MiR-320 regulates cardiomyocyte apoptosis induced by ischemia-reperfusion injury by targeting AKIP1.
ABSTRACT: Background:MicroRNAs play important roles in regulation of the cardiovascular system. The purpose of this study was to investigate microRNA-320 (miR-320) expression in myocardial ischemia-reperfusion (I/R) injury and the roles of miR-320 in cardiomyocyte apoptosis by targeting AKIP1 (A kinase interacting protein 1). Methods:The level of miR-320 was detected using quantitative real-time polymerase chain reaction (qRT-PCR), and cardiomyocyte apoptosis was detected via terminal dUTP nick end-labeling assay. Cardiomyocyte apoptosis and the mitochondrial membrane potential were evaluated via flow cytometry. Bioinformatics tools were used to identify the target gene of miR-320. The expression levels of AKIP1 mRNA and protein were detected via qRT-PCR and Western blot, respectively. Results:Both the level of miR-320 and the rate of cardiomyocyte apoptosis were substantially higher in the I/R group and H9c2 cells subjected to H/R than in the corresponding controls. Overexpression of miR-320 significantly promoted cardiomyocyte apoptosis and increased the loss of the mitochondrial membrane potential, whereas downregulation of miR-320 had an opposite effect. Luciferase reporter assay showed that miR-320 directly targets AKIP1. Moreover, knock down and overexpression of AKIP1 had similar effects on the H9c2 cells subjected to H/R. Conclusions:miR-320 plays an important role in regulating cardiomyocyte apoptosis induced by I/R injury by targeting AKIP1 and inducing the mitochondrial apoptotic pathway.
Project description:A kinase interacting protein 1 (AKIP1) is a molecular regulator of protein kinase A and nuclear factor kappa B signalling. Recent evidence suggests AKIP1 is increased in response to cardiac stress, modulates acute ischemic stress response, and is localized to mitochondria in cardiomyocytes. The mitochondrial function of AKIP1 is, however, still elusive. Here, we investigated the mitochondrial function of AKIP1 in a neonatal cardiomyocyte model of phenylephrine (PE)-induced hypertrophy. Using a seahorse flux analyzer we show that PE stimulated the mitochondrial oxygen consumption rate (OCR) in cardiomyocytes. This was partially dependent on PE mediated AKIP1 induction, since silencing of AKIP1 attenuated the increase in OCR. Interestingly, AKIP1 overexpression alone was sufficient to stimulate mitochondrial OCR and in particular ATP-linked OCR. This was also true when pyruvate was used as a substrate, indicating that it was independent of glycolytic flux. The increase in OCR was independent of mitochondrial biogenesis, changes in ETC density or altered mitochondrial membrane potential. In fact, the respiratory flux was elevated per amount of ETC, possibly through enhanced ETC coupling. Furthermore, overexpression of AKIP1 reduced and silencing of AKIP1 increased mitochondrial superoxide production, suggesting that AKIP1 modulates the efficiency of electron flux through the ETC. Together, this suggests that AKIP1 overexpression improves mitochondrial function to enhance respiration without excess superoxide generation, thereby implicating a role for AKIP1 in mitochondrial stress adaptation. Upregulation of AKIP1 during different forms of cardiac stress may therefore be an adaptive mechanism to protect the heart.
Project description:cAMP-dependent protein kinase (PKA) regulates a myriad of functions in the heart, including cardiac contractility, myocardial metabolism,and gene expression. However, a molecular integrator of the PKA response in the heart is unknown. Here, we show that the PKA adaptor A-kinase interacting protein 1 (AKIP1) is up-regulated in cardiac myocytes in response to oxidant stress. Mice with cardiac gene transfer of AKIP1 have enhanced protection to ischemic stress. We hypothesized that this adaptation to stress was mitochondrial dependent. AKIP1 interacted with the mitochondrial localized apoptosis inducing factor (AIF) under both normal and oxidant stress. When cardiac myocytes or whole hearts are exposed to oxidant and ischemic stress, levels of both AKIP1 and AIF were enhanced. AKIP1 is preferentially localized to interfibrillary mitochondria and up-regulated in this cardiac mitochondrial subpopulation on ischemic injury. Mitochondria isolated from AKIP1 gene transferred hearts showed increased mitochondrial localization of AKIP1, decreased reactive oxygen species generation, enhanced calcium tolerance, decreased mitochondrial cytochrome C release,and enhance phosphorylation of mitochondrial PKA substrates on ischemic stress. These observations highlight AKIP1 as a critical molecular regulator and a therapeutic control point for stress adaptation in the heart.
Project description:Cardiac adaptation to unremitting physiological stress typically involves hypertrophic growth of cardiomyocytes, a compensatory response that often fails and causes heart disease. Gene array analysis identified AKIP1 (A Kinase Interacting Protein 1) as a hypertrophic gene and we therefore hypothesized a potential role in the hypertrophic response. We show for the first time that both AKIP1 mRNA and protein levels increased in hypertrophic cardiomyocytes under conditions of sustained cardiac stress, including pressure overload and after myocardial infarction and in vitro in phenylephrine (PE) stimulated neonatal rat ventricular cardiomyocytes (NRVCs). AKIP1 overexpression in NRVCs markedly stimulated hypertrophic growth responses, including significantly increased cell size, augmented cytoskeletal organization and protein synthesis. Although, AKIP1 was not essential for PE induced hypertrophy in NRVCs, it did potentiate neurohormonal induced protein synthesis. AKIP1 did, however, not induce expression of pathological marker genes like ANP and ?-MHC. ERK and Akt kinase signaling pathways have been linked to hypertrophy and AKIP1 specifically induced phosphorylation of Akt. This phosphorylation of Akt was essential for activation of ribosomal rpS6 and translation elongation factor eEF2 and this readily explains the increased protein synthesis. Akt inhibition fully blocked AKIP1 induced hypertrophy, showing that this pathway is critically involved. In conclusion, our results show that AKIP1 is induced in hypertrophic hearts and can stimulate adaptive cardiomyocyte growth, which involves Akt signaling.
Project description:BACKGROUND:The erythropoietin helix B surface peptide (HBSP) has been shown to have neuroprotective and repair-damaging myocardium effects similar to erythropoietin (EPO). However, the protective mechanism of HBSP on cardiomyocyte hypoxia-reoxygenation (H/R) injury is not clear. METHODS:H9C2 cells were pretreated with HBSP and subjected to hypoxia/reoxygenation (H/R), changes in cell function, autophagy and apoptosis were assessed, respectively. Cells were transfected with miR-21 mimic and miR-NC, and the relative expression of miR-21 and Atg12 were detected by qRT-PCR. The target role of miR-21 and Atg12 was evaluated by dual-luciferase reporter. After transfected with si-Atg12 and si-NC, western blot was used to assess autophagy and apoptosis proteins, flow cytometry assay was used to detect apoptosis rate. RESULTS:We found the expression of miR-21 was significantly down-regulated, accompanied by remarkably activated of autophagy and apoptosis in H9C2 cells during H/R injury. Pleasantly, HBSP pretreatment has a similar effect as transfection of miR-21 mimic, which is to evidently inhibit autophagy and apoptosis by up-regulating miR-21 expression. Moreover, Bioinformatics analysis and luciferase reporter assay revealed that Atg12 was directly bond to miR-21. To further understand whether Atg12 is involved in the process of miR-21 regulating autophagy, si-Atg12 and si-NC were transfected into H9C2 cell, the results showed that knockdown of Atg12 enhances the inhibition autophagy and apoptosis effect of HBSP. CONCLUSION:These results demonstrate that HBSP inhibits myocardial H/R injury induced by autophagy over-activation and apoptosis via miR-21/Atg12 axis.
Project description:The molecular mechanisms underlying anthracyclines-induced cardiotoxicity have not been well elucidated. MiRNAs were revealed dysregulated in the myocardium and plasma of rats received Dox treatment. MicroRNA-34a-5p (miR-34a-5p) was verified increased in the myocardium and plasma of Dox-treated rats, but was reversed in rats received Dox plus DEX treatments. Human miR-34a-5p was also observed increased in the plasma of patients with diffuse large B-cell lymphoma after 9- and 16-week epirubicin therapy. Up-regulation of miR-34a-5p was observed in Dox-induced rat cardiomyocyte H9c2 cells. MiR-34a-5p could augment Bax expression, but inhibited Bcl-2 expression, along with the increases of the activated caspase-3 and mitochondrial potentials in H9C2 cells. MiR-34a-5p was verified to modulate Sirt1 expression post-transcriptionally. In parallel to Sirt1 siRNA, miR-34a-5p could enhance p66shc expression, accompanied by increases of Bax and the activated caspase-3 and a decrease of Bcl-2 in H9c2 cells. Moreover, enforced expression of Sirt1 alleviated Dox-induced apoptosis of H9c2 cells, with suppressing levels of p66shc, Bax, the activated caspase-3 and miR-34a-5p, and enhancing Bcl-2 expression. Therefore, miR-34a-5p enhances cardiomyocyte apoptosis by targeting Sirt1, activation of miR-34a-5p/Sirt1/p66shc pathway contributes to Dox-induced cardiotoxicity, and blockage of this pathway represents a potential cardioprotective effect against anthracyclines.
Project description:Mitochondria-mediated cardiomyocyte apoptosis is involved in myocardial ischemia/reperfusion (MI/R) injury. Clematichinenoside (AR) is a triterpenoid saponin isolated from the roots of Clematis chinensis with antioxidant and anti-inflammatory cardioprotection effects against MI/R injury, yet the anti-apoptotic effect and underlying mechanisms of AR in MI/R injury remain unclear. We hypothesize that AR may improve mitochondrial function to inhibit MI/R-induced cardiomyocyte apoptosis. In this study, we replicated an in vitro H9c2 cardiomyocyte MI/R model by hypoxia/reoxygenation (H/R) treatment. The viability of H9c2 cardiomyocytes was determined by MTT assay; apoptosis was evaluated by flow cytometry and TUNEL experiments; mitochondrial permeability transition pore (mPTP) opening was analyzed by a calcein-cobalt quenching method; and mitochondrial membrane potential (??m) was detected by JC-1. Moreover, we used western blots to determine the mitochondrial cytochrome c translocation to cytosolic and the expression of caspase-3, Bcl-2, and Bax proteins. These results showed that the application of AR decreased the ratio of apoptosis and the extent of mPTP opening, but increased ??m. AR also inhibited H/R-induced release of mitochondrial cytochrome c and decreased the expression of the caspase-3, Bax proteins. Conversely, it remarkably increased the expression of Bcl-2 protein. Taken together, these results revealed that AR protects H9c2 cardiomyocytes against H/R-induced apoptosis through mitochondrial-mediated apoptotic signaling pathway.
Project description:Doxorubicin (DOX) is a broad-spectrum anti-tumor drug, but its cardiotoxicity limits its clinical application. A better understanding of the molecular mechanisms underlying DOX cardiotoxicity will benefit clinical practice and remedy heart failure. Our present study observed that DOX caused cardiomyocyte (H9c2) apoptosis via the induction of abnormal mitochondrial fission. Notably, the expression levels of p21 increased in DOX-treated cardiomyocytes, and the silencing of p21 using siRNA greatly attenuated mitochondrial fission and apoptosis in cardiomyocytes. We also found that miR-499-5p could directly target p21 and attenuated DOX-induced mitochondrial fission and apoptosis. The role of the miR-499-5p-p21 axis in the prevention of DOX cardiotoxicity was also validated in the mice model. DOX treatment induced an upregulation of p21, which induced subsequent abnormal mitochondrial fission and myocardial apoptosis in mouse heart. Adenovirus-harboring miR-499-5p-overexpressing mice exhibited significantly reduced p21 expression, mitochondrial fission and myocardial apoptosis in hearts following DOX administration. The miR-499-5p-overexpressing mice also exhibited improved cardiomyocyte hypertrophy and cardiac function after DOX treatment. However, miR-499-5p was not involved in the DOX-induced apoptosis of cancer cells. Taken together, these findings reveal an emerging role of p21 in the regulation of mitochondrial fission program. miR-499-5p attenuated mitochondrial fission and DOX cardiotoxicity via the targeting of p21. These results provide new evidence for the miR-499-5p-p21 axis in the attenuation of DOX cardiotoxicity. The development of new therapeutic strategies based on the miR-499-5p-p21 axis is a promising path to overcome DOX cardiotoxicity as a chemotherapy for cancer treatment.
Project description:Non-small-cell lung cancer (NSCLC) is one of the leading death-related malignancies worldwide with elusive molecular mechanisms. A-kinase interacting protein 1 (AKIP1) is an important regulator controlling metastasis, lymphangiogenesis and angiogenesis. However, the role of AKIP1 in NSCLC progression is still little known. Here, we found that AKIP1 was overexpressed in NSCLC specimens as well as cell lines. Overexpression of AKIP1 in NSLCC tissues was positively correlated with TNM stage, lymph node metastasis and poor prognosis. Knockdown of AKIP1 inhibited NSCLC cell migration, invasion and epithelial-mesenchymal transition (EMT), as indicated by the up-regulation of mesenchymal markers (fibronectin and vimentin) and down-regulation of epithelial marker E-cadherin, whereas overexpression of AKIP1 showed the opposite effects. Moreover, AKIP1 transactivated Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression via directly binding to ZEB1 promoter, thereby leading to E-cadherin transcriptional repression. Additionally, we observed that the binding efficiency of AKIP1 within ZEB1 promotor was determined by the interaction between AKIP1 and SP1. In conclusion, AKIP1 promoted EMT of NSCLC via transactivating ZEB1, suggesting AKIP1 as a potential therapeutic target.
Project description:miRNAs have been implicated in processing of cardiac hypoxia/reoxygenation (H/R)-induced injury. Recent studies demonstrated that miR-19a might provide a potential cardioprotective effect on myocardial disease. However, the effect of miR-19a in regulating myocardial ischemic injury has not been previously addressed. The present study was to investigate the effect of miR-19a on myocardial ischemic injury and identified the potential molecular mechanisms involved. Using the H/R model of rat cardiomyocytes H9C2 in vitro, we found that miR-19a was in low expression in H9C2 cells after H/R treatment and H/R dramatically decreased cardiomyocyte viability, and increased lactate dehydrogenase (LDH) release and cardiomyocyte apoptosis, which were attenuated by co-transfection with miR-19a mimic. Dual-luciferase reporter assay and Western blotting assay revealed that PTEN was a direct target gene of miR-19a, and miR-19a suppressed the expression of PTEN via binding to its 3'-UTR. We further identified that overexpression of miR-19a inhibited the expression of PTEN at the mRNA and protein levels. Moreover, PTEN was highly expressed in H/R H9C2 cells and the apoptosis induced by H/R was associated with the increase in PTEN expression. Importantly, miR-19a mimic significantly increased p-Akt levels under H/R. In conclusion, our findings indicate that miR-19a could protect against H/R-induced cardiomyocyte apoptosis by inhibiting PTEN /PI3K/p-Akt signaling pathway.
Project description:BACKGROUND:Recent studies have shown that microRNAs (miRNAs) are involved in the process of cardiomyocyte apoptosis. We have previously reported that granulocyte-colony stimulating factor (G-CSF) ameliorated diastolic dysfunction and attenuated cardiomyocyte apoptosis in a rat model of diabetic cardiomyopathy. In this study, we hypothesized a regulatory role of cardiac miRNAs in the mechanism of the anti-apoptotic effect of G-CSF in a diabetic cardiomyopathy rat model. METHODS:Rats were given a high-fat diet and low-dose streptozotocin injection and then randomly allocated to receive treatment with either G-CSF or saline. H9c2 rat cardiomyocytes were cultured under a high glucose (HG) condition to induce diabetic cardiomyopathy in vitro. We examined the extent of apoptosis, miRNA expression, and miRNA target genes in the myocardium and H9c2 cells. RESULTS:G-CSF treatment significantly decreased apoptosis and reduced miR-34a expression in diabetic myocardium and H9c2 cells under the HG condition. G-CSF treatment also significantly increased B-cell lymphoma 2 (Bcl-2) protein expression as a target for miR-34a. In addition, transfection with an miR-34a mimic significantly increased apoptosis and decreased Bcl-2 luciferase activity in H9c2 cells. CONCLUSION:Our results indicate that G-CSF might have an anti-apoptotic effect through down-regulation of miR-34a in a diabetic cardiomyopathy rat model.