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Migratory cells exist in the heart, such as immune cells, fibroblasts, endothelial cells, etc. During myocardium injury, such as ischemia-reperfusion (MIRI), cells migrate to the site of injury to perform repair functions. However, excessive aggregation of these cells may exacerbate damage to the structure and function of the heart, such as acute myocarditis and myocardial fibrosis. Myocardial injury releases exosomes, which are a type of vesicle with signal transduction function and the miRNA carried by exosomes can control cell migration function. Therefore, regulating this migratory cell population through cardiac-derived exosomal miRNA is crucial for protecting and maintaining cardiac function. Through whole transcriptome RNA sequencing, exosomal miRNA sequencing and single-cell dataset analysis, we (1) determined the potential molecular regulatory role of the lncRNA‒miRNA‒mRNA axis in MIRI, (2) screened four important exosomal miRNAs that could be released by cardiac tissue, and (3) screened seven genes related to cell locomotion that are regulated by four miRNAs, among which Tradd and Ephb6 may be specific for promoting migration of different cells of myocardial tissue in myocardial infarct. We generated a core miRNA‒mRNA network based on the functions of the target genes, which may be not only a target for cardiac repair but also a potential diagnostic marker for interactions between the heart and other tissues or organs. In conclusion, we elucidated the potential mechanism of MIRI in cardiac remodeling from the perspective of cell migration, and inhibition of cellular overmigration based on this network may provide new therapeutic targets for MIRI and to prevent MIRI from developing into other diseases., (© 2024. The Author(s).)

Ischemic heart diseases are a major global cause of death, and despite timely revascularization, heart failure due to ischemia-hypoxia reperfusion (IH/R) injury remains a concern. The study focused on the role of Early Growth Response 1 (EGR1) in IH/R-induced apoptosis in human cardiomyocytes (CMs). Human induced pluripotent stem cell (hiPSC)-derived CMs were cultured under IH/R conditions, revealing higher EGR1 expression in the IH/R group through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). Immunofluorescence analysis (IFA) showed an increased ratio of cleaved Caspase-3-positive apoptotic cells in the IH/R group. Using siRNA for EGR1 successfully downregulated EGR1, suppressing cleaved Caspase-3-positive apoptotic cell ratio. Bioinformatic analysis indicated that EGR1 is a plausible target of miR-124-3p under IH/R conditions. The miR-124-3p mimic, predicted to antagonize EGR1 mRNA, downregulated EGR1 under IH/R conditions in qRT-PCR and WB, as confirmed by IFA. The suppression of EGR1 by the miR-124-3p mimic subsequently reduced CM apoptosis. The study suggests that treatment with miR-124-3p targeting EGR1 could be a potential novel therapeutic approach for cardioprotection in ischemic heart diseases in the future., (© 2024. The Author(s).)

Fasting increases susceptibility to acute myocardial ischaemia/reperfusion injury (IRI) but the mechanisms are unknown. Here, we investigate the role of the mitochondrial NAD + -dependent deacetylase, Sirtuin-3 (SIRT3), which has been shown to influence fatty acid oxidation and cardiac outcomes, as a potential mediator of this effect. Fasting was shown to shift metabolism from glucose towards fatty acid oxidation. This change in metabolic fuel substrate utilisation increased myocardial infarct size in wild-type (WT), but not SIRT3 heterozygous knock-out (KO) mice. Further analysis revealed SIRT3 KO mice were better adapted to starvation through an improved cardiac efficiency, thus protecting them from acute myocardial IRI. Mitochondria from SIRT3 KO mice were hyperacetylated compared to WT mice which may regulate key metabolic processes controlling glucose and fatty acid utilisation in the heart. Fasting and the associated metabolic switch to fatty acid respiration worsens outcomes in WT hearts, whilst hearts from SIRT3 KO mice are better adapted to oxidising fatty acids, thereby protecting them from acute myocardial IRI., (© 2022. The Author(s).)

Calpain 1 and 2 (CPN1/2) are calcium-dependent cysteine proteases that exist in cytosol and mitochondria. Pharmacologic inhibition of CPN1/2 decreases cardiac injury during ischemia (ISC)-reperfusion (REP) by improving mitochondrial function. However, the protein targets of CPN1/2 activation during ISC-REP are unclear. CPN1/2 include a large subunit and a small regulatory subunit 1 (CPNS1). Genetic deletion of CPNS1 eliminates the activities of both CPN1 and CPN2. Conditional cardiomyocyte specific CPNS1 deletion mice were used in the present study to clarify the role of CPN1/2 activation in mitochondrial damage during ISC-REP with an emphasis on identifying the potential protein targets of CPN1/2. Isolated hearts from wild type (WT) or CPNS1 deletion mice underwent 25 min in vitro global ISC and 30 min REP. Deletion of CPNS1 led to decreased cytosolic and mitochondrial calpain 1 activation compared to WT. Cardiac injury was decreased in CPNS1 deletion mice following ISC-REP as shown by the decreased infarct size compared to WT. Compared to WT, mitochondrial function was improved in CPNS1 deletion mice following ischemia-reperfusion as shown by the improved oxidative phosphorylation and decreased susceptibility to mitochondrial permeability transition pore opening. H 2 O 2 generation was also decreased in mitochondria from deletion mice following ISC-REP compared to WT. Deletion of CPNS1 also resulted in less cytochrome c and truncated apoptosis inducing factor (tAIF) release from mitochondria. Proteomic analysis of the isolated mitochondria showed that deletion of CPNS1 increased the content of proteins functioning in regulation of mitochondrial calcium homeostasis (paraplegin and sarcalumenin) and complex III activity. These results suggest that activation of CPN1 increases cardiac injury during ischemia-reperfusion by impairing mitochondrial function and triggering cytochrome c and tAIF release from mitochondria into cytosol., (© 2022. The Author(s).)

The return of blood flow to ischemic heart after myocardial infarction causes ischemia-reperfusion injury. There is a clinical need for novel therapeutic targets to treat myocardial ischemia-reperfusion injury. Here we screened for targets for the treatment of ischemia-reperfusion injury using a combination of shRNA and drug library analyses in HL-1 mouse cardiomyocytes subjected to hypoxia and reoxygenation. The shRNA library included lentiviral constructs targeting 4625 genes and the drug library 689 chemical compounds approved by the Food and Drug Administration (FDA). Data were analyzed using protein-protein interaction and pathway analyses. EGFR inhibition was identified as a cardioprotective mechanism in both approaches. Inhibition of EGFR kinase activity with gefitinib improved cardiomyocyte viability in vitro. In addition, gefitinib preserved cardiac contractility in zebrafish embryos exposed to hypoxia-reoxygenation in vivo. These findings indicate that the EGFR inhibitor gefitinib is a potential candidate for further studies of repurposing the drug for the treatment of myocardial infarction., (© 2021. The Author(s).)

Transverse-tubules (T-tubules) play pivotal roles in Ca 2+ -induced, Ca 2+ release and excitation-contraction coupling in cardiomyocytes. The purpose of this study was to uncover mechanisms where sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA2a) improved cardiac function through T-tubule regulation during myocardial ischemia/reperfusion (I/R). SERCA2a protein expression, cytoplasmic [Ca 2+ ] i , calpain activity, junctophilin-2 (JPH2) protein expression and intracellular localization, cardiomyocyte T-tubules, contractility and calcium transients in single cardiomyocytes and in vivo cardiac functions were all examined after SERCA2a knockout and overexpression, and Calpain inhibitor PD150606 (PD) pretreatment, following myocardial I/R. This comprehensive approach was adopted to clarify SERCA2a mechanisms in improving cardiac function in mice. Calpain was activated during myocardial I/R, and led to the proteolytic cleavage of JPH2. This altered the T-tubule network, the contraction function/calcium transients in cardiomyocytes and in vivo cardiac functions. During myocardial I/R, PD pretreatment upregulated JPH2 expression and restored it to its intracellular location, repaired the T-tubule network, and contraction function/calcium transients of cardiomyocytes and cardiac functions in vivo. SERCA2a suppressed calpain activity via [Ca 2+ ] i , and ameliorated these key indices. Our results suggest that SERCA2a ameliorates cardiomyocyte T-tubule remodeling via the calpain/JPH2 pathway, thereby improving cardiac function in myocardial I/R mice.

Suprarenal aortic clamping during abdominal aortic aneurysm (AAA) repair results in ischemia-reperfusion injury (IRI) in local (i.e. kidney) and distant (i.e. heart) tissue. To investigate perioperative approaches that mitigate IRI-induced tissue damage, Wistar rats underwent suprarenal aortic clamping either alone or in combination with short cycles of ischemic conditioning before and/or after clamping. Serum analysis revealed significant reduction in key biochemical parameters reflecting decreased tissue damage at systemic level and improved renal function in conditioned groups compared to controls (p < 0.05), which was corroborated by histolopathological evaluation. Importantly, the levels of DNA damage, as reflected by the biomarkers 8-oxo-G, γH2AX and pATM were reduced in conditioned versus non-conditioned cases. In this setting, NADPH oxidase, a source of free radicals, decreased in the myocardium of conditioned cases. Of note, administration of 5-HD and 8-SPT blocking key protective signaling routes abrogated the salutary effect of conditioning. To further understand the non-targeted effect of IRI on the heart, it was noted that serum TGF-β1 levels decreased in conditioned groups, whereas this difference was eliminated after 5-HD and 8-SPT administration. Collectively, conditioning strategies reduced both renal and myocardial injury. Additionally, the present study highlights TGF-β1 as an attractive target for manipulation in this context.

Thyroid hormone (T3) dyshomeostasis in the cardiac ischemia-reperfusion (IR) setting negatively impacts on mitochondria function and extracellular matrix remodeling. The modulation of cardiac miRNAs may represent the underlying molecular mechanisms, but a systems biology perspective investigating this critical issue in depth is still lacking. A rat model of myocardial IR, with or without an early short-term T3-replacement, was used to predict putative T3-dependent miRNA-gene interactions targeted to mitochondria quality control and wound healing repair. As evidenced by mRNA and miRNA expression profiling, the T3 supplementation reverted the expression of 87 genes and 11 miRNAs that were dysregulated in the untreated group. In silico crossing and functional analysis of the T3-associated differentially expressed transcripts, identified a signature of interconnected miRNA-gene regulatory circuits that confer resistance to noxious cascades of acute stress. In this network the T3-down-regulated Tp53, Jun and Sp1 transcription factors emerge as critical nodes linking intrinsic cell death and oxidative stress pathways to adverse remodeling cascades. The data presented here provide a novel insight into the molecular basis of T3 cardioprotection in the early post-IR phase and highlight the contribution of a previously unappreciated complex T3-regulatory network that may be helpful in translating T3 replacement into clinical practice.

Annexin A5 (AnxA5) is known to have anti-inflammatory and anti-apoptotic properties. Inflammation and apoptosis are key processes in post-ischemic cardiac remodeling. In this study, we investigated the effect of AnxA5 on left ventricular (LV) function and remodeling three weeks after myocardial ischemia-reperfusion (MI-R) injury in hypercholesterolemic ApoE*3-Leiden mice. Using a mouse model for MI-R injury, we demonstrate AnxA5 treatment resulted in a 27% reduction of contrast-enhanced MRI assessed infarct size (IS). End-diastolic and end-systolic volumes were decreased by 22% and 38%, respectively. LV ejection fraction was increased by 29% in the AnxA5 group compared to vehicle. Following AnxA5 treatment LV fibrous content after three weeks was reduced by 42%, which was accompanied by an increase in LV wall thickness of the infarcted area by 17%. Two days and three weeks after MI-R injury the number of cardiac macrophages was significantly reduced in both the infarct area and border zones following AnxA5 treatment compared to vehicle treatment. Finally, we found that AnxA5 stimulation leads to a reduction of IL-6 production in bone-marrow derived macrophages in vitro. AnxA5 treatment attenuates the post-ischemic inflammatory response and ameliorates LV remodeling which improves cardiac function three weeks after MI-R injury in hypercholesterolemic ApoE*3-Leiden mice.

Ischemia reperfusion injuries (IRI) are unavoidable in solid organ transplantation. IRI augments alloimmunity but the mechanisms involved are poorly understood. Herein, we examined the effect of IRI on antigen specific alloimmunity. We demonstrate that ischemia promotes alloimmune activation, leading to more severe histological features of rejection, and increased CD4 + and CD8 + T cell graft infiltration, with a predominantly CD8 + IFNγ + infiltrate. This process is dependent on the presence of alloreactive CD4 + T cells, where depletion prevented infiltration of ischemic grafts by CD8 + IFNγ + T cells. IL-6 is a known driver of ischemia-induced rejection. Herein, depletion of donor antigen-presenting cells reduced ischemia-induced CD8 + IFNγ + allograft infiltration, and improved allograft outcomes. Following prolonged ischemia, accelerated rejection was observed despite treatment with CTLA4Ig, indicating that T cell costimulatory blockade failed to overcome the immune activating effect of IRI. However, despite severe ischemic injury, treatment with anti-IL-6 and CTLA4Ig blocked IRI-induced alloimmune injury and markedly improved allograft survival. We describe a novel pathway where IRI activates innate immunity, leading to upregulation of antigen specific alloimmunity, resulting in chronic allograft injury. Based on these findings, we describe a clinically relevant treatment strategy to overcome the deleterious effect of IRI, and provide superior long-term allograft outcomes.

Urocortin 1 and 2 (Ucn-1 and Ucn-2) have established protective actions against myocardial ischemia-reperfusion (I/R) injuries. However, little is known about their role in posttranscriptional regulation in the process of cardioprotection. Herein, we investigated whether microRNAs play a role in urocortin-induced cardioprotection. Administration of Ucn-1 and Ucn-2 at the beginning of reperfusion significantly restored cardiac function, as evidenced ex vivo in Langendorff-perfused rat hearts and in vivo in rat subjected to I/R. Experiments using microarray and qRT-PCR determined that the addition of Ucn-1 at reperfusion modulated the expression of several miRNAs with unknown role in cardiac protection. Ucn-1 enhanced the expression of miR-125a-3p, miR-324-3p; meanwhile it decreased miR-139-3p. Similarly, intravenous infusion of Ucn-2 in rat model of I/R mimicked the effect of Ucn-1 on miR-324-3p and miR-139-3p. The effect of Ucn-1 involves the activation of corticotropin-releasing factor receptor-2, Epac2 and ERK1/2. Moreover, the overexpression of miR-125a-3p, miR-324-3p and miR-139-3p promoted dysregulation of genes expression involved in cell death and apoptosis (BRCA1, BIM, STAT2), in cAMP and Ca 2+ signaling (PDE4a, CASQ1), in cell stress (NFAT5, XBP1, MAP3K12) and in metabolism (CPT2, FoxO1, MTRF1, TAZ). Altogether, these data unveil a novel role of urocortin in myocardial protection, involving posttranscriptional regulation with miRNAs.

The valosin-containing protein (VCP) participates in signaling pathways essential for cell homeostasis in multiple tissues, however, its function in the heart in vivo remains unknown. Here we offer the first description of the expression, function and mechanism of action of VCP in the mammalian heart in vivo in both normal and stress conditions. By using a transgenic (TG) mouse with cardiac-specific overexpression (3.5-fold) of VCP, we demonstrate that VCP is a new and powerful mediator of cardiac protection against cell death in vivo, as evidenced by a 50% reduction of infarct size after ischemia/reperfusion versus wild type. We also identify a novel role of VCP in preserving mitochondrial respiration and in preventing the opening of mitochondrial permeability transition pore in cardiac myocytes under stress. In particular, by genetic deletion of inducible isoform of nitric oxide synthase (iNOS) from VCP TG mouse and by pharmacological inhibition of iNOS in isolated cardiac myocytes, we reveal that an increase of expression and activity of iNOS in cardiomyocytes by VCP is an essential mechanistic link of VCP-mediated preservation of mitochondrial function. These data together demonstrate that VCP may represent a novel therapeutic avenue for the prevention of myocardial ischemia.

Cardiac ischemia and reperfusion (I/R) injury remains a challenge for clinicians. Ginsenoside Rb1 (Rb1) has been reported to have the ability to attenuate I/R injury, but its effect on energy metabolism during cardiac I/R and the underlying mechanism remain unknown. In this study, we detected the effect of Rb1 on rat myocardial blood flow, myocardial infarct size, cardiac function, velocity of venule red blood cell, myocardial structure and apoptosis, energy metabolism and change in RhoA signaling pathway during cardiac I/R injury. In addition, the binding affinity of RhoA to Rb1 was detected using surface plasmon resonance (SPR). Results showed that Rb1 treatment at 5 mg/kg/h protected all the cardiac injuries induced by I/R, including damaged myocardial structure, decrease in myocardial blood flow, impaired heart function and microcirculation, cardiomyocyte apoptosis, myocardial infarction and release of myocardial cTnI. Rb1 also inhibited the activation of RhoA signaling pathway and restored the production of ATP during cardiac I/R. Moreover, SPR assay showed that Rb1 was able to bind to RhoA in a dose-dependent manner. These results indicate that Rb1 may prevent I/R-induced cardiac injury by regulation of RhoA signaling pathway, and may serve as a potential regime to improve percutaneous coronary intervention outcome.

AS-1, the TIR/BB loop mimetic, plays a protective role in cardiac ischemia/reperfusion (I/R) but the molecular mechanism remains unclear. The muscle specific caveolin3 (Cav-3) and the caveolae have been found to be critical for cardioprotection. This study aimed to evaluate our hypothesis that caveolae and Cav-3 are essential for AS-1-induced cardioprotection against myocardial I/R injury. To address these issues, we analyzed the involvement of Cav-3 in AS-1 mediated cardioprotection both in vivo and in vitro. We demonstrate that AS-1 administration significantly decreased infarct size, improved cardiac function after myocardial I/R and modulated membrane caveolae and Cav-3 expression in the myocardium. For in vitro studies, AS-1 treatment prevented Cav-3 re-distribution induced by H/R injury. In contrast, disruption of caveolae by MCD treatment or Cav-3 knockdown abolished the protection against H/R-induced myocytes injury by AS-1. Our findings reveal that AS-1 attenuates myocardial I/R injury through caveolae and Cav-3 dependent mechanism.

Myocardial ischaemia-reperfusion (I/R) injury is a complex pathophysiological process. Current research has suggested that energy metabolism disorders, of which the abnormal consumption of fatty acids is closely related, compose the main pathological basis for myocardial I/R injury. Lipid droplets (LD) are critical regulators of lipid metabolism by LD-associated proteins. Among the lipid droplet proteins, the perilipin family members regulate lipolysis and lipogenesis through different mechanisms. Plin5, an important perilipin protein, promotes LD generation and lowers fatty acid oxidation, thus protecting the myocardium from lipotoxicity. This study investigated the protective effects of Plin5 in I/R myocardium. Our results indicated that Plin5 deficiency exacerbated the myocardial infarct area, aggravated left ventricular systolic dysfunction, reduced lipid storage, and elevated free fatty acids. Plin5-deficient myocardium exhibited severely damaged mitochondria, elevated reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and decreased superoxide dismutase (SOD) activity. Furthermore, the decreased phosphorylation of PI3K/Akt in Plin5-null cardiomyocytes might contribute to I/R injury aggravation. In conclusion, Plin5, a new regulator of myocardial lipid metabolism, decreases free fatty acid peroxidation by inhibiting the lipolysis of intracellular lipid droplets, thus providing cardioprotection against I/R injury and shedding new light on therapeutic solutions for I/R diseases.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca 2+ -permeable nonselective cation channel and can be activated during ischemia/reperfusion (I/R). This study tested whether blockade of TRPV4 can alleviate myocardial I/R injury in mice. TRPV4 expression began to increase at 1 h, reached statistically at 4 h, and peaked at 24-72 h. Treatment with the selective TRPV4 antagonist HC-067047 or TRPV4 knockout markedly ameliorated myocardial I/R injury as demonstrated by reduced infarct size, decreased troponin T levels and improved cardiac function at 24 h after reperfusion. Importantly, the therapeutic window for HC-067047 lasts for at least 12 h following reperfusion. Furthermore, treatment with HC-067047 reduced apoptosis, as evidenced by the decrease in TUNEL-positive myocytes, Bax/Bcl-2 ratio, and caspase-3 activation. Meanwhile, treatment with HC-067047 attenuated the decrease in the activation of reperfusion injury salvage kinase (RISK) pathway (phosphorylation of Akt, ERK1/2, and GSK-3β), while the activation of survival activating factor enhancement (SAFE) pathway (phosphorylation of STAT3) remained unchanged. In addition, the anti-apoptotic effects of HC-067047 were abolished by the RISK pathway inhibitors. We conclude that blockade of TRPV4 reduces apoptosis via the activation of RISK pathway, and therefore might be a promising strategy to prevent myocardial I/R injury.

Myocardial ischemia/reperfusion (I/R) injury detrimentally alters the prognosis of patients undergoing revascularization after acute myocardial infarction. Our previous study demonstrated that NF-κB-induced autophagy plays a detrimental role in cardiac I/R injury using a rabbit myocardial I/R model. In this study, we sought to explore the specific mechanism of this autophagy-mediated cell damage in an in vitro simulated ischemia/reperfusion (sI/R) model using human umbilical vein endothelial cells. Our current study demonstrates that simulated I/R induces autophagy in a p65-Beclin 1-dependent manner, which can be suppressed with the inhibition of NF-κB. Furthermore, rapamycin which promotes autophagy, exacerbates sI/R-induced cell death. While 3-methyladenine rescues cell damage. Our data thus suggest that I/R promotes NF-κB p65 activity mediated Beclin 1-mediated autophagic flux, thereby exacerbating myocardial injury.

Hyperlipidemia aggravates myocardial ischemia/reperfusion (MI/R) injury through stimulating excessive inflammatory response. Therefore, blockade of inflammatory signal is a potential therapeutic management for MI/R complicated with hyperlipidemia. Hydroxysafflor yellow A (HSYA, a monomer extracted from Carthamus tinctorius L.), was studied in this article to address that the regulation of inflammatory signal would alleviate MI/R combined with hyperlipidemia injury. High-fat diet induced hyperlipidemia worsened MI/R mediated heart injury (elevation of infarct size, CK-MB and LDH activity), activated TLR4 over-expression in hearts, released inflammatory cytokines (LPS, TNF-α and IL-1β) excessively. HSYA administration suppressed the over-expression of TLR4 and alleviated heart damage caused by MI/R complicated with hyperlipidemia. Furthermore, HSYA had little influence on MI/R injury in TLR4-knockout mice, which indicated that HSYA protected MI/R through TLR4 inhibition. In vitro, hypoxia/reoxygenation (H/R) coexisting with LPS model in neonatal rat ventricular myocytes (NRVMs) induced serious damage compared with H/R injury to NRVMs. HSYA decreased excessive secretion of inflammatory cytokines, down-regulated over-expression of TLR4 and NF-κB in H/R + LPS injured NRVMs. In conclusion, HSYA alleviated myocardial inflammatory injury through suppressing TLR4, offering an alternative medication for MI/R associated with hyperlipidemia.

Endothelial damage is a critical mediator of myocardial ischemia/reperfusion (I/R) injury. HSPA12B is an endothelial-cell-specifically expressed heat shock protein. However, the roles of HSPA12B in acute myocardial I/R injury is unknown. Here we reported that myocardial I/R upregulated HSPA12B expression in ventricular tissues, and endothelial overexpression of HSPA12B in transgenic mice (Tg) limited infarct size, attenuated cardiac dysfunction and improved cardiomyocyte survival compared with their wild type littermates. These improvements were accompanied with the diminished myocardial no-reflow phenomenon, decreased microvascular leakage, and better maintained endothelial tight junctions. The I/R-evoked neutrophil infiltration was also suppressed in Tg hearts compared with its wild type (WT) littermates. Moreover, Tg hearts exhibited the enhanced activation of PI3K/Akt//mTOR signaling following I/R challenge. However, pharmacological inhibition of PI3K abolished the HSPA12B-induced cardioprotection against myocardial I/R injury. The data demonstrate for the first time that the endothelial HSPA12B protected hearts against myocardial I/R injury. This cardioprotective action of HSPA12B was mediated, at least in part, by improving endothelial integrity in a PI3K/Akt/mTOR-dependent mechanism. Our study suggests that targeting endothelial HSPA12B could be an alternative approach for the management of patients with myocardial I/R injury.

Overproduction of hydrogen peroxide (H2O2) causes oxidative stress and is the main culprit in the pathogenesis of ischemia/reperfusion (I/R) injury. Suppression of oxidative stress is therefore critical in the treatment of I/R injury. Here, we report H2O2-activatable antioxidant prodrug (BRAP) that is capable of specifically targeting the site of oxidative stress and exerting anti-inflammatory and anti-apoptotic activities. BRAP with a self-immolative boronic ester protecting group was designed to scavenge H2O2 and release HBA (p-hydroxybenzyl alcohol) with antioxidant and anti-inflammatory activities. BRAP exerted potent antioxidant and anti-inflammatory activity in lipopolysaccharide (LPS)- and H2O2-stimulated cells by suppressing the generation of ROS and pro-inflammatory cytokines. In mouse models of hepatic I/R and cardiac I/R, BRAP exerted potent antioxidant, anti-inflammatory and anti-apoptotic activities due to the synergistic effects of H2O2-scavenging boronic esters and therapeutic HBA. In addition, administration of high doses of BRAP daily for 7 days showed no renal or hepatic function abnormalities. Therefore BRAP has tremendous therapeutic potential as H2O2-activatable antioxidant prodrug for the treatment of I/R injuries.

Oxidative stress plays a critical role in ischemia/reperfusion-injury, atherosclerosis, and aging. It causes cell damage that leads to apoptosis via uncertain mechanisms. Because conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation induces extensive apoptosis of cardiac myocytes under normoxia, we hypothesized that a humoral factor released from the hypoxic/reoxygenated cardiac myocytes mediates apoptosis. We identified an apoptosis-inducing humoral factor in the hypoxia/reoxygenation-conditioned medium. Here, we found that eIF5A undergoes tyrosine sulfation in the trans-Golgi and is rapidly secreted from cardiac myocytes in response to hypoxia/reoxygenation; then, eIF5A induces apoptosis by acting as a pro-apoptotic ligand. The apoptosis of cardiac myocytes induced by hypoxia/reoxygenation or ultraviolet irradiation was suppressed by anti-eIF5A neutralizing monoclonal antibodies (mAbs) in vitro. Myocardial ischemia/reperfusion (but not ischemia alone) markedly increased the plasma levels of eIF5A, and treatment with anti-eIF5A neutralizing mAbs significantly reduced myocardial injury. These results identify an important, novel specific biomarker and a critical therapeutic target for oxidative stress-induced cell injury.

Reactive oxygen species (ROS)-induced oxidative stress in cells is an important pathophysiological process during myocardial ischemia/reperfusion (I/R) injury, and the transcription factor Egr-1 is a master switch for various damage pathways during reperfusion injury. An in vitro model of myocardial I/R injury and H9c2 cardiomyoblast cells hypoxia/reoxygenation (H/R) was used to assess whether there is abnormal intracellular ROS/JNK/Egr-1 signaling. We also assessed whether N-n-butyl haloperidol (F2), which exerts protective effects during myocardial I/R injury, can modulate this pathway. H/R induced ROS generation, JNK activation, and increased the expression of Egr-1 protein in H9c2 cells. The ROS scavengers edaravone (EDA) and N-acetyl-L-cysteine (NAC) reduced ROS level, downregulated JNK activation, and Egr-1 expression in H9c2 cells after H/R. The JNK inhibitor SP600125 inhibited Egr-1 overexpression in H9c2 cells caused by H/R. F2 could downregulate H/R-induced ROS level, JNK activation, and Egr-1 expression in H9c2 cells in a dose-dependent manner. The ROS donor hypoxanthine-xanthine oxidase (XO/HX) and the JNK activator ANISO antagonized the effects of F2. Therefore, H/R activates ROS/Egr-1 signaling pathway in H9c2 cells, and JNK activation plays an important role in this pathway. F2 regulates H/R-induced ROS/JNK/Egr-1 signaling, which might be an important mechanism by which it antagonizes myocardial I/R injury.

Autophagy is involved in the development and/or progression of many diseases, including myocardial ischemia/reperfusion (I/R). In this study, we hypothesized a protective role of basic fibroblast growth factor (bFGF) both in vivo and in vitro and demonstrated that excessive autophagy and ubiquitinated protein accumulation is involved in the myocardial I/R model. Our results showed that bFGF improved heart function recovery and increased the survival of cardiomyocytes in myocardial I/R model. The protective effect of bFGF is related to the inhibition of LC3II levels. Additionally, bFGF enhances the clearance of Ub by p62 and increases the survival of H9C2 cells. Moreover, silencing of p62 partially blocks the clearance of Ub and abolishes the anti-apoptosis effect of bFGF. An shRNA against the autophagic machinery Atg7 increased the survival of H9C2 cells co-treated with bFGF and rapamycin. bFGF activates the downstream signaling of the PI3K/Akt/mTOR pathway. These results indicate that the role of bFGF in myocardial I/R recovery is related to the inhibition of excessive autophagy and increased ubiquitinated protein clearance via the activation of PI3K/Akt/mTOR signaling. Overall, our study suggests a new direction for bFGF drug development for heart disease and identifies protein signaling pathways involved in bFGF action.

There is a great need for the development of therapeutic strategies that can target biomolecules to damaged myocardium. Necrosis of myocardium during a myocardial infarction (MI) is characterized by extracellular release of DNA, which can serve as a potential target for ischemic tissue. Hoechst, a histological stain that binds to double-stranded DNA can be conjugated to a variety of molecules. Insulin-like growth factor-1 (IGF-1), a small protein/polypeptide with a short circulating-half life is cardioprotective following MI but its clinical use is limited by poor delivery, as intra-myocardial injections have poor retention and chronic systemic presence has adverse side effects. Here, we present a novel delivery vehicle for IGF-1, via its conjugation to Hoechst for targeting infarcted tissue. Using a mouse model of ischemia-reperfusion, we demonstrate that intravenous delivery of Hoechst-IGF-1 results in activation of Akt, a downstream target of IGF-1 and protects from cardiac fibrosis and dysfunction following MI.