PIAS3 acts as a zinc sensor under zinc deficiency and plays an important role in myocardial ischemia/reperfusion injury.

Alterations in zinc transporter expression in response to zinc loss protect cardiac cells from ischemia/reperfusion (I/R) injury. However, the underlying molecular mechanisms how cardiac cells sense zinc loss remains unclear. Here, we found that zinc deficiency induced ubiquitination and degradation of the protein inhibitor of activated STAT3 (PIAS3), which can alleviate myocardial I/R injury by activating STAT3 to promote the expression of ZIP family zinc transporter genes. The RING finger domain within PIAS3 is vital for PIAS3 degradation, as PIAS3-dRing (missing the RING domain) and PIAS3-Mut (zinc-binding site mutation) were resistant to degradation in the setting of zinc deficiency. Meanwhile, the RING finger domain within PIAS3 is critical for the inhibition of STAT3 activation. Moreover, PIAS3 knockdown increased cardiac Zn2+ levels and reduced myocardial infarction in mouse hearts subjected to I/R, whereas wild-type PIAS3 overexpression, but not PIAS3-Mut, reduced cardiac Zn2+ levels, and exacerbated myocardial infarction. These findings elucidate a unique mechanism of zinc sensing, showing that fast degradation of the zinc-binding regulatory protein PIAS3 during zinc deficiency can correct zinc dyshomeostasis and alleviate reperfusion injury.

Epicardial CCM2 Promotes Cardiac Development and Repair Via its Regulation on Cytoskeletal Reorganization.

The epicardium provides epicardial-derived cells and molecular signals to support cardiac development and regeneration. Zebrafish and mouse studies have shown that ccm2, a cerebral cavernous malformation disease gene, is essential for cardiac development. Endocardial cell-specific deletion of Ccm2 in mice has previously established that Ccm2 is essential for maintenance of the cardiac jelly for cardiac development during early gestation. The current study aimed to explore the function of Ccm2 in epicardial cells for heart development and regeneration. Through genetic deletion of Ccm2 in epicardial cells, our in vivo and ex vivo experiments revealed that Ccm2 is required by epicardial cells to support heart development. Ccm2 regulates epicardial cell adhesion, cell polarity, cell spreading, and migration. Importantly, the loss of Ccm2 in epicardial cells delays cardiac function recovery and aggravates cardiac fibrosis following myocardial infarction. Molecularly, Ccm2 targets the production of cytoskeletal and matrix proteins to maintain epicardial cell function and behaviors. Epicardial Ccm2 plays a critical role in heart development and regeneration via its regulation of cytoskeleton reorganization.

Ischemia-Selective Cardioprotection by Malonate for Ischemia/Reperfusion Injury.

BACKGROUND: Inhibiting SDH (succinate dehydrogenase), with the competitive inhibitor malonate, has shown promise in ameliorating ischemia/reperfusion injury. However, key for translation to the clinic is understanding the mechanism of malonate entry into cells to enable inhibition of SDH, its mitochondrial target, as malonate itself poorly permeates cellular membranes. The possibility of malonate selectively entering the at-risk heart tissue on reperfusion, however, remains unexplored. METHODS: C57BL/6J mice, C2C12 and H9c2 myoblasts, and HeLa cells were used to elucidate the mechanism of selective malonate uptake into the ischemic heart upon reperfusion. Cells were treated with malonate while varying pH or together with transport inhibitors. Mouse hearts were either perfused ex vivo (Langendorff) or subjected to in vivo left anterior descending coronary artery ligation as models of ischemia/reperfusion injury. Succinate and malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS, in vivo by mass spectrometry imaging, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining. RESULTS: Malonate was robustly protective against cardiac ischemia/reperfusion injury, but only if administered at reperfusion and not when infused before ischemia. The extent of malonate uptake into the heart was proportional to the duration of ischemia. Malonate entry into cardiomyocytes in vivo and in vitro was dramatically increased at the low pH (≈6.5) associated with ischemia. This increased uptake of malonate was blocked by selective inhibition of MCT1 (monocarboxylate transporter 1). Reperfusion of the ischemic heart region with malonate led to selective SDH inhibition in the at-risk region. Acid-formulation greatly enhances the cardioprotective potency of malonate. CONCLUSIONS: Cardioprotection by malonate is dependent on its entry into cardiomyocytes. This is facilitated by the local decrease in pH that occurs during ischemia, leading to its selective uptake upon reperfusion into the at-risk tissue, via MCT1. Thus, malonate's preferential uptake in reperfused tissue means it is an at-risk tissue-selective drug that protects against cardiac ischemia/reperfusion injury.

Chlamydia pneumoniae Infection Induces Vascular Smooth Muscle Cell Migration and Atherosclerosis Through Mitochondrial Reactive Oxygen Species-Mediated JunB-Fra-1 Activation.

Infection is closely related to atherosclerosis, which is a major pathological basis for cardiovascular diseases. Vascular smooth muscle cell (VSMC) migration is an important trigger in development of atherosclerosis that is associated with Chlamydia pneumoniae (C. pneumoniae) infection. However, the mechanism of VSMC migration remains unclear, and whether antioxidant could be a therapeutic target for C. pneumoniae infection-induced atherosclerosis also remains unknown. The results showed that C. pneumoniae infection mainly impaired mitochondrial function and increased the level of mitochondrial reactive oxygen species (mtROS). The expressions of protein JunB, Fra-1 and Matrix metalloproteinase 2 (MMP) evidently increased after C. pneumoniae infection, and the interaction between JunB and Fra-1 was also enhanced. After scavenging mtROS by antioxidant Mito-TEMPO, the increasing expressions of JunB, Fra-1, MMP2 and the capacity of VSMC migration induced by C. pneumoniae infection were all inhibited. In comparison with infected ApoE-/- mice, the level of ROS in atherosclerotic lesion in ApoE-/-TLR2-/- mice with C. pneumoniae infection decreased. Knocking out TLR2 suppressed the expressions of JunB, Fra-1 and MMP2 in VSMCs and the formation of atherosclerotic lesion after C. pneumoniae infection. Furthermore, after using small interfering RNA to inhibit the expression of TLR2, the level of mtROS and the expressions of JunB, Fra-1 and MMP2 apparently decreased. Taken together, C. pneumoniae infection may promote VSMC migration and atherosclerosis development by increasing the level of mtROS through TLR2 to activate the JunB-Fra-1/MMP2 signaling pathway. The data provide the first evidence that antioxidant could reduce C. pneumoniae infection-induced VSMC migration and atherosclerosis.

Upregulation of p67phox in response to ischemia/reperfusion is cardioprotective by increasing ZIP2 expression via STAT3.

While the zinc transporter ZIP2 (Slc39a2) is upregulated via STAT3 as an adaptive response to protect the heart from ischemia/reperfusion (I/R) injury, the precise mechanism underlying its upregulation remains unclear. The purpose of this study was to investigate the role of NADPH oxidase (NOX) isoform NOX2-derived ROS in the regulation of ZIP2 expression, focusing on the role of the NOX2 cytosolic factor p67phox. Mouse hearts or H9c2 cells were subjected to I/R. Protein expression was detected with Western blotting. Infarct size was measured with TTC staining. The cardiac-specific p67phox conditional knockout mice (p67phox cKO) were generated by adopting the CRISPR/Cas9 system. I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression was reversed by the ROS scavenger N-acetylcysteine (NAC) and the NOX inhibitor diphenyleneiodonium (DPI). p67phox but not NOX2 expression was increased 30 min after the onset of reperfusion, and downregulation of p67phox by siRNA or cKO invalidated I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression. Both NAC and DPI prevented upregulation of STAT3 phosphorylation and ZIP2 expression induced by overexpression of p67phox, whereas the STAT3 inhibitor stattic abrogated upregulation ZIP2 expression, indicating that the increase of p67phox at reperfusion is an upstream signaling event responsible for ZIP2 upregulation via STAT3. Experiments also showed that chelation of Zn2+ markedly enhanced p67phox and ZIP2 expression as well as STAT3 phosphorylation, whereas supplementation of Zn2+ had the opposite effects, indicating that cardiac Zn2+ loss upon reperfusion triggers p67phox upregulation. Furthermore, ischemic preconditioning (IPC) upregulated ZIP2 via p67phox, and cKO of p67phox aggravated cardiac injury after I/R, indicating that p67phox upregulation is cardioprotective against I/R injury. In conclusion, an increase of p67phox expression in response to Zn2+ is an intrinsic adaptive response to I/R and leads to cardioprotection against I/R by upregulating ZIP2 via STAT3.

Ester Prodrugs of Malonate with Enhanced Intracellular Delivery Protect Against Cardiac Ischemia-Reperfusion Injury In Vivo.

PURPOSE: Mitochondrial reactive oxygen species (ROS) production upon reperfusion of ischemic tissue initiates the ischemia/reperfusion (I/R) injury associated with heart attack. During ischemia, succinate accumulates and its oxidation upon reperfusion by succinate dehydrogenase (SDH) drives ROS production. Inhibition of succinate accumulation and/or oxidation by dimethyl malonate (DMM), a cell permeable prodrug of the SDH inhibitor malonate, can decrease I/R injury. However, DMM is hydrolysed slowly, requiring administration to the heart prior to ischemia, precluding its administration to patients at the point of reperfusion, for example at the same time as unblocking a coronary artery following a heart attack. To accelerate malonate delivery, here we developed more rapidly hydrolysable malonate esters. METHODS: We synthesised a series of malonate esters and assessed their uptake and hydrolysis by isolated mitochondria, C2C12 cells and in mice in vivo. In addition, we assessed protection against cardiac I/R injury by the esters using an in vivo mouse model of acute myocardial infarction. RESULTS: We found that the diacetoxymethyl malonate diester (MAM) most rapidly delivered large amounts of malonate to cells in vivo. Furthermore, MAM could inhibit mitochondrial ROS production from succinate oxidation and was protective against I/R injury in vivo when added at reperfusion. CONCLUSIONS: The rapidly hydrolysed malonate prodrug MAM can protect against cardiac I/R injury in a clinically relevant mouse model.

The zinc transporter ZIP7 (Slc39a7) controls myocardial reperfusion injury by regulating mitophagy.

Whereas elimination of damaged mitochondria by mitophagy is proposed to be cardioprotective, the regulation of mitophagy at reperfusion and the underlying mechanism remain elusive. Since mitochondrial Zn2+ may control mitophagy by regulating mitochondrial membrane potential (MMP), we hypothesized that the zinc transporter ZIP7 that controls Zn2+ levels within mitochondria would contribute to reperfusion injury by regulating mitophagy. Mouse hearts were subjected to ischemia/reperfusion in vivo. Mitophagy was evaluated by detecting mitoLC3II, mito-Keima, and mitoQC. ROS were measured with DHE and mitoB. Infarct size was measured with TTC staining. The cardiac-specific ZIP7 conditional knockout mice (ZIP7 cKO) were generated by adopting the CRISPR/Cas9 system. Human heart samples were obtained from donors and recipients of heart transplant surgeries. KO or cKO of ZIP7 increased mitophagy under physiological conditions. Mitophagy was not activated at the early stage of reperfusion in mouse hearts. ZIP7 is upregulated at reperfusion and ZIP7 cKO enhanced mitophagy upon reperfusion. cKO of ZIP7 led to mitochondrial depolarization by increasing mitochondrial Zn2+ and, accumulation of PINK1 and Parkin in mitochondria, suggesting that the decrease in mitochondrial Zn2+ in response to ZIP7 upregulation resulting in mitochondrial hyperpolarization may impede PINK1 and Parkin accumulation in mitochondria. Notably, ZIP7 is markedly upregulated in cardiac mitochondria from patients with heart failure (HF), whereas mitochondrial PINK1 accumulation and mitophagy were suppressed. Furthermore, ZIP7 cKO reduced mitochondrial ROS generation and myocardial infarction via a PINK1-dependet manner, whereas overexpression of ZIP7 exacerbated myocardial infarction. Our findings identify upregulation of ZIP7 leading to suppression of mitophagy as a critical feature of myocardial reperfusion injury. A timely suppression of cardiac ZIP7 upregulation or inactivation of ZIP7 is essential for the treatment of reperfusion injury.

Zinc transporter SLC39A13/ZIP13 facilitates the metastasis of human ovarian cancer cells via activating Src/FAK signaling pathway.

BACKGROUND: Zinc transporters have been found to be associated with the pathogenesis of numerous human diseases including cancer. As the most lethal gynecologic malignancy, ovarian cancer is characterized by rapid progression and widespread metastases. However, the function and underlying mechanism of zinc transporters in ovarian cancer metastasis remain unclear. METHODS: The relationship between zinc transporter gene expressions and clinical outcomes of ovarian cancer was assessed with the online database Kaplan-Meier plotter ( http://kmplot.com/analysis/ ). Immunohistochemistry was performed to investigate the prognostic importance of ZIP13. The expression of ZIP13 in ovarian cancer cell lines was depleted to explore its effect on proliferation, adhesion, migration, and invasion both in vitro and in vivo assays. RNA-Seq, quantitative RT-PCR, and western blot analysis were performed to explore ZIP13-regulated downstream target genes. RESULTS: The expressions of several zinc transporters were highly associated the clinical outcomes of ovarian cancer patients. Among them, high ZIP13 expression was an independent prognostic factor for poor survival in patients with ovarian cancer. ZIP13 knockout suppressed the malignant phenotypes of ovarian cancer cells both in vitro and in vivo. Further investigation revealed that ZIP13 regulated intracellular zinc distribution and then affected the expressions of genes involved in extracellular matrix organization and cytokine-mediated signaling pathway. This led to the activation of Src/FAK pathway with increased expressions of pro-metastatic genes but decreased expressions of tumor suppressor genes. CONCLUSIONS: ZIP13 is shown to be a novel driver of metastatic progression by modulating the Src/FAK signaling pathway, which may serve as a promising biomarker for prognostic evaluation and targeted therapy in ovarian cancer.

Biomimetic Design of Mitochondria-Targeted Hybrid Nanozymes as Superoxide Scavengers.

Development of enzyme mimics for the scavenging of excessive mitochondrial superoxide (O2 •- ) can serve as an effective strategy in the treatment of many diseases. Here, protein reconstruction technology and nanotechnology is taken advantage of to biomimetically create an artificial hybrid nanozyme. These nanozymes consist of ferritin-heavy-chain-based protein as the enzyme scaffold and a metal nanoparticle core as the enzyme active center. This artificial cascade nanozyme possesses superoxide dismutase- and catalase-like activities and also targets mitochondria by overcoming multiple biological barriers. Using cardiac ischemia-reperfusion animal models, the protective advantages of the hybrid nanozymes are demonstrated in vivo during mitochondrial oxidative injury and in the recovery of heart functionality following infarction via systemic delivery and localized release from adhesive hydrogels (i.e., cardiac patch), respectively. This study illustrates a de novo design strategy in the development of enzyme mimics and provides a promising therapeutic option for alleviating oxidative damage in regenerative medicine.

Endoplasmic Reticulum Stress/Ca2+-Calmodulin-Dependent Protein Kinase/Signal Transducer and Activator of Transcription 3 Pathway Plays a Role in the Regulation of Cellular Zinc Deficiency in Myocardial Ischemia/Reperfusion Injury.

Zinc homeostasis has been known to play a role in myocardial ischemia/reperfusion (I/R) injury, but the precise molecular mechanisms regulating the expression of ZIP transporters during reperfusion are still unclear. The aim of this study was to determine whether ER Stress/CaMKII/STAT3 pathway plays a role in the regulation of cellular zinc homeostasis. Zinc deficiency increased mRNA and protein expressions of the ER stress relevant markers Chop and Bip, and STAT3 phosphorylation in H9c2 or HL-1 cells, an effect that was abolished by ZnCl2. ER calcium concentration [(Ca2+)ER] was decreased and cytosolic calcium concentration [(Ca2+)I] was increased at the condition of normoxia or ischemia/reperfusion, indicating that zinc deficiency triggers ER stress and Ca2+ leak. Further studies showed that upregulation of STAT3 phosphorylation was reversed by Ca2+ chelator, indicating that intracellular Ca2+ is important for zinc deficiency-induced STAT3 activation. In support, zinc deficiency enhanced ryanodine receptors (RyR), a channel in the ER that mediate Ca2+ release, and Ca2+-calmodulin-dependent protein kinase (CaMKII) phosphorylation, implying that zinc deficiency provoked Ca2+ leak from ER via RyR and p-CaMKII is involved in STAT3 activation. Moreover, inhibition of STAT3 activation blocked zinc deficiency induced ZIP9 expression, and resulted in increased Zn2+ loss in cardiomyocytes, further confirming that STAT3 activation during reperfusion promotes the expression of ZIP9 zinc transporter to correct the imbalance in zinc homeostasis. In addition, suppressed STAT3 activation aggravated reperfusion injury. These data suggest that the ER Stress/CaMKII/STAT3 axis may be an endogenous protective mechanism, which increases the resistance of the heart to I/R.

Downregulation of the zinc transporter SLC39A13 (ZIP13) is responsible for the activation of CaMKII at reperfusion and leads to myocardial ischemia/reperfusion injury in mouse hearts.

While Zn2+ dyshomeostasis is known to contribute to ischemia/reperfusion (I/R) injury, the roles of zinc transporters that are responsible for Zn2+ homeostasis in the pathogenesis of I/R injury remain to be addressed. This study reports that ZIP13 (SLC39A13), a zinc transporter, plays a role in myocardial I/R injury by modulating the Ca2+ signaling pathway rather than by regulating Zn2+ transport. ZIP13 is downregulated upon reperfusion in mouse hearts or in H9c2 cells at reoxygenation. Ca2+ but not Zn2+ was responsible for ZIP13 downregulation, implying that ZIP13 may play a role in I/R injury through the Ca2+ signaling pathway. In line with our assumption, knockout of ZIP13 resulted in phosphorylation (Thr287) of Ca2+-calmodulin-dependent protein kinase (CaMKII), indicating that downregulation of ZIP13 leads to CaMKII activation. Further studies showed that the heart-specific knockout of ZIP13 enhanced I/R-induced CaMKII phosphorylation in mouse hearts. In contrast, overexpression of ZIP13 suppressed I/R-induced CaMKII phosphorylation. Moreover, the heart-specific knockout of ZIP13 exacerbated myocardial infarction in mouse hearts subjected to I/R, whereas overexpression of ZIP13 reduced infarct size. In addition, knockout of ZIP13 induced increases of mitochondrial Ca2+, ROS, mitochondrial swelling, decrease in the mitochondrial respiration control rate (RCR), and dissipation of mitochondrial membrane potential (ΔΨm) in a CaMKII-dependent manner. These data suggest that downregulation of ZIP13 at reperfusion contributes to myocardial I/R injury through activation of CaMKII and the mitochondrial death pathway.

Zinc dysregulation in cancers and its potential as a therapeutic target.

Zinc is an essential element and serves as a structural or catalytic component in many proteins. Two families of transporters are involved in maintaining cellular zinc homeostasis: the ZIP (SLC39A) family that facilitates zinc influx into the cytoplasm, and the ZnT (SLC30A) family that facilitates zinc efflux from the cytoplasm. Zinc dyshomeostasis caused by the dysfunction of zinc transporters can contribute to the initiation or progression of various cancers, including prostate cancer, breast cancer, and pancreatic cancer. In addition, intracellular zinc fluctuations lead to the disturbance of certain signaling pathways involved in the malignant properties of cancer cells. This review briefly summarizes our current understanding of zinc dyshomeostasis in cancer, and discusses the potential roles of zinc or zinc transporters in cancer therapy.

[Regulatory effect of the zinc transporter Zip2 on cardiomyocyte mitochondrial respiration function after cardiac ischemia-reperfusion injury in mice].

The aim of the present study was to investigate the effect of zinc transporter Zip2 (SLC39A2) on mitochondrial respiration during myocardial ischemia/reperfusion (I/R) and the underlying mechanisms. An in vivo myocardial I/R model was established in mice by ligation of left anterior descending coronary artery. Cardiac zinc concentration was measured by inductively coupled plasma-optical emission spectrometer (ICP-OES), and the mitochondrial respiratory function and oxidative phosphorylation were determined by high-resolution respirometry (Oxygraph-2K). The phosphorylation levels of STAT3 and ERK in myocardial tissue were detected by Western blot. The results showed that, compared with the sham group, cardiac zinc concentration in myocardium was decreased in wild-type mice and further reduced in Zip2 knockout mice after I/R. Mitochondrial respiratory control rate (RCR) and oxidative phosphorylation were decreased in Zip2 knockout mice and worsened by I/R. Phosphorylation levels of STAT3 (Ser727) and ERK were significantly decreased in Zip2 knockout mice after I/R. In I/R myocardial tissue, STAT3 overexpression significantly improved the mitochondrial respiratory function, while STAT3 dominant negative mutant (STAT3 S727A) inhibited mitochondrial respiratory function. Moreover, the impairment of mitochondrial function by Zip2 knockout was reversed by STAT3 overexpression. These results suggest that Zip2 regulates mitochondrial respiration via phosphorylation of STAT3 during myocardial I/R, which may represent the underlying mechanism of Zip2 cardioprotection against I/R injury.

TLR2/CXCR4 coassociation facilitates Chlamydia pneumoniae infection-induced atherosclerosis.

Chlamydia pneumoniae infection could play a role in atherosclerosis. Toll-like receptor 2 (TLR2) and C-X-C motif chemokine receptor 4 (CXCR4) have been both shown to be involved in atherosclerosis. However, whether and how TLR2/CXCR4 cross talk is involved in C. pneumoniae infection-induced atherosclerosis remains to be determined. Our study aims to demonstrate that C. pneumoniae infection induced the cross talk between TLR2 and CXCR4 to mediate C. pneumoniae infection-induced vascular smooth muscle cell (VSMC) migration and even accelerate atherosclerosis. We first found that C. pneumoniae infection increased the aortic lesion size (en face), cross-sectional lesion area, and lipid content in aortic root lesion, which were both significantly reduced in apolipoprotein E-null (ApoE-/-)TLR2-/- or CXCR4-blocked ApoE-/- mice and were almost reversed in CXCR4-blocked ApoE-/-TLR2-/- mice. Subsequently, our data showed that C. pneumoniae infection-induced increases in VSMC contents in the atherosclerotic lesion were remarkably suppressed in ApoE-/-TLR2-/- mice or CXCR4-blocked ApoE-/- mice, and were further decreased in CXCR4-blocked ApoE-/-TLR2-/- mice. We then demonstrated that the increase in VSMC migratory capacity caused by C. pneumoniae infection was inhibited by either TLR2 or CXCR4 depletion, and downregulating both TLR2 and CXCR4 further decreased C. pneumoniae infection-induced VSMC migration by suppressing the infection-stimulated F-actin reorganization through the inhibition of the phosphorylation of focal adhesion kinase. Taken together, our data indicate that TLR2/CXCR4 coassociation facilitates C. pneumoniae infection-induced acceleration of atherosclerosis by inducing VSMC migration via focal adhesion kinase-mediated F-actin reorganization.NEW & NOTEWORTHY Toll-like receptor 2 (TLR2) and C-X-C motif chemokine receptor 4 (CXCR4) have both been shown to be involved in atherosclerosis. We demonstrate for the first time the presence of TLR2/CXCR4 coassociation during Chlamydia pneumoniae infection-induced atherosclerosis. Amazingly, blocking of both TLR2 and CXCR4 significantly retards and even almost reverses this infection-induced atherosclerosis. Our work reveals new mechanisms about C. pneumoniae infection-induced atherosclerosis and identifies potential new therapeutic targets for the prevention and treatment of atherosclerosis.

The critical role of the zinc transporter Zip2 (SLC39A2) in ischemia/reperfusion injury in mouse hearts.

Although zinc homeostasis has been demonstrated to play a role in myocardial ischemia/reperfusion (I/R) injury, the roles of zinc transporters that are critical for zinc homeostasis in I/R injury are poorly understood. The purpose of this study was to test if Zip2, an important zinc importer, plays a role in I/R injury in mouse hearts and explore the mechanism by which Zip2 expression is regulated. Zip2 expression was increased at reperfusion in in vivo mouse hearts, an effect that was abolished by ZnCl2, indicating Zip2's attempt to compensate for zinc loss at reperfusion. Further studies showed that upregulation of Zip2 expression was reversed by either pharmacological or genetic inhibition of signal transducers and activators of transcription 3 (STAT3), whereas STAT3 overexpression increased Zip2 expression, indicating that STAT3 accounts for Zip2 upregulation. In support, reperfusion enhanced STAT3 phosphorylation (Tyr705), which was blocked by ZnCl2, implying that STAT3 is activated in response to zinc loss. To determine the role of Zip2 in I/R injury, we assessed I/R injury by genetically disrupting Zip2 expression. Knockout of Zip2 genes (Zip2+/- and Zip2-/-) exacerbated I/R injury by increasing infarct size as well as the serum LDH, troponin I (cTnI), and CK-MB activities. In contrast, delivery of Zip2 genes reduced I/R injury. Delivery of STAT3 genes increased STAT3 phosphorylation and reduced I/R injury. However, delivery of the dominant negative STAT3 mutant did not reduce I/R injury. Moreover, delivery of STAT3 genes failed to reduce I/R injury in Zip2-/- mice. Zip2 upregulated upon reperfusion via STAT3 is cardioprotective and this upregulation may serve as an important intrinsic protective mechanism by which the heart is resistant to I/R injury. The factors involved in the zinc homeostasis (zinc and Zip2) are responsible STAT3 activation and its subsequent cardioprotective action.

A collagen hydrogel loaded with HDAC7-derived peptide promotes the regeneration of infarcted myocardium with functional improvement in a rodent model.

Myocardial infarction (MI) leads to the loss of cardiomyocytes, left ventricle (LV) dilation, and cardiac dysfunction, eventually developing into heart failure. Most of the strategies for MI therapy require biomaterials that can support tissue regeneration. In this study, we hypothesized that the extracellular matrix (ECM)-derived collagen I hydrogel loaded with histone deacetylase 7 (HDAC7)-derived-phosphorylated 7-amino-acid peptide (7Ap) could restrain LV remodeling and improve cardiac function after MI. An MI model was established by ligation of the left anterior descending coronary artery (LAD) of C57/B6 mice. The 7Ap-loaded collagen I hydrogel was intramyocardially injected to the infarcted region of the LV wall of the heart. After local delivery, the 7Ap-collagen increased neo-microvessel formation, enhanced stem cell antigen-1 positive (Sca-1+) stem cell recruitment and differentiation, decreased cellular apoptosis, and promoted cardiomyocyte cycle progression. Furthermore, the 7Ap-collagen restricted the fibrosis of the LV wall, reduced the infarct wall thinning, and improved cardiac performance significantly at 2 weeks post-MI. These results highlight the promising implication of 7Ap-collagen as a novel candidate for MI therapy. STATEMENT OF SIGNIFICANCE: The mammalian myocardium has a limited regenerative capability following myocardial infarction (MI). MI leads to extensive loss of cardiomyocytes, thus culminating in adverse cardiac remodeling and congestive heart failure. In situ tissue regeneration through endogenous cell mobilization has great potential for tissue regeneration. A 7-amino-acid-peptide (7A) domain encoded by a short open-reading frame (sORF) of the HDAC7 gene. The phosphorylated from of 7A (7Ap) has been reported to promote in situ tissue repair via the mobilization and recruitment of endogenous stem cell antigen-1 positive (Sca-l+) stem cells. In this study, 7Ap was shown to improve H9C2 cell survival, in vitro. In vivo investigations in a mouse MI model demonstrated that intra-myocardial delivery of 7Ap-loaded collagen hydrogel promoted neovascularization, stimulated Sca-l+ stem cell recruitment and differentiation, reduced cardiomyocyte apoptosis and promoted cell cycle progression. As a result, treated infarcted hearts had increased wall thickness, had improved heart function and exhibited attenuation of adverse cardiac remodeling, observed for up to 2 weeks. Overall, these results highlighted the positive impact of implanting 7Ap-collagen as a novel constituent for MI repair.

A mitochondria-targeted nitric oxide donor triggered by superoxide radical to alleviate myocardial ischemia/reperfusion injury.

A novel mitochondria-targeted superoxide-responsive nitric oxide donor was developed by incorporation of diphenylphosphinyl and triphenylphosphonium groups into diazeniumdiolate, enabling remarkable protection against ischemia/reperfusion injury in H9c2 cells and isolated rat hearts.

A near-infrared ratiometric/turn-on fluorescent probe for in vivo imaging of hydrogen peroxide in a murine model of acute inflammation.

Much attention has been paid to develop optical probes for noninvasive, quantitative, in vivo monitoring of hydrogen peroxide (H2O2) due to its important roles in the initiation and development of numerous diseases. Motivated to meet this need, we herein report the synthesis of a near-infrared (NIR) fluorescent probe (AB1) for H2O2 by modulating intramolecular charge transfer (ICT) process of the dye 9H-1,3-Dichloro-7-hydroxy-9,9-dimethylacridine-2-one (DDAO). The probe AB1 exhibits both a large NIR fluorescence turn-on and a ratiometric response to H2O2 with high sensitivity and specificity. The fluorescence response of AB1 has a good linear relationship with H2O2 over a wide concentration range from 1 µM to 100 µM, thus affording a detection limit of 0.42 µM. Confocal microscopic experiments demonstrated that AB1 could ratiometrically detect exogenous and endogenous H2O2 in living cells. Moreover, owing to the NIR emission of DDAO, the probe was also utilized to image endogenous H2O2 from the peritoneal cavity in a mouse model of lipopolysaccharide-induced acute inflammation, based on the fluorescence turn-on mode. This new probe shows great potential as a reliable chemical tool to study the development and progression of H2O2-associated diseases in living animals.

Remifentanil Induces Cardio Protection Against Ischemia/Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress Through the Maintenance of Zinc Homeostasis.

BACKGROUND: Although it is well known that remifentanil (Rem) elicits cardiac protection against ischemia/reperfusion (I/R) injury, the underlying mechanism remains unclear. This study tested if Rem can protect the heart from I/R injury by inhibiting endoplasmic reticulum (ER) stress through the maintenance of zinc (Zn) homeostasis. METHODS: Isolated rat hearts were subjected to 30 minutes of regional ischemia followed by 2 hours of reperfusion. Rem was given by 3 consecutive 5-minute infusions, and each infusion was followed by a 5-minute drug-free perfusion before ischemia. Total Zn concentrations in cardiac tissue, cardiac function, infarct size, and apoptosis were assessed. H9c2 cells were subjected to 6 hours of hypoxia and 2 hours of reoxygenation (hypoxia/reoxygenation [H/R]), and Rem was given for 30 minutes before hypoxia. Metal-responsive transcription factor 1 (MTF1) overexpression plasmids were transfected into H9c2 cells 48 hours before hypoxia. Intracellular Zn level, cell viability, and mitochondrial injury parameters were evaluated. A Zn chelator N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) or an ER stress activator thapsigargin was administrated during in vitro and ex vivo studies. The regulatory molecules related to Zn homeostasis and ER stress in cardiac tissue, and cardiomyocytes were analyzed by Western blotting. RESULTS: Rem caused significant reversion of Zn loss from the heart (Rem + I/R versus I/R, 9.43 ± 0.55 vs 7.53 ± 1.18; P < .05) by suppressing the expression of MTF1 and Zn transporter 1 (ZnT1). The inhibited expression of ER stress markers after Rem preconditioning was abolished by TPEN. Rem preconditioning improved the cardiac function accompanied by the reduction of infarct size (Rem + I/R versus I/R, 21% ± 4% vs 40% ± 6%; P < .05). The protective effects of Rem could be reserved by TPEN and thapsigargin. Similar effects were observed in H9c2 cells exposed to H/R. In addition, MTF1 overexpression blocked the inhibitory effects of Rem on ZnT1 expression and ER stress at reoxygenation. Rem attenuated the collapse of mitochondrial membrane potential (ΔΨm) and the generation of mitochondrial reactive oxygen species by inhibiting ER stress via cardiac Zn restoration (Rem + H/R versus H/R, 79.57% ± 10.62% vs 58.27% ± 4.32%; P < .05). CONCLUSIONS: Rem maintains Zn homeostasis at reperfusion by inhibiting MTF1 and ZnT1 expression, leading to the attenuation of ER stress and cardiac injury. Our findings provide a promising therapeutic approach for managing acute myocardial I/R injury.