Slack K+ channels attenuate NMDA-induced excitotoxic brain damage and neuronal cell death.

The neuronal Na+ -activated K+ channel Slack (aka Slo2.2, KNa 1.1, or Kcnt1) has been implicated in setting and maintaining the resting membrane potential and defining excitability and firing patterns, as well as in the generation of the slow afterhyperpolarization following bursts of action potentials. Slack activity increases significantly under conditions of high intracellular Na+ levels, suggesting this channel may exert important pathophysiological functions. To address these putative roles, we studied whether Slack K+ channels contribute to pathological changes and excitotoxic cell death caused by glutamatergic overstimulation of Ca2+ - and Na+ -permeable N-methyl-D-aspartic acid receptors (NMDAR). Slack-deficient (Slack KO) and wild-type (WT) mice were subjected to intrastriatal microinjections of the NMDAR agonist NMDA. NMDA-induced brain lesions were significantly increased in Slack KO vs WT mice, suggesting that the lack of Slack renders neurons particularly susceptible to excitotoxicity. Accordingly, excessive neuronal cell death was seen in Slack-deficient primary cerebellar granule cell (CGC) cultures exposed to glutamate and NMDA. Differences in neuronal survival between WT and Slack KO CGCs were largely abolished by the NMDAR antagonist MK-801, but not by NBQX, a potent and highly selective competitive antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, NMDAR-evoked Ca2+ signals did not differ with regard to Slack genotype in CGCs. However, real-time monitoring of K+ following NMDAR activation revealed a significant contribution of this channel to the intracellular drop in K+ . Finally, TrkB and TrkC neurotrophin receptor transcript levels were elevated in NMDA-exposed Slack-proficient CGCs, suggesting a mechanism by which this K+ channel contributes to the activation of the extracellular-signal-regulated kinase (Erk) pathway and thereby to neuroprotection. Combined, our findings suggest that Slack-dependent K+ signals oppose the NMDAR-mediated excitotoxic neuronal injury by promoting pro-survival signaling via the BDNF/TrkB and Erk axis.

Targets of cGMP/cGKI in Cardiac Myocytes.

The 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type I (cGKI aka PKGI) is a major cardiac effector acting downstream of nitric oxide (NO)-sensitive soluble guanylyl cyclase and natriuretic peptides (NPs), which signal through transmembrane guanylyl cyclases. Consistent with the wide distribution of the cGMP-generating guanylyl cyclases, cGKI, which usually elicits its cellular effects by direct phosphorylation of its targets, is present in multiple cardiac cell types including cardiomyocytes (CMs). Although numerous targets of cGMP/cGKI in heart were identified in the past, neither their exact patho-/physiological functions nor cell-type specific roles are clear. Herein, we inform about the current knowledge on the signal transduction downstream of CM cGKI. We believe that better insights into the specific actions of cGMP and cGKI in these cells will help to guide future studies in the search for predictive biomarkers for the response to pharmacological cGMP pathway modulation. In addition, targets downstream of cGMP/cGKI may be exploited for refined and optimized diagnostic and therapeutic strategies in different types of heart disease and their causes. Importantly, key functions of these proteins and particularly sites of regulatory phosphorylation by cGKI should, at least in principle, remain intact, although upstream signaling through the second messenger cGMP is impaired or dysregulated in a stressed or diseased heart state.

Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4-negative mouse hearts.

LIM domain proteins have been identified as essential modulators of cardiac biology and pathology; however, it is unclear which role the cysteine-rich LIM-only protein (CRP)4 plays in these processes. In studying CRP4 mutant mice, we found that their hearts developed normally, but lack of CRP4 exaggerated multiple parameters of the cardiac stress response to the neurohormone angiotensin II (Ang II). Aiming to dissect the molecular details, we found a link between CRP4 and the cardioprotective cGMP pathway, as well as a multiprotein complex comprising well-known hypertrophy-associated factors. Significant enrichment of the cysteine-rich intestinal protein (CRIP)1 in murine hearts lacking CRP4, as well as severe cardiac defects and premature death of CRIP1 and CRP4 morphant zebrafish embryos, further support the notion that depleting CRP4 is incompatible with a proper cardiac development and function. Together, amplified Ang II signaling identified CRP4 as a novel antiremodeling factor regulated, at least to some extent, by cardiac cGMP.-Straubinger, J., Boldt, K., Kuret, A., Deng, L., Krattenmacher, D., Bork, N., Desch, M., Feil, R., Feil, S., Nemer, M., Ueffing, M., Ruth, P., Just, S., Lukowski, R. Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4 negative mouse hearts.

cGMP and mitochondrial K+ channels-Compartmentalized but closely connected in cardioprotection.

The 3',5'-cGMP pathway triggers cytoprotective responses and improves cardiomyocyte survival during myocardial ischaemia and reperfusion (I/R) injury. These beneficial effects were attributed to NO-sensitive GC induced cGMP production leading to activation of cGMP-dependent protein kinase I (cGKI). cGKI in turn phosphorylates many substrates, which eventually facilitate opening of mitochondrial ATP-sensitive potassium channels (mitoKATP ) and Ca2+ -activated potassium channels of the BK type (mitoBK). Accordingly, agents activating mitoKATP or mitoBK provide protection against I/R-induced damages. Here, we provide an up-to-date summary of the infarct-limiting actions exhibited by the GC/cGMP axis and discuss how mitoKATP and mitoBK, which are present at the inner mitochondrial membrane, confer mito- and cytoprotective effects on cardiomyocytes exposed to I/R injury. In view of this, we believe that the functional connection between the cGMP cascade and mitoK+ channels should be exploited further as adjunct to reperfusion therapy in myocardial infarction. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Cysteine-Rich LIM-Only Protein 4 (CRP4) Promotes Atherogenesis in the ApoE-/- Mouse Model.

Vascular smooth muscle cells (VSMCs) can switch from their contractile state to a synthetic phenotype resulting in high migratory and proliferative capacity and driving atherosclerotic lesion formation. The cysteine-rich LIM-only protein 4 (CRP4) reportedly modulates VSM-like transcriptional signatures, which are perturbed in VSMCs undergoing phenotypic switching. Thus, we hypothesized that CRP4 contributes to adverse VSMC behaviours and thereby to atherogenesis in vivo. The atherogenic properties of CRP4 were investigated in plaque-prone apolipoprotein E (ApoE) and CRP4 double-knockout (dKO) as well as ApoE-deficient CRP4 wildtype mice. dKO mice exhibited lower plaque numbers and lesion areas as well as a reduced content of α-smooth muscle actin positive cells in the lesion area, while lesion-associated cell proliferation was elevated in vessels lacking CRP4. Reduced plaque volumes in dKO correlated with significantly less intra-plaque oxidized low-density lipoprotein (oxLDL), presumably due to upregulation of the antioxidant factor peroxiredoxin-4 (PRDX4). This study identifies CRP4 as a novel pro-atherogenic factor that facilitates plaque oxLDL deposition and identifies the invasion of atherosclerotic lesions by VSMCs as important determinants of plaque vulnerability. Thus, targeting of VSMC CRP4 should be considered in plaque-stabilizing pharmacological strategies.

Tissue- and isoform-specific protein complex analysis with natively processed bait proteins.

Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. To date, the analysis of tissue- or even cell- or compartment-specific protein interactions is still relying on the availability of specific antibodies suited for immunoprecipitation. Here, we aimed at establishing a method that allows identification of protein interactions and complexes from intact tissues independent of specific, high affinity antibodies used for protein pull-down and isolation. Tagged bait proteins were expressed in human HEK293T cells and residual interactors removed by SDS. The resulting tag-fusion protein was then used as bait to pull proteins from tissue samples. Tissue-specific interactions were reproducibly identified from porcine retina as well as from retinal pigment epithelium using the ciliary protein lebercilin as bait. Further, murine heart-specific interactors of two gene products of the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 (cGK1) were investigated. Here, specific interactions were associated with the cGK1α and ß gene products, that differ only in their unique amino-terminal region comprising about 100 aa. As such, the new protocol provides a fast and reliable method for tissue-specific protein complex analysis which is independent of the availability or suitability of antibodies for immunoprecipitation. SIGNIFICANCE: Protein-protein interaction in the functional relevant tissue is still difficult due to the dependence on specific antibodies or bait production in bacteria or insect cells. Here, the tagged protein of interest is produced in a human cell line and bound proteins are gently removed using SDS. Because applying the suitable SDS concentration is a critical step, different SDS solutions were tested to demonstrate their influence on interactions and the clean-up process. The established protocol enabled a tissue-specific analysis of the ciliary proteins lebercilin and TMEM107 using pig eyes. In addition, two gene products of the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 showed distinct protein interactions in mouse heart tissue. With the easy, fast and cheap protocol presented here, deep insights in tissue-specific and functional relevant protein complex formation is possible.

Rise of cGMP by partial phosphodiesterase-3A degradation enhances cardioprotection during hypoxia.

3',5'-cyclic guanosine monophosphate (cGMP) is a druggable second messenger regulating cell growth and survival in a plethora of cells and disease states, many of which are associated with hypoxia. For example, in myocardial infarction and heart failure (HF), clinical use of cGMP-elevating drugs improves disease outcomes. Although they protect mice from ischemia/reperfusion (I/R) injury, the exact mechanism how cardiac cGMP signaling is regulated in response to hypoxia is still largely unknown. By monitoring real-time cGMP dynamics in murine and human cardiomyocytes using in vitro and in vivo models of hypoxia/reoxygenation (H/R) and I/R injury combined with biochemical methods, we show that hypoxia causes rapid but partial degradation of cGMP-hydrolyzing phosphodiesterase-3A (PDE3A) protein via the autophagosomal-lysosomal pathway. While increasing cGMP in hypoxia prevents cell death, partially reduced PDE3A does not change the pro-apoptotic second messenger 3',5'-cyclic adenosine monophosphate (cAMP). However, it leads to significantly enhanced protective effects of clinically relevant activators of nitric oxide-sensitive guanylyl cyclase (NO-GC). Collectively, our mouse and human data unravel a new mechanism by which cardiac cGMP improves hypoxia-associated disease conditions.

Cardioprotection by ischemic postconditioning and cyclic guanosine monophosphate-elevating agents involves cardiomyocyte nitric oxide-sensitive guanylyl cyclase.

Aims: It has been suggested that the nitric oxide-sensitive guanylyl cyclase (NO-GC)/cyclic guanosine monophosphate (cGMP)-dependent signalling pathway affords protection against cardiac damage during acute myocardial infarction (AMI). It is, however, not clear whether the NO-GC/cGMP system confers its favourable effects through a mechanism located in cardiomyocytes (CMs). The aim of this study was to evaluate the infarct-limiting effects of the endogenous NO-GC in CMs in vivo. Methods and results: Ischemia/reperfusion (I/R) injury was evaluated in mice with a CM-specific deletion of NO-GC (CM NO-GC KO) and in control siblings (CM NO-GC CTR) subjected to an in vivo model of AMI. Lack of CM NO-GC resulted in a mild increase in blood pressure but did not affect basal infarct sizes after I/R. Ischemic postconditioning (iPost), administration of the phosphodiesterase-5 inhibitors sildenafil and tadalafil as well as the NO-GC activator cinaciguat significantly reduced the amount of infarction in control mice but not in CM NO-GC KO littermates. Interestingly, NS11021, an opener of the large-conductance and Ca2+-activated potassium channel (BK), an important downstream effector of cGMP/cGKI in the cardiovascular system, protects I/R-exposed hearts of CM NO-GC proficient and deficient mice. Conclusions: These findings demonstrate an important role of CM NO-GC for the cardioprotective signalling following AMI in vivo. CM NO-GC function is essential for the beneficial effects on infarct size elicited by iPost and pharmacological elevation of cGMP; however, lack of CM NO-GC does not seem to disrupt the cardioprotection mediated by the BK opener NS11021.