Transcriptional profile of human thymus reveals IGFBP5 is correlated with age-related thymic involution.

Thymus is the main immune organ which is responsible for the production of self-tolerant and functional T cells, but it shrinks rapidly with age after birth. Although studies have researched thymus development and involution in mouse, the critical regulators that arise with age in human thymus remain unclear. We collected public human single-cell transcriptomic sequencing (scRNA-seq) datasets containing 350,678 cells from 36 samples, integrated them as a cell atlas of human thymus. Clinical samples were collected and experiments were performed for validation. We found early thymocyte-specific signaling and regulons which played roles in thymocyte migration, proliferation, apoptosis and differentiation. Nevertheless, signaling patterns including number, strength and path completely changed during aging, Transcription factors (FOXC1, MXI1, KLF9, NFIL3) and their target gene, IGFBP5, were resolved and up-regulated in aging thymus and involved in promoting epithelial-mesenchymal transition (EMT), responding to steroid and adipogenesis process of thymic epithelial cell (TECs). Furthermore, we validated that IGFBP5 protein increased at TECs and Hassall's corpuscle in both human and mouse aging thymus and knockdown of IGFBP5 significantly increased the expression of proliferation-related genes in thymocytes. Collectively, we systematically explored cell-cell communications and regulons of early thymocytes as well as age-related differences in human thymus by using both bioinformatic and experimental verification, indicating IGFBP5 as a functional marker of thymic involution and providing new insights into the mechanisms of thymus involution.

Silencing suppressor of cytokine signaling 3 induces apoptosis and activates the p-STAT3/NF-κ B pathway in hypoxic cultivated H9c2 cells

Suppressor of cytokine signaling 3 (SOCS3) plays a significant role in the process of myocardial adaptation to chronic hypoxia. SOCS3 finely regulates cell signaling cross-talk that occurs between NF-κB and STAT3 during the compensatory protective response. However, the role and mechanism of SOCS3 in hypoxic cardiomyocytes are not fully understood. In the study, we investigated the effect of SOCS3 on the p65 and STAT3 signaling pathways and further examined the potential molecular mechanism involved in regulating apoptosis. Our data showed that SOCS3 silencing could upregulate Ac-p65, p-p65, and p-STAT3 expression in nuclear extracts of H9c2 cells that received hypoxic treatment for 24, 48, and 72 h. SOCS3 silencing also remarkably increased the DNA-binding activity of the p65 motif in hypoxic cultivated H9c2 cells. We also found that SOCS3 knockdown increased cleaved-caspase-3, Bax, and PUMA expression and decreased cleaved PARP and Bcl-2 in expression in hypoxic H9c2 cells. Silencing of SOCS3 caused an increase in LDH leakage from injured cardiomyocytes and reduced cell viability under conditions of hypoxic stress. Furthermore, SOCS3 silencing enhanced the apoptosis of H9c2 cells at 72 h of hypoxia. These findings suggest that knockdown of SOCS3 leads to excessive activation of the NF-κB pathway, which, in turn, might promote apoptosis under conditions of chronic hypoxia. (AU)

Silencing suppressor of cytokine signaling 3 induces apoptosis and activates the p-STAT3/NF-κ B pathway in hypoxic cultivated H9c2 cells

Suppressor of cytokine signaling 3 (SOCS3) plays a significant role in the process of myocardial adaptation to chronic hypoxia. SOCS3 finely regulates cell signaling cross-talk that occurs between NF-κB and STAT3 during the compensatory protective response. However, the role and mechanism of SOCS3 in hypoxic cardiomyocytes are not fully understood. In the study, we investigated the effect of SOCS3 on the p65 and STAT3 signaling pathways and further examined the potential molecular mechanism involved in regulating apoptosis. Our data showed that SOCS3 silencing could upregulate Ac-p65, p-p65, and p-STAT3 expression in nuclear extracts of H9c2 cells that received hypoxic treatment for 24, 48, and 72 h. SOCS3 silencing also remarkably increased the DNA-binding activity of the p65 motif in hypoxic cultivated H9c2 cells. We also found that SOCS3 knockdown increased cleaved-caspase-3, Bax, and PUMA expression and decreased cleaved PARP and Bcl-2 in expression in hypoxic H9c2 cells. Silencing of SOCS3 caused an increase in LDH leakage from injured cardiomyocytes and reduced cell viability under conditions of hypoxic stress. Furthermore, SOCS3 silencing enhanced the apoptosis of H9c2 cells at 72 h of hypoxia. These findings suggest that knockdown of SOCS3 leads to excessive activation of the NF-κB pathway, which, in turn, might promote apoptosis under conditions of chronic hypoxia. (AU)

Silencing suppressor of cytokine signaling 3 induces apoptosis and activates the p-STAT3/NF-κB pathway in hypoxic cultivated H9c2 cells.

Suppressor of cytokine signaling 3 (SOCS3) plays a significant role in the process of myocardial adaptation to chronic hypoxia. SOCS3 finely regulates cell signaling cross-talk that occurs between NF-κB and STAT3 during the compensatory protective response. However, the role and mechanism of SOCS3 in hypoxic cardiomyocytes are not fully understood. In the study, we investigated the effect of SOCS3 on the p65 and STAT3 signaling pathways and further examined the potential molecular mechanism involved in regulating apoptosis. Our data showed that SOCS3 silencing could upregulate Ac-p65, p-p65, and p-STAT3 expression in nuclear extracts of H9c2 cells that received hypoxic treatment for 24, 48, and 72 h. SOCS3 silencing also remarkably increased the DNA-binding activity of the p65 motif in hypoxic cultivated H9c2 cells. We also found that SOCS3 knockdown increased cleaved-caspase-3, Bax, and PUMA expression and decreased cleaved PARP and Bcl-2 in expression in hypoxic H9c2 cells. Silencing of SOCS3 caused an increase in LDH leakage from injured cardiomyocytes and reduced cell viability under conditions of hypoxic stress. Furthermore, SOCS3 silencing enhanced the apoptosis of H9c2 cells at 72 h of hypoxia. These findings suggest that knockdown of SOCS3 leads to excessive activation of the NF-κB pathway, which, in turn, might promote apoptosis under conditions of chronic hypoxia.

Cardiac proteomic profiling suggests that hypertrophic and dilated cardiomyopathy share a common pathogenetic pathway of the calcium signalling pathway.

OBJECTIVE: Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are classified as different diseases but have many similar pathogenic genes and clinical symptoms. Previous research has focused on mutated genes. This study was conducted to identify key molecular mechanisms and explore effective therapeutic targets. METHODS: Myocardial tissue was harvested from patients with HCM (n = 3) or DCM (n = 4) during surgery. Hearts donated by healthy traffic accident victims were treated as controls (n = 4). Total proteins were extracted for liquid chromatography-tandem mass spectrometry. Differentially expressed proteins (DEPs) were annotated via GO and KEGG analyses. Selected distinguishing protein abundance was confirmed by western blotting. RESULTS: Compared with the control group, there were 121 and 76 DEPs in the HCM and DCM groups, respectively. GO terms for these two comparisons are associated with contraction-related components and actin binding. Additionally, the most significantly upregulated and downregulated proteins were periostin and tropomyosin alpha-3 chain in both comparisons. Moreover, when comparing the HCM and DCM groups, we found 60 significant DEPs, and the GO and KEGG terms are related to the calcium signalling pathway. Expression of the calcium regulation-related protein peptidyl-prolyl cis-trans isomerase (FKBP1A) was significantly upregulated in multiple samples. CONCLUSION: HCM and DCM have many mutual pathogenetic pathways. Calcium ion-related processes are among the most significant factors affecting disease development. For HCM and DCM, research on regulating linchpin protein expression or interfering with key calcium-related pathways may be more beneficial than genetic research.

Acute Type A Aortic Dissection and Late Pregnancy: What Should We Do?

ABSTRACT Introduction: Acute type A aortic dissection (AAAD) in late pregnancy is a rare but severe disease. Lack of clinical experience is the main cause of high mortality. This study tries to investigate the multidisciplinary therapeutic strategy for these patients. Case presentation: We reported three patients with AAAD in late pregnancy. Sudden chest pain was the main clinical symptom before operation. All three patients and their newborns survived through multidisciplinary approach in diagnosis and treatment. No serious complications occurred during the mid-term follow-up. Conclusion: Multidisciplinary diagnosis and treatment strategy play a crucial role in saving the lives of pregnant women with AAAD.

Acute Type A Aortic Dissection and Late Pregnancy: What Should We Do?

INTRODUCTION: Acute type A aortic dissection (AAAD) in late pregnancy is a rare but severe disease. Lack of clinical experience is the main cause of high mortality. This study tries to investigate the multidisciplinary therapeutic strategy for these patients. CASE PRESENTATION: We reported three patients with AAAD in late pregnancy. Sudden chest pain was the main clinical symptom before operation. All three patients and their newborns survived through multidisciplinary approach in diagnosis and treatment. No serious complications occurred during the mid-term follow-up. CONCLUSION: Multidisciplinary diagnosis and treatment strategy play a crucial role in saving the lives of pregnant women with AAAD.

Oxidative-Damaged Mitochondria Activate GABARAPL1-Induced NLRP3 Inflammasomes in an Autophagic-Exosome Manner after Acute Myocardial Ischemia.

Objective: This study is aimed at identifying the potential diagnostic markers for circulating endothelial cells (CECs) for acute myocardial ischemia (AMI) and exploring the regulatory mechanisms of the selected biomarker in mitochondrial oxidative damage and vascular inflammation in AMI pathology. Methods: Utilizing the Gene Expression Omnibus dataset GSE66360, we scanned for differentially expressed genes (DEGs) in 49 AMI patients and 50 healthy subjects. To discover possible biomarkers, LASSO regression and support vector machine recursive feature elimination examinations were conducted. Using the GSE60993 and GSE123342 datasets and AMI rat models, the expression levels and diagnostic accuracy of the biomarkers in AMI were thoroughly verified. CIBERSORT was employed to evaluate the compositional patterns of 22 distinct immunological cell percentages in AMI according to combined cohorts. The oxidative-damaged mitochondria were detected by confocal microscopy observation of MitoTracker, ROS-DCFH-DA, and mCherry-GFP-LC3B. Results: In total, 122 genes were identified. The identified DEGs primarily contributed in arteriosclerosis, arteriosclerotic cardiovascular disorders, bacterial infectious disorder, coronary artery disease, and myocardial infarction. Nine features (NR4A2, GABARAPL1 (GEC1), CLEC4D, ITLN1, SNORD89, ZFP36, CH25H, CCR2, and EFEMP1) of the DEGs were shared by two algorithms, and GABARAPL1 (GEC1) was identified and verified as a diagnostic mitochondrial biomarker for AMI. Confocal results showed that there existed mitochondrial damage and oxidative stress in cardiac CMECs after AMI, and the blocked autophagy flux could be released by exosome burst in cardiac CMECs and blood CECs. Immune cell infiltration testing declared that elevated GEC1 expression in blood CECs was linked to the rise of monocytes and neutrophils. Functional tests revealed that high GEC1 expression in CMECs and CECs could activate the vascular inflammatory response by stimulating NLRP3 inflammasome production after AMI. Conclusion: Oxidative-damaged mitochondria in cardiac CMECs activate GEC1-mediated autophagosomes but block autophagy flux after AMI. The exfoliated cardiac CMECs evolve into abnormal blood CECs, and the undegraded GEC1 autophagosomes produce a large number of NLRP3 inflammasomes by exosome burst, stimulating the increase in monocytes and neutrophils and ultimately triggering vascular inflammation after AMI. Therefore, GEC1 in blood CECs is a highly specific diagnostic mitochondrial biomarker for AMI.

Identification of MMP9 as a Novel Biomarker to Mitochondrial Metabolism Disorder and Oxidative Stress in Calcific Aortic Valve Stenosis.

Calcific aortic valve stenosis (CAVS) is the most common heart valve disorder among humans. To date, no effective method has been identified to prevent this disease. Herein, we aimed to identify novel diagnostic and mitochondria-related biomarkers of CAVS, based on two machine learning algorithms. We further explored their association with infiltrating immune cells and studied their potential function in CAVS. The GSE12644, GSE51472, and GSE83453 expression profiles were downloaded from the Gene Expression Omnibus (GEO) repository. The GSE12644 and GSE51472 datasets were integrated to identify differentially expressed genes (DEGs). GSE12644 contains 10 normal and 10 CAVS samples, whereas GSE51472 contains 5 normal and 10 CAVS samples. GO and KEGG assays of DEGs were conducted, and the correlation between matrix metalloproteinase 9 (MMP9) expression and immune cell infiltration was explored, using CIBERSORT. The LASSO regression model and SVM-RFE analysis were used to identify diagnostic genes. The expression of MMP9 in CAVS and non-CAVS samples was measured using RT-PCR, western blotting and immunohistochemistry. A series of functional experiments were performed to explore the potential role of MMP9 in mitochondrial metabolism and oxidative stress during CAVS progression. Twenty-two DEGs were identified, of which six genes (SCG2, PPBP, TREM1, CCL19, WIF1, and MMP9) were ultimately distinguished as diagnostic genes in CAVS. Of these, MMP9 was indicated as a mitochondria-related gene, the expression and diagnostic value of which were further confirmed in the GSE83453 dataset. Correlation analysis revealed a positive correlation between MMP9 and infiltrating immune cells. In our cohort, MMP9 expression was distinctly increased in CAVS samples, and its inhibition attenuated the calcification of valve interstitial cells (VICs) by suppressing mitochondrial damage and oxidative stress. Taken together, our findings suggest MMP9 as a novel mitochondrial dysfunction biomarker and therapeutic target for CAVS.

Hypoxia-primed monocytes/macrophages enhance postinfarction myocardial repair.

Background: Oxygen supplementation in myocardial infarction (MI) remains controversial. Inflammation is widely believed to play a central role in myocardial repair. A better understanding of these processes may lead to the design of novel strategies complementary to MI treatment. Methods: To investigate the role of hypoxia in inflammation and myocardial repair after acute MI, we placed MI mice in a tolerable mild hypoxia (10% O2) chamber for 7 days and then transferred the mice to ambient air for another 3 weeks. Results: We found that the cumulative survival rate of the MI mice under hypoxia was significantly higher than that under oxygen supplementation. Hypoxia promoted postinfarction myocardial repair. Importantly, we found that hypoxia modulated the phenotypic transition of blood monocytes from pro-inflammatory to pro-reparative in a timely manner, leading to the subsequent discontinuation of inflammation in myocardial tissues and promotion of myocardial repair post-MI. Specifically, cultured bone marrow-derived macrophages (BMDMs) primed by hypoxia in vitro exhibited improved reparative capacities and differed from M1 and M2 macrophages through the AMPKα2 signaling pathway. The deletion of AMPKα2 in monocytes/macrophages prevented the phenotypic transition induced by hypoxia and could not promote myocardial repair after MI when transplanted into the myocardium. Conclusions: Taken together, our work demonstrates that hypoxia promotes postinfarction myocardial repair by modulating the blood monocyte/macrophage phenotypic transition from pro-inflammatory to pro-reparative in a timely manner through the AMPKα2 signaling pathway. Hypoxia priming might be an attractive translational strategy for MI treatment by amplifying immune cells during early inflammation and subsequent resolution and repair.

A nomogram prediction model for sternal incision problems.

Presently, the incidence and mortality rates of sternal incision problems (SIPs) after thoracotomy remain high, and no effective preventive measures are available. The data on 23 182 patients at Xinqiao Hospital, Army Medical University treated with median sternotomy from 1 August 2009 to 31 July 2019 were retrospectively reviewed. A prediction model of SIPs after median thoracotomy was established using R software and then validated using the bootstrap method. Next, the validity and accuracy of the model were tested and evaluated. In total, 15 426 cases met the requirements of the present study, among which 309 cases were diagnosed with SIPs, with an incidence rate of 2%. The body mass index (BMI), intensive care unit (ICU) time, diabetes mellitus, and revision for bleeding were identified as independent risk factors for postoperative SIPs. The nomogram model achieved good discrimination (73.9%) and accuracy (70.2%) in predicting the risk of SIPs after median thoracotomy. Receiver operating characteristic curve analysis showed that the area under curve of the model was 0.705 (95% confidence interval [CI]: 0.746-0.803); the Hosmer-Lemeshow test showed that χ2  = 6.987 and P = 0.538, and the fitting degree of the calibration curve was good. Additionally, the clinical decision curve showed that the net benefit of the model was greater than 0, and the clinical application value was high. The nomogram based on BMI, ICU time, diabetes mellitus, and revision for bleeding can predict the individualised risk of SIPs after median sternotomy, showing good discrimination and accuracy, and has high clinical application value. It also provides significant guidance for screening high-risk populations and developing intervention strategies.

Circular RNA circEsyt2 regulates vascular smooth muscle cell remodeling via splicing regulation.

Circular RNAs (circRNAs) have been recently recognized as playing a role in the pathogenesis of vascular remodeling-related diseases by modulating the functions of miRNAs. However, the interplay between circRNAs and proteins during vascular remodeling remains poorly understood. Here, we investigated a previously identified circRNA, circEsyt2, whose expression is known to be upregulated during vascular remodeling. Loss- and gain-of­function mutation analyses in vascular smooth muscle cells (VSMCs) revealed that circEsyt2 enhanced cell proliferation and migration and inhibited apoptosis and differentiation. Furthermore, the silencing of circEsyt2 in vivo reduced neointima formation, while circEsyt2 overexpression enhanced neointimal hyperplasia in the injured carotid artery, confirming its role in vascular remodeling. Using unbiased protein-RNA screening and molecular validation, circEsyt2 was found to directly interact with polyC-binding protein 1 (PCBP1), an RNA splicing factor, and regulate PCBP1 intracellular localization. Additionally, circEsyt2 silencing substantially enhanced p53ß splicing via the PCBP1-U2AF65 interaction, leading to the altered expression of p53 target genes (cyclin D1, p21, PUMA, and NOXA) and the decreased proliferation of VSMCs. Thus, we identified a potentially novel circRNA that regulated vascular remodeling, via altered RNA splicing, in atherosclerotic mouse models.

Mid-term follow-up of aortic valve replacement for bicuspid aortic valve.

OBJECTIVE: The purpose of this study was to evaluate the mid-term outcome of aortic valve replacement for bicuspid aortic valve and tricuspid aortic valve and the related risk factors. METHODS: From January 2014 to June 2019, 177 tricuspid aortic valve patients and 101 bicuspid aortic valve patients who underwent aortic valve replacement in our hospital were collected. 1:1 propensity score matching analysis was used to control the bias in patient selection. The perioperative and follow-up data between the two groups were compared. Independent risk factors which were associated with the continued dilatation of the ascending aorta were identified by univariate or multivariate logistic regression analysis. RESULTS: After the matching procedure, 160 patients were included in the analysis (80 in each group). Baseline characteristics, intraoperative, and perioperative outcomes were similar between the two groups (all p > 0.05). Moreover, 67 patients in the tricuspid aortic valve group and 70 in the bicuspid aortic valve group completed the follow-up. The ascending aorta change, annual change rate, and the proportion of continuous dilation of ascending aorta in bicuspid aortic valve group were significantly higher than those in the tricuspid aortic valve group (p < 0.05). Multivariate logistic regression analysis showed that type 1 in bicuspid aortic valve (OR 5.173; 95% CI 1.772, 15.101; p = 0.003), aortic regurgitation (OR 3.673; 95% CI 1.133, 11.908; p = 0.030), and aortic valve stenosis with regurgitation (OR 6.489; 95% CI 1.726, 24.404; p = 0.006) were independent risk factors for the continued dilatation of the ascending aorta in all AV patients. Furthermore, the multivariate logistic regression analysis showed that type 1 in bicuspid aortic valve (OR 5.157; 95% CI 1.053, 25.272; p = 0.043), age ≥ 40 years (OR 6.956; 95% CI 1.228, 39.410; p = 0.028), and aortic regurgitation (OR 4.322; 95% CI 1.174, 15.911; p = 0.028) were independent risk factors for the continued dilatation of the ascending aorta in bicuspid aortic valve patients. CONCLUSION: Compared with tricuspid aortic valve patients, the ascending aorta of bicuspid aortic valve patients is more likely to continue to enlarge after aortic valve replacement. Type 1 in bicuspid aortic valve, age ≥ 40 years, and aortic regurgitation were the independent risk factors.

Long-term outcomes after aortic root replacement for patients with Marfan syndrome.

BACKGROUND: A diversity of surgical strategies are used to treat Marfan syndrome patients with aortic disease. We sought to evaluate the long-term efficiency of aortic root replacement (ARR) for patients with Marfan syndrome. METHODS: Data were collected from 131 patients with Marfan syndrome and aortic disease who underwent ARR in our center. We retrospectively analyzed the long-term outcomes of these patients, among whom 68 had been diagnosed with aortic aneurysm (AA) and had undergone ARR. The remaining 63 patients had aortic dissection (AD); of these, 35 underwent ARR for limited ascending AD, while the others underwent ARR and total arch replacement combined with frozen elephant trunk (FET). Risk factors for survival and reoperation were identified. RESULTS: The operative mortality rate was 4.58%. Age >40 years was the sole risk factor for operative mortality. During follow-up, 12 deaths occurred. Patients aged <25 years and female patients were more prone to late death than were other patients. Indications for reoperation were noted in 22 patients, and the risk factors were age <30 years and female sex. At 5 years, the survival rate was 92.96%, and the freedom from reoperation rate was 88.36%. At 10 years, the survival rate was 85.25%, and the freedom from reoperation rate was 71.75%. The survival and freedom from reoperation rates were significantly higher in patients with AD than in those with AA. Specifically, chronic AD was a greater risk factor for late survival than was acute AD in patients with Marfan syndrome. CONCLUSIONS: For patients with Marfan syndrome and aortic disease, ARR can be safely performed and results in low operative mortality and favorable long-term survival. Young and female patients have a higher risk for late death and reoperation. To prevent AD, surgical intervention should be promptly implemented following the diagnosis of aortic sinus dilation.

AMPKα2 deficiency exacerbates hypoxia-induced pulmonary hypertension by promoting pulmonary arterial smooth muscle cell proliferation.

Increased evidence indicates that adenosine monophosphate-activated protein kinase (AMPK) plays a vital role in vascular homeostasis, especially under hypoxia, and protects against the progression of pulmonary hypertension (PH). However, the role of AMPK in the pathogenesis of PH remains to be clarified. In the present study, we confirmed that a loss of AMPKα2 exacerbated the development of PH by using hypoxia-induced PH model in AMPKα2 -/- mice. After a 4-week period of hypoxic exposure, AMPKα2 -/- mice exhibited more severe pulmonary vascular remodeling and pulmonary vascular smooth muscle cell (SMC) proliferation when compared with wild type (WT) mice. In vitro, AMPKα2 knockdown promoted the proliferation of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia. This phenomenon was accompanied by upregulated Skp2 and downregulated p27kip1 expression and was abolished by rapamycin, an inhibitor of mTOR. These results indicate that AMPKα2 deficiency exacerbates hypoxia-induced PH by promoting PASMC proliferation via the mTOR/Skp2/p27kip1 signaling axis. Therefore, enhanced AMPKα2 activity might underlie a novel therapeutic strategy for the management of PH.

Apigenin protects against ischemia-/hypoxia-induced myocardial injury by mediating pyroptosis and apoptosis.

Apigenin is a traditional Chinese medicine found in many plants that plays critical roles in several diseases, including cardiovascular diseases. We investigated the protective effect of apigenin against ischemia/hypoxia (I/H)-induced myocardium injury in vitro and explored the potential molecular mechanisms. Together with the flow cytometry results, our results indicated that apigenin attenuated the pyroptosis and apoptosis of myoblastic H9c2 cells that were induced by I/H injury. Furthermore, the pro-inflammatory cytokines (interleukin 18, IL-18, and IL-1ß) were investigated for the roles of apigenin in I/H-induced myocardium injury. It was observed that increases in IL-1ß and IL-18 levels were significantly reduced by apigenin treatment of I/H-induced H9C2 cells. The results demonstrated that apigenin protected H9c2 cells against I/H-induced myocardium injury. The protective effects were most likely related to the reduction of pyroptosis, apoptosis, and pro-inflammatory cytokines.

KLF15 is a protective regulatory factor of heart failure induced by pressure overload.

The aim of the present study was to investigate the protective effect of Kruppel­like factor 15 (KLF15) overexpression on heart failure (HF) induced by left ventricular (LV) pressure overload in mice. Wild­type (WT) mice and cardiac­specific KLF15­overexpressed transgenic (TG) mice were selected as research subjects, and an LV pressure overload model was constructed by ascending aortic constriction surgery. Changes in cardiac morphology and function, and ultrastructure and molecular expression were observed via M­mode echocardiography, histological and immunohistochemical staining, ELISA and western blotting at 2 and 6 weeks of LV overload. WT and TG mice subjected to 2 weeks of overload displayed adaptive LV hypertrophy characterized by ventricular thickness, cardiomyocyte size, ejection fraction and fractional shortening of heart­lung weight ratio and KLF15, and increases in vascular endothelial growth factor (VEGF) expression without other pathological changes. WT mice subjected to 6 weeks of overload displayed enlargement of the LV chamber, severe interstitial remodeling, and HW/LW, cardiac capillary and heart function decline, accompanied by downregulated expression of KLF15 and VEGF, and upregulated expression of connective tissue growth factor, phosphorylated p38 (p­p38) and phosphorylated Smad3 (p­Smad3). In contrast, TG mice exhibited improved resistance to 6 weeks of overload and a slighter molecular expression response compared with WT mice. KLF15 was revealed to be a critical factor regulating the expression of CTGF, VEGF, p­p38 and p­Smad3, and could alleviate the progression from adaptive LV hypertrophy to decompensatory cardiac insufficiency.

Multinucleated polyploid cardiomyocytes undergo an enhanced adaptability to hypoxia via mitophagy.

AIMS: There is a large subpopulation of multinucleated polyploid cardiomyocytes (M*Pc CMs) in the adult mammalian heart. However, the pathophysiological significance of increased M*Pc CMs in heart disease is poorly understood. We sought to determine the pathophysiological significance of increased M*Pc CMs during hypoxia adaptation. METHODS AND RESULTS: A model of hypoxia-induced cardiomyocyte (CM) multinucleation and polyploidization was established and found to be associated with less apoptosis and less reactive oxygen species (ROS) production. Compared to mononucleated diploid CMs (1*2c CMs), tetraploid CMs (4c CMs) exhibited better mitochondria quality control via increased mitochondrial autophagy (mitophagy). RNA-seq revealed Prkaa2, the gene for AMPKα2, was the most obviously up-regulated autophagy-related gene. Knockdown of AMPKα2 increased apoptosis and ROS production and suppressed mitophagy in 4c CMs compared to 1*2c CMs. Rapamycin, an autophagy activator, alleviated the adverse effect of AMPKα2 knockdown. Furthermore, silencing PINK1 also increased apoptosis and ROS in 4c CMs and weakened the adaptive superiority of 4c CMs. Finally, AMPKα2-/- mutant mice exhibited exacerbation of apoptosis and ROS production via decreases in AMPKα2-mediated mitophagy in 4c CMs compared to 1*2c CMs during hypoxia. CONCLUSIONS: Compared to 1*2c CMs, hypoxia-induced 4c CMs exhibited enhanced mitochondria quality control and less apoptosis via AMPKα2-mediated mitophagy. These results suggest that multinucleation and polyploidization allow CM to better adapt to stress via enhanced mitophagy. In addition, activation of AMPKα2 may be a promising target for myocardial hypoxia-related diseases.