The Cardioprotective Effect Of Cyclic GMP And The Role Of Autophagy In Myocardial Ischemia/reperfusion Injury

Part1Cyclic guanosine 3'5'-monophosphate (cGMP) is a second messenger molecule and is generated from the catalytic conversion of guanosine triphosphate (GTP) by the soluble guanylyl cyclase (sGC) and the particulate guanylyl cyclase (pGC) As an important cellular messenger, cGMP has three major identified targets: cGMP-dependent protein kinase or protein kinase G (PKG), cGMP-regulated cAMP phosphodiesterase, and cGMP-gated ion channels. Among these targets, PKG has been identified as the most important downstream signal of cGMP. Under physiological conditions, PKG exerts a beneficial influence on the cardiovascular system through relaxation of smooth muscle cells, inhibition of endothelial permeability, inhibition of platelet activation, and negative inotropic effect on cardiomyocytes. Recent studies have shown that the cGMP/PKG signaling pathway is involved in the cardioprotective mechanism underlying ischemic preconditioning and postconditioning. It has also been reported that inhibition of phosphodiesterase-5 with sildenafil (Viagra) which can augment cGMP accumulation protects cardiomyocytes from ischemia/reperfusion injury via a PKG-dependent mechanism. However, the mechanism underlying the cardioprotective effects mediated by the cGMP/PKG signaling pathway remains to be uncertain.Opening of the mPTP contributes to the pathogenesis of ischemia/reperfusion injury, whereas modulation of the mPTP opening has been proposed to be the common mechanism by which various cardioprotective interventions confer protection against ischemia/reperfusion injury. Although the mPTP has been proposed to be the target of the cGMP/PKG signaling, the impact of the cGMP/PKG on the pore opening is unknown. Studies have shown that glycogen synthase kinase 3β(GSK-3β) may play such a role by interacting with the mPTP after being inactivated by the upstream protective signals. Since sildenafil inactivates GSK-3βvia PKG in cardiomyocytes, it is likely that cGMP may lead to prevention of the mPTP opening by inactivating GSK-3β. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is well known for its negative effect on GSK-3βactivity, and is reported to be activated by sildenafil. Thus, it is possible that the cGMP/PKG signaling inactivates GSK-3βthrough PI3K/Akt. Although Akt activation contributes to cardioprotection of ischemic preconditioning, an excessive activation of Akt also leads to pathologic remodeling and heart failure. Thus, it is intriguing to address the mechanism responsible for the inhibitory effect of cGMP on Akt activity.The purpose of this study was to determine if cGMP prevents the mPTP opening by inactivating GSK-3(3 in cardiac H9c2 cells and to test if cGMP inactivates GSK-3βby altering Akt activity. To test if cGMP could inactivate GSK-3β, we treated H9c2 cells with the cGMP analogue 8-Br-cGMP (500μmol/L) for 30 min and detected GSK-3P phosphorylation at Ser9 with Western blot. Our data showed that 8-Br-cGMP increased GSK-3βphosphorylation in a dose-dependent manner, indicating cGMP can inactivate GSK-3β. Since PKG is the most important downstream signal of cGMP, we examined the potential role of PKG in the action of 8-Br-cGMP by detecting the phosphorylation level of vasodilator-stimulated phosphoprotein (VASP), a major substrate of PKG. The effect of cGMP on GSK-3βactivity was reversed by the selective PKG inhibitor KT5823 (10μmol/L), indicating that PKG accounts for the action of cGMP. Experiments with cell lysates showed that 8-Br-cGMP alone did not alter but together with PKG la markedly enhanced GSK-3βphosphorylation, confirming that cGMP inhibits GSK-3βthrough PKG. In support, in vitro experiments revealed that interaction of 8-Br-cGMP with PKG la markedly enhanced phosphorylation of purified GSK-3β. This result also indicates that GSK-3βis a direct substrate of PKG. These results clearly demonstrate that cGMP inactivates GSK-3βvia PKG. To determine if cGMP can prevent the mPTP opening, we examined the effect of 8-Br-cGMP on oxidant-induced loss ofΔΨm by monitoring changes in TMRE fluorescence with confocal microscopy. cGMP prevented the loss of TMRE fluorescence caused by H2O2, indicating that cGMP can suppress the mPTP opening. cGMP failed to preserve TMRE fluorescence in cells transfected with the constitutively active GSK-3β(GSK-3P-S9A) mutant plasmid, suggesting that cGMP modulates the mPTP opening by inactivating GSK-3β. To test if Akt activation contributes to the inhibitory effect of cGMP on GSK-3βactivity, we measured Akt phosphorylation at Ser473 with Western blot. To our great surprise,8-Br-cGMP did not increase but decreased Akt phosphorylation under physiological conditions, indicating that an accumulation of cGMP rapidly inactivates Akt. To confirm this finding, we further tested if the non-specific inhibitor of phosphodiesterases isobutylmethylxanthine (IBMX,200μmol/L) which maintains intracellular cGMP accumulation by inhibiting enzymatic hydrolysis of cGMP, could also mimic the effect of cGMP to inhibit Akt phosphorylation. IB MX markedly reduced Akt phosphorylation but enhanced VASP phosphorylation, indicating that IBMX indeed negatively regulates Akt activity via cGMP. Further experiments showed that 8-Br-cGMP suppresses the effect of insulin on Akt phosphorylation, suggesting that cGMP not only suppresses the basal Akt activity but also impedes the ligand-induced Akt activation. Because an excessive activation of Akt may lead to cardiac hypertrophy and heart failure, our finding suggests that cGMP may prevent hypertrophy and heart failure. The inhibitory effect of cGMP on Akt phosphorylation was not reversed by both the PKG inhibitor KT5823 and PKG siRNA, suggesting that PKG is not required for the negative regulatory action of cGMP on Akt signaling, although it serves as the main downstream signal of cGMP to prevent the mPTP opening by inactivating GSK-3β.Akt is activated by phosphorylation of Thr308 and Ser473, and dephosphorylation of these residues by Ser/Thr protein phosphatases inactivates the kinase. Thus, we tested if cGMP could inactivate Akt by activating Ser/Thr protein phosphatases. Calyculin A (5 nmol/L), a cell permeable Ser/Thr protein phosphatase inhibitor, dramatically increased Akt phosphorylation, an effect that was partially but significantly blocked by 8-Br-cGMP, indicating that cGMP is able to activate Ser/Thr protein phosphatases, which may lead to inactivation of Akt. To corroborate this finding, we further detected the activity of protein phosphatase 2A (PP2A), a major Ser/Thr protein phosphatase.8-Br-cGMP significantly increased PP2A activity, indicating that cGMP may inhibit Akt activity by activating PP2A.Summary 1. Cyclic GMP prevents the mPTP opening by inactivating GSK-3βvia PKG in H9c2 cells, which accounts for the cardioprotective of cGMP;2. Cyclic GMP negatively regulates Akt activity;3. Cyclic GMP inactivates Akt through activation of PP2A but not through PKG;4.Cyclkic GMP has duel roles in cardiac survival. Part1Ischemic heart disease (IHD) is the leading cause of morbidity and mortality worldwide. The fundamental cause of IHD is myocardial ischemia. Reperfusion therapies cause myocardial contractile dysfunction and tissue damage in the ischemic zone, so-called myocardial reperfusion injury. Development of effective therapies to protect reperfusion injury is an important issue of the medical community. The mechanism underlying myocardial reperfusion injury is complicated and remains unclear, although free radicals, calcium overload, defects in energy metabolism, inflammation, and apoptosis have been proposed to account for the injury. Recent studies indicate that autophagy may play a role in myocardial reperfusion injury. Autophagy occurs constitutively in eukaryotic cells and is a process involving the degradation of cell's own components through the lysosomal machinery. Autophagy occurs under normal conditions, but is also upregulated in response to stress such as starving and hypoxia. Although autophagy can promote cell survival, it may also cause cell death (autophagic cell death or type II programmed cell death). While studies have shown that ischemia/reperfusion can trigger autophagy, the exact induction timing and role of autophagy are unclear. Understanding of the detailed time course for the induction and progression of autophagy during ischemia/reperfusion is critical for the development of preventive and therapeutic interventions for reperfusion injury. Autophagy is regulated by the class I and III of PI3K. The class I inhibits autophagy through mammalian target of rapamycin (mTOR) (the negative pathway), whereas the class III PI3K activates autophagy by increasing Beclin-1 expression, so-called the positive pathway or non mTOR-dependent pathway. LC3, the rat microtubule-associated protein 1 light chain 3, a mammalian homologue of yeast ATG8, is associated with autophagy. There are two forms of LC3, namely, LC3-Ⅰ(cytosol form) and LC3-Ⅱ(processed form). The ratio of LC3-Ⅱ/LC3-Ⅰis correlated with the extent of autophagosome formation and thus was suggested as an excellent marker of autophagy. Investigation of the regulatory mechanism of autophagy occurring during reperfusion is important for understanding of the mechanism underlying reperfusion injury and will help discover effective therapeutic intervention to prevent reperfusion injury. Based on the above theoretical interpretations, the current study investigated the time course of autophagy induction, the exact role of autophagy during reperfusion injury, and the regulatory mechanism underlying the induction of autophagy.Isolated rat hearts were perfused on a Langendorff apparatus and subjected to 30 min regional ischemia followed by 2 h of reperfusion. Biopsies were collected from the risk zones during ischemia (0,10,20 and 30 min of ischemia) and reperfusion (10,30,60, and 120 min after the onset of reperfusion), and autophagy was determined by the ratio of LC3-Ⅱto LC3-Ⅰwith Western blotting. We found that although LC3-Ⅱ/LC3-Ⅰratio had a tendency to increase during ischemia, there was no statistical difference when compared to the sham group, indicating that autophagy is not prominent during ischemia. In contrast, LC3-Ⅱ/LC3-Ⅰratio was markedly increased upon reperfusion, implying that autophagy may play a role in reperfusion injury.Since autophagy may play a role during reperfusion, we treated rat hearts with 3-MA and NEC A starting 5 min before the onset of reperfusion for 35 min. Myocardial samples were collected form the risk zones 10,30,60, and 120 min after the onset of reperfusion. Autophagy was determined by the ratio of LC3-Ⅱto LC3-Ⅰwith Western blotting, whereas myocardial infarction was measured with TTC staining. Our data showed that 3-MA not only inhibited autophagy during reperfusion but also reduced infarct size, suggesting that autophagy plays a detrimental role in reperfusion injury. Similarly, NEC A also reduced both autophagy and myocardial infarction, indicating that inhibition of autophagy may contribute to the protective effect of NEC A on reperfusion injury.To investigate the mechanism underlying the induction of autophagy, we detected Beclin 1 expression and mTOR activities during ischemia and reperfusion with Western blotting. Either ischemia or reperfusion did not enhance Beclin 1 expression, suggesting Beclin 1 may not be critical for the formation of autophagy. Since Beclin 1 is a critical gene for the positive pathway, our observation suggests that the induction of autophagy during reperfusion is not dependent on the positive pathway in isolated rat hearts. In contrast, the activity of mTOR was decreased during both ischemia and reperfusion, indicating that an inhibition of the negative pathway may account for autophagy in the setting of ischemia/reperfusion.Summary1. Autophagy is induced upon reperfusion but not during ischemia in isolated rat hearts;2. Autophagy plays a detrimental role during ischemia/reperfusion;3. Inhibition of the negative pathway but not the positive pathway account for the formation of autophagy in isolated rat hearts.