We aimed to test if stimulation of both adenosine A2A and A2B receptors is required to produce an effective cardioprotection against reperfusion injury. Isolated rat hearts were subjected to 30 min regional ischemia followed by 2 h of reperfusion. The adenosine A1/A2 receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size, an effect that was reversed by both the adenosine A2A antagonist SCH58261 and the A2B antagonist MRS1706. The A2B agonist BAY 60-6583 but not the selective A2A agonist CGS21680 reduced infarct size. Interestingly, a combination of BAY 60-6583 and CGS21680 further reduced infarct size. These results suggest that both A2A and A2B receptors are involved in NECA’s anti-infarct effect at reperfusion. NECA attenuated mitochondrial swelling upon reperfusion and this was blocked by both SCH58261 and MRS1706, indicating that activation of A2 receptors with NECA can modulate reperfusion-induced mitochondrial permeability transition pore (mPTP) opening. In support, NECA also prevented oxidant-induced loss of mitochondrial membrane potential (ΔΨm) and matrix Ca2+ overload in cardiomyocytes via both the A2 receptors. In addition, NECA increased mitochondrial glycogen synthase kinase 3β (GSK-3β) phosphorylation upon reperfusion and this was again blocked by SCH58261 and MRS1706. In conclusion, A2A and A2B receptors work in concert to prevent reperfusion injury in rat hearts treated with NECA. NECA may protect the heart by modulating the mPTP opening through inactivating mitochondrial GSK-3β. A simultaneous stimulation of A2A and A2B receptors at reperfusion is required to produce a strong cardioprotection against reperfusion injury.

The purpose of this study was to determine whether exogenous zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via glycogen synthase kinase-3beta (GSK-3beta). The treatment of cardiac H9c2 cells with ZnCl2 (10 microM) in the presence of zinc ionophore pyrithione for 20 min significantly enhanced GSK-3beta phosphorylation at Ser9, indicating that exogenous zinc can inactivate GSK-3beta in H9c2 cells. The effect of zinc on GSK-3beta activity was blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 but not by the mammalian target of rapamycin (mTOR) inhibitor rapamycin or the PKC inhibitor chelerythrine, implying that PI3K but not mTOR or PKC accounts for the action of zinc. In support of this interpretation, zinc induced a significant increase in Akt but not mTOR phosphorylation. Further experiments found that zinc also increased mitochondrial GSK-3beta phosphorylation. This may indicate an involvement of the mitochondria in the action of zinc. The effect of zinc on mitochondrial GSK-3beta phosphorylation was not altered by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. Zinc applied at reperfusion reduced cell death in cells subjected to simulated ischemia/reperfusion, indicating that zinc can prevent reperfusion injury. However, zinc was not able to exert protection in cells transfected with the constitutively active GSK-3beta (GSK-3beta-S9A-HA) mutant, suggesting that zinc prevents reperfusion injury by inactivating GSK-3beta. Cells transfected with the catalytically inactive GSK-3beta (GSK-3beta-KM-HA) also revealed a significant decrease in cell death, strongly supporting the essential role of GSK-3beta inactivation in cardioprotection. Moreover, zinc prevented oxidant-induced mPTP opening through the inhibition of GSK-3beta. Taken together, these data suggest that zinc prevents reperfusion injury by modulating the mPTP opening through the inactivation of GSK-3beta. The PI3K/Akt signaling pathway is responsible for the inactivation of GSK-3beta by zinc.

Our previous study demonstrated that zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via Akt and glycogen synthetase kinase 3beta (GSK-3beta). We aimed to address the mechanism by which zinc activates Akt. Treatment of H9c2 cells with ZnCl(2) (10 microM) in the presence of the zinc ionophore pyrithione (4 microM) for 20 min enhanced Akt phosphorylation (Ser(473)), indicating that zinc can rapidly activate Akt. Zinc did not alter either phosphatase and tensin homolog deleted on chromosome 10 (PTEN) phosphorylation and total PTEN protein levels or PTEN oxidation, implying that PTEN may not play a role in the action of zinc. However, zinc-induced Akt phosphorylation was blocked by both the nonselective receptor tyrosine kinase (RTK) inhibitor genistein and the selective insulin-like growth factor-1 RTK (IGF-1RTK) inhibitor AG1024, indicating that zinc activates Akt via IGF-1RTK. Zinc-induced phosphorylation of protein tyrosine and Ser/Thr was also abolished by AG1024. In addition, zinc markedly enhanced phosphorylation of IGF-1 receptor (IGF-1R), which was again reversed by genistein and AG1024. A confocal imaging study revealed that AG1024 abolished the preventive effect of zinc on oxidant-induced mPTP opening, confirming that IGF-1RTK plays a role in zinc-induced cardioprotection. Furthermore, zinc decreased the activity of protein phosphatase 2A (PP2A), a major protein Ser/Thr phosphatase, implying that protein Ser/Thr phosphatases may also play a role in the action of zinc on Akt activity. Taken together, these findings demonstrate that exogenous zinc activates Akt via IGF-1RTK and prevents the mPTP opening in cardiac cells. Inactivation of Ser/Thr protein phosphatases may also contribute to zinc-induced Akt activation.

Exogenous zinc can protect cardiac cells from reperfusion injury, but the exact roles of endogenous zinc in the pathogenesis of reperfusion injury and in adenosine A 2 receptor activation-induced cardioprotection against reperfusion injury remain unknown. Adenosine A 1 /A 2 receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size in isolated rat hearts subjected to 30 min ischemia followed by 2 h of reperfusion. This effect of NECA was partially but significantly blocked by the zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and ZnCl 2 given at reperfusion mimicked the effect of NECA by reducing infarct size. Total tissue zinc concentrations measured with inductively coupled plasma optical emission spectroscopy (ICPOES) were decreased upon reperfusion in rat hearts and this was reversed by NECA. NECA increased intracellular free zinc during reperfusion in the heart. Confocal imaging study showed a rapid increase in intracellular free zinc in isolated rat cardiomyocytes treated with NECA. Further experiments revealed that NECA increased total zinc levels upon reperfusion in mitochondria isolated from isolated hearts. NECA attenuated mitochondrial swelling upon reperfusion in isolated hearts and this was inhibited by TPEN. Similarly, NECA prevented the loss of mitochondrial membrane potential (ΔΨm) caused by oxidant stress in cardiomyocytes. Finally, both NECA and ZnCl 2 inhibited the mitochondrial metabolic activity. NECA-induced cardioprotection against reperfusion injury is mediated by intracellular zinc. NECA prevents reperfusion-induced zinc loss and relocates zinc to mitochondria. The inhibitory effects of zinc on both the mPTP opening and the mitochondrial metabolic activity may account for the cardioprotective effect of NECA.

Inhibition of PDEs prevents the mPTP opening by inactivating GSK-3β through PKA and PKG. GSK-3β is a common downstream target of PKA and PKG. Inhibition of PDEs may be a useful approach to prevent reperfusion injury.

No abstract

Fire has shaped global ecosystems for millennia by directly killing organisms and indirectly altering habitats and resources. All terrestrial ecosystems, including fire‐prone ecosystems, rely on soil‐inhabiting fungi, where they play vital roles in ecological processes. Yet our understanding of how fire regimes influence soil fungi remains limited and our knowledge of these interactions in semiarid landscapes is virtually absent. We collected soil samples and vegetation measurements from sites across a gradient in time‐since‐fire ages (0–75 years‐since‐fire) and fire frequency (burnt 0–5 times during the recent 29‐year period) in a semiarid heathland of south‐eastern Australia. We characterized fungal communities using ITS amplicon‐sequencing and assigned fungi taxonomically to trophic guilds. We used structural equation models to examine direct, indirect and total effects of time‐since‐fire and fire frequency on total fungal, ectomycorrhizal, saprotrophic and pathogenic richness. We used multivariate analyses to investigate how total fungal, ectomycorrhizal, saprotrophic and pathogenic species composition differed between post‐fire successional stages and fire frequency classes. Time‐since‐fire was an important driver of saprotrophic richness; directly, saprotrophic richness increased with time‐since‐fire, and indirectly, saprotrophic richness declined with time‐since‐fire (resulting in a positive total effect), mediated through the impact of fire on substrates. Frequently burnt sites had lower numbers of saprotrophic and pathogenic species. Post‐fire successional stages and fire frequency classes were characterized by distinct fungal communities, with large differences in ectomycorrhizal species composition. Understanding the complex responses of fungal communities to fire can be improved by exploring how the effects of fire flow through ecosystems. Diverse fire histories may be important for maintaining the functional diversity of fungi in semiarid regions.

Myocardial ischemia/reperfusion causes zinc depletion and supplementation of zinc can attenuate ischemia/reperfusion injury. The purpose of this study was to determine the role of the intracellular homeostasis in the cardioprotective mechanism of ischemic postconditioning. Isolated rat hearts were subjected to 30 min regional ischemia followed by 2 h of reperfusion. Postconditioning was elicited by six cycles of 10 second reperfusion and 10 seconds ischemia. Zinc concentration was measured with inductively coupled plasma optical spectroscopy (ICPOES). Upon reperfusion cytosolic zinc levels were dramatically decreased but this was prevented by postconditioning, indicating that postconditioning can prevent reperfusion-induced zinc loss. Moreover, postconditioning could also attenuate mitochondrial zinc loss upon reperfusion. In agreement with these observations, the anti-infarct effect of postconditioning was reversed by the zinc chelator N,N,N’,N’-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and exogenous zinc given at reperfusion mimicked the effect of postconditioning by reducing infarct size, suggesting that intracellular zinc account for the cardioprotective effect of postconditioning. Finally, postconditioning enhanced phosphorylation of Akt, ERK, and GSK-3β upon reperfusion, and these effects of postconditioning were inhibited by TPEN, indicating that zinc may mediate the cardioprotective effect of postconditioning by activating the RISK pathway. In conclusion, these data suggest that postconditioning may protect the heart by maintaining intracellular zinc homeostasis and activation of the RISK pathway may account for zinc-mediated cardioprotection of postconditioning.