alteration, RNA expression profiling and DNA methylation of HMGB1 in more than 30 types of tumours. It is worth noting that HMGB1 is overexpressed in malignant tissues, including lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), pancreatic adenocarcinoma (PAAD) and thymoma (THYM). Interestingly, there is a positive correlation between the high expression of HMGB1 and the high survival prognosis of THYM. Finally, this study comprehensively evaluates the genetic variation of HMGB1 in human malignant tumours. As a prospective biomarker of COVID-19, the role that HMGB1 plays in THYM is highlighted.

Atherosclerosis is one of the primary causes that lead to cardiovascular disease. LncRNAs have been regarded as key modulators in many pathological processes. The study aims to identify the regulatory role of LncRNA fetal-lethal noncoding developmental regulatory RNA (FENDRR) in atherosclerosis. Cell viability proliferation, cell cycle and cell apoptosis were evaluated by Cell Counting Kit-8 (CCK-8) assay flow cytometric analysis and western blot analysis. Quantitative real-time PCR (qRT-PCR) was carried out to determine FENDRR expression in PDGF-BB/TNF-αinduced VSMCs. Levels of TNF-α, IL-1, IL-6, MCP-1 and ICAM-1 were investigated by enzyme-linked immunosorbent assay (ELISA). The results showed that cell viability was enhanced and FENDRR expression was downregulated after VSMCs were induced by platelet derived growth factor BB (PDGF-BB) or tumor necrosis factor a (TNF-α). Cell proliferation was inhibited by FENDRR overexpression in a time-dependent manner in PDGF-BB or TNF-αinduced VSMCs. Moreover, FENDRR overexpression blocked cell cycle, suppressed the generations of TNF-α, IL-1, IL-6, MCP-1 and ICAM-1, and facilitated cell apoptosis in VSMCs induced by PDGF-BB or TNF-α. These findings indicate the functional role of LncRNA FENDRR in atherosclerosis that attenuates cell proliferation and accelerates cell apoptosis.

Recent studies have revealed that exercise has myocardial protective effects, but the exact mechanism remains unclear. Studies have increasingly found that peptides play a protective role in myocardial ischaemia‐reperfusion (I/R) injury. However, little is known about the role of exercise‐induced peptides in myocardial I/R injury. To elucidate the effect of exercise‐induced peptide EIP‐22 in myocardial I/R injury, we first determined the effect of EIP‐22 on hypoxia/reperfusion (H/R)‐ or H2O2‐induced injury via assessing cell viability and lactate dehydrogenase (LDH) level. In addition, reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) was assessed by fluorescence microscope. Meanwhile, Western blot and TUNEL methods were used to detect apoptosis level. Then, we conducted mice I/R injury model and verified the effect of EIP‐22 by measuring cardiac function, evaluating heart pathology and detecting serum LDH, CK‐MB and cTnI level. Finally, the main signalling pathway was analysed by RNA‐seq. In vitro, EIP‐22 treatment significantly improved cells viabilities and MMP and attenuated the LDH, ROS and apoptosis level. In vivo, EIP‐22 distinctly improved cardiac function, ameliorated myocardial infarction area and fibrosis and decreased serum LDH, CK‐MB and cTnI level. Mechanistically, JAK/STAT signalling pathway was focussed by RNA‐seq and we confirmed that EIP‐22 up‐regulated the expression of p‐JAK2 and p‐STAT3. Moreover, AG490, a selective inhibitor of JAK2/STAT3, eliminated the protective roles of EIP‐22. The results uncovered that exercise‐induced peptide EIP‐22 protected cardiomyocytes from myocardial I/R injury via activating JAK2/STAT3 signalling pathway and might be a new candidate molecule for the treatment of myocardial I/R injury.

Background and objective: As a result of the pandemic, family physicians face the additional challenge of navigating COVID-19. The aim of this study was to provide simulated training for best-practice management of COVID-19 presentations for residency program trainees in Shanghai, China. Methods: A simulated suspected COVID-19 case was designed on the basis of a real patient. The simulation included: pre-and post-simulation surveys, a PowerPoint presentation, simulation practice, debriefing and reflection. Improvement in survey outcomes was assessed using a paired t-test. Results: A total of 25 trainees participated in the simulation, consisting of first-, second-and third-year family medicine residents. Significant improvement was observed in their knowledge of COVID-19, and sub-analysis showed that all three grades of residents improved their knowledge significantly. Ninety-six per cent of participants believed the simulation was very helpful. Discussion: The simulation scenario improves crisis management skills for family physicians managing the high risk of transmission of respiratory infectious diseases. Higher-order learning outcomes will be explored in future training programs.

Recent studies have revealed that proper exercise can reduce the risk of chronic disease and is beneficial to the body. Peptides have been shown to play an important role in various pathological processes, including cardiovascular diseases. However, little is known about the role of exercise-induced peptides in cardiovascular disease. We aimed to explore the function and mechanism of TAG-23 peptide in reperfusion injury and oxidative stress. Treatment with TAG-23 peptide significantly improved cell viability, the mitochondrial membrane potential, and ROS levels and reduced LDH release, the apoptosis rate and caspase 3 activation in vitro. In vivo, TAG-23 ameliorated MI and heart failure induced by I/R or DOX treatment. Pull-down assays showed that TAG-23 can bind to PKG . The TAG-23-PKG complex inhibited PKG degradation through the UPS. We also identified cCbl as the E3 ligase of PKG and found that the interaction between these proteins was impaired by TAG-23 treatment. In addition, we provided evidence that TAG-23 mediated Lys48-linked polyubiquitination and subsequent proteasomal degradation. Our results reveal that a novel exercise-induced peptide, TAG-23, can inhibit PKG degradation by serving as a competitive binding peptide to attenuate the formation of the PKG–cCbl complex. Treatment with TAG-23 may be a new therapeutic approach for reperfusion injury. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00878-4.

Background Right-sided accessory pathway (RAP) ablation is sometimes challenging. Objective Our study aimed to demonstrate an alternative ablation approach to RAPs under the tricuspid valve, especially when the conventional ablation attempts at the atrial side failed. Methods Twelve patients with RAPs were enrolled, 8 of whom had previously failed ablation. With the help of a long sheath, the under-valve approach was attempted in 3 patients during tachycardia, in 2 patients during ventricular pacing, and in 7 patients during sinus rhythm. Three-dimensional electroanatomic mapping was performed in 3 patients during their repeat procedures. Results The acute outcomes of the procedures in all patients were successful. Patients were free of tachycardia or recurrence of accessory pathway conduction during a median follow-up of 12.5 months (range 7–45 months). No complications were found during the procedure or follow-up period. Conclusion Radiofrequency ablation under the tricuspid valve to eliminate RAPs is feasible because of its stable contact and the accurate ablation of the ventricular insertion site. It provides an alternative approach to tough RAP ablation.

In previous studies, we have demonstrated that long noncoding RNA uc.4 may influence the cell differentiation through the TGF-β signaling pathway, suppressed the heart development of zebrafish and resulting cardiac malformation. DNA methylation plays a significant role in the heart development and disordered of DNA methylation may cause disruption of control of gene promoter. In this study, methylated DNA immunoprecipitation was performed to identify the different expression levels of methylation regions. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were also performed to identify the possible biological process and pathway that uc.4 may join, associated with Rap1 signaling pathway, gonadotropin-releasing hormone signaling pathway, and Calcium signaling pathway. We found that the distribution of differentially methylated regions peaks was mainly located in intergenic and intron regions. Altogether, our result showed that differentially methylated genes are significantly expressed in uc.4-overexpression cells, providing valuable data for further exploration of the role of uc.4 in heart development.

Oxidative stress serves a key role in doxorubicin (DOX)-induced cardiotoxicity. The peptide Szeto-Schiller (SS)31 is an efficacious antioxidant with the capacity to reduce mitochondrial reactive oxygen species (ROS) levels and scavenge free radicals. Although SS31 is involved in the pathophysiological process of various cardiovascular diseases, the role of SS31 in DOX-induced cardiotoxicity remains unclear. To explore the effects of SS31 in DOX-induced cardiotoxicity, the present study first constructed DOX-induced cardiotoxicity models, in which H9c2 cells were incubated with 1 μM DOX for 24 h and C57BL/6 mice were administered DOX (20 mg/kg cumulative dose). The results of various assays in these models demonstrated that SS31 exhibited a cardioprotective effect in vitro and in vivo by attenuating the level of ROS, stabilizing the mitochondrial membrane potential and ameliorating myocardial apoptosis as well as fibrosis following treatment with DOX. Mechanistically, the results of the present study revealed that the p38 MAPK signaling pathway was inhibited by SS31 in DOX-treated H9c2 cells, which was associated with the cardioprotective function of SS31. In addition, P79350, a selective agonist of p38 MAPK, reversed the protective effects of SS31. Taken together, these results demonstrated the effects of SS31 on ameliorating DOX-induced cardiotoxicity and indicated its potential as a drug for the treatment of DOX-induced cardiotoxicity.

Vascular endothelial dysfunction is a vital pathological change in hypertension, which is mainly caused by apoptosis and oxidative stress injury of vascular endothelial cells. Peptidomics is a method for the direct analysis of small bioactive peptides in various biological samples using liquid chromatography‑mass spectrometry (MS)/MS. Given the advantages of the low molecular weight, optimum targeting and easy access to cells, peptides have attracted extensive attention in the field of drug research. However, to the best of our knowledge, little is currently known regarding the role of peptides in vascular endothelial injury. In order to investigate the peptides involved in vascular endothelial protection, MS was used to analyze the peptide profiles in the supernatant of human umbilical vein endothelial cells (HUVECs) stimulated by Ang II. The results revealed that 211 peptides were identified, of which six were upregulated and 13 were downregulated when compared with the control group. Subsequently, the present study analyzed the physical and chemical properties and biological functions of identified peptides by bioinformatics, and successfully screened a peptide (LLQDSVDFSLADAINTEFK) named VMP‑19 that could alleviate the apoptosis and oxidative stress injury of HUVECs induced by Ang II. In conclusion, to the best of our knowledge, the present study was the first to use peptidomics to analyze the peptide profiles of supernatant secreted by HUVECs, and revealed that the novel peptide VMP‑19 could protect HUVECs from apoptosis and oxidative stress injury. The results of the present study could provide novel insights into treatment strategies for hypertension.

Background: It has been acknowledged that endocrine activity is associated with the function of multiple systems in vivo. The apelin-13 peptide has been demonstrated to play a crucial role in physiological and pathological processes. However, the function of apelin-13 peptide in doxorubicin (DOX)-induced cardiotoxicity is unknown. Methods: We explored the function and mechanism of apelin-13 peptide in apoptosis and oxidative stress by cell counting kit-8 (CCK-8) assay, trypan blue staining, TUNEL, lactate dehydrogenase (LDH), mitochondrial membrane potential assay kit with JC-1 (JC-1) and western blot in vitro. Then we verified the effect of apelin-13 in vivo by detecting serum apelin-13, CKMB, LDH, cardiac troponin I (cTnI) and cardiac troponin T (cTnT). EF, FS and LVEDs were used to identify the structural modification by echocardiography. Sirius red staining and HE staining assay were used to detecting the myocardial fibers alteration under apelin-13 treatment.Results: Treatment with apelin-13 peptide significantly enhanced cell viability, mitochondrial membrane potential, but reduced LDH release, rate of apoptotic cells and activation of caspase-3 in vitro. In mice, apelin-13 alleviated the heart dysfunction induced by DOX. 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) inhibited the effect of extracellular signal-related kinases (ERK), phosphatidylinositol 3 kinases (PI3K) and protein kinase B (AKT) proteins phosphorylation expression compared with DOX.Conclusion: The apelin-13 and apelin receptor (APJ) interaction on the cell membrane inhibits apoptosis through the ERK/mitogen-activated protein kinase (MAPK) and PI3K/AKT signaling pathways. Our research gives a first glimpse on the biological function and mechanism of apelin-13 on cardiotoxicity.

This study aimed to investigate the regulatory role of differentially-expressed circular RNAs (circRNAs) in mouse cardiomyocytes during doxorubicin (DOX)-induced cardiotoxicity.Two groups of mice were injected with equal volumes (0.1 mL) of normal saline and DOX. Mouse heart tissue was isolated and digested for total RNA extraction and then subjected to next-generation RNA-sequencing. Expression profiles of circRNAs and circRNA-miRNA-mRNA networks were also constructed. Overall, 48 upregulated and 16 downregulated circRNAs were found to be statistically significant (p 0.05) in the DOX-injected group. Bioinformatics analysis revealed several potential biological pathways that might be related to apoptosis caused by DOX-induced cardiotoxicity. In addition, using qRT-PCR, we found that a circRNA coded by the Arhgap12 gene, termed circArhgap12, was upregulated in the mouse heart tissue upon DOX intervention. CircArhgap12 enhanced apoptotic cell rate, as assessed using terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, and increased reactive oxygen species and malondialdehyde release as well as superoxide dismutase and caspase-3 activation. Using a luciferase reporter assay, we found that circArhgap12 could sponge miR-135a-5p. In rat primary cardiomyocytes, we found that si-circArhgap12 promoted apoptosis and oxidative stress by sponging the miR-135a-5p inhibitor. Using bioinformatics analysis and luciferase reporter assay, we found that miR-135a-5p might have a potential target site for ADCY1 mRNA.Our research demonstrated that the expression profile of circRNAs was modified significantly and that circArhgap12 might play a competitive role among endogenous RNAs in mouse cardiomyocytes during DOX-induced cardiotoxicity.Our study may provide a preliminary understanding of DOX-induced cardiotoxicity modulated by circRNA and its competing endogenous RNAs network.

Doxorubicin (DOX) is limited due to dose-dependent cardiotoxicity. Peptidomics is an emerging field of proteomics that has attracted much attention because it can be used to study the composition and content of endogenous peptides in various organisms. Endogenous peptides participate in various biological processes and are important sources of candidates for drug development. To explore peptide changes related to DOX-induced cardiotoxicity and to find peptides with cardioprotective function, we compared the expression profiles of peptides in the hearts of DOX-treated and control mice by mass spectrometry. The results showed that 236 differential peptides were identified upon DOX treatment, of which 22 were upregulated and 214 were downregulated. Next, we predicted that 31 peptides may have cardioprotective function by conducting bioinformatics analysis on the domains of each precursor protein, the predicted score of peptide biological activity, and the correlation of each peptide with cardiac events. Finally, we verified that a peptide (SPFYLRPPSF) from Cryab can inhibit cardiomyocyte apoptosis, reduce the production of reactive oxygen species, improve cardiac function, and ameliorate myocardial fibrosis in vitro and vivo. In conclusion, our results showed that the expression profiles of peptides in cardiac tissue change significantly upon DOX treatment and that these differentially expressed peptides have potential cardioprotective functions. Our study suggests a new direction for the treatment of DOX-induced cardiotoxicity.

Objectives Oxidative stress plays an important role in myocardial ischemia-reperfusion (I/R) injury. And pNaKtide is known to inhibit Na/K-ATPase/Src/reactive oxygen species (ROS) amplification signaling. Accordingly, we aimed to investigate the effect of pNaKtide on myocardial I/R injury. Methods We first determine the effect of pNaKtide on hypoxia- or cobalt chloride-induced injury in embryonic heart-derived H9c2 cells via measuring lactate dehydrogenase (LDH) level and trypan blue stain assay. In addition, TUNEL stain assay and western blot analysis of cleaved-PARP and cleaved-caspase3 were performed to detect apoptosis level. Meanwhile, ROS accumulation was assessed by dichlorofluorescin diacetate (DCFH-DA) assay. Then we conducted cell counting kit-8 (CCK-8) and flow cytometry to examine cell proliferation and cell cycle respectively. We next generated rat I/R model and determined the effect of pNaKtide by measuring serum LDH and evaluating heart pathology. At last, the activities of Src and ERK1/2 were examined via western blot to clarify molecular mechanism. Results In vitro, pNaKtide exposure significantly attenuated the H9c2 cells death and ROS accumulation induced by hypoxia or cobalt chloride. And no significant effect was detected on cell cycle and proliferation upon pNaKtide administration. In vivo, pNaKtide distinctly decreased serum LDH level and ameliorated I/R induced myocardial injury in the rats. Western blot analysis revealed pNaKtide decreased Src and ERK1/2 activities robustly. Conclusions The results provided evidence that pNaKtide exhibited cardioprotective effect against hypoxia-induced injury in vitro and in vivo. And pNaKtide might be a potential molecular for therapy of I/R related heart disease.

Neonatal mouse hearts have completely regenerative capability after birth, but the ability to regenerate rapidly lost after 7 days, the mechanism has not been clarified. Previous studies have shown that mRNA profile of adult mouse changed greatly compared to neonatal mouse. So far, there is no research of peptidomics related to heart regeneration. In order to explore the changes of proteins, enzymes, and peptides related to the transient regeneration, we used comparative petidomics technique to compare the endogenous peptides in the mouse heart of postnatal 1 and 7 days. In final, we identified 236 differentially expressed peptides, 169 of which were upregulated and 67 were downregulated in the postnatal 1 day heart, and also predicted 36 functional peptides associated with transient regeneration. The predicted 36 candidate peptides are located in the important domains of precursor proteins and/or contain the post-transcriptional modification (PTM) sites, which are involved in the biological processes of cardiac development, cardiac muscle disease, cell proliferation, necrosis, and apoptosis. In conclusion, for the first time, we compared the peptidomics profiles of neonatal heart between postnatal 1 day and postnatal 7 day. This study provides a new direction and an important basis for the mechanism research of transient regeneration in neonatal heart. J. Cell. Biochem. 118: 2828-2840, 2017. © 2017 Wiley Periodicals, Inc.

// Yu-Mei Chen 1, * , Hua Li 2, * , Yi Fan 2 , Qi-Jun Zhang 2 , Xing Li 2 , Li-Jie Wu 2 , Zi-jie Chen 2 , Chun Zhu 3 , Ling-Mei Qian 2 1 Department of Emergency, Zhongshan Hospital, Fudan University, Shanghai 200032, P.R. China 2 Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, P. R. China 3 Department of Pediatrics, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu 210004, P. R. China * These authors contributed equally to this work Correspondence to: Ling-Mei Qian, email: lmqian@njmu.edu.cn Chun Zhu, email: zhifangxibao@163.com Keywords: neonatal mouse, heart, regeneration, lncRNAs Received: August 15, 2016 Accepted: February 20, 2017 Published: March 03, 2017 ABSTRACT Previous studies have shown that mammalian cardiac tissue has a regenerative capacity. Remarkably, neonatal mice can regenerate their cardiac tissue for up to 6 days after birth, but this capacity is lost by day 7. In this study, we aimed to explore the expression pattern of long noncoding RNA (lncRNA) during this period and examine the mechanisms underlying this process. We found that 685 lncRNAs and 1833 mRNAs were differentially expressed at P1 and P7 by the next-generation high-throughput RNA sequencing. The coding genes associated with differentially expressed lncRNAs were mainly involved in metabolic processes and cell proliferation, and also were potentially associated with several key regeneration signalling pathways, including PI3K-Akt, MAPK, Hippo and Wnt. In addition, we identified some correlated targets of highly-dysregulated lncRNAs such as Igfbp3, Trnp1, Itgb6, and Pim3 by the coding-noncoding gene co-expression network. These data may offer a reference resource for further investigation about the mechanisms by which lncRNAs regulate cardiac regeneration.

Background: Acute Myocardial Infarction (AMI) is a life-threatening cardiovascular disease involving disruption of blood flow to the heart, consequent tissue damage, and sometimes death. Peptidomics, an emerging branch of proteomics, has attracted wide attention. Methods: A comparative peptidomic profiling was used to explore changes induced by acute ischemic-hypoxia in primary cultured neonatal rat myocardial cells. Analysis of six-plex tandem mass tag (TMT) labelled peptides was performed using nanoflow liquid chromatography coupled online with an LTQ-Orbitrap Velos mass spectrometer. Results: A total of 220 differentially expressed peptides originating from 119 proteins were identified, of which 37 were upregulated and 183 were downregulated in cardiomyocytes exposed to hypoxia/ischemia conditions. Many of the identified peptides were derived from functional domains of proteins closely associated with cardiomyocyte structure or AMI. Conclusion: Numerous peptides may be involved in process of AMI. These results pave the way for future functional studies of the identified peptides.

Congenital heart disease (CHD) is the most common type of birth defect, and the leading cause of fetal mortality. The long noncoding RNA (lncRNA) uc.457 is differentially expressed in cardiac tissue from patients with a ventricular septal defect; however, its role in cardiac development and CHD remains unknown. In the present study, the role of uc.457 in the differentiation and maturation of cardiomyocytes was investigated. Bioinformatics approaches were employed to analyze putative transcription factor (TF) regulation, histone modifications and the biological functions of uc.457. Subsequently, uc.457 overexpression and small interfering RNA‑mediated knockdown were performed to evaluate the functional role of the lncRNA in the dimethyl sulfoxide‑induced differentiation of P19 cells into cardiomyocytes. Bioinformatics analyses predicted that uc.457 binds to TFs associated with cardiomyocyte growth and cardiac development. Cell Counting Kit‑8 assays demonstrated that uc.457 overexpression inhibited cell proliferation, whereas knockdown of uc.457 enhanced the proliferation of differentiating cardiomyocytes. Additionally, reverse transcription‑quantitative polymerase chain reaction and western blot analyses revealed that overexpression of uc.457 suppressed the mRNA and protein expression of histone cell cycle regulation defective homolog A, natriuretic peptide A, cardiac muscle troponin T and myocyte‑specific enhancer factor 2C. Collectively, the results indicated that overexpression of uc.457 inhibited the differentiation and proliferation of cardiomyocytes, suggesting that dysregulated uc.457 expression may be associated with CHD.

In our previous study, the upregulation of microRNA (miR)-29c was identified in the mother of a fetus with a congenital heart defect. However, the functional and regulatory mechanisms of miR‑29c in the development of the heart remain to be elucidated. In the present study, the role and mechanism of miR‑29c inhibition in heart development were investigated in an embryonic carcinoma cell model. Inhibition of miR‑29c promoted proliferation, and suppressed the apoptosis and differentiation of P19 cells. It was also demonstrated that Wingless‑related MMTV integration site 4 (Wnt4) was a target of miR‑29c, determined using bioinformatic analysis combined with luciferase assays. The inhibition of miR‑29c stimulated the WNT4/β‑catenin pathway, promoting proliferation of the P19 cells, but suppressing their differentiation into cardiomyocytes. Furthermore, the inhibition of miR‑29c promoted the expression of B cell lymphoma‑2 and inhibited cell apoptosis. These results demonstrate the significance of miR‑29c in the process of cardiac development and suggest that miR-29c dysregulation may be associated with the occurrence of CHD. Thus, miR-29c may have therapeutic potential in the future.

Background: Ventricular septal defect (VSD) is one of the most common congenital heart diseases and to date the role of peptides in human amniotic fluid in the pathogenesis of VSD have been rarely investigated. Methods: To gain insight into the mechanisms of protein and peptides in cardiovascular development, we constructed a comparative peptidomic profiling of human amniotic fluid between normal and VSD fetuses using a stable isobaric labeling strategy involving tandem mass tag reagents, followed by nano liquid chromatography tandem mass spectrometry. Results: We identified and quantified 692 non-redundant peptides, 183 of which were differentially expressed in the amniotic fluid of healthy and VSD fetuses; 69 peptides were up regulated and 114 peptides were down regulated. These peptides were imported into the Ingenuity Pathway Analysis (IPA) and identified putative roles in cardiovascular system morphogenesis and cardiogenesis. Conclusion: We concluded that 35 peptides located within the functional domains of their precursor proteins could be candidate bioactive peptides for VSD. The identified peptide changes in amniotic fluid of VSD fetuses may advance our current understanding of congenital heart disease and these peptides may be involved in the etiology of VSD.

Compared to healthy controls, microRNA-29c (miR-29c) is highly expressed in the heart during progression towards ventricular septal defect. However, studies on miR-29c function in heart development are scarce. We investigated the role of miR-29c in P19 cell proliferation, apoptosis, and differentiation and the underlying mechanisms. We evaluated proliferation and cell cycle progression, detected morphological changes; apoptosis rate; BAX, BCL2, GATA binding protein 4 (GATA4), cardiac troponin T (cTnT), and myocyte enhancer factor 2C (MEF2C) expression; and caspase-3, -8, and -9 activity in miR-29c-overexpressing P19 cells, and investigated whether WNT4 was a miR-29c target. MiR-29c-overexpressing cells had decreased proliferation, increased G1 cells, and significantly higher apoptotic rate than the controls. Expression of the apoptosis-related BAX and BCL2 genes and caspase-3, -8, and -9 activity were significantly increased in miR-29c-overexpressing cells. Expression of the cardiac-specific markers GATA4, cTnT, and MEF2C revealed promoted differentiation in miR-29c-overexpressing cells compared to the controls. Luciferase assay confirmed that WNT4 is a miR-29c target. Wnt4 and β-catenin expression was decreased in miR-29c-overexpressing cells. MiR-29c inhibits P19 cell proliferation and promotes apoptosis and differentiation, possibly by suppressing Wnt4 signaling, whose deregulation contributes to congenital heart disease development.

In previous studies, we have demonstrated the function of uc.167 in the heart development. DNA methylation plays a crucial role in regulating the expression of developmental genes during embryonic development. In this study, the methylomic landscape was investigated in order to identify the DNA methylation alterations. Methylated DNA immunoprecipitation (MeDIP) was performed to examine the differences in methylation status of overexpressed uc.167 in P19 cells. GO and KEGG pathway analyses of differentially methylated genes were also conducted. We found that the distribution of differentially methylated regions (DMRs) peaks in different components of genome was mainly located in intergenic regions and intron. The biological process associated with uc.167 was focal adhesion and Rap1 signaling pathway. MEF2C was significantly decreased in uc.167 overexpressed group, suggesting that uc.167 may influence the P19 differentiation through MEF2C reduction. Taken together, our findings revealed that the effect of uc.167 on P19 differentiation may be attributed to the altered methylation of specific genes.

Acute myocardial infarction (AMI) is a life‑threatening disease and seriously influences patient quality of life. Long non‑coding RNAs (lncRNAs), an emerging class of non‑coding genes, have attracted attention in research, however, whether lncRNAs serve a function in acute ischemic hypoxia remains to be elucidated. In the present study, an lncRNA microarray was used to analyze differential lncRNA expression in acute ischemic hypoxia. A total of 323 lncRNAs were identified, 168 of which were upregulated and 155 of which were downregulated. Gene Ontology and Pathway analyses were also used to identify the potential functions of dysregulated lncRNAs; it was predicted that these dysregulated lncRNAs may contribute to the initiation of AMI. It was demonstrated that an lncRNA termed sloyfley may influence acute ischemic hypoxia through its neighboring gene Peg3, which has been linked to brain ischemia hypoxia. In summary, the present study identified numerous lncRNAs, which may provide further opportunities for the development of novel therapeutic strategies.

Background/Aims: PID1 was originally described as an insulin sensitivity relevance protein, which is also highly expressed in heart tissue. However, its function in the heart is still to be elucidated. Thus this study aimed to investigate the role of PID1 in the heart in response to hypertrophic stimuli. Methods: Samples of human failing hearts from the left ventricles of dilated cardiomyopathy (DCM) patients undergoing heart transplants were collected. Transgenic mice with cardiomyocyte-specific overexpression of PID1 were generated, and cardiac hypertrophy was induced by transverse aortic constriction (TAC). The extent of cardiac hypertrophy was evaluated by echocardiography as well as pathological and molecular analyses of heart samples. Results: A significant increase in PID1 expression was observed in failing human hearts and TAC-treated wild-type mouse hearts. When compared with TAC-treated wild-type mouse hearts, PID1-TG mouse showed a significant exacerbation of cardiac hypertrophy, fibrosis, and dysfunction. Further analysis of the signaling pathway in vivo suggested that these adverse effects of PID1 were associated with the inhibition of AKT, and activation of MAPK pathway. Conclusion: Under pathological conditions, over-expression of PID1 promotes cardiac hypertrophy by regulating the Akt and MAPK pathway.

Induced pluripotent stem cells (iPSc) hold significant promise for the development of cardiac regenerative therapy for myocardial infarction (MI). However, preclinical optimization and validation of large-animal models will be required before iPSc used clinically. Therefore, we aim to investigate the therapeutic potential of iPSc transplantation for MI and relative mechanisms in a post-infarcted swine model. Left anterior descending coronary artery was balloon-occluded after percutaneous transluminal angiography to generate MI (60-min no-flow ischemia). Animals were then divided into Sham, PBS control, and iPS experimental groups. The cardiac function and LV structural were assessed by dual-source computed tomography. Terminal deoxynucleotidyl nick end labeling, histology, and immunofluorescence were used to examine the effect of transplanted iPS cells on apoptosis, fibrosis, and hypertrophy. At 6 weeks, LV structural abnormality and cardiac dysfunction were less pronounced in iPSc group than in PBS group, and these improvements were accompanied by reduction of scar size. iPSc transplantation was associated with significant increase of vascular density and reduced myocardial apoptosis in the border zone of infarction, which was accompanied by the reduction in fibrosis degree. Moreover, proangiogenic and antiapoptotic factors were increased significantly in iPS group compared with PBS group. Cardiomyocyte hypertrophy was significantly attenuated by iPSc transplantation. In conclusion, these results suggested that transplantation of iPSc may result in functional recovery by promoting angiogenesis, inhibiting apoptosis, and ameliorating cardiac remodeling. This proof of concept study may provide a basis for an autologous iPSc-based therapy of MI.

MicroRNAs (miRNAs) are small noncoding RNAs that are involved in key biological processes, including development, differentiation, and regeneration. The global miRNA expression profile that regulates the regenerative potential of the neonatal mouse heart has not been reported. We performed deep sequencing to determine the genome-wide miRNA expression profile of the neonatal mouse heart at three key ages (1, 6, and 7 days). The miRNAs at least 1.4-fold differentially expressed between the three time points were selected for further analysis. Two miRNAs (mmu-miR-22-5p and mmu-miR-338-3p) were significantly upregulated, and nine miRNAs (mmu-miR-324-5p, mmu-miR-337-5p, mmu-miR-339-5p, mmu-miR-365-1-5p, mmu-miR-500-3p, mmu-miR-505-5p, mmu-miR-542-5p, mmu-miR-668-3p, and mmu-miR-92a-1-5p) were significantly downregulated in cardiac tissue of 7-day-old mice compared to 1- and 6-day-old mice. The expression patterns of five significantly different miRNAs were verified by quantitative real-time PCR. Furthermore, the potential targets of these putative miRNAs were suggested using miRNA target prediction tools. The candidate target genes are involved in the myocardial regenerative process, with a prominent role for the Notch signaling pathway. Our study provides a valuable resource for future investigation of the biological function of miRNAs in heart regeneration.

Neural remodeling after myocardial infarction (MI) may cause malignant ventricular arrhythmia, which is the main cause of sudden cardiac death following MI. Herein, we aimed to examine whether induced pluripotent stem cells (iPSc) transplantation can ameliorate neural remodeling and reduce ventricular arrhythmias (VA) in a post-infarcted swine model. Left anterior descending coronary arteries were balloon-occluded to generate MI. Animals were then divided into Sham, PBS control, and iPS groups. Dynamic electrocardiography programmed electric stimulation were performed to evaluate VA. The spatial distribution of vascularization, Cx43 and autonomic nerve regeneration were evaluated by immunofluorescence staining. Associated protein expression was detected by Western blotting. Likewise, we measured the enzymatic activities of superoxide dismutase and content of malondialdehyde. Six weeks later, the number of blood vessels increased significantly in the iPSc group. The expression of vascular endothelial growth factor and connexin 43 in the iPS group was significantly higher than the PBS group; however, the levels of nerve growth factor and tyrosine hydroxylase were lower. The oxidative stress was ameliorated by iPSc transplantation. Moreover, the number of sympathetic nerves in the iPSc group was reduced, while the parasympathetic nerve fibers had no obvious change. The transplantation of iPSc also significantly decreased the low-/high-frequency ratio and arrhythmia score of programmed electric stimulation-induced VA. In conclusion, iPSc intramyocardial transplantation reduces vulnerability to VAs, and the mechanism was related to the remodeling amelioration of autonomic nerves and gap junctions. Moreover, possible mechanisms of iPSc transplantation in improving neural remodeling may be related to attenuated oxidative stress and inflammatory response.

Background Ventricular septal defect (VSD) is a highly prevalent fetal congenital heart defect, which can become spontaneously closed during infancy. The current study aims to characterize fetal VSDs that were subsequently spontaneously closed in the first 2 years of life in eastern China. Methods Between January 2011 and December 2013, 257 fetal patients diagnosed with isolated VSD by fetal echocardiography at Nanjing Maternity and Child Health Care Hospital, China, were enrolled in the study. Subjects were divided into three groups: group 1 = persistent VSD; group 2 = closed after birth; group 3 = closed during gestation. Fetal echocardiography data, physical features at birth and follow-up outcomes for 2 years were compared to identify factors contributing to spontaneous closure (SC) of VSD. A predictive formula was applied to patients admitted to hospital in the first quarter of 2014 (n = 23) for validation. Results SC occurred in 42.8% patients. Birth weight (3.095 ± 0.774, 3.174 ± 0.535, 3.499 ± 0.532 kg in groups 1, 2 and 3, respectively) and defect diameter (3.422 ± 0.972, 2.426 ± 0.599, 2.292 ± 0.479 mm, in groups 1, 2 and 3, respectively) showed statistically significant differences between the three groups (P = 0.004 and P = 0.000, respectively). Receiver operating characteristic (ROC) curves identified cut-off value for the defect diameter as 2.55 mm, and logistic regression analysis identified the SC probability = (1 + exp -[-2.151 − 0.716*birth weight + 1.393*diameter])-1. Results indicated that male fetuses, full-term birth, muscular VSD, and defects without blood flow crossing the septum, have higher incidence of SC. Conclusions The major determinants of SC of isolated VSD are birth weight and diameter of the defect. In addition, VSD location may also affect the SC incidence.

Background/Aims: Previous studies have indicated that long non-coding RNAs (lncRNA) are related to the occurrence and development of many human diseases, such as cancer and the HELLP and the brachydactyly syndromes. However, studies of LncRNA in heart failure have not yet been reported. Here, we investigated cardiac lncRNA expression profiles in the myocardial-specific knockout pdk1 gene (KO) mouse model of heart failure. Methods: Cardiac samples were obtained from PDK1 KO and WT mice on postnatal (P) day 8 (P8) and day 40 (P40), and lncRNA expression profiles were analyzed by sequencing and screening using the Arraystar mouse lncRNA microarray. Quantitative real-time PCR analysis of these lncRNAs confirmed the identity of some genes. Results: Comparisons of the KO and control groups showed fold changes of >1.5 in the expression levels of 2,024 lncRNAs at P8, while fold changes of >2 in the expression levels of 4,095 lncRNAs were detected at P40. Nineteen lncRNAs were validated by RT-PCR. Bioinformatic and pathway analyses indicated that mkk7, a sense overlap lncRNA, may be involved in the pathological processes of heart failure through the MAPK signaling pathway. Conclusion: These data reveal differentially expressed lncRNA in mice with a myocardial-specific deletion of the pdk1 gene, which may provide new insights into the mechanism of heart failure in PDK1 knockout mice.

Fatty acid-binding protein 3 (FABP3) is a low molecular weight protein with distinct tissue distribution, which may play an important role in fatty acid transport, cell growth, cellular signaling, and gene transcription. We have previously shown FABP3 was more highly expressed in myocardium with ventricular septal defects than in normal myocardium and furthermore, that overexpression of FABP3 causes mitochondrial dysfunction and induces apoptosis in the P19 mouse teratocarcinoma cell line (P19), which is a suitable model for the investigation of cardiac differentiation at the molecular and functional levels. α-Lipoic acid (α-LA), a natural dithiol compound with antioxidant properties, has been reported to protect mitochondrial function in cells. In this study, we established an FABP3-overexpressing P19 cell line for the investigation of the impact of α-LA on mitochondrial impairment and apoptosis in these cells. Mitochondrial morphology was evaluated by transmission electron microscopy, while the effects of α-LA on reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), intracellular ATP content and the amount of mitochondrial DNA were analyzed by flow cytometry, a commercially available assay and quantitative real-time PCR, respectively. The results revealed that α-LA ameliorated mitochondrial deformation and decreased intracellular ROS production. Furthermore, the MMP, intracellular ATP synthesis and the amount of mitochondrial DNA were also increased. Most significantly, α-LA was shown to reverse apoptosis. Collectively, our results indicate that abnormalities in FABP3 expression contribute to mitochondrial dysfunction and apoptosis, and that α-LA represents a suitable candidate for development as a treatment for apoptosis-related congenital cardiac malformations.

MicroRNA (miR)-19b is part of the miR-17-92 cluster associated with cardiac development. Here, we investigated the effects of overexpressing miR-19b on proliferation, differentiation, apoptosis, and regulation of the Wnt/β-catenin signaling pathway in the multipotent murine P19 cell line that can be induced to undergo cardiogenesis. P19 cells were transfected with the miR-19b plasmid or empty vector, and miR-19b overexpression was verified by Quantitative Real-Time PCR (qPCR). The miR-19b or vector control stable cell lines were selected using Blasticidin S HCl, and their proliferation, cell cycle, and apoptosis levels were analyzed using the Cell Counting Kit-8 and flow cytometry. P19 cell differentiation markers, apoptosis-related genes (bax, bcl-2), and Wnt/β-catenin signaling pathway-related genes were detected by qPCR, the corresponding proteins by Western blot. Expression of the Wnt pathway and differentiation marker proteins was also verified by immunofluorescence. Morphological changes associated with apoptosis were observed by electron microscopy and Hoechst staining. On the basis of these results, we demonstrated that miR-19b overexpression promoted proliferation and differentiation but inhibited apoptosis in P19 cells; Wnt and β-catenin expressions were decreased, while that of GSK3β was increased with miR-19b overexpression. Overexpression of miR-19b inhibited activation of the Wnt/β-catenin signaling pathway in P19 cells, which may regulate cardiomyocyte differentiation. Our findings may bring new insights into the mechanisms underlying cardiac diseases and suggest that miR-19b is a potential new therapeutic target for cardiovascular diseases.

Neonatal mouse hearts have completely regenerative capability after birth, but the ability to regenerate rapidly lost after 7 days, the mechanism has not been clarified. Previous studies have shown that mRNA profile of adult mouse changed greatly compared to neonatal mouse. So far, there is no research of peptidomics related to heart regeneration. In order to explore the changes of proteins, enzymes, and peptides related to the transient regeneration, we used comparative petidomics technique to compare the endogenous peptides in the mouse heart of postnatal 1 and 7 days. In final, we identified 236 differentially expressed peptides, 169 of which were upregulated and 67 were downregulated in the postnatal 1 day heart, and also predicted 36 functional peptides associated with transient regeneration. The predicted 36 candidate peptides are located in the important domains of precursor proteins and/or contain the post-transcriptional modification (PTM) sites, which are involved in the biological processes of cardiac development, cardiac muscle disease, cell proliferation, necrosis, and apoptosis. In conclusion, for the first time, we compared the peptidomics profiles of neonatal heart between postnatal 1 day and postnatal 7 day. This study provides a new direction and an important basis for the mechanism research of transient regeneration in neonatal heart. J. Cell. Biochem. 118: 2828-2840, 2017. © 2017 Wiley Periodicals, Inc.

In our previous study we screened thousands of lncRNAs for their relationship with ventricular septal defect. Among these lncRNAs, uc.167 attracted our attention for its high level of conservation and that it was antisense to the Mef2c gene, which encodes myocyte enhancer factor 2C. This study aims to investigate the role of uc.167 during cardiomyocyte maturation in P19 cells induction and possible mechanism. The uc.167 expression level in human heart tissue of ventricular septum defect (VSD) was evaluated by qRT-PCR. The UCSC database was searched to investigate the bioinformatics of uc.167. We constructed overexpression vector of uc.167 and Mef2c. To detect proliferation and apoptosis, we combined cell cycle analysis and CCK8, Hoechst staining, flow cytometry and caspase-3 assays, respectively. The cardiomyogenesis related RNAs (cTnT, GATA4, and Mef2c) and proteins were detected by qRT-PCR and Western blotting. In this study, we found that uc.167 expression was significantly increased in VSD heart tissues. uc.167 is on the opposite strand to the coding gene Mef2c. The expression model of Mef2c and uc.167 showed an opposite correlation in the embryonic development and process of differentiation of P19 cells into cardiomyocytes. Overexpression of uc.167 inhibited proliferation but promoted apoptosis in P19 cells compared with the vector group, and those relative mRNAs and proteins decreased during the differentiation process. Whereas, co-expression of Mef2c and uc.167 can partially reverse the negative effects of uc.167 on proliferation, apoptosis and differentiation. Taken together, our findings suggest that uc.167 contributes to the development potential of VSD and may constitute a potential therapeutic target in this disease. uc.167 influences cell proliferation, apoptosis and differentiation of P19 cell by regulating Mef2c.

Fatty acid binding protein 3 (FABP3) (also known as H-FABP) is a member of the intracellular lipid-binding protein family, and is mainly expressed in cardiac muscle tissue. The in vivo function of FABP3 is proposed to be in fatty acid metabolism, trafficking, and cell signaling. Our previous study found that FABP3 is highly regulated in patients with ventricular septal defect (VSD), and may play a significant role in the development of human VSD. In the present study, we aimed to investigate the impact of FABP3 knockdown by RNA interference (RNAi) on apoptosis and mitochondrial function of embryonic carcinoma (P19) cells. The results revealed that downregulated FABP3 expression promoted apoptosis, and resulted in mitochondrial deformation, increased mitochondrial membrane potential (MMP), and decreased intracellular ATP synthesis. In addition, the knockdown of FABP3 also led to excess intracellular ROS production. However, there was no obvious influence on the amount of mitochondrial DNA. Collectively, our results indicated that FABP3 knockdown promoted apoptosis and caused mitochondrial dysfunction in P19 cells, which might be responsible for the development of human VSD.

Ventricular septal defect (VSD) is the most comon form of congenital heart disease (CHD). DNA hypermethylation analysis may provide an insight into the molecular features and pathogenesis of this heart disease. Although aberrant DNA hypermethylation is implicated in the pathophysiology of this heart disease, only a limited number of genes are known to be epigenetically altered in VSD. We previously identified regulation of the NOX5 gene by hypermethylation in VSD fetuses by promoter methylation microarrays. This study was designed to detect the expression of NOX5 mRNA in VSD and normal fetuses. We also verified the results of promoter methylation microarrays by methylation-specific PCR. DNA extraction and nested methylation-specific PCR were performed on myocardial tissue samples from 21 VSD and 15 normal fetuses. The primers specific for methylated vs. unmethylated DNA were designed and amplified by nested PCR. The products were visualized on agarose gel. Hypermethylation of the NOX5 promoter was more frequent in VSD fetuses (66.67%) than in normal fetuses (20%). There was a significant concordance between NOX5 methylation and a decrease in its mRNA expression. Taken together, our results demonstrate that hypermethylation of the NOX5 gene may be involved in the pathogenesis of VSD.

Congenital heart disease (CHD) is the most common type of birth defect, but its underlying molecular mechanisms remain unidentified. Previous studies determined that Homo sapiens LYR motif containing 1 (LYRM1) is a novel nucleoprotein expressed at the highest level in adipose tissue and in high levels in heart tissue. The LYRM1 gene may play an important role in the development of the human heart. This study was designed to identify the biological characteristics of the LYRM1 gene in heart development. On the basis of expression-specific differentiation markers identified with quantitative real-time RT-PCR and the morphology of LYRM1-overexpressing cells during differentiation, ectopic expression was not found to significantly affect differentiation of P19 cells into cardiomyocytes. MTT assays and cell cycle analysis showed that LYRM1 dramatically increases the proliferation of P19 cells. Furthermore, data from annexin V-FITC binding and caspase-3 activity revealed that LYRM1 can inhibit the apoptosis of P19 cells. Our data suggest that LYRM1 might have the potential to modulate cell growth, apoptosis, and heart development.

In a previous study, we screened thousands of long non-coding RNAs (lncRNAs) to assess their potential relationship with congenital heart disease (CHD). In this study, uc.4 attracted our attention because of its high level of evolutionary conservation and its antisense orientation to the CASZ1 gene, which is vital for heart development. We explored the function of uc.4 in cells and in zebrafish, and describe a potential mechanism of action. P19 cells were used to investigate the function of uc.4. We studied the effect of uc.4 overexpression on heart development in zebrafish. The overexpression of uc.4 influenced cell differentiation by inhibiting the TGF-beta signaling pathway and suppressed heart development in zebrafish, resulting in cardiac malformation. Taken together, our findings show that uc.4 is involved in heart development, thus providing a potential therapeutic target for CHD.

NYGGF4 is a recently discovered gene that is involved in obesity-associated insulin resistance. The exact mechanism by which NYGGF4 induces insulin resistance has not yet been fully elucidated. In this study, we demonstrated that the overexpression of NYGGF4 in 3T3-L1 adipocytes decreased mitochondrial mass, mitochondrial DNA, and intracellular ATP synthesis. In addition, NYGGF4 overexpression also led to an imbalance of the mitochondrial dynamics and excess intracellular ROS production. Collectively, our results indicated that the overexpression of NYGGF4 caused mitochondrial dysfunction in adipocytes, which might be responsible for the development of NYGGF4-induced insulin resistance.

To explore the effects of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the embryonic carcinoma (P19) cell model of cardiac differentiation. Knockdown of LYRM1 using small interfering RNA (siRNA) was confirmed by quantitative real-time PCR. Cell Counting Kit-8(CCK-8) proliferation assays and cell cycle analysis demonstrated that LYRM1 gene silencing significantly inhibited P19 cell proliferation. Flow cytometry and measurement of their caspase-3 activities revealed that knockdown of LYRM1 increased P19 cell apoptosis. Observation of morphological changes using an inverted microscope and expression analysis of specific differentiation marker genes using quantitative real-time PCR and Western blotting revealed that knockdown of LYRM1 significantly inhibited the differentiation of P19 cells into cardiomyocytes. Furthermore, real-time quantitative PCR applied to detect mitochondrial DNA (mtDNA) copy number implied that there was no significant difference in the LYRM1 knockdown group compared with the control group. Cellular ATP production investigated by luciferase-based luminescence assay was dramatically decreased in differentiated cells transfected with LYRM1 RNAi. Fluorescence microscopy and flow cytometery were used to detect the reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) showed that the level of ROS was dramatically increased and MMP was obviously decreased in differentiated cells transfected with LYRM1 RNAi. Collectively, knockdown of LYRM1 promoted apoptosis and suppressed proliferation and differentiation in P19 cells. In addition, knockdown of LYRM1 induced mitochondrial impairment in P19 cells during differentiation, which was reflected by decreased ATP synthesis, lower MMP and increased ROS levels.