Destabilization of the cytosolic calcium level and the death of cardiomyocytes in the presence of derivatives of long-chain fatty acids

By means of fluorescent microscopy, long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, were shown to exert the most toxic effect on rat ventricular cardiomyocytes. The addition of 20–50 μM acylcarnitines increased calcium concentration in cytoplasm ([Ca²⁺]_i) and caused cell death after a lag-period of 4–8 min. This effect was independent of extracellular calcium level and Ca²⁺ inhibitors of L-type channels. Free myristic and palmitic acids at concentrations of 300–500 μM had little effect on [Ca²⁺]_i within 30 min. We suggest that the toxic effect is due to the activation of calcium channels of sarcoplasmic reticulum by acylcarnitines and/or arising acyl-CoA. Mitochondria play a role of calcium-buffer system under these conditions. The calcium capacity of the buffer determines the duration of the lag-period. Phosphate increases the calcium capacity of mitochondria and the lag-period. In the presence of rotenone and oligomycin, the elevation of [Ca²⁺]_i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondria leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.     

Regulation of potential-dependent L-type Ca2+ currents by agmatine. Imidazoline receptors in isolated cardiomyocytes

The main goal of the present work was to study the mechanisms of voltage-gated L-type Ca²⁺ currents regulation by agmatine in isolated cardiomyocytes and to determine whether agmatine is involved in mediating the “arginine paradox”. It was shown that agmatine at concentrations from 200 μM to 15 mM inhibited L-type Ca²⁺ currents in isolated cardiomyocytes in a dose-dependent manner. The selective antagonists of α₂-adrenoceptors (α₂-ARs), yohimbine and rauwolscine, did not modulate the effect of agmatine. In contrast, efaroxan and idazoxan known to antagonize both α₂-ARs and type 1 imidazoline receptors (I₁Rs) decreased the efficiency of agmatine almost twofold. The NO synthase inhibitor 7NI insignificantly influenced the suppressive action of agmatine on L-type Ca²⁺ currents, whereas the protein kinase C inhibitor, calphostin C, markedly reduced the effects of agmatine. Arginine did not affect L-type Ca²⁺ currents in the presence of agmatine and vice versa. These data suggest that agmatine is not involved in mediating the “arginine paradox” and that its effects are not due to the activation of endothelial NO synthase (eNOS) followed by cGMP-dependent inhibition of L-type Ca²⁺ current. Most likely, agmatine acts via I₁Rs coupled with the signaling pathway that involves the activation of protein kinase C. Previously nothing was known about possible localization of I₁Rs in isolated cardiomyocytes. Consistently, we have shown that single cardiomyocytes express the nischarin genes homologous to the IRAS gene, which is considered in the modern literature as the major candidate for the gene encoding I₁Rs. To the best our knowledge, this is the first demonstration of I₁Rs expression at the level of individual cells, including cardiomyocytes.     

Identifying a Kinase Network Regulating FGF14:Nav1.6 Complex Assembly Using Split-Luciferase Complementation

Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and diseased brain.     

Modulation of L-type Ca2+ currents and intracellular calcium by agmatine in rat cardiomyocytes

It is shown that agmatine inhibits L-type Ca²⁺ currents in isolated cardiomyocytes of rats in a dose-dependent manner. The inhibitory analysis indicates that imidazoline receptors of type I (I₁Rs) rather than α₂-adrenoceptors (α₂-ARs) are implicated in mediating the effects of agmatine. Agmatine affects the dynamics of intracellular Ca²⁺ concentration changes in spontaneously active cardiomyocytes. The averaged intracellular Ca²⁺ concentration ([Ca²⁺]_in) varied biphasically, depending on the agmatine dose: at 1–500 μM, agmatine decreased [Ca²⁺]_in; at 500 μM-2 mM, [Ca²⁺]_in remained unchanged, and at concentrations above 2 mM agmatine caused an increase of [Ca²⁺]_in. The effects of low agmatine concentrations were inhibited by 7NI, an inhibitor of NO synthases (NOS), as well as by the inhibitors of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) thapsigargin and cyclopiazonic acid. In contrast, ODQ, a blocker of NO-sensitive guanylate cyclase, and the antagonist of I₁Rs efaroxan were ineffective. At low concentrations agmatine did not affect the increase in [Ca²⁺]_in induced by stimulating doses of ryanodine (40 nM). In addition, agmatine at low doses was found to markedly stimulate NO production. When efaroxan (10 μM) or ryanodine (200 μM) were added to the bath to inhibit I₁Rs and ryanodine receptors (RyRs), respectively, [Ca²⁺]_in became much less sensitive to millimolar agmatine. In contrast to low concentrations (100 μM), high agmatine doses (10–15 mM) did not stimulate the NO synthesis but were effective as NOS inducer in cells pretreated with efaroxan. The selective I₁R agonist rilmenidine increased [Ca²⁺]_in in a dose-dependent manner. The effect of rilmenidine was similar to that of agmatine at high doses and was abolished by RyRs inhibition. Our findings indicate that in spontaneously active cardiomyocytes agmatine at low concentrations decreases [Ca²⁺]_in, does not stimulate I₁Rs but most likely enhances NO synthase followed by an increase in SERCA activity due to the direct nitrosylation of SERCA and/or phospholamban. The effects of high agmatine doses are apparently mediated by I₁Rs and involve RyRs.     

“Arginine paradox” in cardiomyocytes of Sprague Dawley and spontaneously hypertensive rats: α<Subscript>2</Subscript>-adrenoreceptor-mediated regulation of L-type Ca<Superscript>2+</Superscript> currents by <Emphasis Type="Italic">L</Emphasis>-arginine

The “arginine paradox” in cardiomyocytes isolated from the left ventricle of Spraque Dawlay (SD) and spontaneously hypertensive rats (SHR) was studied. With 1 mM L-arginine in the bath, the addition of 5 mM L-arginine to incubation medium increased NO production and inhibited amplitude of L-type Ca²⁺ currents in SD cardiomyocytes. A variety of compounds, including the antagonist of α₂-adrenoceptors yohimbine and inhibitors of PI3 kinase (wortmanine), NO synthase (7NI), and cGMP-dependent protein kinase (KT5823), dramatically weakened the inhibitory effects of 5 mM L-arginine on Ca²⁺ currents. The agonist of α₂-adrenoceptors guanabenz acetate increased NO production and inhibited Ca²⁺ currents, while wortmanine, 7NI, and KT5823 antagonized guanabenz. In SHR cardiomyocytes, the “arginine paradox” was not observed: 5 mM L-arginine affected neither NO production nor Ca²⁺ currents. Consistently, guanabenz acetate did not alter NO production and inhibited Ca²⁺ currents to a much smaller extent in SHR cardiomyocytes as compared to SD cardiomyocytes. Taken together, the data of the inhibitory analysis suggest that millimolar L-arginine serves as an agonist of α₂-adrenoceptors, which are coupled to PI3K-Akt pathway as well as downstream NO-cGMP pathway to control activity of L-type Ca²⁺ channels, thus providing new insights into the “arginine paradox” in cardiomyocytes.     

Role of the NO-cGMP cascade in regulation of L-type Ca2+ currents in isolated cardiomyocytes

Regulatory mechanisms of voltage-dependent L-type Ca²⁺ channels involving the cyclic nucleotide system of mammalian cardiomyocytes have been studied. Activation of cGMP-dependent phosphorylation in the presence of 1 mM arginine in all experimental media resulted in inhibition of amplitudes of basal L-type Ca²⁺ currents in rat ventricular myocytes. Effects of compounds regulating the activity of different compoments of the NO-cGMP cascade on L-type Ca²⁺ currents were investigated. It was found that endogenous (arginine, 5 mM) and exogenous (sodium nitroprusside, 1 mM) NO sources decreased the Ca²⁺ current amplitude by 30 ± 10%. The nonspecific NO synthase blocker 7NI (2 μM) abolished the effect of arginine, while the soluble guanylyl cyclase blocker ODQ (50 μM) eliminated the effects of both arginine and sodium nitroprusside. The fact that inhibitory effects of arginine, sodium nitroprusside and 8Br-cGMP disappeared in the presence of the protein kinase G blocker KT5823 (0.5, 1 μM) provides direct evidence in favor of activating effect of these compounds on PKG-dependent phosphorylation. Inhibition of L-type Ca²⁺ currents can also be due to activation of phosphodiesterase II. However, the selective phosphodiesterase II blocker EHNA (30 μM) failed to abolish inhibitory effects of arginine and sodium nitroprusside on Ca²⁺ currents. Isoproterenol (0.1 μM)-activated L-type Ca²⁺ currents were only partly blocked by acetylcholine (0.1 mM). Contrary to basal currents, the NO-cGMP cascade agonists arginine and sodium nitroprusside (SNP), like 8Br-cGMP, had no effect on isoproterenol-induced currents. Full inhibition of isoproterenol-induced currents was achieved through combination of acetylcholine with NO-cGMP cascade agonists.     

The Fibroblast Growth Factor 14·Voltage-gated Sodium Channel Complex Is a New Target of Glycogen Synthase Kinase 3 (GSK3)

The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na⁺ (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na⁺ currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions.     

Extra virgin olive oil improves synaptic activity,short‐term plasticity,memory, and neuropathology in a tauopathy model

In recent years, increasing evidence has accumulated supporting the health benefits of extra virgin olive oil (EVOO). Previous studies showed that EVOO supplementation improves Alzheimer's disease (AD)‐like amyloidotic phenotype of transgenic mice. However, while much attention has been focused on EVOO‐mediated modulation of Aβ processing, its direct influence on tau metabolism in vivo and synaptic function is still poorly characterized. In this study, we investigated the effect of chronic supplementation of EVOO on the phenotype of a relevant mouse model of tauopathy, human transgenic tau mice (hTau). Starting at 6 months of age, hTau mice were fed chow diet supplemented with EVOO or vehicle for additional 6 months, and then the effect on their phenotype was assessed. At the end of the treatment, compared with control mice receiving EVOO displayed improved memory and cognition which was associated with increased basal synaptic activity and short‐term plasticity. This effect was accompanied by an upregulation of complexin 1, a key presynaptic protein. Moreover, EVOO treatment resulted in a significant reduction of tau oligomers and phosphorylated tau at specific epitopes. Our findings demonstrate that EVOO directly improves synaptic activity, short‐term plasticity, and memory while decreasing tau neuropathology in the hTau mice. These results strengthen the healthy benefits of EVOO and further support the therapeutic potential of this natural product not only for AD but also for primary tauopathies.     

Quantitative Proteomics Reveals Protein–Protein Interactions with Fibroblast Growth Factor 12 as a Component of the Voltage-Gated Sodium Channel 1.2 (Nav1.2) Macromolecular Complex in Mammalian Brain

Voltage-gated sodium channels (Nav1.1–Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein–protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.Voltage-gated sodium channels (Nav)¹ are transmembrane proteins consisting of a pore-forming α subunit (Nav1.1-Nav1.9) and one or more accessory β-subunits (β₁–β₄) (1–3). Predominately clustered at the axonal initial segment (AIS), the α subunit alone is necessary and sufficient for channel assembly and the initiation and propagation of action potentials following membrane depolarization (4). Although the α subunit is functional on its own, it is the transient and stable protein–protein interactions that modulate subcellular trafficking, compartmentalization, functional expression, and fine-tune the channel biophysical properties (5–9). Thus, the Nav channel and the protein constituents that comprise the protein–protein interaction network are all part of a macromolecular complex that modulates the spatiotemporal dynamics of neuronal input and output playing a critical role in synaptic transmission, signal integration, and neuronal plasticity. Perturbations in this protein–protein interaction network can lead to deficits in neuronal excitability, and eventually neurodegeneration and cell death (10–15).Given the relevance of these interactions for the native channel activity and its overall role in controlling brain circuits, it is increasingly important to uncover these associations. Antibody-based affinity purification (AP) combined with mass spectrometry (MS) is widely used for the enrichment and analysis of target proteins and constituents of their protein–protein interactions as it can be performed at near physiological conditions and preserves post-translational modifications relevant to protein complex organization (16–19). Differential mass spectrometry provides an unbiased method for the efficient, MS-based measurement of relative protein fold changes across multiple complex biological samples. This technology has been successfully applied to a number of ion channels (20–26), but—to the best of our knowledge—not to the study of any member of the Nav channel family. Using a target-directed AP approach combined with quantitative MS, we identified proteins constituting the putative interactome of Nav1.2, one of three dominant Nav channel isoforms in the mammalian brain, from native tissue (1, 2, 4, 8). Among these putative interactors, the fibroblast growth factor 12 (FGF12), a member of the intracellular FGF family (5, 13, 14), stood out as one of the most abundant coprecipitating proteins with ∼30-fold enrichment over other interactors. With a combination of confocal microscopy in brain tissue, reconstitution of the interactor in a heterologous systems and electrophysiological assays, we provide validation for FGF12 as a bona fide relevant component of the Nav1.2 proteome and a modulator of Nav1.2-encoded currents. Altogether, the identified channel/protein interaction between FGF12 and Nav1.2 provides new insights for structural and functional interpretation of neuronal excitability, synaptic transmission, and plasticity in the normal and diseased brain.