Hypoxia-induced Cardiomyocyte Research Articles

Leonurine Alleviates Hypoxia-Induced Myocardial Damage by Regulating miRNAs

Objective miRNAs as pharmaco-targets have been investigated in multifarious diseases. Our study aimed to determine whether leonurine was a potential cardioprotective agent by targeting miRNAs in hypoxia-stimulated mice and H9c2 cardiomyocytes. Methods Cell proliferation and apoptosis were examined by CCK-8 and TUNEL assay in hypoxia-stimulated rat H9c2 cardiomyocytes. miRNAs expression levels in cardiomyocytes in response to hypoxia stimulation were detected by RT-qPCR. Mice with myocardial injury were induced by chronic intermittent hypoxia stimulation. Results Leonurine alleviated hypoxia-induced cardiac hypertrophy in mice. Moreover, up-regulation of miR-31 and down-regulation of miR-210 in hypoxia-stimulated mice were reversed by leonurine administration. Leonurine exhibited cardioprotective activity in an vitro cell model of hypoxia-stimulated rat H9c2 cardiomyocytes, reflecting that the compound improved hypoxia-induced growth inhibition and apoptosis of cardiomyocytes. TUNEL assay revealed that transfection of miR-31 inhibitors or miR-210 mimics abrogated hypoxia-induced cardiomyocyte apoptosis. In contrast to that, miR-31 mimics or miR-210 inhibitors counteracted the anti-apoptotic effect of leonurine on hypoxia-treated rat H9c2 cardiomyocytes. Conclusion Our findings suggest that miR-31 and miR-210 as the upstream regulators of leonurine are involved in hypoxia-induced cardiomyocyte apoptosis. Leonurine can target miRNAs to protect against hypoxia-induced myocardial damage. miRNAs as potential drug targets may provide prospective therapeutic strategies for the treatment of myocardial damage.

Hypoxia-induced miR-27 and miR-195 regulate ATP consumption, viability, and metabolism of rat cardiomyocytes by targeting PPARγ and FASN expression.

This study examined whether hypoxia-induced microRNA (miRNA) upregulation was related to the inhibition of chondriosome aliphatic acid oxidation in myocardial cells under anoxia. We showed that anoxia induced high expression of hypoxia-inducible factor-1-alpha, muscle carnitine palmitoyltransferase I, and vascular endothelial growth factor in cardiomyocytes. Meanwhile, miR-27 and miR-195 were also upregulated in hypoxia-induced cardiomyocytes. Furthermore, hypoxia induction led to reductions in the adenosine triphosphate (ATP) consumption rate and oxidative metabolism as well as an increase in cardiomyocyte glycolysis. Metabolic reprogramming was reduced by hypoxia, as evidenced by the downregulation of sirtuin 1, forkhead box protein O1, sterol regulatory element-binding protein 1c, ATP citrate lyase, acetyl-coenzyme A carboxylase 2, adiponutrin, adipose triglyceride lipase, and glucose transporter type 4, while miR-27 and miR-195 inhibition partially recovered the expression of these transcription factors. In addition, hypoxia induction reduced cell viability and survival by triggering apoptosis; however, miR-27 and miR-195 inhibition partially increased cell viability. Moreover, miR-27 and miR-195 targeted the 3’untranslated regions of two key lipid-associated metabolic players, peroxisome proliferator-activated receptor gamma and fatty acid synthase. In conclusion, miR-27 and miR-195 are related to hypoxia-mediated ATP levels, glycolysis, oxidation, cell survival, and a cascade of transcription factors that control metabolism in cardiomyocytes.

HIF-1/AKT Signaling-Activated PFKFB2 Alleviates Cardiac Dysfunction and Cardiomyocyte Apoptosis in Response to Hypoxia.

Myocardial infarction (MI) is the most prevalent disease with severe mortality, and hypoxia-induced cardiac injury and cardiomyocyte apoptosis are the significant and harmful consequences of this disease. The cross talk between hypoxia signaling and glycolysis energy flux plays a critical role in modulating MI-related heart disorder. However, the underlying mechanism remains unclear. Here, we aimed to explore the effect of a key glycolytic enzyme of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 (PFKFB2) on cardiac dysfunction and apoptosis in response to hypoxia. Our data demonstrated that the mRNA and protein expression of PFKFB2 were significantly elevated in the MI mice. The MI treatment promoted the activation of PFKFB2 in vivo, as presented by the remarkably increased phosphorylation levels of PFKFB2. PFKFB2 depletion enhanced MI-induced cardiac dysfunction and cardiomyocyte apoptosis in the MI mouse model. Moreover, hypoxia treatment dramatically upregulated the expression and activation of PFKFB2 in a time-dependent manner in cardiomyocytes. Hypoxia-stimulated PFKFB2 relieved hypoxia-induced cardiomyocyte apoptosis in vitro. PFKFB2 activated the fructose-2, 6-bisphosphate (Fru-2, 6-p2) /PFK/anaerobic adenosine triphosphate (ATP) glycolysis energy flux in response to hypoxia in cardiomyocytes. Mechanically, hypoxia-activated PFKFB2 by stimulating the hypoxia-inducible factor 1 (HIF-1) /ATK signaling. Thus, we conclude that HIF-1/AKT axis-activated PFKFB2 alleviates cardiac dysfunction and cardiomyocyte apoptosis in response to hypoxia. Our finding presents a new insight into the mechanism by which HIF-1/AKT/PFKFB2 signaling modulates MI-related heart disorder under the hypoxia condition, providing potential therapeutic targets and strategy for hypoxia-related myocardial injury.

MiR-3574 ameliorates intermittent hypoxia-induced cardiomyocyte injury through inhibiting Axin1.

Objective: miRNAs play critical roles in the regulation of many cardiovascular diseases. However, its role and potential mechanism in cardiac injury caused by obstructive sleep apnea (OSA) remain poorly elucidated. In the present study, we aimed to investigate the effects of miR-3574 on cardiomyocyte injury under intermittent hypoxia (IH).Results: We confirmed that IH inhibited cell viability, induced cell apoptosis and suppressed miR-3574 expression in the H9c2. miR-3574 overexpression could ameliorate the effects of IH on the cell viability and cell apoptosis in the H9c2. Axin1 was a target gene of miR-3574, and miR-3574 overexpression reduced the expression of Axin1. miR-3574 could inhibit the IH-induced cardiomyocyte injury via downregulating Axin1. However, Axin1 could partially reverse these effects of miR-3574.Conclusion: Our study first reveals that miR-3574 could alleviate IH-induced cardiomyocyte injury by targeting Axin1, which may function as a novel and promising therapy target for OSA-associated cardiovascular diseases.Methods: H9c2 were exposed to IH condition. CCK-8 assay was applied to determine cell viability of H9c2. qRT-PCR was conducted to measure the expression level of mRNA and miRNA. Western blot assay was then performed to detect the protein levels. Finally, we used dual-luciferase reporter assay identify the potential target of miR-3574.

Circ_0023461 Silencing Protects Cardiomyocytes from Hypoxia‐Induced Dysfunction through Targeting miR‐370‐3p/PDE4D Signaling

Background Acute myocardial infarction (AMI) is a common cardiovascular disease with high disability and mortality. Circular RNAs (circRNAs) are implicated in the pathomechanism of multiple human diseases, including AMI. This study intended to explore the function and working mechanism of a novel circRNA circ_0023461 in hypoxia-induced cardiomyocytes. Methods Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were implemented to detect RNA and protein expression. Cell counting kit-8 (CCK8) assay and 5-ethynyl-2′-deoxyuridine (Edu) assay were conducted to analyze cell viability and proliferation ability. Cell migration and apoptosis were assessed by Transwell assay and flow cytometry. Cell oxidative stress was analyzed using the commercial kits. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze cell inflammation. Cell glycolytic metabolism was evaluated using the commercial kits. Dual-luciferase reporter assay and RNA pull-down assay were conducted to verify the intermolecular interactions. Results circ_0023461 expression was upregulated in AMI patients and hypoxia-induced AC16 cells. Hypoxia restrained the viability, proliferation, migration, and glycolysis and induced the apoptosis, oxidative stress, and inflammation of AC16 cells, and these effects were attenuated by the silence of circ_0023461. MicroRNA-370-3p (miR-370-3p) was verified as a target of circ_0023461, and circ_0023461 silencing-mediated protective effects in hypoxia-induced cardiomyocytes were partly alleviated by the knockdown of miR-370-3p. miR-370-3p interacted with the 3′ untranslated region (3′ UTR) of phosphodiesterase 4D (PDE4D), and PDE4D overexpression partly reversed miR-370-3p overexpression-induced protective effects in hypoxia-induced cardiomyocytes. circ_0023461 can upregulate PDE4D expression by acting as a molecular sponge for miR-370-3p in AC16 cells. Conclusion circ_0023461 knockdown attenuated hypoxia-induced dysfunction in AC16 cells partly by targeting the miR-370-3p/PDE4D axis.

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

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

LRG1 promotes hypoxia-induced cardiomyocyte apoptosis and autophagy by regulating hypoxia-inducible factor-1α

ABSTRACT Cardiomyocyte apoptosis and autophagy play important roles in acute myocardial infarction (AMI), but the effect of leucine-rich alpha-2-glycoprotein 1 (LRG1) on the apoptosis and autophagy of H9c2 has not yet been reported. It was found through differential gene analysis and LASSO analysis that LRG1 was the key gene in AMI. In this study, western blot was applied to detect the protein expression of Bax, Bcl2, LC3, p62, LRG1 and hypoxia-inducible factor-1α (HIF-1α); CCK-8 assay was employed to detect cell viability; Annexin V-FITC/PI staining was adopted to evaluate apoptosis, and immunofluorescence assay was applied to detect autophagy. Under hypoxia conditions in H9c2 cells, LRG1 protein levels were increased, the cell activity was decreased, and apoptosis and autophagy were promoted; the downregulated LRG1 significantly enhanced cell viability but inhibited apoptosis and autophagy. When knocking down HIF-1α in the overexpressed LRG1 cells, the effects of LRG1 were reversed under hypoxia condition. In conclusion, LRG1/HIF-1α promoted H9c2 cell apoptosis and autophagy in hypoxia, potentially providing new ideas for the determination and treatment of AMI. Abbreviation: LRG1: Leucine-rich alpha-2-glycoprotein 1; LRR: leucine-rich repeat; HIF-1α: Hypoxia-inducible factor-1α; AMI: acute myocardial infarction

Long non-coding RNA plasmacytoma variant translocation 1 linked to hypoxia-induced cardiomyocyte injury of H9c2 cells by targeting miR-135a-5p/forkhead box O1 axis.

BackgroundMyocardial infarction occurs due to insufficient (ischemia) blood supply to heart for long time; plasmacytoma variant translocation 1 (PVT1) is a long non-coding RNAs (lncRNAs) involved in the pathogenesis of various diseases, including heart disease; However, few studies have explored its role. The present study evaluated the effects of lncRNA PVT1 on hypoxic rat H9c2 cells.MethodsHypoxic injury was examined by measuring cell viability and apoptosis by using cell counting kit-8 activity and flow cytometry assays. Gene expressions after hypoxia were estimated by quantitative real time polymerase chain reaction and the signaling pathway were explored by Western blot analysis. RNA immunoprecipitation and luciferase reporter assays were applied to examine the interactions among genes. Data were analyzed using t-test with one-way or two-way analysis of variance.ResultsThe lncRNA PVT1 is up-regulated in hypoxia-stressed H9c2 cells and knockdown of PVT1 mitigates hypoxia-induced injury in H9c2 cells. PVT1 acts as a sponge for miR-135a-5p and knockdown of PVT1 attenuated the increased hypoxia-induced injury by up-regulating miR-135a-5p. Forkhead box O1 (FOXO1) was identified as a target of miR-135a-5p, and the expression was negatively regulated by miR-135a-5p. The exploration of the underlying mechanism demonstrated that knockdown of FOXO1 reversed PVT1/miR-135a-5p mediated hypoxia-induced injury in H9c2 cells.ConclusionsPVT1 plays a crucial role in hypoxia-injured H9c2 cells through sponging miR-135a-5p and then positively regulating FOXO1.

HCINAP serves a critical role in hypoxia‑induced cardiomyocyte apoptosis via modulating lactate production and mitochondrial‑mediated apoptosis signaling.

Acute myocardial infarction (AMI) is a major cause of heart failure and is associated with insufficient myocardial oxygen supply. However, the molecular mechanisms underlying hypoxia-induced cardiomyocyte apoptosis are not completely understood. In the present study, the role of human coilin interacting nuclear ATPase protein (hCINAP) in cardiomyocytes was investigated. AC16 cells were divided into the following four groups: i) Small interfering (si)RNA-control (Ctrl); (ii) siRNA-hCINAP; (iii) empty vector; and (iv) hCINAP-Flag. Protein expression was assessed using western blotting. MTT and apoptosis assays were conducted to detect cell viability and apoptosis, respectively. CCK8 assays and apoptosis assays were used to detect cell viability and apoptosis, respectively. hCINAP promoter activity was examined by luciferase reporter assay. hCINAP expression was induced in a hypoxia-inducible factor-1α-dependent manner under hypoxic conditions. Compared with the siRNA-Ctrl group, hCINAP knockdown inhibited apoptosis, whereas compared with the vector group, hCINAP overexpression increased apoptosis under hypoxic conditions. Mechanistically, compared with the siRNA-Ctrl group, hCINAP knockdown decreased hypoxia-induced lactate accumulation via regulating lactate dehydrogenase A activity. Moreover, the results indicated that hCINAP was associated with mitochondrial-mediated apoptosis via Caspase signaling. Collectively, the present study suggested that hCINAP was an important regulator in hypoxia-induced apoptosis and may serve as a promising therapeutic target for AMI.

Abstract 15756: Vascular Guidance of Cardiomyocyte Proliferation During Growth and Regeneration

Introduction: Stimulating cardiomyocyte (CM) proliferation is a major strategy for achieving therapeutic heart regeneration. However, heart regeneration requires coordinated interactions of multiple cell types. Because a hallmark of advanced heart failure is vascular rarefaction, the requirement of cardiac endothelial cells (CECs) for cardiac growth and regeneration is of particular importance. Hypothesis: We hypothesized that CECs are required for CM proliferation during growth and regeneration. Methods and Results: We performed a large-scale histologic assessment of neonatal mouse hearts and found the rate of CEC proliferation to shadow CM proliferation over the first 10 days of life. Using a nearest neighbor analysis, we found the fraction of proliferating CECs to be significantly enriched around cycling CMs compared to non-cycling CMs, suggesting that CEC and CM expansion are coupled within a myovascular niche. Single cell sequencing of neonatal mouse hearts after cryoinjury revealed that a majority of these proliferating CECs also express Vegfr2 . To functionally link CEC and CM proliferation, we generated Cdh5-CreER T2 ; Vefgr2 flox/flox mice to genetically delete Vegfr2 from CECs. Compared to mice with intact Vegfr2 , loss of Vegfr2 from CECs in neonatal mice leads to loss of CECs and severely dampens CM proliferation by 4 days (7.01±0.88% vs 0.39±0.35%, p = 7.4x10-4, n = 9),. Interestingly, CM proliferation is attenuated when Vegfr2 is deleted from CECs despite an increase of hypoxia indicators in CMs, signifying that hypoxia-induced CM proliferation is dependent on CECs. In contrast to CEC depletion, treatment of cryoinjured neonatal hearts with AAV encoding the master angiogenic factor, Vegfa can enhance heart regeneration with increased CM cycling in the borderzone (12.6±2.2% vs 5.4±0.4%, p = 0.02, n = 8), reduced scarring of the left ventricle (3.4±1.4% vs 7.6±1.2%, p = 03, n = 16), and improved fractional shortening (51.7±2.5% vs 36.7±4.3%, p = 0.007, n = 14). Conclusions: CEC and CM expansion are spatiotemporally coupled in a myovascular niche during cardiac growth. CECs play a critical role to support CM proliferation and are likely to provide instructive cues that may be leveraged for therapeutic heart regeneration.

Abstract 12889: Clathrin-mediated Endocytosis Plays a Key Role in Stem Cell Therapy for Ischemic Heart Disease

Background and Aim: Cell therapy represents a potential frontier to transform the treatment and prognosis of heart failure and acute myocardial infarction (AMI). Extracellular vesicles (EVs) have come out as key mediators of intercellular communication to improve cardiac function in cell therapy. However, the mechanisms by which cardiomyocytes respond to EVs remain unclear. Here, we sought to clarify the effect of cardiomyocyte endocytosis on AMI. Methods and Results: Treatment with the culture supernatant of adipose-derived regenerative cells (ADRCs) prevented cardiomyocyte cell damage under conditions of hypoxia in vitro. In vivo, the injection of ADRCs in the heart simultaneous with coronary artery ligation decreased infarct area in the heart in a dose-dependent manner. In addition, the injection of ADRCs prevented cardiac rupture after AMI and significantly reduced apoptosis in cardiomyocytes at 1 and 2 days. To examine the efficacy of cardiomyocyte endocytosis, cardiomyocytes were cultured with EVs previously isolated from ADRC culture supernatant and labeled with the fluorescent dye, PKH67. Notably, hypoxic culture conditions increased the intensity of the labeled EVs in cardiomyocytes. An inhibitor of clathrin-mediated endocytosis (chlorpromazine), but not inhibitors of caveolae-mediated endocytosis, micropinocytosis, and phagocytosis (genistein, cytochalasin D, or amiloride), significantly suppressed the ADRC culture supernatant-induced decreases of hypoxia-damaged cardiomyocytes. Chlorpromazine treatment also decreased hypoxia-induced cardiomyocyte capture of labeled EVs secreted from ADRCs. To investigate whether communication between cardiomyocytes and ADRCs affords cardiac protection through clathrin-mediated endocytosis in vivo, mice were treated with ADRCs alone or in combination with chlorpromazine following coronary artery ligation. Chlorpromazine treatment failed to show ADRC-induced cardiac protection such as prevention of cardiac rupture and anti-apoptotic effect. Conclusions: Our results indicate that EVs secreted from ADRCs prevent cardiac rupture after AMI and that clathrin-mediated endocytosis plays a critical role in transporting the EVs into cardiomyocytes.

Circ_0010729 knockdown protects cardiomyocytes against hypoxic dysfunction via miR-370-3p/TRAF6 axis.

Few studies have addressed the mechanism by which circ_0010729 regulates hypoxia-induced cell injury in cardiovascular diseases. However, its role and its regulatory mechanism in myocardial infarction remain to be explored. Cell viability, cycle, apoptosis, and migration were analyzed using cell counting kit-8 assay, flow cytometry, caspase-3 activity assay kit and transwell assay, respectively. Tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentrations were examined by enzyme-linked immunosorbent assay. Glucose metabolism was calculated by detecting ATP production, glucose uptake and lactate production. Levels of circ_0010729, miR-370-3p and TNF Receptor Associated Factor 6 (TRAF6) were detected using quantitative real-time polymerase chain reaction or western blot. The direct interaction between circ_0010729 and TRAF6 or miR-370-3p was verified using dual-luciferase reporter assay and RNA immunoprecipitation assay. Under hypoxia condition, cardiomyocytes suffered from cell viability suppression, cell cycle arrest, cell apoptosis promotion, migration reduction, increase of inflammatory factor IL-6 and TNF-α, as well as glycolysis inhibition. Circ_0010729 expression was up-regulated in the cardiomyocytes at different hypoxia-exposed time points. Circ_0010729 knockdown protected cardiomyocytes against hypoxic dysfunction, while circ_0010729 overexpression showed inverse effects. MiR-370-3p was confirmed to directly bind to circ_0010729 or TRAF6. MiR-370-3p inhibition attenuated the protective effects of circ_0010729 knockdown on hypoxia-modulated cardiomyocyte dysfunction. MiR-370-3p restoration protected cardiomyocytes against hypoxic injury via targeting TRAF6. Besides, circ_0010729 indirectly regulated TRAF6 expression via miR-370-3p. This study demonstrated that circ_0010729 knockdown attenuated hypoxia-induced cardiomyocyte dysfunction via miR-370-3p/TRAF6 axis, indicating a potential therapeutic target for myocardial infarction.

Downregulation of FOXO6 alleviates hypoxia-induced apoptosis and oxidative stress in cardiomyocytes by enhancing Nrf2 activation via upregulation of SIRT6.

Forkhead box protein O6 (FOXO6) has been recently identified as a novel regulator of oxidative stress in multiple pathological processes. However, whether FOXO6 participates in the regulation of oxidative stress of myocardial infarction is unclear. The present study was performed to evaluate the potential role of FOXO6 in regulating hypoxia-induced apoptosis and oxidative stress in cardiomyocytes in vitro. Our results demonstrated that FOXO6 expression was highly elevated in cardiomyocytes exposed to hypoxia. Downregulation of FOXO6 expression by the siRNA-mediated gene knockdown in hypoxia-exposed cardiomyocytes increased cell viability, while repressing apoptosis and reactive oxygen species (ROS) production. In contrast, overexpression of FOXO6 enhanced the sensitivity of cardiomyocytes to hypoxia-induced injury. Further, in-depth research revealed that knockdown of FOXO6 promoted the expression of sirtuin6 (SIRT6) and enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signaling. Moreover, SIRT6 inhibition markedly blocked the FOXO6 knockdown-induced promotion effect on Nrf2 activation. In addition, Nrf2 inhibition partially reversed the FOXO6 knockdown-mediated protective effect against hypoxia-induced cardiomyocyte injury. Taken together, the findings of our study demonstrate that knockdown of FOXO6 is capable of protecting cardiomyocytes from hypoxia-induced apoptosis and oxidative stress by enhancing Nrf2 activation via upregulation of SIRT6. Our study highlights a potential role of FOXO6 in myocardial infarction and suggests it as an attractive target for cardioprotection.

Inhibition of sphingomyelin synthase 2 relieves hypoxia-induced cardiomyocyte injury by reinforcing Nrf2/ARE activation via modulation of GSK-3β.

Sphingomyelin synthase 2 (SMS2) is a vital contributor to tissue injury and affects various pathological processes. However, whether SMS2 participates in the modulation of cardiac injury in myocardial infarction has not been determined. This study aimed to evaluate the potential role of SMS2 in the regulation of cardiomyocyte injury induced by hypoxia, an in vitro model for studying myocardial infarction. Our data revealed that SMS2 expression was significantly upregulated in cardiomyocytes in response to hypoxia. Loss-of-function experiments revealed that knockdown of SMS2 markedly restored the viability of cardiomyocytes impaired by hypoxia, and attenuated hypoxia-evoked apoptosis and reactive oxygen species (ROS) generation. In contrast, cardiomyocytes that highly expressed SMS2 were more sensitive to hypoxia-induced injury. Moreover, SMS2 deficiency enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling through inactivation of glycogen synthase kinase-3β. Notably, suppression of Nrf2 markedly abrogated SMS2 knockdown-mediated cardioprotective effects on hypoxia-exposed cardiomyocytes. Our results illustrate that downregulation of SMS2 exerts a cardioprotective function by protecting cardiomyocytes from hypoxia-induced apoptosis and oxidative stress through enhancement of Nrf2 activation. Our study indicates a potential role of SMS2 in the modulation of cardiac injury, which may contribute to the progression of myocardial infarction.

ABHD15 promotes cell viability, glycolysis, and inhibits apoptosis in cardiomyocytes under hypoxia

Background and aimsMyocardial infarction (MI) has been an important heart disease affecting human health. The aim of this study was to investigate the regulatory effect of abhydrolase domain containing 15 (ABHD15) on hypoxic cardiomyocytes. Methods and resultsHypoxic cardiomyocytes are commonly used as an vitro model for the study of MI. We found that cardiomyocyte viability was decreased under hypoxia, but cell glucose uptake, insulin receptor phosphorylation level and apoptosis were increased. Interestingly, ABHD15 expression was up-regulated in hypoxia-induced cardiomyocytes. Then, we identified the function of ABHD15 in hypoxic cardiomyocytes by using ABHD15 overexpression vector or short interfering RNA (siRNA) against ABHD15. The results showed that overexpression of ABHD15 promoted hypoxic cardiomyocyte viability, glucose uptake and IR phosphorylation (p-IR), and inhibited cell apoptosis. However, knockdown of ABHD15 attenuated hypoxic cardiomyocyte viability, glucose uptake and IR phosphorylation, and promoted apoptosis. Moreover, we found that ABHD15 promoted glucose transporter 4 (GLUT4) expression, translocation and enhance rate-limiting enzyme activation of glycolysis, thereby affecting glucose uptake. Furthermore, our study suggested that ABHD15 may affect the viability and apoptosis of hypoxic cardiomyocytes through IR/Ras/Raf/ERK/MEK and IR/PI3K/AKT/Bcl2/Bad/caspase9 signaling pathways, respectively. When the phosphorylation of IR, Raf or ERK was blocked by inhibitors, the protective effect of overexpressing ABHD15 on the viability of hypoxic cardiomyocytes was eliminated. Furthermore, inhibiting the phosphorylation of IR, AKT or Bcl2 abolished the inhibitory effect of overexpressing ABHD15 on hypoxic cardiomyocyte apoptosis. ConclusionABHD15 regulated myocardial cell viability, glycolysis, and apoptosis under hypoxia, providing a novel potential therapeutic strategy for MI.

Circ_0010729 regulates hypoxia-induced cardiomyocyte injuries by activating TRAF5 via sponging miR-27a-3p

Ischemic cardiomyopathy is a severe cardiovascular disease with high mortality. Circular RNAs (circRNAs) are widely regulated in diverse human diseases, including Ischemic cardiomyopathy. This study aimed to investigate a novel functional mechanism of circRNA circ_0010729 in hypoxia-induced cardiomyocyte injuries. Human cardiomyocytes (AC16) were exposed to hypoxia to mimic ischemic cardiomyopathy in vitro. Cell viability, apoptosis/necrosis and glycolysis progress, were determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, flow cytometry assay and glycolysis stress test, respectively. Cell apoptosis was also assessed by the activity of cleaved caspase-3/7. The levels of glycolysis-related proteins and tumor necrosis factor receptor-associated factor 5 (TRAF5) were examined by western blot. The expression of circ_0010729 and miR-27a-3p was measured by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The prediction about the targeted relationship was verified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay. As a result, hypoxia treatment inhibited cell viability, induced cell apoptosis and blocked glycolysis, however, these injuries were alleviated by circ_0010729 knockdown. MiR-27a-3p was targeted by circ_0010729, and miR-27a-3p inhibition reversed the role of circ_0010729 knockdown, leading to the deterioration of cell injuries. Further, TRAF5 was a target of miR-27a-3p, and circ_0010729 upregulated the expression of TRAF5 by sponging miR-27a-3p. MiR-27a-3p restoration enhanced cell viability, depleted cell apoptosis and promoted glycolysis of hypoxia-induced AC16 cells, while these effects were abolished by TRAF5 overexpression. In conclusion, circ_0010729 knockdown alleviated hypoxia-induced AC16 cell injuries by mediating the miR-27a-3p/TRAF5 axis.

FABP4 inhibitor BMS309403 protects against hypoxia-induced H9c2 cardiomyocyte apoptosis through attenuating endoplasmic reticulum stress.

Acute myocardial infarction is characterized by ischaemia‐induced cardiomyocyte apoptosis, in which the endoplasmic reticulum (ER) stress plays an important role. The fatty acid‐binding protein‐4 (FABP4) has been implicated in regulating ER stress and apoptosis. Yet, whether FABP4 is involved in modulating cardiomyocyte apoptosis remains unclarified. By applying an in vitro model of hypoxia‐induced apoptosis of H9c2 cardiomyocytes, we found that FABP4 expression was elevated upon hypoxia stimulation, which was further demonstrated to be transcriptionally activated by the hypoxia‐inducible factor 1a (HIF‐1α). In addition, the pharmacological inhibition of FABP4 with BMS309403 protected against hypoxia‐induced apoptosis in cardiomyocytes, indicating that FABP4 induction is detrimental for cardiomyocyte survival under hypoxic condition. Moreover, BMS309403 attenuated ER stress in cardiomyocytes exposed to hypoxia, which, however, was reversed by tunicamycin, an ER stress activator. More importantly, the protective effect of BMS309403 on cardiomyocytes vanished in the presence of tunicamycin. Thus, these observations establish that FABP4 inhibitor BMS309403 reduces hypoxia‐induced cardiomyocyte apoptosis through attenuating excessive ER stress, implying that FABP4 inhibition may be of clinical benefit for MI treatment.

Opa1 Reduces Hypoxia-Induced Cardiomyocyte Death by Improving Mitochondrial Quality Control.

Mitochondrial dysfunction contributes to cardiovascular disorders, especially post-infarction cardiac injury, through incompletely characterized mechanisms. Among the latter, increasing evidence points to alterations in mitochondrial quality control, a range of adaptive responses regulating mitochondrial morphology and function. Optic atrophy 1 (Opa1) is a mitochondrial inner membrane GTPase known to promote mitochondrial fusion. In this study, hypoxia-mediated cardiomyocyte damage was induced to mimic post-infarction cardiac injury in vitro. Loss- and gain-of-function assays were then performed to evaluate the impact of Opa1 expression on mitochondrial quality control and cardiomyocyte survival and function. Hypoxic stress reduced cardiomyocyte viability, impaired contractile/relaxation functions, and augmented the synthesis of pro-inflammatory mediators. These effects were exacerbated by Opa1 knockdown, and significantly attenuated by Opa1 overexpression. Mitochondrial quality control was disturbed by hypoxia, as reflected by multiple mitochondrial deficits; i.e., increased fission, defective fusion, impaired mitophagy, decreased biogenesis, increased oxidative stress, and blunted respiration. By contrast, overexpression of Opa1 normalized mitochondrial quality control and sustained cardiomyocyte function. We also found that ERK, AMPK, and YAP signaling can regulate Opa1 expression. These results identify Opa1 as a novel regulator of mitochondrial quality control and highlight a key role for Opa1 in protecting cardiomyocytes against post-infarction cardiac injury.

FOXD3-AS1 Knockdown Suppresses Hypoxia-Induced Cardiomyocyte Injury by Increasing Cell Survival and Inhibiting Apoptosis via Upregulating Cardioprotective Molecule miR-150-5p In Vitro.

Congenital heart disease (CHD) is the most common type of human innate malformation in fetuses. LncRNAs have been pointed to play critical regulatory roles in various types of cardiac development and diseases including CHD. Our study aimed to explore the effects of lncRNA forkhead box D3 antisense RNA 1 (FOXD3-AS1) on hypoxia-induced injury in AC16 cardiomyocytes and the related molecular mechanism. In vitro cell model of CHD was established by stimulating AC16 cells with hypoxia (1% O2). Expression of FOXD3-AS1 and miR-150-5p was detected by qRT-PCR. Hypoxia-induced injury was evaluated by detecting cell survival, lactate dehydrogenase (LDH) release, apoptosis, and caspase-3/7 activity using MTT, LDH assay, flow cytometry analysis, and caspase-3/7 activity assay, respectively. The regulatory relationship between FOXD3-AS1 and miR-150-5p was explored by luciferase reporter assay, RNA immunoprecipitation (RIP), and qRT-PCR. Results showed that hypoxia exposure caused an upregulation of FOXD3-AS1 and a downregulation of miR-150-5p in AC16 cells. Knockdown of FOXD3-AS1 attenuated reduction of cell survival and increase of LDH release, apoptosis, caspase-3/7 activity, and Bcl-2 associated X (Bax) expression induced by hypoxia in AC16 cells. Notably, we demonstrated that FOXD3-AS1 directly interacted with miR-150-5p to inhibit its expression. miR-150-5p knockdown reinforced the reduction of survival and induction of apoptosis by hypoxia and attenuated the effects of FOXD3-AS1 silencing on the same parameters in AC16 cells. In conclusion, FOXD3-AS1 knockdown protected AC16 cardiomyocytes from hypoxia-induced injury by increasing cell survival and inhibiting apoptosis through upregulating miR-150-5p.

Therapeutic potential of tricarboxylic acid cycle metabolite itaconate in cardiovascular diseases.

Therapeutic potential of tricarboxylic acid cycle metabolite itaconate in cardiovascular diseases.