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.

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.

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.

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.

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.

[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.

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.

Zinc improves mitochondrial respiratory function and prevents mitochondrial ROS generation at reperfusion by phosphorylating STAT3 at Ser727.

Serine 727 (Ser727) phosphorylation of STAT3 plays a role in the regulation of mitochondrial respiration. This study aimed to test if zinc could regulate mitochondrial respiration through phosphorylation of STAT3 at Ser727 in the setting of ischemia/reperfusion in the heart. Under normoxic conditions, treatment of isolated rat hearts with ZnCl2 increased cytosolic STAT3 phosphorylation at Ser727 followed by phospho-STAT3 translocation to mitochondria. In isolated rat hearts subjected to 30 min regional ischemia followed by 20 min of reperfusion, ZnCl2 given 5 min before the onset of reperfusion also increased mitochondrial phospho-STAT3. ZnCl2 enhanced ERK phosphorylation and PD98059 reversed the effect of ZnCl2 on STAT3 phosphorylation. ZnCl2 improved the mitochondrial oxidative phosphorylation at reperfusion. This effect was abolished by STAT3S727A, a mutant in which Ser727 is replaced with alanine, in H9c2 cells subjected to hypoxia/reoxygenation. In addition, ZnCl2 increased the mRNA level of the complex I subunit ND6, which was also reversed by STAT3S727A. Moreover, ZnCl2 attenuated mitochondrial ROS generation and dissipation of mitochondrial membrane potential (ΔΨm) at reoxygenation through Ser727 phosphorylation. Finally, ZnCl2 suppression of succinate dehydrogenase (SDH) activity upon the onset of reperfusion was nullified by the Ser727 mutation. In conclusion, zinc improves cardiac oxidative phosphorylation and inhibits mitochondrial ROS generation at reperfusion by increasing mitochondrial STAT3 phosphorylation at Ser727 via ERK. The preservation of ND6 mtDNA and the inhibition of SDH activity may account for the role of STAT3 in the beneficial action of zinc on the mitochondrial oxidative phosphorylation and ROS generation at reperfusion.

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.

The Relationship Between Serum Zinc Levels, Cardiac Markers and the Risk of Acute Myocardial Infarction by Zinc Quartiles.

BACKGROUND: Zinc is one of the most important microelements in the body and zinc homeostasis plays a critical role in maintaining cellular structure and function. Zinc dyshomeostasis can lead to many diseases, such as cardiovascular disease. Our aim was to investigate whether there is a relationship between zinc and cardiac markers, and the risk of acute myocardial infarction (AMI) by zinc quartiles. METHODS: We enrolled a total of 529 patients and measured their serum zinc levels and cardiac markers. We performed further studies after dividing subjects into four groups according to their concentrations of zinc by quartile to clarify the relationship between zinc levels and risk of increased acute myocardial infarction prevalence rate. RESULTS: We observed that there was a significant inverse linear relationship between zinc and Lg(creatine kinase) (p=0.011), Lg(creatine kinase-MB) (p=0.002) and Lg(cardiac troponin T) (p=0.045). In addition, the acute myocardial infarction prevalence rates were 28.8%, 24.8%, 20.5%, and 18.2% by patients with zinc quartiles, respectively. Multivariate logistic regression analysis showed that the odds ratio between the lowest and highest zinc quartile groups was 1.92 (1.019-3.604) (p<0.05). CONCLUSIONS: The present study revealed a relationship between serum zinc levels in that zinc levels were significantly inversely correlated with serum creatine kinase (CK), creatine kinase-MB (CKMB) and cardiac troponin T (cTnT) levels. Furthermore, we found that the prevalence rate of acute myocardial infarction decreased with increasing zinc quartiles.

Chlamydia pneumoniae infection promotes vascular endothelial cell angiogenesis through an IQGAP1-related signaling pathway.

Chlamydia pneumoniae (C. pneumoniae) infection plays a potential role in angiogenesis. However, it is still an enigma how C. pneumoniae is involved in this process. Therefore, we investigated the effect of C. pneumoniae infection on angiogenesis, and then explored the roles of IQGAP1-related signaling in C. pneumoniae infection-induced angiogenesis. C. pneumoniae infection significantly enhanced angiogenesis as assessed by the tube formation assay possibly by inducing vascular endothelial cell (VEC) migration in the wound healing and Transwell migration assays. Subsequently, immunoprecipitation, Western blot and tube formation assay results showed that the phosphorylation of both IQGAP1 and N-WASP was required for the angiogenesis induced by C. pneumoniae infection. Our co-immunoprecipitation study revealed that IQGAP1 physically associated with N-WASP after C. pneumoniae infection of VECs. Actin polymerization assay further showed that in C. pneumoniae-infected VECs, both IQGAP1 and N-WASP were recruited to filamentous actin, and shared some common compartments localized at the leading edge of lamellipodia, which was impaired after the depletion of IQGAP1 by using the small interference RNA. Moreover, the knockdown of IQGAP1 also significantly decreased N-WASP phosphorylation at Tyr256 induced by C. pneumoniae infection. We conclude that C. pneumoniae infection promotes VEC migration and angiogenesis presumably through the IQGAP1-related signaling pathway.

Mitochondrial events responsible for morphine's cardioprotection against ischemia/reperfusion injury.

Morphine may induce cardioprotection by targeting mitochondria, but little is known about the exact mitochondrial events that mediate morphine's protection. We aimed to address the role of the mitochondrial Src tyrosine kinase in morphine's protection. Isolated rat hearts were subjected to 30 min ischemia and 2h of reperfusion. Morphine was given before the onset of ischemia. Infarct size and troponin I release were measured to evaluate cardiac injury. Oxidative stress was evaluated by measuring mitochondrial protein carbonylation and mitochondrial ROS generation. HL-1 cells were subjected to simulated ischemia/reperfusion and LDH release and mitochondrial membrane potential (ΔΨm) were measured. Morphine reduced infarct size as well as cardiac troponin I release which were aborted by the selective Src tyrosine kinase inhibitors PP2 and Src-I1. Morphine also attenuated LDH release and prevented a loss of ΔΨm at reperfusion in a Src tyrosine kinase dependent manner in HL-1 cells. However, morphine failed to reduce LDH release in HL-1 cells transfected with Src siRNA. Morphine increased mitochondrial Src phosphorylation at reperfusion and this was abrogated by PP2. Morphine attenuated mitochondrial protein carbonylation and mitochondrial superoxide generation at reperfusion through Src tyrosine kinase. The inhibitory effect of morphine on the mitochondrial complex I activity was reversed by PP2. These data suggest that morphine induces cardioprotection by preventing mitochondrial oxidative stress through mitochondrial Src tyrosine kinase. Inhibition of mitochondrial complex I at reperfusion by Src tyrosine kinase may account for the prevention of mitochondrial oxidative stress by morphine.

[Injurious effect of zinc deficiency on cardiomyocytes].

The aim of the present study was to investigate the effect of zinc deficiency on cardiomyocyte survival and the underlying mechanisms. Simulated zinc deficiency model was developed in H9c2 cardiac cells with zinc chelator N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). MTT assay was used to evaluate cell viability. Morphological changes of the cells were observed by optical microscope. Lacate dehydrogenase (LDH) levels of the cells were determined with LDH assay kit. Mitochondrial membrane potential (ΔΨ) was measured with confocal microscope using JC-1 dye. Intracellular reactive oxygen species (ROS) levels were determined by DCFH-DA staining. PD98059 (an inhibitor of ERK), SNAP, which can activate ERK, and the ROS scavenger, MPG, were respectively used to investigate mechanism of signal transduction. The phosphorylation of ERK was detected by Western blot. The results showed that TPEN significantly induced the cell morphological damage and the loss of ΔΨ, increased LDH leakage, and promoted ROS generation. In the H9c2 cells, TPEN significantly inhibited ERK phosphorylation and decreased cell viability, which was potentiated by PD98059, whereas both SNAP and MPG reversed the inhibitory effects of TPEN. These data suggest that zinc deficiency leads to the injury in H9c2 cardiac cells through down-regulating ERK pathway. Increased intracellular ROS may account for the effect of zinc deficiency.

The Critical Roles of Zinc: Beyond Impact on Myocardial Signaling.

Zinc has been considered as a vital constituent of proteins, including enzymes. Mobile reactive zinc (Zn(2+)) is the key form of zinc involved in signal transductions, which are mainly driven by its binding to proteins or the release of zinc from proteins, possibly via a redox switch. There has been growing evidence of zinc's critical role in cell signaling, due to its flexible coordination geometry and rapid shifts in protein conformation to perform biological reactions. The importance and complexity of Zn(2+) activity has been presumed to parallel the degree of calcium's participation in cellular processes. Whole body and cellular Zn(2+) levels are largely regulated by metallothioneins (MTs), Zn(2+) importers (ZIPs), and Zn(2+) transporters (ZnTs). Numerous proteins involved in signaling pathways, mitochondrial metabolism, and ion channels that play a pivotal role in controlling cardiac contractility are common targets of Zn(2+). However, these regulatory actions of Zn(2+) are not limited to the function of the heart, but also extend to numerous other organ systems, such as the central nervous system, immune system, cardiovascular tissue, and secretory glands, such as the pancreas, prostate, and mammary glands. In this review, the regulation of cellular Zn(2+) levels, Zn(2+)-mediated signal transduction, impacts of Zn(2+) on ion channels and mitochondrial metabolism, and finally, the implications of Zn(2+) in health and disease development were outlined to help widen the current understanding of the versatile and complex roles of Zn(2+).

Zinc plays a critical role in the cardioprotective effect of postconditioning by enhancing the activation of the RISK pathway in rat hearts.

This study investigated if zinc plays a role in postconditioning-induced cardioprotection in rat hearts. Isolated rat hearts were subjected to 30 min regional ischemia followed by 2h of reperfusion. Postconditioning was elicited by 6 cycles of 10s reperfusion and 10s ischemia. Cytosolic zinc concentrations were measured with inductively coupled plasma optical emission spectroscopy (ICPOES). Infarct size was assessed by triphenyltetrazolium chloride staining. Cytosolic zinc concentrations were decreased dramatically upon reperfusion in the control hearts. In contrast, postconditioning increased cytosolic zinc levels at reperfusion. The anti-infarct effect of postconditioning was inhibited by the selective zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN). Postconditioning significantly increased phosphorylation levels of the reperfusion injury salvage kinases (RISK) including Akt (Ser(473)), extracellular signal-regulated kinase1/2 (ERK1/2) (Thr(202)/Tyr(204)), and glycogen synthase kinase-3ß (GSK-3ß) (Ser(9)) at reperfusion, which were nullified by TPEN. Postconditioning decreased the activity of protein phosphatase 2A (PP2A) in a zinc-dependent manner. Knockdown of the zinc transporter Zip2 inhibited the protective effect of postconditioning on hypoxia/reoxygenation injury in H9c2 cells. These results suggest that zinc plays an important role in the cardioprotective effect of postconditioning presumably by enhancing the activation of the RISK pathway. Zip2 and inactivation of PP2A by zinc may, at least in part, account for the activation of the RISK pathway.

MuRF1 activity is present in cardiac mitochondria and regulates reactive oxygen species production in vivo.

MuRF1 is a previously reported ubiquitin-ligase found in striated muscle that targets troponin I and myosin heavy chain for degradation. While MuRF1 has been reported to interact with mitochondrial substrates in yeast two-hybrid studies, no studies have identified MuRF1's role in regulating mitochondrial function to date. In the present study, we measured cardiac mitochondrial function from isolated permeabilized muscle fibers in previously phenotyped MuRF1 transgenic and MuRF1-/- mouse models to determine the role of MuRF1 in intermediate energy metabolism and ROS production. We identified a significant decrease in reactive oxygen species production in cardiac muscle fibers from MuRF1 transgenic mice with increased α-MHC driven MuRF1 expression. Increased MuRF1 expression in ex vivo and in vitro experiments revealed no alterations in the respiratory chain complex I and II function. Working perfusion experiments on MuRF1 transgenic hearts demonstrated significant changes in glucose oxidation. However, total oxygen consumption was decreased [corrected]. This data provides evidence for MuRF1 as a novel regulator of cardiac ROS, offering another mechanism by which increased MuRF1 expression may be cardioprotective in ischemia reperfusion injury, in addition to its inhibition of apoptosis via proteasome-mediate degradation of c-Jun. The lack of mitochondrial function phenotype identified in MuRF1-/- hearts may be due to the overlapping interactions of MuRF1 and MuRF2 with energy regulating proteins found by yeast two-hybrid studies reported here, implying a duplicity in MuRF1 and MuRF2's regulation of mitochondrial function.

NecroX-5 suppresses sodium nitroprusside-induced cardiac cell death through inhibition of JNK and caspase-3 activation.

Although sodium nitroprusside (SNP) is an effective hypotensive drug and is often used in pediatric intensive care units and to treat acute heart failure, clinical application of SNP is limited by its cardiotoxicity. NecroX-5 (NX-5) was recently developed and has the capacity to inhibit necrotic cell death. No current literature addresses whether NX-5 suppresses SNP-induced cell death or its mechanism of action. We have investigated the protective role of NX-5 against SNP-induced cell death in cardiomyocyte-like H9c2 cells. SNP treatment induced severe cell death, possibly through phosphorylation of stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) and activation of the apoptotic signaling pathway, including downregulation of Bcl-2 and cleavage of caspase-3. However, NX-5 suppresses SNP-induced cell death through inhibition of JNK activation and suppression of both downregulation of Bcl-2 protein expression and caspase-3 cleavage. These findings will provide insights and facilitate development of antidotes to SNP toxicity in cardiac cells.

Kobophenol A inhibits sodium nitroprusside-induced cardiac H9c2 cell death through suppressing activation of JNK and preserving mitochondrial anti-apoptotic Bcl-2 and Mcl-1.

Sodium nitroprusside (SNP) releases nitric oxide (NO), a powerful vasodilator, and thus widely used in intensive care unit for treating hypertension emergency. However, cardiac toxicity after SNP administration is a clinical problem. For finding a natural compound that suppressing SNP-induced cardiac toxicity, we tested the protective potential of kobophenol A (Kob A), purified from the root of Caragana sinica, against the toxic effects of SNP. The severe cardiac H9c2 cell death was induced by SNP (2 mM) treatment. Kob A ameliorated SNP-induced cardiac H9c2 cell death, and this protective effect of Kob A may be related to the inhibition of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase activation following SNP administration. In addition, the downregulation of cellular Bcl-2 and Mcl-1 levels by SNP exposure was strongly abrogated in the presence of Kob A. These biological properties of Kob A might provide insights into developing new cardioprotectant against SNP-induced cardiac cell death.

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.