ATP-sensitive potassium (K_ATP) channels couple cell metabolic status to membrane excitability and are crucial for stress adaptation and cytoprotection in the heart. Atrial natriuretic peptide (ANP), a cardiac peptide important for cardiovascular homeostasis, also exhibits cytoprotective features including protection against myocardial ischemia-reperfusion injuries. However, how ANP modulates cardiac K_ATP channels is largely unknown. In the present study we sought to address this issue by investigating the role of ANP signaling in functional modulation of sarcolemmal K_ATP (sarcK_ATP) channels in ventricular myocytes freshly isolated from adult rabbit hearts. Single-channel recordings were performed in combination with pharmacological approaches in the cell-attached patch configuration. Bath application of ANP markedly potentiated sarcK_ATP channel activities induced by metabolic inhibition with sodium azide, whereas the K_ATP-stimulating effect of ANP was abrogated by selective inhibition of the natriuretic peptide receptor type A (NPR-A), cGMP-dependent protein kinase (PKG), reactive oxygen species (ROS), extracellular signal-regulated protein kinase (ERK)1/2, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), or the ryanodine receptor (RyR). Blockade of RyRs also nullified hydrogen peroxide (H₂O₂)-induced stimulation of sarcK_ATP channels in intact cells. Furthermore, single-channel kinetic analyses revealed that ANP enhanced the function of ventricular sarcK_ATP channels through destabilizing the long closures and facilitating the opening transitions, without affecting the single-channel conductance. In conclusion, here we report that ANP positively modulates the activity of ventricular sarcK_ATP channels via an intracellular signaling mechanism consisting of NPR-A, PKG, ROS, ERK1/2, CaMKII, and RyR2. This novel mechanism may regulate cardiac excitability and contribute to cytoprotection, in part, by opening myocardial K_ATP channels.

Background

Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (K(ATP)) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue.

Methods and findings

Single-channel recordings of cardiac K(ATP) channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type K(ATP)) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H(2)O(2) scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H(2)O(2) also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of K(ATP) channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions.

Conclusion

The present study provides novel evidence that PKG exerts dual regulation of cardiac K(ATP) channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H(2)O(2) in particular), calmodulin and CaMKII, alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins. The novel cGMP/PKG/ROS/calmodulin/CaMKII signaling pathway may regulate cardiomyocyte excitability by opening K(ATP) channels and contribute to cardiac protection against ischemia-reperfusion injury.

Increased expression of vascular cell adhesion molecule (VCAM)-1 on the activated arterial endothelial cell (EC) surface critically contributes to atherosclerosis which may in part be regulated by epigenetic mechanisms. This study investigated whether and how the clinically available histone deacetylases 1 and 2 (HDAC1/2) inhibitor drug Romidepsin epigenetically modulates VCAM-1 expression to suppress atherosclerosis. Methods: VCAM-1 expression was analyzed in primary human aortic EC (HAEC) treated with Romidepsin or transfected with HDAC1/2-targeting siRNA. Methylation of GATA6 promoter region was examined with methylation-specific PCR assay. Enrichment of STAT3 to GATA6 promoter was detected with chromatin immunoprecipitation. Lys685Arg mutation was constructed to block STAT3 acetylation. The potential therapeutic effect of Romidepsin on atherosclerosis was evaluated in Apoe ^-/- mice fed with a high-fat diet. Results: Romidepsin significantly attenuated TNFα-induced VCAM-1 expression on HAEC surface and monocyte adhesion through simultaneous inhibition of HDAC1/2. This downregulation of VCAM-1 was attributable to reduced expression of transcription factor GATA6. Romidepsin enhanced STAT3 acetylation and its binding to DNA methyltransferase 1 (DNMT1), leading to hypermethylation of the GATA6 promoter CpG-rich region at +140/+255. Blocking STAT3 acetylation at Lys685 disrupted DNMT1-STAT3 interaction, decreased GATA6 promoter methylation, and reversed the suppressive effects of HDAC1/2 inhibition on GATA6 and VCAM-1 expression. Finally, intraperitoneal administration of Romidepsin reduced diet-induced atherosclerotic lesion development in Apoe ^-/- mice, accompanied by a reduction in GATA6/VCAM-1 expression in the aorta. Conclusions: HDAC1/2 contributes to VCAM-1 expression and atherosclerosis by suppressing STAT3 acetylation-dependent GATA6 promoter methylation. These findings may provide a rationale for HDAC1/2-targeting therapy in atherosclerotic heart disease.

The ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of KATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e., the principal ventricular-type KATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+ / calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-α,β-d-glucopyranose-2-)-N2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcKATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcKATP channel activity in intact cardiomyocytes, but the H2O2-induced KATP channel stimulation was obliterated when ERK1/2 or CaMKII activity was suppressed, implying that H2O2 is positioned upstream of ERK1/2 and CaMKII for K(ATP) channel modulation. Furthermore, genetic ablation (i.e., knockout) of CaMKIIδ, the predominant cardiac CaMKII isoform, diminished ventricular sarcK(ATP) channel stimulation elicited by activation of PKG, unveiling CaMKIIδ as a crucial player. Additionally, evidence from kinase activity and Western blot analyses revealed that activation of NO-PKG signalling augmented CaMKII activity in rabbit ventricular myocytes and, importantly, CaMKII activation by PKG occurred in an ERK1/2-dependent manner, placing ERK1/2 upstream of CaMKII. Taken together, these findings suggest that NO modulates ventricular sarcK(ATP) channels via a novel sGC-cGMP-PKG-ROS(H2O2)-ERK1/2-calmodulin-CaMKII (δ isoform in particular) signalling cascade, which heightens K(ATP) channel activity by destabilizing the long closed states while facilitating closed-to-open state transitions. This pathway may contribute to regulation of cardiac excitability and cytoprotection against ischaemia-reperfusion injury, in part, by opening myocardial sarcK(ATP) channels.

Aims

Small conductance Ca(2+)-activated K(+) channels (K(Ca)2 or SK channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca(2+) (Ca(i)²⁺) with membrane potentials. We have recently reported the functional expression of SK channels in human and mouse cardiac myocytes. Additionally, we have found that the channel is highly expressed in atria compared with the ventricular myocytes. We demonstrated that human cardiac myocytes expressed all three members of SK channels (SK1, 2, and 3); moreover, the different members are capable of forming heteromultimers. Here, we directly tested the contribution of SK3 to the overall repolarization of atrial action potentials.

Methods and results

We took advantage of a mouse model with site-specific insertion of a tetracycline-based genetic switch in the 5' untranslated region of the KCNN3 (SK3 channel) gene (SK3(T/T)). The gene-targeted animals overexpress the SK3 channel without interfering with the normal profile of SK3 expression. Whole-cell, patch-clamp techniques show a significant shortening of the action potential duration mainly at 90% repolarization (APD90) in atrial myocytes from the homozygous SK3(T/T) animals. Conversely, treatment with dietary doxycycline results in a significant prolongation of APD90 in atrial myocytes from SK3(T/T) animals. We further demonstrate that the shortening of APDs in SK3 overexpression mice predisposes the animals to inducible atrial arrhythmias.

Conclusion

SK3 channel contributes importantly towards atrial action potential repolarization. Our data suggest the important role of the SK3 isoform in atrial myocytes.

Clinically used inhibitors of mammalian target of rapamycin (mTOR) negatively impacts endothelial-dependent vasodilatation (EDD) through unidentified mechanisms. Here we show that either the endothelium-specific deletion of Mtor to inhibit both mTOR complexes, or depletion of Raptor or Rictor to disrupt mTORC1 or mTORC2, causes impaired EDD, accompanied by reduced NO in the serum of mice. Consistently, inhibition of mTOR decreases NO production by human and mouse EC. Specifically, inhibition of mTORC1 suppresses eNOS gene expression, due to impairment in p70S6K-mediated posttranscriptional regulation of the transcription factor KLF2 expression. In contrast to mTORC1 inhibition, a positive-feedback between MAPK (p38 and JNK) activation and Nox2 upregulation contributes to the excessive generation of reactive oxygen species (ROS), which causes eNOS uncoupling and decreased NO bioavailability in mTORC2-inhibited EC. Adeno-associated virus-mediated EC-specific overexpression of KLF2 or suppression of Nox2 restores EDD function in endothelial mTORC1- or mTORC2-inhibited mice.

Schizophrenia is a debilitating psychiatric disorder with approximately 1% lifetime risk globally. Large-scale schizophrenia genetic studies have reported primarily on European ancestry samples, potentially missing important biological insights. Here, we report the largest study to date of East Asian participants (22,778 schizophrenia cases and 35,362 controls), identifying 21 genome-wide-significant associations in 19 genetic loci. Common genetic variants that confer risk for schizophrenia have highly similar effects between East Asian and European ancestries (genetic correlation = 0.98 ± 0.03), indicating that the genetic basis of schizophrenia and its biology are broadly shared across populations. A fixed-effect meta-analysis including individuals from East Asian and European ancestries identified 208 significant associations in 176 genetic loci (53 novel). Trans-ancestry fine-mapping reduced the sets of candidate causal variants in 44 loci. Polygenic risk scores had reduced performance when transferred across ancestries, highlighting the importance of including sufficient samples of major ancestral groups to ensure their generalizability across populations.