Search

Long-term potentiation (LTP) and long-term depression (LTD) are key forms of synaptic plasticity in the hippocampus. LTP and LTD are believed to underlie the processes occurring during learning and memory. Search of mechanisms responsible for switching from LTP to LTD and vice versa is an important fundamental task. Protein synthesis blockers (PSB) are widely used in models of memory impairment and LTP suppression. Here, we found that blockade of serine/threonine phosphatases 1 (PP1) and 2A (PP2A) with the specific blockers, calyculin A (CalyA) or okadaic acid (OA), and simultaneous blockade of the protein translation by anisomycin or cycloheximide leads to a switch from PSB-impaired LTP to LTD. PP1/PP2A-dependent LTD was extremely sensitive to the intensity of the test stimuli, whose increase restored the field excitatory postsynaptic potentials (fEPSP) to the values corresponding to control LTP in the non-treated slices. PP1/PP2A blockade affected the basal synaptic transmission, increasing the paired-pulse facilitation (PPF) ratio, and restored the PSB-impaired PPF 3 h after tetanus. Prolonged exposure to anisomycin led to the NO synthesis increase (measured using fluorescent dye) both in the dendrites and somata of CA1, CA3, dentate gyrus (DG) hippocampal layers. OA partially prevented the NO production in the CA1 dendrites, as well in the CA3 and DG somas. Direct measurements of changes in serine/threonine phosphatase (STPP) activity revealed importance of the PP1/PP2A-dependent component in the late LTP phase (L-LTP) in anisomycin-treated slices. Thus, serine/threonine phosphatases PP1/PP2A influence both basal synaptic transmission and stimulation-induced synaptic plasticity.

The search for strategies for strengthening the synaptic efficiency in Aβ

Alzheimer’s disease (AD) is the most common cause of senile dementia and one of the greatest medical, social, and economic challenges. According to a dominant theory, amyloid-β (Aβ) peptide is a key AD pathogenic factor. Aβ-soluble species interfere with synaptic functions, aggregate gradually, form plaques, and trigger neurodegeneration. The AD-associated pathology affects numerous systems, though the substantial loss of cholinergic neurons and α7 nicotinic receptors (α7AChR) is critical for the gradual cognitive decline. Aβ binds to α7AChR under various experimental settings; nevertheless, the functional significance of this interaction is ambiguous. Whereas the capability of low Aβ concentrations to activate α7AChR is functionally beneficial, extensive brain exposure to high Aβ concentrations diminishes α7AChR activity, contributes to the cholinergic deficits that characterize AD. Aβ and snake α-neurotoxins competitively bind to α7AChR. Accordingly, we designed a chemically modified α-cobratoxin (mToxin) to inhibit the interaction between Aβ and α7AChR. Subsequently, we examined mToxin in a set of original in silico, in vitro, ex vivo experiments, and in a murine AD model. We report that mToxin reversibly inhibits α7AChR, though it attenuates Aβ-induced synaptic transmission abnormalities, and upregulates pathways supporting long-term potentiation and reducing apoptosis. Remarkably, mToxin demonstrates no toxicity in brain slices and mice. Moreover, its chronic intracerebroventricular administration improves memory in AD-model animals. Our results point to unique mToxin neuroprotective properties, which might be tailored for the treatment of AD. Our methodology bridges the gaps in understanding Aβ-α7AChR interaction and represents a promising direction for further investigations and clinical development. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-020-02270-0.

The sinoatrial node (SAN) of the heart produces rhythmic action potentials, generated via calcium signaling within and among pacemaker cells. Our previous work has described the SAN as composed of a hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)-expressing pacemaker cell meshwork, which merges with a network of connexin 43This study sought to describe the 3-dimensional cytoarchitecture of the mouse SAN, including autonomic innervation, peripheral glial cells, and pacemaker cells.The cytoarchitecture of SAN whole-mount preparations was examined by three-dimensional confocal laser-scanning microscopy of triple immunolabeled with combinations of antibodies for HCN4, S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), choline acetyltransferase, or vesicular acetylcholine transporter, and tyrosine hydroxylase, and transmission electron microscopy.The SAN exhibited heterogeneous autonomic innervation, which was accompanied by a web of peripheral glial cells and a novel S100BThe autonomic plexus, peripheral glial cell web, and a novel S100B

Imidazoline receptor of the first type (I1R) in addition to the established inhibition of sympathetic neurons may mediate the direct control of myocellular functions. Earlier, we revealed that I1-mediated signaling in the normotensive rat cardiomyocytes suppresses the nitric oxide production by endothelial NO synthase, impairs sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity, and elevates intracellular calcium in the cytosol. Also, I1-agonists counteract β-adrenoceptor stimulation effects in respect to voltage-gated calcium currents. This study ascertains the I1R signal transduction in the normotensive Wistar and SHR cardiomyocytes. Reduction of Ca2+-currents by rilmenidine, a specific agonist of I1R, ensued from the phosphatidylcholine-specific phospholipase C-mediated activation of protein kinase C. There is a stimulation of serine/threonine phosphatase activity. In SHR cardiomyocytes, both the rilmenidine, and putative endogenous ligand, agmatine, almost twofold less effectively reduced L-type of Ca2+-currents. Average mRNA level of Nischarin, established functional component of I1R, is slightly decreased in SHR, as well as the intracellular Nischarin pool immunolabeled in the cytosol of SHR cardiomyocytes. Disturbance of I1R signal transduction in SHR may aggravate the development of this cardiovascular pathology.

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is an imaging approach that enables analysis of the 3D architecture of cells and tissues at resolutions that are 1–2 orders of magnitude higher than that possible with light microscopy. The slow speeds of data collection and manual segmentation are two critical problems that limit the more extensive use of FIB-SEM technology. Here, we present an easily accessible robust method that enables rapid, large-scale acquisition of data from tissue specimens, combined with an approach for semi-automated data segmentation using the open-source machine learning Weka segmentation software, which dramatically increases the speed of image analysis. We demonstrate the feasibility of these methods through the 3D analysis of human muscle tissue by showing that our process results in an improvement in speed of up to three orders of magnitude as compared to manual approaches for data segmentation. All programs and scripts we use are open source and are immediately available for use by others.

Recently identified imidazoline receptors of the first type (I1Rs) on the cardiomyocyte's sarcolemma open a new field in calcium signaling research. In particular, it is interesting to investigate their functional interaction with other well-known systems, such as β-adrenergic receptors. Here we investigated the effects of I1Rs activation on L-type voltage-gated Ca2+-currents under catecholaminergic stress induced by the application of β-agonist, isoproterenol. Pharmacological agonist of I1Rs (I1-agonist), rilmenidine, and the putative endogenous I1-ligand, agmatine, have been shown to effectively reduce Ca2+-currents potentiated by isoproterenol. Inhibitory analysis shows that the ability to suppress voltage-gated Ca2+-currents by rilmenidine and agmatine is fully preserved in the presence of the protein kinase A blocker (PKA), which indicates a PKA-independent mechanism of their action. The blockade of NO synthase isoforms with 7NI does not affect the intrinsic effects of agmatine and rilmenidine, which suggests NO-independent signaling pathways triggered by I1Rs. A nonspecific serine/threonine protein phosphatase (STPP) inhibitor, calyculin A, abrogates effects of rilmenidine or agmatine on the isoproterenol-induced Ca2+-currents. Direct measurements of phosphatase activity in the myocardial tissues showed that activation of the I1Rs leads to stimulation of STPP, which could be responsible for the I1-agonist influences. Obtained data clarify peripheral effects that occur during activation of the I1Rs under endogenous catecholaminergic stress, and can be used in clinical practice for more precise control of heart contractility in some cardiovascular pathologies.

For several decades, the ability of protein synthesis inhibitors (PSI) to suppress the long-term potentiation (LTP) of hippocampal responses is known. It is considered that mechanisms of such impairment are related to a cessation of translation and a delayed depletion of the protein pool required for maintenance of synaptic plasticity. The present study demonstrates that cycloheximide or anisomycin applications reduce amplitudes of the field excitatory postsynaptic potentials as well as the presynaptically mediated form of plasticity, the paired-pulse facilitation after LTP induction in neurons of the CA1 area of hippocampus. We showed that nitric oxide signaling could be one of the pathways that cause the LTP decrease induced by cycloheximide or anisomycin. Inhibitor of the NO synthase, L-NNA or the NO scavenger, PTIO, rescued the late-phase LTP and restored the paired-pulse facilitation up to the control levels. For the first time we have directly measured the nitric oxide production induced by application of the translation blockers in hippocampal neurons using the NO-sensitive dye DAF-FM. Inhibitory analysis demonstrated that changes during protein synthesis blockade downstream the NO signaling cascade are cGMP-independent and apparently are implemented through degradation of target proteins. Prolonged application of the NO donor SNAP impaired the LTP maintenance in the same manner as PSI.

The search for strategies for strengthening the synaptic efficiency in Aβ25-35-treated slices is a challenge for the compensation of amyloidosis-related pathologies. Here, we used the recording of field excitatory postsynaptic potentials (fEPSPs), nitric oxide (NO) imaging, measurements of serine/threonine protein phosphatase (STPP) activity, and the detection of the functional mitochondrial parameters in suspension of brain mitochondria to study the Aβ25-35-associated signaling in the hippocampus. Aβ25-35 aggregates shifted the kinase–phosphatase balance during the long-term potentiation (LTP) induction in the enhancement of STPP activity. The PP1/PP2A inhibitor, okadaic acid, but not the PP2B blocker, cyclosporin A, prevented Aβ25-35-dependent LTP suppression for both simultaneous and delayed enzyme blockade protocols. STPP activity in the Aβ25-35-treated slices was upregulated, which is reverted relative to the control values in the presence of PP1/PP2A but not in the presence of the PP2B blocker. A selective inhibitor of stress-induced PP1α, sephin1, but not of the PP2A blocker, cantharidin, is crucial for Aβ25-35-mediated LTP suppression prevention. A mitochondrial Na+/Ca2+ exchanger (mNCX) blocker, CGP37157, also attenuated the Aβ25-35-induced LTP decline. Aβ25-35 aggregates did not change the mitochondrial transmembrane potential or reactive oxygen species (ROS) production but affected the ion transport and Ca2+-dependent swelling of organelles. The staining of hippocampal slices with NO-sensitive fluorescence dye, DAF-FM, showed stimulation of the NO production in the Aβ25-35-pretreated slices at the dendrite-containing regions of CA1 and CA3, in the dentate gyrus (DG), and in the CA1/DG somata. NO scavenger, PTIO, or nNOS blockade by selective inhibitor 3Br-7NI partly restored the Aβ25-35-induced LTP decline. Thus, hippocampal NO production could be another marker for the impairment of synaptic plasticity in amyloidosis-related states, and kinase–phosphatase balance management could be a promising strategy for the compensation of Aβ25-35-driven deteriorations.

Protein kinase C is the superfamily of intracellular effector molecules which control crucial cellular functions. Here, we for the first time did the percentage estimation of all known PKC and PKC-related isozymes at the individual cadiomyocyte level. Broad spectrum of PKC transcripts is expressed in the left ventricular myocytes. In addition to the well-known ‘heart-specific’ PKCα, cardiomyocytes have the high expression levels of PKCN1, PKCδ, PKCD2, PKCe. In general, we detected all PKC isoforms excluding PKCη. In cardiomyocytes PKC activity tonically regulates voltage-gated Ca2+-currents, intracellular Ca2+ level and nitric oxide (NO) production. Imidazoline receptor of the first type (I1R)-mediated induction of the PKC activity positively modulates Ca2+ release through ryanodine receptor (RyR), increasing the Ca2+ leakage in the cytosol. In cardiomyocytes with the Ca2+-overloaded regions of > 9–10 μm size, the local PKC-induced Ca2+ signaling is transformed to global accompanied by spontaneous Ca2+ waves propagation across the entire cell perimeter. Such switching of Ca2+ signaling in cardiac cells can be important for the development of several cardiovascular pathologies and/or myocardial plasticity at the cardiomyocyte level.

True hibernators are remarkable group of mammals whose hearts are resistant to such stressors as deep hypothermia, ischemia, arrhythmia. Capability of cardiac cells from hibernating species to effectively rule Ca2+ homeostasis during torpor is poorly studied. Better understanding of these mechanisms could allow to introduce new strategies for improvement the cardiac performance and may be useful for cardiovascular medicine. Here for the first time we have shown that the regulation of Ca2+ handling and thereby cardiomyocyte contractility by endogenous neurotransmitter agmatine occurs through the modulation of calcium-sensing receptor (CaSR). In isolated cardiocytes of hibernating ground squirrels generating stationary Ca2+ transients in the absence of actual myocellular excitation, low doses of this polyamine (up to 500 μM) induce the Gβγ-dependent activation of PI3-kinase with subsequent stimulation of Akt-kinase and nitric oxide (NO) production by endothelial NO-synthase (eNOS). NO production abolishes Ca2+ oscillations in virtue of the enhancement of Ca2+ reuptake by sarco(endo)plasmic Ca2+ ATPase (SERCA). Simultaneously, the activation of phospholipase A2 (PLA2) and arachidonic-acid dependent Ca2+ entry occur providing replenishment of Ca2+ store. High concentrations of agmatine (> 2 mM) induce other CaSR-mediated pathways involving phospholipase C (PLC) pathway, the formation of inositoltriphosphate (IP3) and diacylglicerol (DAG) followed by induction of their targets: IP3 receptors and protein kinase C isoforms (PKC), respectively. Furthermore, it is also responsible for the stimulation of PLA2 and elevation of intracellular calcium caused by arachidonic acid-regulated Ca2+-permeable (ARC) channels. Additionally, there is a potent store-operated Ca2+ entry (SOC) in cardiomyocyte. Negative (NPS 2143) and positive (R 568) allosteric modulators of CaSR recapitulate effects of low and high agmatine doses on Ca2+ handling and NO synthesis. These facts and the alteration of agmatine influence in response to an increase of extracellular Ca2+, which is the direct agonist of CaSR, may confirm the participation of CaSR in regulation of Ca2+ handling and excitability of cardiomyocytes by agmatine.

Current understanding of how cardiac pacemaker cells operate is based mainly on studies in isolated single sinoatrial node cells (SANC), specifically those that rhythmically fire action potentials similar to the in vivo behavior of the intact sinoatrial node. However, only a small fraction of SANC exhibit rhythmic firing after isolation. Other SANC behaviors have not been studied. Here, for the first time, we studied all single cells isolated from the sinoatrial node of the guinea pig, including traditionally studied rhythmically firing cells (‘rhythmic SANC’), dysrhythmically firing cells (‘dysrhythmic SANC’) and cells without any apparent spontaneous firing activity (‘dormant SANC’). Action potential-induced cytosolic Ca(2+) transients and spontaneous local Ca(2+) releases (LCRs) were measured with a 2D camera. LCRs were present not only in rhythmically firing SANC, but also in dormant and dysrhythmic SANC. While rhythmic SANC were characterized by large LCRs synchronized in space and time towards late diastole, dys-rhythmic and dormant SANC exhibited smaller LCRs that appeared stochastically and were widely distributed in time. β-adrenergic receptor (βAR) stimulation increased LCR size and synchronized LCR occurrences in all dysrhythmic and a third of dormant cells (25 of 75 cells tested). In response to βAR stimulation, these dormant SANC developed automaticity, and LCRs became coupled to spontaneous action potential-induced cytosolic Ca(2+) transients. Conversely, dormant SANC that did not develop automaticity showed no significant change in average LCR characteristics. The majority of dysrhythmic cells became rhythmic in response to βAR stimulation, with the rate of action potential-induced cytosolic Ca(2+) transients substantially increasing. In summary, isolated SANC can be broadly categorized into three major populations: dormant, dysrhythmic, and rhythmic. We interpret our results based on simulations of a numerical model of SANC operating as a coupled-clock system. On this basis, the two previously unstudied dysrhythmic and dormant cell populations have intrinsically partially or completely uncoupled clocks. Such cells can be recruited to fire rhythmically in response to βAR stimulation via increased rhythmic LCR activity and ameliorated coupling between the Ca(2+) and membrane clocks.

Arginine is one of the most versatile semi-essential amino acids. Further to the primary role in protein biosynthesis, arginine is involved in the urea cycle, and it is a precursor of nitric oxide. Arginine deficiency is associated with neurodegenerative diseases such as Parkinson’s, Huntington’s and Alzheimer’s diseases (AD). In this study, we administer arginine intracerebroventricularly in a murine model of AD and evaluate cognitive functions in a set of behavioral tests. In addition, the effect of arginine on synaptic plasticity was tested electrophysiologically by assessment of the hippocampal long-term potentiation (LTP). The effect of arginine on β amyloidosis was tested immunohistochemically. A role of arginine in the prevention of cytotoxicity and apoptosis was evaluated in vitro on PC-12 cells. The results indicate that intracerebroventricular administration of arginine improves spatial memory acquisition in 3xTg-AD mice, however, without significantly reducing intraneuronal β amyloidosis. Arginine shows little or no impact on LTP and does not rescue LTP deterioration induced by Aβ. Nevertheless, arginine possesses neuroprotective and antiapoptotic properties.

Poly-arginine peptides are a promising class of bioactive compounds that are capable of crossing the blood-brain barrier (BBB) and present neuroprotective properties. In this study, we test the activity of poly-arginine peptides in a triple-transgenic mouse model of Alzheimer’s disease. To identify the best candidate, we examined the relative neuroprotective efficacy of the compounds with various lengths (R7, R9, and R11) via assessment of memory acquisition, long-term hippocampal potentiation (LTP), and cytotoxicity. Also, we explored the expression profiles of hundreds of key cell signaling proteins, and perform a high content antibody microarray comparative analysis of brain samples. The chronically treated animals with poly-arginine R9 show significantly improved acquisition of memory. This compound rescues hippocampal LTP deteriorated by Aβ at a better rate than other agents tested in this study and induces cellular pathways involved in neuroprotection and neuroplasticity. The treatment escalates the expression levels of Synapsin Ia in the mice hippocampi; however, it has no significant effect upon the rate of beta-amyloidosis. Poly-arginine R9 peptide is a well-tolerated compound that crosses the BBB and presents unique neuroprotective qualities. The substance halters the development of AD symptoms in a murine model and can be recommended for clinical investigation.

Spontaneous Ca

Aims to investigate α2-AR subtype distribution and the relationship between receptor amounts and their functionality in normotensive and spontaneously hypertensive rats. Methods experiments were performed on left ventricular cardiomyocytes isolated from Wistar rats and SHR (2–2.5 months). Molecular routine tools (RT-PCR, Western blotting, immunocytochemistry) were used for semi-quantitative estimation of α2-AR subtypes. Fluorescence of both the Ca2+-dependent and NO-sensitive probes were used to define functionality of α2-AR, evaluated by changes in the dynamics of spontaneous Ca2+-transients and NO production in cardiomyocytes in response to the α2-AR agonist application. Results percentage of the three known α2-AR subtypes in Wistar and SHR cardiomyocytes is not principally different. Total amounts of α2A-AR subtype in SHR increases, for both the sarcolemmal and intracellular receptor pools. Total number of α2B-AR is also significantly higher in hypertensive rats with an increase in the sarcolemmal, but not the intracellular immunoreactivity. For α2C-AR subtype, no significant differences between Wistar and SHR were identified, despite the fact that its amounts in cardiomyocytes are somewhat higher than the other two subtypes. Notwithstanding the increased expression of α2-AR subtypes in SHR, α2-AR-agonist guanabenz was ineffective in suppression of spontaneous Ca2+-transients, as well as the lowering of free calcium levels in the cytosol. Guanabenz-induced NO synthesis is well correlated with the Ca2+-loading into sarcoplasmic reticulum and actually decreased in SHR cardiomyocytes. Conclusion data indicate α2-AR dysfunction and ineffectiveness of α2-AR-mediated signaling pathways in this model of cardiovascular pathologies. Results can be used for clinical practice for more effective control of cardiovascular functions in various disease states.

Imidazoline receptor of the first type (I

α2-Adrenoceptors (α2-AR) found in the cardiomyocyte's sarcolemma represent a very important negative feedback for control of myocardial contractility by endogenous catecholamines. Earlier, we showed that the endogenous neurotransmitter agmatine in micromolar concentrations via α2-AR activates the nitric oxide (NO) synthesis, enhancing the Ca2+ pumping into sarcoplasmic reticulum (SR). In the millimolar doses it inhibits Ca2+ sequestration by SR Ca2+ ATPase (SERCA), acting through the first type of imidazoline receptors. Here, we study the functional activity of agmatine, as well as a specific α2-agonist, guanabenz, in respect to spontaneous Ca2+-transients in SHR cardiomyocytes of the early age (2–2.5 months), and adulthood animals (8–9 months). α2-mediated cardioprotective effect was almost twofold decreased in SHR cardiac cells compared to normotensive rats of the corresponding age, despite the fact that both α2A- and α2B-AR protein levels were significantly increased in SHR cardiomyocytes. NO-mediated facilitation of SERCA activity is substantially reduced in SHR cardiomyocytes vs. normotensive rats. These data suggest that the SHR phenotype starting from early age shows signs of the impaired sarcolemmal α2-AR signaling, which can aggravate the development of this cardiovascular pathology.

Spontaneous Ca2+-transient (wave) generation in isolated cardiomyocytes is well established phenomenon which poses a lot of questions about myocardial excitability. Current studies of spontaneous Ca2+-activity in cardiac cells mainly relate to the kinetic characteristics, classification and simulation of Ca2+-events through ryanodine receptor (RyR) activity modeling. Here, for the first time we pay attention to the Ca2+-transients having stationary kinetics for correct estimation of the sarcoplasmic reticulum Ca2+ transport. In cardiomyocytes generating such type of Ca2+-transients, the averaged intracellular calcium ([Ca2+]in) fluorescence practically does not change in time. Stationary Ca2+-transients are observed in different animal models (Wistar, SHR, ground squirrels) revealing a common cardiomyocyte phenomenon. They somewhat depend on external Ca2+ ([Ca2+]ex) because the [Ca2+]ex lowering to 1 μM in the presence of EGTA disrupts Ca2+-wave propagation. At the same time, spontaneous Ca2+-transients do not associated with the forward or reverse mode of Na+/Ca2+ exchanger (NCX), but partially modulated by the L-type Ca2+-channels. Among the sarcoplasmic reticulum targets, RyR and SERCA are crucial for Ca2+-wave generation and sustained self-oscillation activity. Analysis of the spontaneous wave kinetics reveals that both slopes of the rising wave front and the wave front decline are gradually changed during propagation, which well correlates with the RyR and SERCA activity, respectively. On the contrary, in the electrical field-stimulated myocytes, both slope factors are sharply changed corresponding to ‘all-or-nothing’ rule, which is fundamental principle for action potential in cardiomyocytes. Furthermore, stimulation of single cardiomyocyte using local electrode appears the deterioration in the [Ca2+]in utilization from the cytosol, which limits the time of observation during the protocol. Obtained data suggest that stationary spontaneous Ca2+-transients occurring without actual myocellular excitation represent useful and precise tools for estimation of the sarcoplasmic reticulum Ca2+-transport.

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is an imaging approach that enables analysis of the 3D architecture of cells and tissues at resolutions that are 1-2 orders of magnitude higher than that possible with light microscopy. The slow speeds of data collection and analysis are two critical problems that limit more extensive use of FIB-SEM technology. Here, we present a robust method that enables rapid, large-scale acquisition of data from tissue specimens, combined with an approach for automated data segmentation using machine learning, which dramatically increases the speed of image analysis. We demonstrate the feasibility of these methods through the 3D analysis of human muscle tissue by showing that our process results in an improvement in speed of up to three orders of magnitude as compared to manual approaches for data segmentation. All programs and scripts we use are open source and are immediately available for use by others.Impact StatementThe high-throughput, easy-to-use and versatile segmentation pipeline described in our manuscript will enable rapid, large-scale statistical analysis of sub-cellular structures in tissues.

The spontaneous rhythmic action potentials generated by the sinoatrial node (SAN), the primary pacemaker in the heart, dictate the regular and optimal cardiac contractions that pump blood around the body. Although the heart rate of humans is substantially slower than that of smaller experimental animals, current perspectives on the biophysical mechanisms underlying the automaticity of sinoatrial nodal pacemaker cells (SANCs) have been gleaned largely from studies of animal hearts. Using human SANCs, we demonstrated that spontaneous rhythmic local Ca2+ releases generated by a Ca2+ clock were coupled to electrogenic surface membrane molecules (the M clock) to trigger rhythmic action potentials, and that Ca2+-cAMP-protein kinase A (PKA) signaling regulated clock coupling. When these clocks became uncoupled, SANCs failed to generate spontaneous action potentials, showing a depolarized membrane potential and disorganized local Ca2+ releases that failed to activate the M clock. β-Adrenergic receptor (β-AR) stimulation, which increases cAMP concentrations and clock coupling in other species, restored spontaneous, rhythmic action potentials in some nonbeating "arrested" human SANCs by increasing intracellular Ca2+ concentrations and synchronizing diastolic local Ca2+ releases. When β-AR stimulation was withdrawn, the clocks again became uncoupled, and SANCs reverted to a nonbeating arrested state. Thus, automaticity of human pacemaker cells is driven by a coupled-clock system driven by Ca2+-cAMP-PKA signaling. Extreme clock uncoupling led to failure of spontaneous action potential generation, which was restored by recoupling of the clocks. Clock coupling and action potential firing in some of these arrested cells can be restored by β-AR stimulation-induced augmentation of Ca2+-cAMP-PKA signaling.

We present a new model for etiology of Alzheimer's disease (AD) which postulates early involvement of specialized neuroprotective mechanisms in the pathology of AD. These neuroprotective mechanisms work in concert to regulate metabolic homeostasis in healthy neuronal cells, but contribute to the distinctive cytopathic phenotype of neuronal degeneration in AD. According to this model, two molecular/genetic hallmarks of AD, amyloid-β (Aβ) deposition and tau hyperphosphorylation, are associated with neuronal mechanisms for dissipating thermal energy associated with high levels of protein synthesis in highly temperature-sensitive neuronal cells. Development of effective methods of AD treatment will require a better understanding of how this neuronal defense system is activated in response to cytopathological triggers in sporadic AD. The cause and effect link between synthesis and processing of amyloid-β protein precursor (AβPP) and the AD terminal phenotype of neurofibrillary tangles and neuron loss involve the formation of Aβ peptides that accumulate as oligomers, cannot be controlled by neurons, and are toxic to the surrounding neuronal membranes. We analyze experimental and clinical studies that have investigated the correlation between phosphorylation of some transport proteins and increased synthesis of proteins in neurons. We also review the evidence related to the possibility that protein hyperphosphorylation may be a byproduct of energetic imbalances in AD cells associated with high levels of protein synthesis, and that activation of defense systems, through which energy-rich molecules are eliminated from the site of protein synthesis and are sequestered to the peripheral neuronal areas, may bring about some of the distinctive morphological features of AD.

Evidence suggests that intracellular Ca(2+) levels and contractility of cardiomyocytes can be modulated by targeting receptors other than already identified adrenergic or non-adrenergic sarcolemmal receptors. This study uncovers the presence in myocardial cells of adrenergic α2 (α2-AR) and imidazoline I1 (I1R) receptors. In isolated left ventricular myocytes generating stationary spontaneous Ca(2+) transients in the absence of triggered action potentials, the prototypic agonist of both receptors agmatine can activate corresponding signaling cascades with opposing outcomes on nitric oxide (NO) synthesis and intracellular Ca(2+) handling. Specifically, activation of α2-AR signaling through PI3 kinase and Akt/protein kinase B stimulates NO production and abolishes Ca(2+) transients, while targeting of I1R signaling via phosphatidylcholine-specific phospholipase C (PC-PLC) and protein kinase C (PKC) suppresses NO synthesis and elevates averaged intracellular Ca(2+). We identified that endothelial NO synthase (eNOS) is a major effector for both signaling cascades. According to the established eNOS transitions between active (Akt-dependent) and inactive (PKC-dependent) conformations, we suggest that balance between α2-AR and I1R signaling pathways sets eNOS activity, which by defining operational states of myocellular sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) can adjust Ca(2+) re-uptake and thereby cardiac inotropy. These results indicate that the conventional catalog of cardiomyocyte sarcolemmal receptors should be expanded by the α2-AR and I1R populations, unveiling previously unrecognized targets for endogenous ligands as well as for existing and potential pharmacological agents in cardiovascular medicine.

The self-Assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Local Ca Releases (LCRs) are crucial events involved in cardiac pacemaker cell function. However, specific algorithms for automatic LCR detection and analysis have not been developed in live, spontaneously beating pacemaker cells. Here we measured LCRs using a high-speed 2D-camera in spontaneously contracting sinoatrial (SA) node cells isolated from rabbit and guinea pig and developed a new algorithm capable of detecting and analyzing the LCRs spatially in two-dimensions, and in time. Our algorithm tracks points along the midline of the contracting cell. It uses these points as a coordinate system for affine transform, producing a transformed image series where the cell does not contract. Action potential-induced Ca transients and LCRs were thereafter isolated from recording noise by applying a series of spatial filters. The LCR birth and death events were detected by a differential (frame-to-frame) sensitivity algorithm. An LCR was detected when its signal changes sufficiently quickly within a sufficiently large area. The LCR is considered to have died when its amplitude decays substantially, or when it merges into the rising whole cell Ca transient. Our algorithm provides major LCR parameters such as period, signal mass, duration, and path area. As LCRs propagate within cells, the algorithm identifies splitting and merging behaviors, indicating the importance of Ca-induced-Ca-release for the fate of LCRs and for generating a powerful ensemble Ca signal. Thus, our new computer algorithms eliminate motion artifacts and detect 2D local spatiotemporal Ca release events from recording noise and global signals. While the algorithms detect LCRs in sinoatrial nodal cells, they have the potential to be used in other applications in biophysics and cell physiology, for example, to detect Ca wavelets (abortive waves), sparks and embers in muscle cells and Ca puffs and syntillas in neurons., Comment: 43 pages, 7 figures, 2 tables, 5 videos (with web links)

Intracellular Local Ca releases (LCRs) from sarcoplasmic reticulum (SR) regulate cardiac pacemaker cell function by activation of electrogenic Na/Ca exchanger (NCX) during diastole. Prior studies demonstrated the existence of powerful compensatory mechanisms of LCR regulation via a complex local cross-talk of Ca pump, release and NCX. One major obstacle to study these mechanisms is that LCR exhibit complex Ca release propagation patterns (including merges and separations) that have not been characterized. Here we developed new terminology, classification, and computer algorithms for automatic detection of numerically simulated LCRs and examined LCR regulation by SR Ca pumping rate (Pup) that provides a major contribution to fight-or-flight response. In our simulations the faster SR Ca pumping accelerates action potential-induced Ca transient decay and quickly clears Ca under the cell membrane in diastole, preventing premature releases. Then the SR generates an earlier, more synchronized, and stronger diastolic LCR signal activating an earlier and larger inward NCX current. LCRs at higher Pup exhibit larger amplitudes and faster propagation with more collisions to each other. The LCRs overlap with Ca transient decay, causing an elevation of the average diastolic [Ca] nadir to ~200 nM (at Pup = 24 mM/s). Background Ca (in locations lacking LCRs) quickly decays to resting Ca levels (, Author summary Life’s vital rhythm, the heartbeat is guaranteed by specialized, cardiac pacemaker cells. Based upon the Hodgkin-Huxley membrane excitation theory and its application to cardiac cells, the cardiac pacemaker clock has been, for a long time, to be considered essentially a surface membrane oscillator, i.e. a membrane clock, driven by an ensemble of time- and voltage-dependent ion channels. More recent studies, however, discovered a tight integration of the membrane clock with an intracellular Ca oscillator (dubbed Ca clock). The Ca clock generates rhythmic, diastolic locally propagating Ca releases from sarcoplasmic reticulum, a cell major Ca store, that is refilled with Ca via a Ca pump. The released Ca activates Na/Ca exchanger that generates an inward current and accelerates the diastolic depolarization. Despite their importance, the local releases have not been systematically studied. Here we developed a new computer algorithm for automatic detection, classification, and analysis of local Ca releases generated by a recent computational model of a sinoatrial node cell. Then we investigated how the releases are regulated by the Ca pump, a major functional component of Ca clock. We discovered counterintuitive behaviors stabilizing diastolic Ca signal mass and ensuring fail-safe pacemaker operation at various pumping rates.

Sustained cardiac adrenergic stimulation has been implicated in the development of heart failure and ventricular dysrhythmia. Conventionally, α2 adrenoceptors (α2-AR) have been assigned to a sympathetic short-loop feedback aimed at attenuating catecholamine release. We have recently revealed the expression of α2-AR in the sarcolemma of cardiomyocytes and identified the ability of α2-AR signaling to suppress spontaneous Ca2+ transients through nitric oxide (NO) dependent pathways. Herein, patch-clamp measurements and serine/threonine phosphatase assay revealed that, in isolated rat cardiomyocytes, activation of α2-AR suppressed L-type Ca2+ current (ICaL) via stimulation of NO synthesis and protein kinase G- (PKG) dependent activation of phosphatase reactions, counteracting isoproterenol-induced β-adrenergic activation. Under stimulation with norepinephrine (NE), an agonist of β- and α-adrenoceptors, the α2-AR antagonist yohimbine substantially elevated ICaL at NE levels >10nM. Concomitantly, yohimbine potentiated triggered intracellular Ca2+ dynamics and contractility of cardiac papillary muscles. Therefore, in addition to the α2-AR-mediated feedback suppression of sympathetic and adrenal catecholamine release, α2-AR in cardiomyocytes can govern a previously unrecognized local cardiomyocyte-delimited stress-reactive signaling pathway. We suggest that such aberrant α2-AR signaling may contribute to the development of cardiomyopathy under sustained sympathetic drive. Indeed, in cardiomyocytes of spontaneously hypertensive rats (SHR), an established model of cardiac hypertrophy, α2-AR signaling was dramatically reduced despite increased α2-AR mRNA levels compared to normal cardiomyocytes. Thus, targeting α2-AR signaling mechanisms in cardiomyocytes may find implications in medical strategies against maladaptive cardiac remodeling associated with chronic sympathoadrenal stimulation.

Spontaneous, rhythmic LCRs are an important factor of pacemaker function of SA node cells (SANC). LCRs operate within the context of a coupled-clock system involving interactions of plasma membrane and sarcoplasmic reticulum (SR)-based calcium clocks. To date, the functional importance of this coupled-clock system has been demonstrated in rabbit SANC that beat spontaneously and rhythmically. The contribution of LCRs to action potential firing in guinea pig SANC, however, is controversial.We quantified the entire ensemble of LCRs in healthy appearing, single guinea pig SANC prior to and in the presence of beta-adrenergic stimulation (isoproterenol, 1µM). LCRs were measured by a high-speed 2D-camera and analyzed by a novel computer program capable of identifying LCRs in video-recordings.Although nine of the 22 SANC examined did not exhibit Ca2+ transients, LCRs were present in all cells. LCRs were smaller and more intense in cells without Ca2+ transients than in those with Ca2+ transients (rhythmic or dysrhythmic). Isoproterenol increased LCR size and induced rhythmic Ca2+ transients in majority of cells (6/9) without Ca2+ transients prior to isoproterenol. SANC that exhibited Ca2+ transients prior to isoproterenol generated approximately 80 LCRs/s. Cells in which Ca2+ transients were rhythmic had greater LCR size and amplitude than those with dysrhythmic Ca2+ transients. In cells with dysrhythmic Ca2+ transients, isoproterenol increased LCR size and amplitude and decreased LCR period, concurrently increasing the Ca2+ transient rhythmicity and frequency via presenting a larger and earlier occurring net Ca2+ signal to electrogenic Na+/Ca2+exchanger. Thus, all guinea pig SANC do indeed exhibit LCRs that contribute to generation of rhythmic action potential-induced Ca2+ transients.

Local calcium releases (LCRs) observed in cardiac pacemaker cells have a complex spatiotemporal structure that has never been studied. We developed a computer algorithm for automatic detection and classification of LCRs in simulations of rabbit sinoatrial-node cells (using our recent 3D-model) to get new insights into pacemaker cell operation, specifically, the role of sarcoplasmic reticulum calcium pumping rate (Pup).Identified release events that share a common intensity level are categorized as a release cluster, i.e. a complex release with multiple intensity peaks. These complex LCRs tend to live longer and propagate farther via calcium-induced-calcium release, thus occupying larger areas. Release events that don’t share any intensity level with other events are calcium sparks that do not live for a long time and do not propagate. Collisions and splits of LCRs are handled as follows. When an LCR separates into different parts, all parts are still considered part of the LCR. On the other hand, when an LCR collides with another, the one with the weaker signal mass is considered dead and the one with the larger signal mass takes its signal mass as its own. An LCR may also die by stochastic attrition when all its components fade out.Under voltage clamp, LCR areas and signal masses were paradoxically smaller at larger Pup, likely reflecting uptake of cytosolic calcium before it can propagate. Under spontaneous beating conditions, however, higher Pup greatly increased diastolic LCR signal mass and beating rate as predicted by the coupled-clock theory. Interestingly, the total integral of all LCRs during diastolic depolarization in both cases remained almost the same as longer integration time with smaller events is comparable to shorter time with larger events.

Previous confocal microscopy studies of SA-node cells revealed (in tangential sections) hierarchical clustering of calcium release channels (RyRs). Further numerical model simulations demonstrated that such hierarchy can enable propagating Local-Calcium-Releases (LCRs), crucial events of pacemaker cell operation. RyR distributions in 3D remain, however, unknown, leaving substantial uncertainty in constructing realistic models of SA-node cells. Here we approached the problem by super-resolution structured illumination microscopy (SIM) achieving approximately double the resolution of conventional microscopy to measure both 3D-distribution of RyRs and 2D-calcium dynamics in isolated rabbit SA-node cells. Each 3D-distribution of RyR clusters was reconstructed with 150 nm z-sectioning to capture the entire cell (bottom-to-top). We developed a novel computer algorithm to detect and characterize fluorescence signals of RyR clusters, including their sizes (volumes) and genuine distances to their nearest neighbors in 3D. We analyzed 8 cells with RyR localization mainly at the cell surface and found from 1107 to 2420 (mean=2070) RyR clusters in each cell. Distributions for nearest neighbor (center-to-center) distance were essentially (>95%) within the range 200-800 nm (mean=405 nm) that is substantially smaller than we previously reported (mean∼800 nm), indicating that many small clusters (lacking in confocal scans) now become resolved by SIM and also many neighboring clusters previously missed in 2D-projections now become revealed by the new 3D-analysis. Our cell-mid-section SIM measurements of calcium dynamics (using fluo-4) revealed that LCRs occur mainly within ∼1µm under the cell surface, in agreement with immunofluorescence data on RyR cluster localization. The new SIM results on Ca dynamics and anatomical information are included in new numerical modeling of SA-node cell. The larger number of clusters and shorter distances facilitate calcium-induced-calcium-release and LCR propagation observed experimentally.

Background: Intracellular Ca2+ dynamics contribute to cardiac pacemaker cell function via activation of electrogenic Na+/Ca2+ exchanger during diastolic depolarization. Two fundamental mechanisms have been implicated for the diastolic Ca2+ signaling: LCRs and the decaying AP-induced Ca2+ transients. Their relative contributions, however, are unknown.Methods: We evaluated the relative contributions based on simultaneous high-speed camera recordings of Ca2+ signal, alongside membrane potential recordings by perforated patch clamp in single isolated rabbit SA node cells prior to and during beta-adrenergic receptor stimulation with isoproterenol (100 nM). We obtained further insights by employing simulations of our recent numerical model featuring local Ca2+ dynamics in 3D in these cells.Results: All cells tested (n=5) exhibited both LCRs and AP-induced-transient residuals during diastolic depolarization. Individual LCRs have been characterized in the 2D movies using our novel computer algorithm (see our poster on automated analysis of LCRs). The values of LCR signal mass and Ca2+ transient residuals were integrated within the entire cell perimeter in the focal plane of video-recording and compared. Both our analysis of the experimental data and model simulations demonstrated that LCRs provide a major part of the net Ca2+ diastolic signal, both prior to and during isoproterenol exposure. The relative contribution of LCRs, however, substantially increased after isoproterenol application. Specifically, while the contribution of the transient residuals into the net diastolic Ca2+ signal prior to isoproterenol is about 30%, the net signal following isoproterenol becomes almost completely driven by the LCRs, Ca2+ transient decays are accelerated and almost fully complete prior the maximum diastolic depolarization.Conclusions: Both LCRs and local Ca2+ transient residuals contribute to diastolic depolarization. As pacemaker rate increases LCR signal waxes, but the contribution of Ca2+ transient residuals wanes.

LCRs are crucial events of cardiac pacemaker cell function. However, specific algorithms for their automatic detection and analysis have not been developed in 2D+time for live spontaneously beating pacemaker cells. Here we measured calcium signals by a high-speed 2D-camera in isolated rabbit SA-node cells. Our new algorithm tracks points along the midline of the moving/contracting cell. It uses these points as a coordinate system for affine transform, producing a transformed image series where the cell does not move/contract. AP-induced calcium transients and LCRs were isolated from noise by applying a series of spatial filters. We detected LCR birth/death events by a differential (frame-to-frame) sensitivity algorithm applied to each pixel (cell location). An LCR is detected when its signal changes sufficiently quickly within a sufficiently large area. The LCR dies when its amplitude decays substantially or when it merges into a rising transient.The algorithm is capable of rapidly analyzing long recordings, e.g. we detected and analyzed as many as 1746 individual LCR in 81 consecutive cycles from one cell. On average, there were 21.6 LCR events per diastolic period (SD=8.0), ranging between 8 and 48, with mean LCR duration being 28.4 ms.Ultimately our algorithm finds not only known key LCR parameters such as period, signal mass, and duration, but also novel parameters crucial for further biophysical insights such as LCR birth and death statistics, propagation path and “hot-spots” (areas with higher LCR activity). As the LCRs travel in live cells, the algorithm identified their splits and merges, validating predictions of our recent 3D-numerical model of SA-node cell and indicating importance of locally propagating calcium-induced-calcium release for the LCR fate and power critical for the diastolic depolarization acceleration via electrogenic sodium/calcium exchanger.

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.