A novel conceptual model of heart rate autonomic modulation based on a small-world modular structure of the sinoatrial node.

The present view on heartbeat initiation is that a primary pacemaker cell or a group of cells in the sinoatrial node (SAN) center paces the rest of the SAN and the atria. However, recent high-resolution imaging studies show a more complex paradigm of SAN function that emerges from heterogeneous signaling, mimicking brain cytoarchitecture and function. Here, we developed and tested a new conceptual numerical model of SAN organized similarly to brain networks featuring a modular structure with small-world topology. In our model, a lower rate module leads action potential (AP) firing in the basal state and during parasympathetic stimulation, whereas a higher rate module leads during ß-adrenergic stimulation. Such a system reproduces the respective shift of the leading pacemaker site observed experimentally and a wide range of rate modulation and robust function while conserving energy. Since experimental studies found functional modules at different scales, from a few cells up to the highest scale of the superior and inferior SAN, the SAN appears to feature hierarchical modularity, i.e., within each module, there is a set of sub-modules, like in the brain, exhibiting greater robustness, adaptivity, and evolvability of network function. In this perspective, our model offers a new mainframe for interpreting new data on heterogeneous signaling in the SAN at different scales, providing new insights into cardiac pacemaker function and SAN-related cardiac arrhythmias in aging and disease.

Amyloid Aß25-35 Aggregates Say 'NO' to Long-Term Potentiation in the Hippocampus through Activation of Stress-Induced Phosphatase 1 and Mitochondrial Na+/Ca2+ Exchanger.

The search for strategies for strengthening the synaptic efficiency in Aß25-35-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 Aß25-35-associated signaling in the hippocampus. Aß25-35 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 Aß25-35-dependent LTP suppression for both simultaneous and delayed enzyme blockade protocols. STPP activity in the Aß25-35-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 Aß25-35-mediated LTP suppression prevention. A mitochondrial Na+/Ca2+ exchanger (mNCX) blocker, CGP37157, also attenuated the Aß25-35-induced LTP decline. Aß25-35 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 Aß25-35-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 Aß25-35-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 Aß25-35-driven deteriorations.

The Heart's Pacemaker Mimics Brain Cytoarchitecture and Function: Novel Interstitial Cells Expose Complexity of the SAN.

BACKGROUND: 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 43+/F-actin+ cells. It is also known that sympathetic and parasympathetic innervation create an autonomic plexus in the SAN that modulates heart rate and rhythm. However, the anatomical details of the interaction of this plexus with the pacemaker cell meshwork have yet to be described. OBJECTIVES: This study sought to describe the 3-dimensional cytoarchitecture of the mouse SAN, including autonomic innervation, peripheral glial cells, and pacemaker cells. METHODS: 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. RESULTS: The SAN exhibited heterogeneous autonomic innervation, which was accompanied by a web of peripheral glial cells and a novel S100B+/GFAP- interstitial cell population, with a unique morphology and a distinct distribution pattern, creating complex interactions with other cell types in the node, particularly with HCN4-expressing cells. Transmission electron microscopy identified a similar population of interstitial cells as telocytes, which appeared to secrete vesicles toward pacemaker cells. Application of S100B to SAN preparations desynchronized Ca2+ signaling in HCN4-expressing cells and increased variability in SAN impulse rate and rhythm. CONCLUSIONS: The autonomic plexus, peripheral glial cell web, and a novel S100B+/GFAP- interstitial cell type embedded within the HCN4+ cell meshwork increase the structural and functional complexity of the SAN and provide a new regulatory pathway of rhythmogenesis.

Functional Heterogeneity of Cell Populations Increases Robustness of Pacemaker Function in a Numerical Model of the Sinoatrial Node Tissue.

Each heartbeat is initiated by specialized pacemaker cells operating within the sinoatrial node (SAN). While individual cells within SAN tissue exhibit substantial heterogeneity of their electrophysiological parameters and Ca cycling, the role of this heterogeneity for cardiac pacemaker function remains mainly unknown. Here we investigated the problem numerically in a 25 × 25 square grid of connected coupled-clock Maltsev-Lakatta cell models. The tissue models were populated by cells with different degree of heterogeneity of the two key model parameters, maximum L-type Ca current conductance (g CaL ) and sarcoplasmic reticulum Ca pumping rate (P up ). Our simulations showed that in the areas of P up -g CaL parametric space at the edge of the system stability, where action potential (AP) firing is absent or dysrhythmic in SAN tissue models populated with identical cells, rhythmic AP firing can be rescued by populating the tissues with heterogeneous cells. This robust SAN function is synergistic with respect to heterogeneity in g CaL and P up and can be further strengthened by clustering of cells with similar properties. The effect of cell heterogeneity is not due to a simple summation of activity of intrinsically firing cells naturally present in heterogeneous SAN; rather AP firing cells locally and critically interact with non-firing/dormant cells. When firing cells prevail, they recruit many dormant cells to fire, strongly enhancing overall SAN function; and vice versa, prevailing dormant cells suppress AP firing in cells with intrinsic automaticity and halt SAN function. The transitions between firing and non-firing states of the system are sharp, resembling phase transitions in statistical physics. Furthermore, robust function of heterogeneous SAN tissue requires weak cell coupling, a known property of the central area of SAN where cardiac impulse emerges; stronger cell coupling reduces AP firing rate and ultimately halts SAN automaticity at the edge of stability.

Serine/Threonine Phosphatases in LTP: Two B or Not to Be the Protein Synthesis Blocker-Induced Impairment of Early Phase.

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.

PP1/PP2A phosphatase inhibition-induced metaplasticity in protein synthesis blocker-treated hippocampal slices: LTP and LTD, or There and Back again.

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.

Protein kinase C-mediated calcium signaling as the basis for cardiomyocyte plasticity.

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, PKCε. 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.

Cardiomyocytes generating spontaneous Ca2+-transients as tools for precise estimation of sarcoplasmic reticulum Ca2+ transport.

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.

Social heterogeneity drives complex patterns of the COVID-19 pandemic: insights from a novel Stochastic Heterogeneous Epidemic Model (SHEM).

In todays absence of a vaccine and impactful treatments, the most effective way to combat the virus is to find and implement mitigation strategies. An invaluable resource in this task is numerical modeling that can reveal key factors in COVID-19 pandemic development. On the other hand, it has become evident that regional infection curves of COVID-19 exhibit complex patterns which often differ from curves predicted by forecasting models. The wide variations in attack rate observed among different social strata suggest that this may be due to social heterogeneity not accounted for by regional models. We investigated this hypothesis by developing and using a new Stochastic Heterogeneous Epidemic Model (SHEM) that focuses on subpopulations that are vulnerable in the sense of having an increased likelihood of spreading infection among themselves. We found that the isolation or embedding of vulnerable sub-clusters in a major population hub generated complex stochastic infection patterns which included multiple peaks and growth periods, an extended plateau, a prolonged tail, or a delayed second wave of infection. Embedded vulnerable groups became hotspots that drove infection despite efforts of the main population to socially distance, while isolated groups suffered delayed but intense infection. Amplification of infection by these hotspots facilitated transmission from one urban area to another, causing the epidemic to hopscotch in a stochastic manner to places it would not otherwise reach, resembling a microcosm of the situation worldwide as of September 2020. Our results suggest that social heterogeneity is a key factor in the formation of complex infection propagation patterns. Thus, the mitigation of vulnerable groups is essential to control the COVID-19 pandemic worldwide. The design of our new model allows it to be applied in future studies of real-world scenarios on any scale, limited only by computing memory and the ability to determine the underlying topology and parameters.

Upregulation of α2-adrenoceptor synthesis in SHR cardiomyocytes: Recompense without sense - Increased amounts, impaired commands.

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.

Disturbance of I1-imidazoline receptor signal transduction in cardiomyocytes of Spontaneously Hypertensive Rats.

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 ß-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.

Semi-automated 3D segmentation of human skeletal muscle using Focused Ion Beam-Scanning Electron Microscopic images.

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.

LTP suppression by protein synthesis inhibitors is NO-dependent.

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.

Heterogeneity of calcium clock functions in dormant, dysrhythmically and rhythmically firing single pacemaker cells isolated from SA node.

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 Ca2+ transients and spontaneous local Ca2+ 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, dysrhythmic and dormant SANC exhibited smaller LCRs that appeared stochastically and were widely distributed in time. ß-adrenergic receptor (ßAR) 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 ßAR stimulation, these dormant SANC developed automaticity, and LCRs became coupled to spontaneous action potential-induced cytosolic Ca2+ 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 ßAR stimulation, with the rate of action potential-induced cytosolic Ca2+ 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 ßAR stimulation via increased rhythmic LCR activity and ameliorated coupling between the Ca2+ and membrane clocks.

Agmatine modulates calcium handling in cardiomyocytes of hibernating ground squirrels through calcium-sensing receptor signaling.

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 Gßγ-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.

A coupled-clock system drives the automaticity of human sinoatrial nodal pacemaker cells.

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. ß-Adrenergic receptor (ß-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 ß-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 ß-AR stimulation-induced augmentation of Ca2+-cAMP-PKA signaling.

Stabilization of diastolic calcium signal via calcium pump regulation of complex local calcium releases and transient decay in a computational model of cardiac pacemaker cell with individual release channels.

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 (<100 nM) at high Pup, but remained elevated during slower decay at low Pup. Release propagation is facilitated at higher Pup by a larger LCR amplitude, whereas at low Pup by higher background Ca. While at low Pup LCRs show smaller amplitudes, their larger durations and sizes combined with longer transient decay stabilize integrals of diastolic Ca and NCX current signals. Thus, the local interplay of SR Ca pump and release channels regulates LCRs and Ca transient decay to insure fail-safe pacemaker cell operation within a wide range of rates.

Computer algorithms for automated detection and analysis of local Ca2+ releases in spontaneously beating cardiac pacemaker cells.

Local Ca2+ 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. In the present study 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 Ca2+ 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 applied to each pixel (cell location). 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 Ca2+ transient. Ultimately, our algorithm provides major LCR parameters such as period, signal mass, duration, and propagation path area. As the LCRs propagate within live cells, the algorithm identifies splitting and merging behaviors, indicating the importance of locally propagating Ca2+-induced-Ca2+-release for the fate of LCRs and for generating a powerful ensemble Ca2+ signal. Thus, our new computer algorithms eliminate motion artifacts and detect 2D local spatiotemporal events from recording noise and global signals. While the algorithms were developed to 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 Ca2+ wavelets (abortive waves), sparks and embers in muscle cells and Ca2+ puffs and syntillas in neurons.

Sarcolemmal α2-adrenoceptors control protective cardiomyocyte-delimited sympathoadrenal response.

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 ß-adrenergic activation. Under stimulation with norepinephrine (NE), an agonist of ß- 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.

Alpha-2 adrenoceptors and imidazoline receptors in cardiomyocytes mediate counterbalancing effect of agmatine on NO synthesis and intracellular calcium handling.

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.