Simulated Ischemia-reperfusion Research Articles

SIRT3 and RORα are two prospective targets against mitophagy during simulated ischemia/reperfusion injury in H9c2 cells

Autophagy during myocardial ischemia/reperfusion (MI/R) exacerbates cardiomyocyte injury. Melatonin (Mel) alleviates myocardial damage by regulating mitochondrial function and mitophagy, but the role of mitophagy in melatonin-induced cardioprotection remains unclear. This study aimed to explore the roles of sirtuin3 (SIRT3) and retinoid‐related orphan nuclear receptor‐α (RORα) in mitophagy during simulated ischemia reperfusion (SIR) in H9c2 cells. Our data showed that mitophagy was excessively activated after SIR injury, which was consistent with reduced cell survival, enhanced oxidative responses and mitochondrial dysfunction in H9c2 myocytes. Melatonin greatly enhanced cell viability, reduced oxidative stress and improved mitochondrial function. The effects of melatonin protection were involved in excessive mitophagy inhibition, as demonstrated by the reduced levels of mitophagy-linked proteins, including Parkin, Beclin1, NIX and BNIP3, and the LC3 II/LC3 I ratio and elevations in p62. Additionally, the decreases in SIRT3 and RORα in H9c2 myocytes after SIR were reversed by melatonin, and the above effects of melatonin were eliminated by small interfering RNA (siRNA)-mediated knockdown of SIRT3 and RORα. In brief, SIRT3 and RORα are two prospective targets in the cardioprotection of melatonin against mitophagy during SIR in H9c2 myocytes.

Thyroid hormone protects human lung epithelial cells from cold preservation and warm reperfusion-induced injury

BackgroundCellular stress associated with static-cold storage (SCS) and warm reperfusion of donor lungs can contribute to ischemia–reperfusion (IR) injury during transplantation. Adding cytoprotective agents to the preservation solution may be conducive to reducing graft deterioration and improving post-transplant outcomes.MethodsSCS and warm reperfusion were simulated in human lung epithelial cells (BEAS-2B) by exposing cells to low potassium dextran glucose solution at 4 °C for different periods and then switching back to serum-containing culture medium at 37 °C. Transcriptomic analysis was used to explore potential cytoprotective agents. Based on its results, cell viability, caspase activity, cell morphology, mitochondrial function, and inflammatory gene expression were examined under simulated IR conditions with or without thyroid hormones (THs).ResultsAfter 18 h SCS followed by 2 h warm reperfusion, genes related to inflammation and cell death were upregulated, and genes related to protein synthesis and metabolism were downregulated in BEAS-2B cells, which closely mirrored gene profiles found in thyroid glands of mice with congenital hypothyroidism. The addition of THs (T3 or T4) to the preservation solution increases cell viability, inhibits activation of caspase 3, 8 and 9, preserves cell morphology, enhances mitochondrial membrane potential, reduces mitochondrial superoxide production, and suppresses inflammatory gene expression.ConclusionAdding THs to lung preservation solutions may protect lung cells during SCS by promoting mitochondrial function, reducing apoptosis, and inhibiting pro-inflammatory pathways. Further in vivo testing is warranted to determine the potential clinical application of adding THs as therapeutics in lung preservation solutions.

Low-intensity pulsed ultrasound of different intensities differently affects myocardial ischemia/reperfusion injury by modulating cardiac oxidative stress and inflammatory reaction.

The prevalence of ischemic heart disease has reached pandemic levels worldwide. Early revascularization is currently the most effective therapy for ischemic heart diseases but paradoxically induces myocardial ischemia/reperfusion (MI/R) injury. Cardiac inflammatory reaction and oxidative stress are primarily involved in the pathology of MI/R injury. Low-intensity pulsed ultrasound (LIPUS) has been demonstrated to reduce cell injury by protecting against inflammatory reaction and oxidative stress in many diseases, including cardiovascular diseases, but rarely on MI/R injury. This study was designed to clarify whether LIPUS alleviates MI/R injury by alleviating inflammatory reaction and oxidative stress. Simultaneously, we have also tried to confirm which intensity of the LIPUS might be more suitable to ameliorate the MI/R injury, as well as to clarify the signaling mechanisms. MI/R and simulated ischemia/reperfusion (SI/R) were respectively induced in Sprague Dawley rats and human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). LIPUS treatment, biochemical measurements, cell death assay, estimation of cardiac oxidative stress and inflammatory reaction, and protein detections by western blotting were performed according to the protocol. In our study, both in vivo and in vitro, LIPUS of 0.1 W/cm2 (LIPUS0.1) and 0.5 W/cm2 (LIPUS0.5) make no significant difference in the cardiomyocytes under normoxic condition. Under the hypoxic condition, MI/R injury, inflammatory reaction, and oxidative stress were partially ameliorated by LIPUS0.5 but were significantly aggravated by LIPUS of 2.5 W/cm2 (LIPUS2.5) both in vivo and in vitro. The activation of the apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) pathway in cardiomyocytes with MI/R injury was partly rectified LIPUS0.5 both in vivo and in vitro. Our study firstly demonstrated that LIPUS of different intensities differently affects MI/R injury by regulating cardiac inflammatory reaction and oxidative stress. Modulations on the ASK1/JNK pathway are the signaling mechanism by which LIPUS0.5 exerts cardioprotective effects. LIPUS0.5 is promising for clinical translation in protecting against MI/R injury. This will be great welfare for patients suffering from MI/R injury.

Metformin protects against retinal ischemia/reperfusion injury through AMPK-mediated mitochondrial fusion

Retinal ischemia/reperfusion (I/R) injury is a common pathological process responsible for cellular damage in glaucoma, diabetic retinopathy and hypertensive retinopathy. Metformin is a biguanide drug that exerts strong effects on multiple diseases. This study aims to evaluate the protective effect of metformin against retinal I/R injury and its underlying mechanism. I/R induced reduction in retina thickness and cell number in ganglion cell layer, and metformin alleviated I/R-induced retinal injury. Both retinal I/R and simulated ischemia/reperfusion (SIR) in R28 cells down-regulated expression of mitochondrial fusion protein Mfn2 and OPA1, which led to mitochondrial fission. Metformin also alleviated damage in R28 cells, and reversed the alteration in Mfn2 and OPA1, mitochondrial fission and mitochondrial membrane potential (MMP) disruption-induced by I/R or SIR as well. Intriguingly, inhibition of AMPK by compound C or siRNA prevented metformin-mediated up-regulation of Mfn2 and OPA1. Compound C and knockdown of Mfn2 or OPA1 dramatically alleviated the protective effect of metformin against intracellular ROS generation, MMP disruption, mitochondrial fission and loss of RGCs in ganglion cell layer induced by SIR or I/R. Moreover, scavenging mitochondrial ROS (mito-ROS) by mito-TEMPO exerted the similar protection against I/R-induced retinal injury or SIR-induced damage in R28 cells as metformin. Our data show for the first time that metformin protects against retinal I/R injury through AMPK-mediated mitochondrial fusion and the decreased mito-ROS generation. These findings might also repurpose metformin as a therapeutic agent for retinal I/R injury.

Penehyclidine Hydrochloride Protects Rat Cardiomyocytes from Ischemia- Reperfusion Injury by Platelet-derived Growth Factor-B.

The lack of effective treatments for myocardial ischemiareperfusion (MI-R) injury severely restricts the effectiveness of the treatment of ischemic heart disease. In the present research, we aimed to investigate the protective effect and molecular mechanism of penehyclidine hydrochloride (PHC) on MI-R cells. Cell viability was quantified using CCK8. Cell apoptosis was analyzed using flow cytometry. Western blot and Elisa assays were used for the detection of target proteins. PHC pretreatment attenuated the inhibition of cell viability and decreased the percentage of apoptosis induced by simulated ischemia reperfusion (SIR). Platelet-derived growth factor B (PDGF-B) and its downstream AKT pathway were activated in PHC pretreated cells. After siRNAPDGF- B transfection, cell viability was inhibited and apoptosis was activated in PHC pretreated SIR cells, suggesting that PHC protected cells from SIR. PDGF-B knockdown also increased the levels of CK, LDH, IL-6 and TNF-α in PHC pretreated SIR cells. The effect of AKT inhibitor on H9C2 cells was consistent with that of PDGF-B knockdown. PHC pretreatment can protect cardiomyocytes from the decrease of cell activity and the increase of apoptosis caused by reperfusion through up-regulating PDGF-B to activate PI3K pathway. Our study indicates that PHC is a potential drug to protect cells from reperfusion injury and PDGF-B is a potential target for preventing MI-R injury.

Role of autophagy in simulated ischemic-reperfused left atrial myocardium

Background and aimAutophagy has recently emerged as a potential and promising therapeutic approach to maintain cardiac cellular homeostasis. The aim of the present study was to investigate the role of autophagy in the ischemic-reperfused atrial myocardium. MethodsIsolated rat left atria subjected to simulated ischemia-reperfusion were used. The bathing medium contained either 10 mM d-glucose or 10 mM d-glucose and 1.2 mM palmitate. 3-methyladenine (3-MA) was used as pharmacological autophagy inhibitor. ResultsLC3-II/LC3-I ratio, an indicator of autophagosome formation, was significantly enhanced during reperfusion, this increase being slowed by the exposure to high palmitate concentration and prevented by 3-MA. Beclin-1 was significantly increased during reperfusion period in both metabolic conditions, and pharmacological inhibition of AMPK partially prevented LC3-II/LC3-I ratio increase. Autophagy inhibition significantly increased mitochondrial damage and impaired mitochondrial ATP synthesis rate at reperfusion. Tissue ATP content recovery and contractile reserve were also reduced during this period, these effects being more pronounced either in 3-MA treated atria and ischemic-reperfused atria incubated with palmitate. Moreover, severe tachyarrhythmias were observed in the presence of 3-MA, in both metabolic conditions. This phenomenon was partially prevented by mitochondrial inner membrane ion channels blocker, PK11195. ConclusionPresent study provides new insights into the role of autophagy in ischemic-reperfused atrial myocardium. The observation of greater deterioration in mitochondrial structure and function when this process was inhibited, suggests an association between autophagy and the structural and functional preservation of mitochondria. Exogenous metabolic substrates, to which the myocardium is exposed during ischemia-reperfusion, might not affect this process.

Bergenin alleviates myocardial ischemia‐reperfusion injury via SIRT1 signaling

Myocardial ischemia-reperfusion (MI/R) is a major risk factor for cardiovascular disease. At present, reducing oxidative stress and apoptosis is a crucial therapeutic strategy for ameliorating MI/R injury. However, there is a lack of drugs targeting oxidative stress and apoptosis for the clinical therapy of MI/R. Bergenin is a reportedly effective agent with antioxidative and antiapoptotic activity against acute injury. Nevertheless, the roles and potential mechanisms of bergenin against MI/R injury remain unknown. Here, we hypothesized that bergenin attenuated MI/R-induced apoptosis and reactive oxygen species (ROS) production via SIRT1. Mice were subjected to MI/R and treated with bergenin, after which the cardiac function, cardiomyocyte apoptosis, LDH release, and MDA content were evaluated. In vitro, myocardial injury model of H9c2 cells was induced by simulated ischemia/reperfusion (SI/R), apoptosis and oxidative stress was decreased after treated with bergenin. Bergenin significantly reduced myocardial apoptosis and ROS generation in vitro and improved cardiac function in vivo. Intriguingly, bergenin remarkably decreased apoptosis in cardiac tissue accompanied by SIRT1 upregulation following MI/R injury. Further studies showed that inhibiting SIRT1 blocked bergenin’s beneficial impact against apoptosis following SI/R injury through excessive oxidative stress and depression of the Bcl2 to Bax ratio. Collectively, these findings indicate that bergenin alleviates MI/R injury by ameliorating myocardial apoptosis and oxidative damage via the SIRT1 signaling pathway.

Role of cardiac mitofusins in cardiac conduction following simulated ischemia–reperfusion

Mitochondrial dysfunction induced by acute cardiac ischemia–reperfusion (IR), may increase susceptibility to arrhythmias by perturbing energetics, oxidative stress production and calcium homeostasis. Although changes in mitochondrial morphology are known to impact on mitochondrial function, their role in cardiac arrhythmogenesis is not known. To assess action potential duration (APD) in cardiomyocytes from the Mitofusins-1/2 (Mfn1/Mfn2)-double-knockout (Mfn-DKO) compared to wild-type (WT) mice, optical-electrophysiology was conducted. To measure conduction velocity (CV) in atrial and ventricular tissue from the Mfn-DKO and WT mice, at both baseline and following simulated acute IR, multi-electrode array (MEA) was employed. Intracellular localization of connexin-43 (Cx43) at baseline was evaluated by immunohistochemistry, while Cx-43 phosphorylation was assessed by Western-blotting. Mfn-DKO cardiomyocytes demonstrated an increased APD. At baseline, CV was significantly lower in the left ventricle of the Mfn-DKO mice. CV decreased with simulated-ischemia and returned to baseline levels during simulated-reperfusion in WT but not in atria of Mfn-DKO mice. Mfn-DKO hearts displayed increased Cx43 lateralization, although phosphorylation of Cx43 at Ser-368 did not differ. In summary, Mfn-DKO mice have increased APD and reduced CV at baseline and impaired alterations in CV following cardiac IR. These findings were associated with increased Cx43 lateralization, suggesting that the mitofusins may impact on post-MI cardiac-arrhythmogenesis.

Salidroside attenuates myocardial ischemia/reperfusion injury via AMPK-induced suppression of endoplasmic reticulum stress and mitochondrial fission

Ischemic heart disease (IHD) is the primary cause of death worldwide. Salidroside (Sal), the major active compound derived from Rhodiola rosea, is believed to have cardioprotective effects. AMP-activated protein kinase (AMPK), is a pivotal AMP-activated protein kinase in energy metabolism. Whether Sal plays an anti-endoplasmic reticulum stress/mitochondrial fission role through AMPK remains elusive. In this study, we established a myocardial ischemia/reperfusion (I/R) rat model. Rat hearts exposed to Sal with or without compound C were then subjected to I/R. Further, H9c2 cardiomyocytes were subjected to simulated ischemia/reperfusion (SIR) by hypoxia-reoxygenation. The rats and cardiomyocytes were pretreated with Sal, followed by Compound C and AMPK-siRNA to block AMPK activity. We found that Sal significantly ameliorated cardiac function, mitigated infarct size and serum content of lactate dehydrogenase and creatine kinase, improved mitochondrial function, and reduced mitochondrial fission and apoptosis. Furthermore, in cultured H9c2 cardiomyocytes, Sal increased the cell viability and inhibited SIR-induced myocardial apoptosis and mitochondrial fission. Furthermore, the translocation of Drp1 from the cytoplasm to mitochondria induced by salidroside was confirmed both in vivo and in vitro. However, the use of Compound C or AMPK siRNA to block AMPK activity leads to blockade of the protective effects of Sal. In summary, protects against myocardial I/R by activating the AMPK signaling pathway, inhibiting ER stress, and reducing mitochondrial fission and apoptosis.

5-Methyltetrahydrofolate Attenuates Oxidative Stress and Improves Kidney Function in Acute Kidney Injury through Activation of Nrf2 and Antioxidant Defense.

Oxidative stress is a major mediator of adverse outcomes in acute kidney injury (AKI). Deficiency of micronutrients, such as folate, is common in AKI. Our previous study reported that AKI impaired kidney reabsorption of folate and decreased plasma folate level in rats. The present study investigated the effect of 5-methyltetrahydrofolate (5-MTHF), a biologically active form of folate/folic acid, on AKI-impaired kidney function and oxidative stress. Sprague-Dawley rats developed AKI after kidney ischemia (45 min) and reperfusion (24 h). Injection of 5-MTHF (3 µg/kg body weight) improved kidney function and attenuated oxidative stress with a restoration of glutathione and a reduction of lipid peroxidation in the kidney. Injection of 5-MTHF activated transcription factor Nrf2 and increased the expression of glutathione synthesizing enzymes, superoxide dismutase-1 and heme oxygenase-1 in the kidney. Simulated ischemia-reperfusion through hypoxia-reoxygenation increased oxidative stress in proximal tubular cells. Incubation of cells with 5-MTHF alleviated cell injury and increased antioxidant enzyme expression and intracellular glutathione levels. Inhibition of Nrf2 expression through siRNA transfection abolished the effect of 5-MTHF against oxidative stress. These results suggest that low-dose folic acid can improve kidney function through activation of Nrf2 and restoration of antioxidant defence. Micronutrient supplements may improve clinical outcomes in AKI.

Effect of Pre‐Conditioning with Copolymer‐Based Cell Membrane Stabilizer Poloxamer 188 on Adult Mouse Coronary Artery Endothelial Layer Integrity after Prolonged Hypoxia/Reoxygenation

Prolonged ischemia followed by reperfusion can cause significant tissue damage and cell death. This ischemia/reperfusion (IR) injury can be alleviated by administration of Copolymer‐based Cell Membrane Stabilizers (CCMS), like the tri‐block Poloxamer (P)188, thought to be able to seal gaps in cell membranes and/or tight junctions and, thus, repair leaks attributable to severe cellular stress. We have previously tested post‐conditioning, i.e. the administration of P188 upon reperfusion/reoxygenation, to attenuate IR injury in various models. Here, we hypothesized that pre‐conditioning, i.e. its administration before simulated ischemia (hypoxia), has a protective effect on endothelial cell layer integrity as assessed by Trans‐Endothelial Electrical Resistance (TEER).Confluent adult mouse coronary artery endothelial cell (ECs) layers were pre‐treated with 0, 30, 100, 300, 1,000, or 2,000 µM P188 during plating of cells before simulated IR with a 24‐hour hypoxic followed by a 2‐hour reoxygenation (HR) period. EC monolayer barrier integrity was assessed by TEER.In contrast to mere post‐conditioning, pre‐conditioning dose‐dependently attenuated HR injury compared to control/normoxia (Fig 1) even after prolonged HR for 24+2 hrs.These results suggest that CCMS may indeed elicit its cardioprotective effect, in part, through protection of the EC layer. However, more research is needed to determine optimal dosage and timing for CCMS administration.

Parathyroid Hormone-Related Peptide and Its Analog, Abaloparatide, Attenuate Lethal Myocardial Ischemia-Reperfusion Injury.

Parathyroid hormone-related peptide (PTHrP) is well-known to play a role in bone formation, and abaloparatide, an analog of PTHrP(1-34), is approved for the treatment of osteoporosis in post-menopausal women. PTHrP has also been reported to have cardiovascular effects, with recent data demonstrating that exogenously administered PTHrP can limit the death of isolated cardiomyocytes subjected to oxidative stress via upregulation of classic ‘survival kinase’ signaling. Our aim in the current study was to extend this concept and, employing both in vitro and in vivo models, establish whether PTHrP(1-36) and abaloparatide are cardioprotective in the setting of lethal myocardial ischemia-reperfusion injury. We report that preischemic administration of PTHrP(1-36) and abaloparatide attenuated cell death in HL-1 cardiomyocytes subjected to simulated ischemia-reperfusion, an effect that was accompanied by the augmented expression of phospho-ERK and improved preservation of phospho-Akt, and blocked by co-administration of the MEK-ERK inhibitor PD98059. Moreover, using the translationally relevant swine model of acute coronary artery occlusion-reperfusion, we make the novel observation that myocardial infarct size was significantly reduced in pigs pretreated with PTHrP(1-36) when compared with placebo-controls (13.1 ± 3.3% versus 42.0 ± 6.6% of the area of at-risk myocardium, respectively; p < 0.01). Taken together, these data provide the first evidence in support of the concept that pretreatment with PTHrP(1-36) and abaloparatide renders cardiomyocytes resistant to lethal myocardial ischemia-reperfusion injury.

Antioxidant Activity Of Rat Liver With A Low Resistance To Hypoxia After Systemic Ischemia Reperfusion

Objective — To assess the antioxidant activity of rat liver after systemic ischemia reperfusion (IRP). Material and Methods — The study was conducted on 70 male rats. For all animals of the treatment group (n=35) under ether anesthesia, we were stopping stopping systemic circulation for five minutes. After that, the animals were given an external cardiac massage and artificial lung ventilation. We did not perform circulatory arrest after ether anesthesia in animals of the control group (n=35). In all animals, we were measuring the levels of serum hormones (corticosterone, aldosterone), the content of glucocorticoid and mineralocorticoid receptors in liver homogenates, and the activity of enzymes of the antioxidant system (superoxide dismutase and catalase). We were making control measurements on days 1, 3, 5, 7, 14, 21, and 35 after the simulated IRP. Results — On day 1 after simulation of IRP development, the levels of cortisol and aldosterone in the serum of treatment group rats were significantly higher, by 14.3% and 33.5%, respectively, compared with the control group. In response to stress (IRP), we observed the highest concentration of cortisol in the blood of treatment group rats on day 3 (p=0.0002), which decreased afterwards. On day 1 after IRP, there was a reduction in the activity of superoxide dismutase and catalase in treatment group rats, by 50.3% and by 29%, respectively (p&lt;0.0001). The lowest antioxidant activity in the rat liver after IRP was observed on days 3-7. Conclusion — Systemic IRP is associated with pronounced changes in the dynamics of corticosteroid receptors in the liver, which leads to a reduction in the activity of key antioxidant enzymes.

Abstract 11710: Poloxamer 188 Protects Coronary Artery Endothelial Cells After Extended Hypoxia and Reoxygenation in an in vitro Model of Simulated Ischemia/Reperfusion Injury

Reperfusion restores blood flow after myocardial ischemia but can cause additional cellular injury by the sudden reintroduction of oxygen and nutrients. There is still no effective remedy for myocardial ischemia/reperfusion (IR) injury. Our previous study using cardiomyocytes (CMs) found that, after 3 hrs hypoxia followed by 2 hrs reoxygenation, viability decreased, and release of lactate dehydrogenase (LDH), calcium influx, membrane leakage (insertion of fluorescent probe FM1-43) significantly increased, indicating that cell membrane function was negatively affected. This was attenuated by the triblock copolymer Poloxamer (P)188. Here, we first hypothesized that endothelial cells are also susceptible to simulated IR injury, albeit requiring longer hypoxia times. We further hypothesized that P188 can also attenuate simulated IR injury in endothelial cells when given upon reoxygenation. Mouse coronary artery endothelial cells (MCAECs) were exposed to different durations of hypoxia (2, 3, 12 and 24 hrs) in serum- and glucose-free media +/- reoxygenation for 2 hrs in regular media. P188 was administered upon reoxygenation at 0, 100, 300 or 1,000 μM in experiments of 24 hrs hypoxia / 2 hrs reoxygenation. LDH release was measured and compared to appropriately timed normoxic control experiments. Reoxygenation and hypoxia times significantly longer than 3 hrs were required to elicit sufficient injury (panel A). When P188 was given upon reoxygenation after 24 hrs hypoxia, it dose-dependently attenuated LDH release (panel B). These findings contrast to the higher susceptibility of CMs to IR injury that only allowed shorter hypoxia durations. They also confirm a protective effect of P188 on the endothelium, not just on CMs. These findings have important implications for co-culture models with MCAECs and CMs to elucidate the interplay of both cell types on each other when studying mechanisms of cardioprotective strategies and compounds like P188.

Tubeimoside I Ameliorates Myocardial Ischemia-Reperfusion Injury through SIRT3-Dependent Regulation of Oxidative Stress and Apoptosis

Myocardial ischemia-reperfusion injury (MIRI) is a phenomenon that reperfusion leads to irreversible damage to the myocardium and increases mortality in acute myocardial infarction (AMI) patients. There is no effective drug to treat MIRI. Tubeimoside I (TBM) is a triterpenoid saponin purified from Chinese traditional medicine tubeimu. In this study, 4 mg/kg TBM was given to mice intraperitoneally at 15 min after ischemia. And TBM treatment improved postischemic cardiac function, decreased infarct size, diminished lactate dehydrogenase release, ameliorated oxidative stress, and reduced apoptotic index. Notably, ischemia-reperfusion induced a significant decrease in cardiac SIRT3 expression and activity, while TBM treatment upregulated SIRT3's expression and activity. However, the cardioprotective effects of TBM were largely abolished by a SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP). This suggests that SIRT3 plays an essential role in TBM's cardioprotective effects. In vitro, TBM also protected H9c2 cells against simulated ischemia/reperfusion (SIR) injury by attenuating oxidative stress and apoptosis, and siSIRT3 diminished its protective effects. Taken together, our results demonstrate for the first time that TBM protects against MIRI through SIRT3-dependent regulation of oxidative stress and apoptosis. TBM might be a potential drug candidate for MIRI treatment.

LncRNA TUG1 aggravates cardiomyocyte apoptosis and myocardial ischemia/reperfusion injury.

Cardiomyocyte apoptosis is a fundamental pathogenic factor leading to myocardial ischemia/reperfusion (MI/R) injury. The long non-coding RNA (IncRNA) TUG1 regulates apoptosis in various cell types. We report here that TUG1 expression is induced in mouse heart following MI/R injury as well as in cardiomyocytes subjected to simulated ischemia/reperfusion (SI/R) in vitro. Clinically, TUG1 expression is also elevated in plasma from patients with acute myocardial infarction (AMI), which implies its potential application as a disease biomarker. Functionally, TUG1 overexpression promotes, and its knockdown reduces SI/R-induced lactate dehydrogenase (LDH) release and caspase-3 activity in cardiomyocytes in vitro, illustrating that TUG1 exacerbates SI/R-induced apoptosis. Furthermore, in vivo, TUG1 aggravates MI/R injury in a mouse model, and subsequent observations show concurrent increased apoptosis of cardiomyocytes. Hence, this study unveils a clinical relevance and functional role of TUG1 in MI/R injury, and also implicates that targeting TUG1 may have therapeutic effects in treating MI/R injury.

Expression of atrial‑fetal light chains in cultured human cardiomyocytes after chemical ischemia‑reperfusion injury.

Atrial light chains (ALC1) are naturally present in adult heart atria, while ventricular light chains (VLC1) are predominant in ventricles. Degradation of VLC1 and re-expression of ALC1 in heart ventricles are associated with heart disorders in response to pressure overload. The aim of the current study was to investigate changes in myosin light chain expression after simulated ischemia and simulated reperfusion (sI/sR). Human cardiomyocytes (HCM) isolated from adult heart ventricles were subjected to chemical ischemia. The control group was maintained under aerobic conditions. Myocyte injury was determined by testing lactate dehydrogenase (LDH) activity. The gene expression of ALC1, VLC1 and MMP-2 were assessed by reverse transcription-quatitive PCR. Additionally, protein synthesis was measured using ELISA kits and MMP-2 activity was measured by zymography. The results revealed that LDH activity was increased in sI/sR cell-conditioned medium (P=0.02), confirming the ischemic damage of HCM. ALC1 gene expression and content in HCM were also increased in the sI/sR group (P=0.03 and P<0.001, respectively), while VLC1 gene expression after sI/sR was decreased (P=0.008). Furthermore, MMP-2 gene expression and synthesis were lower in the sI/sR group when compared with the aerobic control group (P<0.001 and P=0.03, respectively). MMP-2 activity was also increased in sI/sR cell-conditioned medium (P=0.006). In conclusion, sI/sR treatment led to increased ALC1 and decreased VLC1 expression in ventricular cardiomyocytes, which may constitute an adaptive mechanism to altered conditions and contribute to the improvement of heart function.

Ischemia-Reperfusion Injury Reduces Kidney Folate Transporter Expression and Plasma Folate Levels.

Acute or chronic kidney disease can cause micronutrient deficiency. Patients with end-stage renal disease, kidney transplantation or on dialysis have reduced circulating levels of folate, an essential B vitamin. However, the molecular mechanism is not well understood. Reabsorption of folate in renal proximal tubules through folate transporters is an important process to prevent urinary loss of folate. The present study investigated the impact of acute kidney injury (AKI) on folate transporter expression and the underlying mechanism. AKI was induced in Sprague-Dawley rats that were subjected to kidney ischemia (45 min)-reperfusion (24 h). Both male and female rats displayed kidney injury and low plasma folate levels compared with sham-operated rats. The plasma folate levels were inversely correlated to plasma creatinine levels. There was a significant increase in neutrophil gelatinase-associated lipocalin (NGAL) and IL-6 mRNA expression in the kidneys of rats with ischemia-reperfusion, indicating kidney injury and increased inflammatory cytokine expression. Ischemia-reperfusion decreased mRNA and protein expression of folate transporters including folate receptor 1 (FOLR1) and reduced folate carrier (RFC); and inhibited transcription factor Sp1/DNA binding activity in the kidneys. Simulated ischemia-reperfusion through hypoxia-reoxygenation or Sp1 siRNA transfection in human proximal tubular cells inhibited folate transporter expression and reduced intracellular folate levels. These results suggest that ischemia-reperfusion injury downregulates renal folate transporter expression and decreases folate uptake by tubular cells, which may contribute to low folate status in AKI. In conclusion, ischemia-reperfusion injury can downregulate Sp1 mediated-folate transporter expression in tubular cells, which may reduce folate reabsorption and lead to low folate status.

The peroxisomal enzyme, FAR1, is induced during ER stress in an ATF6-dependent manner in cardiac myocytes.

Although peroxisomes have been extensively studied in other cell types, their presence and function have gone virtually unexamined in cardiac myocytes. Here, in neonatal rat ventricular myocytes (NRVM) we showed that several known peroxisomal proteins co-localize to punctate structures with a morphology typical of peroxisomes. Surprisingly, we found that the peroxisomal protein, fatty acyl-CoA reductase 1 (FAR1), was upregulated by pharmacological and pathophysiological ER stress induced by tunicamycin (TM) and simulated ischemia-reperfusion (sI/R), respectively. Moreover, FAR1 induction in NRVM was mediated by the ER stress sensor, activating transcription factor 6 (ATF6). Functionally, FAR1 knockdown reduced myocyte death during oxidative stress induced by either sI/R or hydrogen peroxide (H2O2). Thus, Far1 is an ER stress-inducible gene, which encodes a protein that localizes to peroxisomes of cardiac myocytes, where it reduces myocyte viability during oxidative stress. Since FAR1 is critical for plasmalogen synthesis, these results imply that plasmalogens may exert maladaptive effects on the viability of myocytes exposed to oxidative stress.NEW & NOTEWORTHY The peroxisomal enzyme, FAR1, was shown to be an ER stress- and ATF6-inducible protein that localizes to peroxisomes in cardiac myocytes. FAR1 decreases myocyte viability during oxidative stress.

Role of AMPK in the protective effects exerted by triiodothyronine in ischemic-reperfused myocardium.

Recent studies have provided evidence that triiodothyronine (T3) might play an effective role in the recovery of ischemic myocardium, through the preservation of mitochondrial function and the improvement of energy substrate metabolism. To this respect, it has been suggested that T3 could activate AMP-activated protein kinase (AMPK), the cellular 'fuel-gauge' enzyme, although its role has yet to be elucidated. The aim of the present study was to investigate the effects produced by acute treatment with T3 (60 nM) and the pharmacological inhibition of AMPK by compound C on isolated rat left atria subjected to 75 min simulated ischemia-75 min reperfusion. Results showed that T3 increased AMPK activation during simulated ischemia-reperfusion, while compound C prevented it. At the end of simulated reperfusion, acute T3 treatment increased contractile function recovery and cellular viability conservation. Mitochondrial ultrastructure was better preserved in the presence of T3 as well as mitochondrial ATP production rate and tissue ATP content. Calcium retention capacity, a parameter widely used as an indicator of the resistance of mitochondrial permeability transition pore (MPTP) to opening, and GSK-3β phosphorylation, a master switch enzyme that limits MPTP opening, were increased by T3 administration. All these beneficial effects exerted by T3 acute treatment were prevented when compound C was co-administrated. The present study provided original evidence that T3 enhances intrinsic activation of AMPK during myocardial ischemia-reperfusion, being this enzyme involved, at least in part, in the protective effects exerted by T3, contributing to mitochondrial structure and function preservation, post-ischemic contractile recovery and conservation of cellular viability.