Spexin inhibits excessive autophagy-induced ferroptosis to alleviate doxorubicin-induced cardiotoxicity by upregulating Beclin 1.

BACKGROUND AND PURPOSE: Doxorubicin is widely used in the treatment of malignant tumours, but doxorubicin-induced cardiotoxicity severely limits its clinical application. Spexin is a neuropeptide that acts as a novel biomarker in cardiovascular disease. However, the effects of spexin on doxorubicin-induced cardiotoxicity is unclear. EXPERIMENTAL APPROACH: We established a model of doxorubicin-induced cardiotoxicity both in vivo and in vitro. Levels of cardiac damage in mice was assessed through cardiac function assessment, determination of serum cardiac troponin T and CKMB levels and histological examination. CCK8 and PI staining were used to assess the doxorubicin-induced toxicity in cultures of cardiomyocytes in vitro. Ferroptosis was assessed using FerroOrange staining, determination of MDA and 4-HNE content and ferroptosis-associated proteins SLC7A11 and GPX4. Mitochondrial membrane potential and lipid peroxidation levels were measured using TMRE and C11-BODIPY 581/591 probes, respectively. Myocardial autophagy was assessed by expression of P62 and Beclin1. KEY RESULTS: Spexin treatment improved heart function of mice with doxorubicin-induced cardiotoxicity, and attenuated doxorubicin-induced cardiotoxicity by decreasing iron accumulation, abnormal lipid metabolism and inhibiting ferroptosis. Interestingly, doxorubicin caused excessive autophagy in cardiomyocyte in culture, which could be alleviated by treatment with spexin. Knockdown of Beclin 1 eliminated the protective effects of spexin in mice with DIC. CONCLUSION AND IMPLICATIONS: Spexin ameliorated doxorubicin-induced cardiotoxicity by inhibiting excessive autophagy-induced ferroptosis, suggesting that spexin could be a drug candidate against doxorubicin-induced cardiotoxicity. Beclin 1 might be critical in mediating the protective effect of spexin against doxorubicin-induced cardiotoxicity.

A novel function of claudin-5 in maintaining the structural integrity of the heart and its implications in cardiac pathology.

This study aims to investigate the role of claudin-5 (Cldn5) in cardiac structural integrity. Proteomic analysis was performed to screen the protein profiles in enlarged left atrium from atrial fibrillation (AF) patients. Cldn5 shRNA adeno-associated virus (AAV) or siRNA was injected into the mouse left ventricle or added into HL1 cells respectively to knockdown Cldn5 in cardiomyocytes to observe whether the change of Cldn5 influences cardiac morphology and function, and affects those protein expressions stem from the proteomic analysis. Mitochondrial density and membrane potential were also measured by Mitotracker staining and JC-1 staining under the confocal microscope in HL1 cells. Cldn5 was reduced in cardiomyocytes from the left atrial appendage of AF patients compared to non-AF donors. Proteomic analysis showed 83 proteins were less abundant and 102 proteins were more abundant in AF patients. KEGG pathway analysis showed less abundant CACNA2D2, CACNB2, MYL2 and MAP6 were highly associated with dilated cardiomyopathy. Cldn5 shRNA AAV injection caused severe cardiac atrophy, dilation and myocardial dysfunction in mice. The decreases in mitochondrial numbers and mitochondrial membrane potentials in HL1 cells were observed after Cldn5 knockdown. We demonstrated for the first time the mechanism of Cldn5 downregulation-induced myocyte atrophy and myocardial dysfunction might be associated with the downregulation of CACNA2D2, CACNB2, MYL2 and MAP6, and mitochondrial dysfunction in cardiomyocytes.

Research progress on activation transcription factor 3: A promising cardioprotective molecule.

Activation transcription factor 3 (ATF3), a member of the ATF/cyclic adenosine monophosphate response element binding family, can be induced by a variety of stresses. Numerous studies have indicated that ATF3 plays multiple roles in the development and progression of cardiovascular diseases, including atherosclerosis, hypertrophy, fibrosis, myocardial ischemia-reperfusion, cardiomyopathy, and other cardiac dysfunctions. In past decades, ATF3 has been demonstrated to be detrimental to some cardiac diseases. Current studies have indicated that ATF3 can function as a cardioprotective molecule in antioxidative stress, lipid metabolic metabolism, energy metabolic regulation, and cell death modulation. To unveil the potential therapeutic role of ATF3 in cardiovascular diseases, we organized this review to explore the protective effects and mechanisms of ATF3 on cardiac dysfunction, which might provide rational evidence for the prevention and cure of cardiovascular diseases.

Compound Danshen Dripping Pills pretreatment protects the heart from ischemia/reperfusion injury by enhancing autophagic flux

Abstract Compound Danshen Dripping Pills (CDDPs) have been used in clinical treatment to protect the heart from ischemia/reperfusion (IR) injury for many years. However, the underlying mechanism implicated in the protective effects remains to be explored. Here, we determined the effects of CDDPs in Sprague-Dawley rats with the IR model. Cardiac function in vivo was assessed by echocardiography. Transmission electron microscopy, histological and immunohistochemical techniques, Western blotting and recombinant adeno-associated virus 9 transfection were used to illustrate the effects of CDDPs on IR and autophagy. Our results showed that pretreatment with CDDPs decreased the level of serum myocardial enzymes and infarct size in rats after IR. Apoptosis evaluation showed that CDDPs significantly ameliorated the cardiac apoptosis level after IR. Meanwhile, CDDPs pretreatment increased myocardial autophagic flux, with upregulation of LC3B, downregulation of p62, and increased autophagosomes and autolysosomes. Moreover, the autophagic flux inhibitor chloroquine could increase IR injury, while CDDPs could partially reverse the effects. Furthermore, our results showed that the activation of AMPK/mTOR was involved in the cardioprotective effect exerted by CDDPs. Herein, we suggest that CDDPs partially protect the heart from IR injury by enhancing autophagic flux through the activation of AMPK/mTOR.

A Novel Role of Claudin-5 in Prevention of Mitochondrial Fission Against Ischemic/Hypoxic Stress in Cardiomyocytes.

BACKGROUND: Downregulation of claudin-5 in the heart is associated with the end-stage heart failure. However, the underlying mechanism ofclaudin-5 is unclear. Here we investigated the molecular actions of claudin-5 in perspective of mitochondria in cardiomyocytes to better understand the role of claudin-5 in cardioprotection during ischemia. METHODS: Myocardial ischemia/reperfusion (I/R; 30 min/24 h) and hypoxia/reoxygenation (H/R; 24 h/4 h) were used in this study. Confocal microscopy and transmission electron microscope (TEM) were used to observe mitochondrial morphology. RESULTS: Claudin-5 was detected in murine heart tissue and neonatal rat cardiomyocytes (NRCM). Its protein level was severely decreased after myocardial I/R or H/R. Confocal microscopy showedclaudin-5 presented in the mitochondria of NRCM. H/R-induced claudin-5 downregulation was accompanied by mitochondrial fragmentation. The mitofusin 2 (Mfn2) expressionwas dramatically decreased while the dynamin-related protein (Drp) 1 expression was significantly increased after H/R. The TEM indicatedH/R-induced mitochondrial swelling and fission. Adenoviral claudin-5 overexpression reversed these structural disintegration of mitochondria. The mitochondria-centered intrinsic pathway of apoptosis triggered by H/R and indicated by the cytochrome c and cleaved caspase 3 in the cytoplasm of NRCMs was also reduced by overexpressing claudin-5. Claudin-5 overexpression in mouse heart also significantly decreased cleaved caspase 3 and the infarct size in ischemic heart with improved systolic function. CONCLUSION: We demonstrated for the first time the presence of claudin-5 in the mitochondria in cardiomyocytes and provided the firm evidence for the cardioprotective role of claudin-5 in the preservation of mitochondrial dynamics and cell fate against hypoxia- or ischemia-induced stress.

Neuregulin­1, a microvascular endothelial­derived protein, protects against myocardial ischemia­reperfusion injury (Review).

As regards acute myocardial infarction, great success has been achieved in therapies that reduce the effects of myocardial ischemic injury, while few interventions have achieved satisfactory outcomes for myocardial ischemia­reperfusion (IR) injury. Thus, new research is urgently required to achieve breakthroughs in promising treatments. Neuregulin­1 (NRG­1), which is an endothelium­derived protein and the ligand of ErbB receptors, exerts cardioprotective effects and is rapidly upregulated during IR. NRG­1/ErbB activates several downstream signaling pathways in response to myocardial IR injury. Previous studies have revealed the protective effects of NRG­1 during heart failure, and numerous experiments have explored the mechanisms underlying the NRG­1­induced cardioprotective effects against myocardial IR injury. In the present review, the progress made in the research of NRG­1 as a cardioprotective agent during IR and related conditionings is summarized. Furthermore, the potential benefits of NRG­1 against myocardial IR injury are listed with the prospective use of NRG­1 in clinical applications.

Resveratrol pretreatment alleviates myocardial ischemia/reperfusion injury by inhibiting STIM1-mediated intracellular calcium accumulation.

Previous studies have shown that stromal interaction molecule1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) contributes to intracellular Ca2+ accumulation in H9C2 cells subjected to hypoxia/reoxygenation(H/R) injury. The aim of the present study was to investigate the effect of resveratrol on STIM1-mediated intracellular Ca2+ accumulation and subsequent cell death in the context of myocardial ischemia/reperfusion (I/R) injury. C57 BL/6 mice were fed with either saline or resveratrol (50 mg/kg daily for 2 weeks) and then subjected to myocardial I/R injury. TTC/Evans Blue staining and TUNEL assay were performed to quantify the infarct size and apoptosis index. The cardiac function was evaluated by echocardiography. Neonatal rat ventricular cardiomyocytes (NRVCs) underwent hypoxia/reoxygenation (H/R) to establish the in vitro model. To achieve over-expression, NRVCs were transfected with STIM1-adenovirus vector. Apoptosis was analyzed by TUNEL assay. Cell viability was measured using MTS assay and cell necrosis was determined by LDH release assay. Intracellular Ca2+ concentration was detected by laser scanning confocal microscopy using a Fluo-3AM probe. Resveratrol significantly reduced apoptosis, decreased infarct size, and improved cardiac function in mice subjected to myocardial I/R injury. In NRVCs, resveratrol also downregulated STIM1 expression accompanied by decreased intracellular Ca2+ accumulation elicited by H/R injury. In addition, resveratrol reduced cell apoptosis, upregulated the Bcl-2, decreased Bax, and cleaved caspase-3 expression. Furthermore, the effects of resveratrol on STIM1-mediated intracellular Ca2+ accumulation, apoptotic proteins, and H/R-induced cell injury were exacerbated by STIM1 over-expression and were partly abolished by SOCE inhibitor SKF96365 in NRVCs in vitro. Our findings demonstrate that resveratrol exerts anti-apoptotic activity and improves cardiac functional recovery following myocardial I/R by inhibiting STIM1-induced intracellular Ca2+ accumulation.

Honokiol post-treatment ameliorates myocardial ischemia/reperfusion injury by enhancing autophagic flux and reducing intracellular ROS production.

Honokiol (HKL) is a natural low-molecular-weight biphenolic compound derived from the bark of magnolia trees. Previous studies indicate that HKL exerts potent cardioprotective effects on ischemia/reperfusion (I/R) injury; however, evidence of the further relationship between HKL posttreatment and myocardial I/R injury has not been clearly found. In our study, we explored the protective effect of HKL post treatment on myocardial I/R injury in C57BL/6 mice. We also demonstrated that HKL significantly reduced cellular reactive oxygen species production and attenuated mitochondrial damage in neonatal rat cardiomyocytes exposed to hypoxia/reoxygenation (H/R). In addition, HKL was found to enhance autophagy during I/R or H/R; these effects could be partially blocked by the autophagic flux inhibitor chloroquine. Moreover, our results suggested that enhanced autophagic flux is associated with the Akt signaling pathway. Collectively, our results indicate that HKL posttreatment alleviates myocardial I/R injury and suggest a critical cardioprotective role of HKL in promoting autophagic flux.