The activation of LBH-CRYAB signaling promotes cardiac protection against I/R injury by inhibiting apoptosis and ferroptosis.

Myocardial ischemia-reperfusion (I/R) injury stands out among cardiovascular diseases, and current treatments are considered unsatisfactory. For cardiomyocytes (CMs) in ischemic tissues, the upregulation of Limb-bud and Heart (LBH) and αB-crystallin (CRYAB) and their subsequent downregulation in the context of cardiac fibrosis have been verified in our previous research. Here, we focused on the effects and mechanisms of activated LBH-CRYAB signaling on damaged CMs during I/R injury, and confirmed the occurrence of mitochondrial apoptosis and ferroptosis during I/R injury. The application of inhibitors, ectopic expression vectors, and knockout mouse models uniformly verified the role of LBH in alleviating both apoptosis and ferroptosis of CMs. p53 was identified as a mutual downstream effector for both LBH-CRYAB-modulated apoptosis and ferroptosis inhibition. In mouse models, LBH overexpression was confirmed to exert enhanced cardiac protection against I/R-induced apoptosis and ferroptosis, suggesting that LBH could serve as a promising target for the development of I/R therapy.

Fucoxanthin alleviated myocardial ischemia and reperfusion injury through inhibition of ferroptosis via the NRF2 signaling pathway.

Background: Myocardial ischemia and reperfusion injury (MIRI) is a severe complication of revascularization therapy in patients with myocardial infarction. Therefore, there is an urgent requirement to find more therapeutic solutions for MIRI. Recently, ferroptosis, which is characterized by lipid peroxidation, was considered a critical contributor to MIRI. Fucoxanthin (FX), a natural antioxidant carotenoid, which is abundant in brown seaweed, exerts protective effects under various pathological conditions. However, whether FX alleviates MIRI is unclear. This study aims to clarify the effects of FX on MIRI. Methods: Mice with left anterior descending artery ligation and reperfusion were used as in vivo models. Neonatal rat cardiomyocytes (NRCs) induced with hypoxia and reperfusion were used as in vitro models. TTC-Evans blue staining was performed to validate the infarction size. Transmission electron microscopy was employed to detect mitochondrial injury in cardiomyocytes. In addition, 4 weeks after MIRI, echocardiography was performed to measure cardiac function; fluorescent probes and western blots were used to detect ferroptosis. Results: TTC-Evans blue staining showed that FX reduced the infarction size induced by MIRI. Transmission electron microscopy showed that FX ameliorated the MIRI-induced myofibril loss and mitochondrion shrinkage. Furthermore, FX improved LVEF and LVFS and inhibited myocardial hypertrophy and fibrosis after 4 weeks in mice with MIRI. In the in vitro study, calcein AM/PI staining and TUNEL staining showed that FX reduced cell death caused by hypoxia and reperfusion treatment. DCFH-DA and MitoSOX probes indicated that FX inhibited cellular and mitochondrial reactive oxygen species (ROS). Moreover, C11-BODIPY 581/591 staining, ferro-orange staining, MDA assay, Fe2+ assay, 4-hydroxynonenal enzyme-linked immunosorbent assay, and western blot were performed and the results revealed that FX ameliorated ferroptosis in vitro and in vivo, as indicated by inhibiting lipid ROS and Fe2+ release, as well as by modulating ferroptosis hallmark FTH, TFRC, and GPX4 expression. Additionally, the protective effects of FX were eliminated by the NRF2 inhibitor brusatol, as observed from western blotting, C11-BODIPY 581/591 staining, and calcein AM/PI staining, indicating that FX exerted cardio-protective effects on MIRI through the NRF2 pathway. Conclusion: Our study showed that FX alleviated MIRI through the inhibition of ferroptosis via the NRF2 signaling pathway.

Angiotensin IV ameliorates doxorubicin-induced cardiotoxicity by increasing glutathione peroxidase 4 and alleviating ferroptosis.

BACKGROUND: Doxorubicin (DOX)-induced cardiotoxicity is an important cause of poor prognosis in cancer patients treated with DOX. Angiotensin IV (Ang IV) has multiple protective effects against cardiovascular diseases, including diabetic cardiomyopathy and myocardial infarction, but its role in DOX-induced cardiotoxicity is currently unclear. In this study, we investigated the effects of Ang IV on DOX-induced cardiotoxicity. METHODS: The viability of primary cardiomyocytes was measured by Cell Counting Kit-8 assays and Hoechst 33342/propidium iodide staining in vitro. ELISAs (serum cTnT and CK-MB) and echocardiography were performed to assess myocardial injury and cardiac function in vivo. Phalloidin staining, haematoxylin and eosin staining and wheat germ agglutinin staining were conducted to detect cardiomyocyte atrophy. We also performed C11 BODIPY staining, measured the levels of Ptgs2 and malondialdehyde and detected the concentrations of ferrous ions, glutathione and oxidized glutathione to indicate ferroptosis. RESULTS: Ang IV not only attenuated DOX-induced atrophy and cardiomyocyte injury in vitro but also alleviated myocardial injury and improved cardiac function in DOX-treated mice in vivo. Moreover, Ang IV reversed DOX-induced downregulation of glutathione peroxidase 4 (GPX4) and inhibited ferroptosis both in vitro and in vivo. Knockdown of GPX4 by siRNA abolished the cardioprotective effects of Ang IV. Furthermore, Ang IV increased GPX4 levels and ameliorated ferroptosis in RAS-selective lethal 3-treated primary cardiomyocytes. CONCLUSIONS: Ang IV ameliorates DOX-induced cardiotoxicity by upregulating GPX4 and inhibiting ferroptosis. Ang IV may be a promising candidate to protect against DOX-induced cardiotoxicity in the future.

MicroRNA-574-5p targeting HOXC6 expression inhibits the hepatocyte lipid uptake to alleviate non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is one of the main causes of liver disease that has reached its last stage. Over the past few years, evidence for miRNAs' centrality in NAFLD pathogenesis has accumulated. According to some studies, miR-574-5p plays a role in lipid metabolism. However, research on the relationship between miR-574-5p and NAFLD is lacking. For in vivo experiments, we induced the NAFLD mice model with a high-fat diet (HFD). AgomiR-574-5p was injected intravenously into HFD-fed mice for eight weeks, and qPCR was used to identify the expression of miR-574-5p in the serum. In in vitro experiments, The treatment of L-O2 cells with a miR-574-5p mimic resulted in a significant reduction in lipid deposition, suggesting that miR-574-5p can inhibit lipid accumulation and lipid formation induced by OA. The dual-luciferase reporter gene assay revealed that miR-574-5p targets the 3' UTR region of HOXC6 directly. We discovered that OA-induced lipid accumulation in hepatocytes might be mediated through the miR-574-5p-HOXC6 signaling axis. Additional research is required in order to determine the specific mechanism by which HOXC6 downstream pathways are involved in the miR-574-5p induced lipid uptake.

Amine oxidase copper-containing 3 aggravates cardiac remodelling by generating hydrogen peroxide after myocardial infarction.

Amine oxidase copper-containing 3 (AOC3) is a member of the semicarbazide-sensitive amine oxidase enzyme family. It acts as an ectoenzyme catalysing the oxidative deamination of primary amines and generating hydrogen peroxide (H2 O2 ). While AOC3 is implicated in cardiovascular diseases such as atherosclerosis, its role in cardiac remodelling after myocardial infarction (MI) is unclear. In this study, we first confirmed a long-term upregulation of AOC3 in both cardiac myofibroblasts after MI in vivo and angiotensin II (ANGII)-treated cardiac fibroblasts in vitro. AOC3 knockdown not only inhibited the activation of cardiac fibroblasts induced by ANGII but also alleviated cardiac fibrosis in mice after MI. Using sh-AOC3 lentiviruses, exogenous recombinant AOC3 (r-AOC3), semicarbazide (an AOC3 inhibitor), and catalase (a hydrogen peroxide scavenger) treatments, we also demonstrated that AOC3 promoted H2 O2 generation, increased oxidative stress, and enhanced ERK1/2 activation, which were responsible for the activation of cardiac fibroblasts. In particular, AOC3 knockdown also improved cardiac function and hypertrophy after MI. Through a coculture system, we confirmed that AOC3 expressed on cardiac myofibroblasts was able to enhance oxidative stress and induce hypertrophy of cardiomyocytes by promoting H2 O2 generation. Similarly, r-AOC3 promoted H2 O2 generation and resulted in oxidative stress and hypertrophy of cardiomyocytes, which were almost inhibited by both semicarbazide and catalase. In conclusion, AOC3 plays a critical role in cardiac fibrosis and hypertrophy after MI by promoting the generation of H2 O2 . AOC3 is a promising therapeutic target against cardiac remodelling. © 2023 The Pathological Society of Great Britain and Ireland.

Trimethylamine-N-oxide (TMAO) promotes balloon injury-induced neointimal hyperplasia via upregulating Beclin1 and impairing autophagic flux.

BACKGROUND AND AIMS: TMAO is a microbiota-dependent metabolite associated with increased risk of various cardiovascular diseases. However, the relationship between TMAO and vascular injury-related neointimal hyperplasia is unclear. This study aimed to explore whether TMAO promotes neointimal hyperplasia after balloon injury and elucidate the underlying mechanism. METHODS AND RESULTS: Through hematoxylin and eosin staining and immunohistochemistry staining, we found that supplementary TMAO promoted balloon injury-induced neointimal hyperplasia, while reducing TMAO by antibiotic administration produced the opposite result. TMAO showed limited effect on rat aortic vascular smooth muscle cells (RAOSMCs) proliferation and migration. However, TMAO notably induced dysfunction of rat aortic vascular endothelial cells (RAOECs) in vitro and attenuated reendothelialization of carotid arteries after balloon injury in vivo. Autophagic flux was measured by fluorescent mRFP-GFP-LC3, transmission electron microscopy, and western blot. TMAO impaired autophagic flux, as evidenced by the accumulation of p62 and LC3II and high autophagosome to autolysosome ratios. Furthermore, we confirmed that Beclin1 level increased in TMAO-treated RAOECs and carotid arteries. Knocking down Beclin1 alleviated TMAO-induced autophagic flux impairment and neointimal hyperplasia. CONCLUSIONS: TMAO promoted neointimal hyperplasia through Beclin1-induced autophagic flux blockage, suggesting that TMAO is a potential target for improvement of vascular remodeling after injury.

An injectable co-assembled hydrogel blocks reactive oxygen species and inflammation cycle resisting myocardial ischemia-reperfusion injury.

The overproduction of reactive oxygen species (ROS) and burst of inflammation following cardiac ischemia-reperfusion (I/R) are the leading causes of cardiomyocyte injury. Monotherapeutic strategies designed to enhance anti-inflammatory or anti-ROS activity explicitly for treating I/R injury have demonstrated limited success because of the complex mechanisms of ROS production and induction of inflammation. Intense oxidative stress leads to sustained injury, necrosis, and apoptosis of cardiomyocytes. The damaged and necrotic cells can release danger-associated molecular patterns (DAMPs) that can cause the aggregation of immune cells by activating Toll-like receptor 4 (TLR4). These immune cells also promote ROS production by expressing NADPH oxidase. Finally, ROS production and inflammation form a vicious cycle, and ROS and TLR4 are critical nodes of this cycle. In the present study, we designed and prepared an injectable hydrogel system of EGCG@Rh-gel by co-assembling epigallocatechin-3-gallate (EGCG) and the rhein-peptide hydrogel (Rh-gel). The co-assembled hydrogel efficiently blocked the ROS-inflammation cycle by ROS scavenging and TLR4 inhibition. Benefited by the abundant noncovalent interactions of π-π stacking and hydrogen bonding between EGCG and Rh-gel, the co-assembled hydrogel had good mechanical strength and injectable property. Following the injection EGCG@Rh-gel into the damaged region of the mice's heart after I/R, the hydrogel enabled to achieve long-term sustained release and treatment, improve cardiac function, and significantly reduce the formation of scarring. Further studies demonstrated that these beneficial outcomes arise from the reduction of ROS production, inhibition of inflammation, and induction of anti-apoptosis in cardiomyocytes. Therefore, EGCG@Rh-gel is a promising drug delivery system to block the ROS-inflammation cycle for resisting myocardial I/R injury. STATEMENT OF SIGNIFICANCE: 1. Monotherapeutic strategies designed to enhance anti-inflammatory or anti-ROS effects for treating I/R injury have demonstrated limited success because of the complex mechanisms of ROS and inflammation. 2. ROS production and inflammation form a vicious cycle, and ROS and TLR4 are critical nodes of this cycle. 3. Here, we designed an injectable hydrogel system of EGCG@Rh-gel by co-assembling epigallocatechin-3-gallate (EGCG) and a rhein-peptide hydrogel (Rh-gel). EGCG@Rh-gel efficiently blocked the ROS-inflammation cycle by ROS scavenging and TLR4 inhibition. 4. EGCG@Rh-gel achieved long-term sustained release and treatment, improved cardiac function, and significantly reduced the formation of scarring after I/R. 5. The beneficial outcomes arise from reducing ROS production, inhibiting inflammation, and inducing anti-apoptosis in cardiomyocytes.

Elabela ameliorates doxorubicin-induced cardiotoxicity by promoting autophagic flux through TFEB pathway.

Doxorubicin (DOX) is a widely used and effective antineoplastic drug; however, its clinical application is limited by cardiotoxicity. A safe and effective strategy to prevent from doxorubicin-induced cardiotoxicity (DIC) is still beyond reach. Elabela (ELA), a new APJ ligand, has exerted cardioprotective effect against multiple cardiovascular diseases. Here, we asked whether ELA alleviates DIC. Mice were injected with DOX to established acute DIC. In vivo studies were assessed with echocardiography, serum cTnT and CK-MB, HW/BW ratio and WGA staining. Cell death and atrophy were measured by AM/PI staining and phalloidin staining respectively in vitro. Autophagic flux was monitored with Transmission electron microscopy in vivo, as well as LysoSensor and mRFP-GFP-LC3 puncta in vitro. Our results showed that ELA improved cardiac dysfunction in DIC mice. ELA administration also attenuated cell death and atrophy in DOX-challenged neonatal rat cardiomyocytes (NRCs). Additionally, we found that ELA restored DOX-induced autophagic flux blockage, which was evidenced by the reverse of p62 and LC3II, improvement of lysosome function and accelerated degradation of accumulated autolysosomes. Chloroquine, a classical autophagic flux inhibitor, blunted the improvement of ELA on cardiac dysfunction. At last, we revealed that ELA reversed DOX-induced downregulation of transcription factor EB (TFEB), and silencing TFEB by siRNA abrogated the effects of ELA on autophagic flux as well as cell death and atrophy in NRCs. In conclusion, this study indicated that ELA ameliorated DIC through enhancing autophagic flux via activating TFEB. ELA may become a potential target against DIC.

Nkx2-3 induces autophagy inhibiting proliferation and migration of vascular smooth muscle cells via AMPK/mTOR signaling pathway.

Vascular remodeling and restenosis are common complications after percutaneous coronary intervention. Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in intimal hyperplasia-induced vascular restenosis. NK2 Homeobox 3 (Nkx2-3), a critical member of Nkx family, is involved in tissue differentiation and organ development. However, the role of Nkx2-3 in VSMCs proliferation and migration remains unknown. In this study, we used carotid balloon injury model and platelet-derived growth factor-BB (PDGF)-treated VSMCs as in vivo and in vitro experimental models. EdU assay and CCK-8 assay were used to detect cell proliferation. Migration was measured by scratch test. Hematoxylin and eosin staining and immunohistochemistry staining were used to evaluate the intimal hyperplasia. The autophagy level was detected by fluorescent mRFP-GFP-LC3 in vitro and by transmission electron microscopy in vivo. It was shown that Nkx2-3 was upregulated both in balloon injured carotid arteries and PDGF-stimulated VSMCs. Adenovirus-mediated Nkx2-3 overexpression inhibited intimal hyperplasia after balloon injury, and suppressed VSMCs proliferation and migration induced by PDGF. Conversely, silencing of Nkx2-3 by small interfering RNA exaggerated proliferation and migration of VSMCs. Furthermore, we found that Nkx2-3 enhanced autophagy level, while the autophagy inhibitor 3-MA eliminated the inhibitory effect of Nkx2-3 on VSMCs proliferation and migration both in vivo and in vitro. Moreover, Nkx2-3 promoted autophagy in VSMCs by activating the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway. These results demonstrated for the first time that Nkx2-3 inhibited VSMCs proliferation and migration through AMPK/mTOR-mediated autophagy.

Nkx2-5 Regulates the Proliferation and Migration of H9c2 Cells.

BACKGROUND The protein NKX2-5 affects mammalian heart development. In mice, the disruption of Nkx2-5 has been associated with arrhythmias, abnormal myocardial contraction, abnormal cardiac morphogenesis, and death. However, the details of the mechanisms are unclear. This study was designed to investigate them. MATERIAL AND METHODS Rat cardiomyocytes from the H9c2 cell line were used in our study. First, we knocked down Nkx2-5 in the H9c2 cells and then validated consequent changes in cell proliferation and migration. We then used RNA sequencing to determine the changes in transcripts. Finally, we validated these results by quantitative reverse transcription-polymerase chain reaction. RESULTS We confirmed that Nkx2-5 regulates the proliferation and migration of H9c2 cells. In our experiments, Nkx2-5 regulated the expression of genes related to proliferation, migration, heart development, and disease. Based on bioinformatics analysis, knockdown of Nkx2-5 caused differential expression of genes involved in cardiac development, calcium ion-related biological activity, the transforming growth factor (TGF)-ß signaling pathway, pathways related to heart diseases, the MAPK signaling pathway, and other biological processes and signaling pathways. CONCLUSIONS Nkx2-5 may regulate proliferation and migration of the H9c2 cells through the genes Tgfb-2, Bmp10, Id2, Wt1, Hey1, and Cacna1g; rno-miR-1-3p; the TGF­ß signaling pathway; the MAPK signaling pathway; as well as other genes and pathways.

Cardioprotective effects of melatonin: Focusing on its roles against diabetic cardiomyopathy.

Melatonin is a pineal-produced indole known for its anti-aging, antiapoptotic and antioxidant properties. In past decades, the protective potentials of melatonin for cardiovascular diseases, such as atherosclerosis and myocardial infarction, have been widely revealed, triggering more investigations focused on other cardioprotective effects of melatonin. Recently, the roles of melatonin in diabetic cardiomyopathy (DCM) have attracted increased attention. In this regard, researchers found that melatonin attenuated cardiac fibrosis and hypertrophy, thus interrupting the development of DCM. Retinoid-related orphan receptor α is a key melatonin receptor that contributed to the cardioprotective effect of melatonin in hearts with DCM. For the downstream mechanisms, the inhibition of mammalian STE20-like kinase 1 plays a pivotal role, which exerts antiapoptotic and proautophagic effects, thus enhancing cardiac tolerance in high-glucose conditions. In addition, other signalling mechanisms, such as sirtuin-1/peroxisome proliferator-activated receptor gamma-coactivator alpha and endoplasmic reticulum-related signalling, are also involved in the protective effects of melatonin on cardiomyocytes under diabetic conditions. This review will focus on the protective signalling mechanisms regulated by melatonin and provide a better understanding of the therapeutic applications of melatonin signalling in DCM.

Resveratrol and Diabetic Cardiomyopathy: Focusing on the Protective Signaling Mechanisms.

Diabetic cardiomyopathy (DCM) is a common cardiovascular complication of diabetic mellitus that is characterized by diastolic disorder in the early stage and clinical heart failure in the later stage. Presently, DCM is considered one of the major causes of death in diabetic patients. Resveratrol (RSV), a naturally occurring stilbene, is widely reported as a cardioprotective substance in many heart diseases. Thus far, the specific roles of RSV in DCM prevention and treatment have attracted great attention. Here, we discuss the roles of RSV in DCM by focusing its downstream targets from both in vivo and in vitro studies. Among such targets, Sirtuins 1/3 and AMP-activated kinase have been identified as key mediators that induce cardioprotection during hyperglycemia. In addition, many other signaling molecules (e.g., forkhead box-O3a and extracellular regulated protein kinases) are also regulated in the presence of RSV and exert beneficial effects such as opposing oxidative stress, inflammation, and apoptosis in cardiomyocytes exposed to high-glucose conditions. The beneficial potential of an RSV/stem cell cotherapy is also reviewed as a promising therapeutic strategy for preventing the development of DCM.

Heat shock protein 70: A promising therapeutic target for myocardial ischemia-reperfusion injury.

Acute myocardial infarction is a major cause of death worldwide. The most important therapy for limiting ischemic injury and infarct size is timely and efficient myocardial reperfusion treatment, which may instead induce cardiomyocyte necrosis due to myocardial ischemia-reperfusion (I/R) injury. Heat shock protein 70 (HSP70), a stress-inducible protein, is overexpressed during myocardial I/R. The induced HSP70 is shown to regulate several intracellular proteins (e.g., transcription factors, enzymes, and apoptosis-related proteins) and signaling pathways (e.g., c-Jun N-terminal kinase pathway and extracellular-signal-regulated kinase 1/2 pathway), forming a complicated network that contributes to reducing reactive oxygen species accumulation, improving calcium homeostasis, inhibiting cellular apoptosis, thereby enhancing the stress adaption of myocardium to I/R injury. In addition, the extracellular HSP70, which is released from injured cardiomyocytes during I/R, acts as a proinflammatory mediator that results in cell death, while the intracellular HSP70 exerts antiinflammatory effects by suppressing proinflammatory signaling pathways. Notably, HSP70 is induced and contributes to the cardioprotection in several types of preconditioning and postconditioning. Meanwhile, it is shown that the cardioprotective effectiveness of preconditioning-induced HSP70 (e.g., hyperthermia preconditioning-induced HSP70) can be impaired by certain pathological conditions, such as hyperlipidemia and hyperglycemia. Thus, we highlight the widespread cardioprotective involvement of HSP70 in preconditioning and postconditioning and elucidate how HSP70-mediated cardioprotection is impaired in these pathological conditions. Furthermore, several therapeutic potentials of HSP70 against myocardial I/R injury and potential directions for future studies are also provided in this review.

The Role of Peroxisome Proliferator-Activated Receptor γ in Mediating Cardioprotection Against Ischemia/Reperfusion Injury.

Myocardial infarction (MI) is a serious cardiovascular disease resulting in high rates of morbidity and mortality. Although advances have been made in restoring myocardial perfusion in ischemic areas, decreases in cardiomyocyte death and infarct size are still limited, attributing to myocardial ischemia/reperfusion (I/R) injury. It is necessary to develop therapies to restrict myocardial I/R injury and protect cardiomyocytes against further damage after MI. Many studies have suggested that peroxisome proliferator-activated receptor γ (PPARγ), a ligand-inducible nuclear receptor that predominantly regulates glucose and lipid metabolism, is a promising therapeutic target for ameliorating myocardial I/R injury. Thus, this review focuses on the role of PPARγ in cardioprotection during myocardial I/R. The cardioprotective effects of PPARγ, including attenuating oxidative stress, inhibiting inflammatory responses, improving glucose and lipid metabolism, and antagonizing apoptosis, are described. Additionally, the underlying mechanisms of cardioprotective effects of PPARγ, such as regulating the expression of target genes, influencing other transcription factors, and modulating kinase signaling pathways, are further discussed.

Age-Dependent Alpha-Synuclein Accumulation and Phosphorylation in the Enteric Nervous System in a Transgenic Mouse Model of Parkinson's Disease.

The enteric nervous system (ENS) controls the function of the gastrointestinal tract and has been implicated in various diseases, including Parkinson's disease (PD). PD is a neurodegenerative disease with Lewy bodies (LBs) and Lewy neurites (LNs) as the main pathological features. In addition to the typical motor symptoms in PD, attention has been drawn to non-motor symptoms, such as constipation, implying dysfunction of the ENS. In the present study, we characterized the age-dependent morphological alterations and aggregation of α-synuclein (α-syn), the primary protein component in LBs and LNs, in the ENS in an α-syn transgenic mouse model. We found that the expression and accumulation of α-syn increased gradually in neurons of Meissner's and Auerbach's plexuses of the gastrointestinal tract with age (from 1 week to 2 years). In addition, α-syn was increasingly phosphorylated at the serine 129 residue, reflecting pathological alterations of the protein over time. Furthermore, α-syn was present in different subtypes of neurons expressing vasoactive intestinal polypeptide, neuronal nitric oxide synthase, or calretinin. The results indicated that BAC-α-Syn-GFP transgenic mice provide a unique model in which to study the relationship between ENS and PD pathogenesis.