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Purpose. To study morphotype and neurodynamic peculiarities and their intersystem relationship in students with different BMI levels living in Khanty-Mansi Autonomous Okrug – Yugra. Materials and methods. The research covered 174 first-year students of Surgut State Pedagogical University: 33 male and 141 female students. The study was conducted on a voluntary basis, with written informed consent to be examined. Anthropometric indices, body composition (Tanita BC-601), neurodynamic parameters (“NS-PsychoTest”) were evaluated. Statistical analysis was performed using the application package Statistica 7.0 (StatSoft, USA). Results. Students with different BMI levels did not differ significantly in body length. The differences in mean body weight of the examined students (with low and high BMI values) were due to the combined increase of fat mass (49.6%) and muscle mass (34.3%) in the males and the predominant increase of fat mass (96.7%) in the females, as well as to a decrease of body water content (by 23.4% in the females and 12.1% in the males). The neurodynamic characteristics of the students surveyed reflect statistically greater velocity-strength performance in young males, with less reliability in the executive systems of neurodynamic reactions. The obtained results indicate a strong correlation between morphotype and neurodynamic indices in students with low BMI (R=0,77; χ2=78,527, p=0,016), and a moderate strength relationship in the groups with medium and high BMI (R=0,64; χ2=84,688, p=0,036 and R=0,61; χ2=78,040, p=0,028 respectively). Conclusion. Values of the morphological indices of the examined students reflect the prevalence of meso- and macrosomatotypes. There is a prevalence of anthropometric indices and higher velocity and force indices of neurodynamic in males in comparison with females. In the complex of morphological and neurodynamic processes, the studied students act as a heterogeneous group, the heterogeneity of which is due not only to age-sex-specific morphotype and neurodynamic, but also to their joint interdependence.

A transparent Yb:YMgB5O10 single crystal with dimensions up to 25 × 23 × 25 mm and weight 10.337 g was grown using a high-temperature solution growth on dipped seeds technique with a K2Mo3O10 solvent. The Yb3+ concentration was calculated to be 4.7 at.% (NYb = 3.71 × 1020 atoms/cm3) with a distribution coefficient Kd of 0.59. The grown crystal was characterized by means of PXRD, TGA-DSC and ATR-FTIR techniques. The spectroscopic characteristics of the Yb:YMgB5O10 crystal were demonstrated. Absorption cross-section spectra were produced. The luminescence spectra of the Yb:YMgB5O10 crystal were measured in the spectral range of 950–1100 nm. The luminescence kinetics of the 2F5/2 energy level were investigated and the lifetime was determined.

Objectives This study sought to identify subcellular Ca2+ signals within and among cells comprising the sinoatrial node (SAN) tissue. Background The current paradigm of SAN impulse generation: 1) is that full-scale action potentials (APs) of a common frequency are initiated at 1 site and are conducted within the SAN along smooth isochrones; and 2) does not feature fine details of Ca2+ signaling present in isolated SAN cells, in which small subcellular, subthreshold local Ca2+ releases (LCRs) self-organize to generate cell-wide APs. Methods Immunolabeling was combined with a novel technique to detect the occurrence of LCRs and AP-induced Ca2+ transients (APCTs) in individual pixels (chronopix) across the entire mouse SAN images. Results At high magnification, Ca2+ signals appeared markedly heterogeneous in space, amplitude, frequency, and phase among cells comprising an HCN4+/CX43− cell meshwork. The signaling exhibited several distinguishable patterns of LCR/APCT interactions within and among cells. Rhythmic APCTs that were apparently conducted within the meshwork were transferred to a truly conducting HCN4−/CX43+ network of striated cells via narrow functional interfaces where different cell types intertwine, that is, the SAN anatomic/functional unit. At low magnification, the earliest APCT of each cycle occurred within a small area of the HCN4 meshwork, and subsequent APCT appearance throughout SAN pixels was discontinuous and asynchronous. Conclusions The study has discovered a novel, microscopic Ca2+ signaling paradigm of SAN operation that has escaped detection using low-resolution, macroscopic tissue isochrones employed in prior studies: synchronized APs emerge from heterogeneous subcellular subthreshold Ca2+ signals, resembling multiscale complex processes of impulse generation within clusters of neurons in neuronal networks.

Excitation–contraction coupling kinetics is dictated by the action potential rate of sinoatrial-nodal cells. These cells generate local Ca releases (LCRs) that activate Na/Ca exchanger current, which accelerates diastolic depolarization and determines the pace. LCRs are generated by clusters of ryanodine receptors, Ca release units (CRUs), residing in the sarcoplasmic reticulum. While CRU distribution exhibits substantial heterogeneity, its functional importance remains unknown. Using numerical modeling, here we show that with a square lattice distribution of CRUs, Ca-induced-Ca-release propagation during diastolic depolarization is insufficient for pacemaking within a broad range of realistic ICaL densities. Allowing each CRU to deviate randomly from its lattice position allows sparks to propagate, as observed experimentally. As disorder increases, the CRU distribution exhibits larger empty spaces and simultaneously CRU clusters, as in Poisson clumping. Propagating within the clusters, Ca release becomes synchronized, increasing action potential rate and reviving pacemaker function of dormant/nonfiring cells. However, cells with fully disordered CRU positions could not reach low firing rates and their β-adrenergic–receptor stimulation effect was substantially decreased. Inclusion of Cav1.3, a low-voltage activation L-type Ca channel isoform into ICaL, strongly increases recruitment of CRUs to fire during diastolic depolarization, increasing robustness of pacemaking and complementing effects of CRU distribution. Thus, order/disorder in CRU locations along with Cav1.3 expression regulates pacemaker function via synchronization of CRU firing. Excessive CRU disorder and/or overexpression of Cav1.3 boosts pacemaker function in the basal state, but limits the rate range, which may contribute to heart rate range decline with age and disease.

Excitation-contraction coupling kinetics are dictated by the rate and rhythm of the excitations generated by sinoatrial-nodal cells. These cells generate local Ca releases (LCRs) that activate Na/Ca exchanger current, which accelerates diastolic depolarization and determines the rate and rhythm of the excitations. The LCRs are generated by clusters of ryanodine receptors, Ca release units (CRUs), residing in the sarcoplasmic reticulum. While the spatial CRU distribution in pacemaker cells exhibits substantial heterogeneity, it remains unknown if it has any functional importance. Using numerical modeling, here we showed that with a square lattice distribution of CRUs, Ca-induced-Ca-release propagation during diastolic depolarization is insufficient for pacemaking within a broad lower range of realistic ICaL densities. Allowing each CRU to deviate from its original lattice position fundamentally changes the model behavior: during diastolic depolarization sparks propagate, forming LCRs observed experimentally. As disorder in the CRU positions increases, the CRU distribution exhibits larger empty spaces but simultaneously CRU clusters, as in Poisson clumping. Propagating within the clusters, Ca release becomes synchronized, increasing AP firing rate and reviving pacemaker function within lower ICaL densities at which cells with lattice CRU distribution were dormant/non-firing. However, cells with fully disordered CRU positions cannot reach low firing rates and their β-adrenergic receptor stimulation effect was substantially decreased. Thus, order/disorder in CRU locations regulates Ca release propagation and could be harnessed by pacemaker cells to regulate their function. Excessive disorder is expected to limit heart rate range that may contribute to heart rate range decline with age and in disease.SummaryThe present numerical modeling study shows that disorder in locations of Ca release units in cardiac pacemaker cells has substantial functional impact by creating release clusters, similar to Poisson clumping, and opportunity of Ca release to propagate within the clusters.

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3′,5′-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2–3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner, (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site, (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load, (4) augmented L-type Ca2+ current amplitude, (5) augmented LCR’s characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

In our previous study, we analyzed heart rate variability and heart rate from a large variety of cardiac preparations (including humans, living animals, Langendorff-perfused isolated hearts, and single sinoatrial nodal cells) in diverse species, combining our data with those of previously published articles. The analysis revealed that regardless of conditions, heart rate variability (for the purposes of the study assessed as standard deviation of beat-to-beat intervals) vs. heart rate follows a universal exponential decay-like relationship. Numerical simulations of diastolic interval variability by adding a randomly fluctuating term (Iper) to net current revealed a similar relationship. In the present study, using a Taylor series, we found that this relationship is, in fact, inverse square, and we derive an explicit formula for the standard deviation (sd) of the cycle length (CL) as a function of heart rate (HR) with biophysically meaningful parameters: sd(CL)=sd(Iper)*(60,000/mean(HR) -APD)^2/(ΔV*C), where CL is in ms, HR in beats per minute, Iper in pA, APD in ms is an average AP duration of pacemaker cells, C in pF is cell membrane capacitance, and ΔV is the magnitude of diastolic depolarization in mV. This relationship gives direct insight into heart rate variability mechanisms at the basic level of individual pacemaker cells, i.e. their intrinsic CL variability linked to stochastic operation of ion channels (both Ca release and cell membrane channels) generating Iper. Our explicit formula may be also used for a more precise biomedical interpretation of heart rate variability after respective corrections for heart rate.

Ca2+ and Vm transitions occurring throughout action potential (AP) cycles in sinoatrial nodal (SAN) cells are cues that (1) not only regulate activation states of molecules operating within criticality (Ca2+ domain) and limit-cycle (Vm domain) mechanisms of a coupled-clock system that underlies SAN cell automaticity, (2) but are also regulated by the activation states of the clock molecules they regulate. In other terms, these cues are both causes and effects of clock molecular activation (recursion). Recently, we demonstrated that Ca2+ and Vm transitions during AP cycles in single SAN cells isolated from mice, guinea pigs, rabbits, and humans are self-similar (obey a power law) and are also self-similar to trans-species AP firing intervals (APFIs) of these cells in vitro, to heart rate in vivo, and to body mass. Neurotransmitter stimulation of β-adrenergic receptor or cholinergic receptor–initiated signaling in SAN cells modulates their AP firing rate and rhythm by impacting on the degree to which SAN clocks couple to each other, creating the broad physiologic range of SAN cell mean APFIs and firing interval variabilities. Here we show that Ca2+ and Vm domain kinetic transitions (time to AP ignition in diastole and 90% AP recovery) occurring within given AP, the mean APFIs, and APFI variabilities within the time series of APs in 230 individual SAN cells are self-similar (obey power laws). In other terms, these long-range correlations inform on self-similar distributions of order among SAN cells across the entire broad physiologic range of SAN APFIs, regardless of whether autonomic receptors of these cells are stimulated or not and regardless of the type (adrenergic or cholinergic) of autonomic receptor stimulation. These long-range correlations among distributions of Ca2+ and Vm kinetic functions that regulate SAN cell clock coupling during each AP cycle in different individual, isolated SAN cells not in contact with each other. Our numerical model simulations further extended our perspectives to the molecular scale and demonstrated that many ion currents also behave self-similar across autonomic states. Thus, to ensure rapid flexibility of AP firing rates in response to different types and degrees of autonomic input, nature “did not reinvent molecular wheels within the coupled-clock system of pacemaker cells,” but differentially engaged or scaled the kinetics of gears that regulate the rate and rhythm at which the “wheels spin” in a given autonomic input context.

Aim. The Federal State Educational Standard of Higher Education prescribes providing the educational process in accordance with the individual and typological features of students. The nature of adaptation shifts in students depends on ethnic, ecological, and morphological indicators determining the functional status of the body. Its functional status and functional abilities conditioned by academic and professional activities are determined by the cerebral process, which influences the efficiency of adaptation and cognitive performance in general. The article deals with studying the features of psychophysiological potential in Russian and Kazakh first-year female students during their adaptation to studying at the pedagogical university. Materials and methods. We conducted the diagnostics of the functional status of the central nervous system and cognitive performance in first-year female students at pedagogical universities. The average age of participants was 17.9 years. The total sample (n = 120) consisted of the Russian and Kazakh students living in Kostanay (Kazakhstan) and Chelyabinsk (Russia). A simple eye motor response (SEMR) and a complex eye motor response (CEMR) to a light stimulus were measured with the NS-Psychotest equipment. The calculated data are the following: the functional status of the system; response stability; level of functional abilities. These data were used as a basis for the assessment of cognitive performance in the participants of the study. Results. The results obtained characterize the neuro-dynamic basis of first-year students’ adaptation expressed in the average level of CNS activity, mobility of nervous processes, and optimal functional abilities. In the conditions of the average intragroup expression of the functional criteria of the central nervous system better cognitive processes for the processing of sensory information were revealed in students from Chelyabinsk compared to Kostanay. Conclusion. The analysis of the data obtained revealed the regional specifics of the average values of chronoreflexometry in first-year students, namely a significantly higher level of neural interaction and sensorimotor processing in first-year students from Chelyabinsk.

OBJECTIVES: The purpose of this study was to discover regulatory universal mechanisms of normal automaticity in sinoatrial nodal (SAN) pacemaker cells that are self-similar across species. BACKGROUND: Translation of knowledge of SAN automaticity gleaned from animal studies to human dysrhythmias (e.g., “sick sinus” syndrome [SSS]) requiring electronic pacemaker insertion has been suboptimal, largely because heart rate varies widely across species. METHODS: Subcellular Ca(2+) releases, whole cell action potential (AP)–induced Ca(2+) transients, and APs were recorded in isolated mouse, guinea pig, rabbit, and human SAN cells. Ca(2+)-Vm kinetic parameters during phases of AP cycles from their ignition to recovery were quantified. RESULTS: Although both AP cycle lengths (APCLs) and Ca(2+)-Vm kinetic parameters during AP cycles differed across species by 10-fold, trans-species scaling of these during AP cycles and scaling of these to APCL in cells in vitro, electrocardiogram RR intervals in vivo, and body mass (BM) were self-similar (obeyed power laws) across species. Thus, APCL in vitro, heart rate in vivo, and BM of any species can be predicted by Ca(2+)-Vm kinetics during AP cycles in SAN cells measured in any single species in vitro. CONCLUSIONS: In designing optimal heart rate to match widely different BM and energy requirements from mice to humans, nature did not “reinvent pacemaker cell wheels,” but differentially scaled kinetics of gears that regulate the rates at which the “wheels spin.” This discovery will facilitate the development of novel pharmacological and biological pacemakers featuring a normal, wide-range rate regulation in animal models and the translation of these to humans to target recalcitrant human SSS. (J Am Coll Cardiol EP 2021;7:1331–1344) Published by Elsevier on behalf of the American College of Cardiology Foundation.

BackgroundTranslation of knowledge of sinoatrial nodal “SAN” automaticity gleaned from animal studies to human dysrhythmias, e.g. “Sick Sinus” Syndrome (SSS) requiring electronic pacemaker insertion has been sub-optimal, largely because heart rate (HR) varies widely across species.ObjectivesTo discover regulatory universal mechanisms of normal automaticity in SAN pacemaker cells that are self-similar across species.MethodSub-cellular Ca2+ releases, whole cell AP-induced Ca2+ transients and APs were recorded in isolated mouse, guinea-pig, rabbit and human SAN cells. Parametric Ca2+ and Vm Kinetic Transitions (PCVKT) during phases of AP cycles from their ignition to recovery were quantified.ResultsAlthough both action potential cycle lengths (APCL) and PCVKT during AP cycles differed across species by ten-fold, trans-species scaling of PCVKT during AP cycles and scaling, of PCVKT to APCL in cells in vitro, EKG RR intervals in vivo, and BM were self-similar (obeyed power laws) across species. Thus, APCL in vitro, HR in vivo, and BM of any species can be predicted by PCVKT during AP cycles in SAN cells measured in any single species in vitro.ConclusionsIn designing optimal HR to match widely different BM and energy requirements from mice to humans, nature did not “reinvent pacemaker cell wheels”, but differentially scaled kinetics of gears that regulate the rates at which the “wheels spin”. This discovery will facilitate the development of novel pharmalogic therapies and biologic pacemakers featuring a normal, wide-range rate regulation in animal models and the translation of these to humans to target recalcitrant human SSS.Condensed AbstractStudies in animal models are an important facet of cardiac arrhythmia research. Because HR differs by over ten-fold between some animals and humans, translation of knowledge about regulatory mechanisms of SAN normal automaticity gleaned from studies in animal models to target human SSS has been sub-optimal. Our findings demonstrating that trans-species self-similarity of sub-cellular and cellular mechanisms that couple Ca2+ to Vm during AP cycles can predict heart rate in vivo from mice to humans will inform on the design of novel studies in animal models and facilitate translation of this knowledge to target human disease.

Ca2+ and Vm transitions occurring throughout AP cycles in sinoatrial nodal (SAN) cells are cues that: (1) not only regulate activation states of molecules operating within criticality (Ca2+ domain) and limit-cycle (Vm domain) mechanisms of a coupled-clock system that underlies SAN cell automaticity; (2) but are also regulated by the activation states of the clock molecules they regulate. In other terms, these cues are both causes and effects of clock molecular activation (recursion). Recently, we demonstrated that Ca2+ and Vm transitions during AP cycles in single SAN cells isolated from mice, guinea pigs, rabbits and humans are self-similar (obey a power law) and are also self-similar to trans-species AP firing intervals of these cells in vitro, to heart rate in vivo, and to body mass.Neurotransmitter stimulation of β adrenergic receptor or cholinergic receptor initiated signaling in SAN cells modulates their AP firing rate and rhythm by impacting on the degree to which SAN clocks couple to each other, creating the broad physiologic range of SAN cell mean AP firing intervals and firing interval variabilities. Here we show that Ca2+ and Vm domain kinetic transitions (time to AP ignition in diastole and 90% AP recovery) occurring within given AP, the mean AP firing intervals, and AP firing interval variabilities within time-series of APs in 230 individual SAN cells are self-similar (obey power laws). In other terms, these long-range correlations inform on self-similar distributions of order among SAN cells across the entire broad physiologic range of SAN AP firing intervals, regardless of whether autonomic receptors of these cells are stimulated or not, and regardless of the type (adrenergic or cholinergic) of autonomic receptor stimulation. These long-range correlations among distributions of Ca2+ and Vm kinetic functions that regulate SAN cell clock coupling during each AP cycle in different individual, isolated SAN cells not in contact with each other. Our numerical model simulations further extended our perspectives to the molecular scale and demonstrated that many ion currents also behave self-similar across autonomic states.Thus, to ensure rapid flexibility of AP firing rates in response to different types and degrees of autonomic input, nature “did not reinvent molecular wheels within the coupled-clock system of pacemaker cells”, but differentially engaged or scaled the kinetics of gears that regulate the rate and rhythm at which the “wheels spin” in a given autonomic input context.

BackgroundThe current paradigm of Sinoatrial Node (SAN) impulse generation: (i) is that full-scale action potentials (APs) of a common frequency are initiated at one site and are conducted within the SAN along smooth isochrones; and (ii) does not feature fine details of Ca2+ signalling present in isolated SAN cells, in which small subcellular, subthreshold local Ca2+ releases (LCRs) self-organize to generate cell-wide APs.ObjectivesTo study subcellular Ca2+ signals within and among cells comprising the SAN tissue.MethodsWe combined immunolabeling with a novel technique to detect the occurrence of LCRs and AP-induced Ca2+ transients (APCTs) in individual pixels (chonopix) across the entire mouse SAN images.ResultsAt high magnification, Ca2+ signals appeared markedly heterogeneous in space, amplitude, frequency, and phase among cells comprising an HCN4+/CX43- cell meshwork. The signalling exhibited several distinguishable patterns of LCR/APCT interactions within and among cells. Apparently conducting rhythmic APCTs of the meshwork were transferred to a truly conducting HCN4-/CX43+ network of straited cells via narrow functional interfaces where different cell types intertwine, i.e. the SAN anatomical/functional unit. At low magnification, the earliest APCT of each cycle occurred within a small area of the HCN4 meshwork and subsequent APCT appearance throughout SAN pixels was discontinuous.ConclusionsWe have discovered a novel, microscopic Ca2+ signalling paradigm of SAN operation that has escaped detection using low-resolution, macroscopic tissue isochrones employed in prior studies: APs emerge from heterogeneous subcellular subthreshold Ca2+ signals, resembling multiscale complex processes of impulse generation within clusters of neurons in neuronal networks.Condensed abstractBy combining immunolabeling with a novel optical technique we detected markedly heterogenous Ca2+signals within and among cell clusters of an HCN4+/CX43- meshwork in mouse sinoatrial node. These Ca2+ signals self-organized and transferred, throughout the node, to projections from an HCN4-/CX43+ network connected to a highly organized, rapidly conducting part of the CX43+ network. Thus, APs emerge from heterogeneous, subthreshold Ca2+ signaling not detected in low-resolution macroscopic isochrones. Our discovery requires a fundamental paradigm shift from concentric impulse propagation initiated within a leading site, to a multiscale/complex process, resembling the emergence of organized signals from heterogeneous local signals within neuronal networks.

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.

Coupling of an intracellular Ca2+ clock to surface membrane ion channels, i.e., a “membrane clock, ” via coupling of electrochemical Na+ and Ca2+ gradients ( ENa and ECa, respectively) has been theorized to regulate sinoatrial nodal cell (SANC) normal automaticity. To test this hypothesis, we measured responses of [Na+]i, [Ca2+]i, membrane potential, action potential cycle length (APCL), and rhythm in rabbit SANCs to Na+/K+ pump inhibition by the digitalis glycoside, digoxigenin (DG, 10–20 μmol/l). Initial small but significant increases in [Na+]i and [Ca2+]i and reductions in ENa and ECa in response to DG led to a small reduction in maximum diastolic potential (MDP), significantly enhanced local diastolic Ca2+ releases (LCRs), and reduced the average APCL. As [Na+]i and [Ca2+]i continued to increase at longer times following DG exposure, further significant reductions in MDP, ENa, and ECa occurred; LCRs became significantly reduced, and APCL became progressively and significantly prolonged. This was accompanied by increased APCL variability. We also employed a coupled-clock numerical model to simulate changes in ENa and ECa simultaneously with ion currents not measured experimentally. Numerical modeling predicted that, as the ENa and ECa monotonically reduced over time in response to DG, ion currents ( ICaL, ICaT, If, IKr, and IbNa) monotonically decreased. In parallel with the biphasic APCL, diastolic INCX manifested biphasic changes; initial INCX increase attributable to enhanced LCR ensemble Ca2+ signal was followed by INCX reduction as ENCX ( ENCX = 3 ENa − 2 ECa) decreased. Thus SANC automaticity is tightly regulated by ENa, ECa, and ENCX via a complex interplay of numerous key clock components that regulate SANC clock coupling.

Heart muscle contraction is normally activated by a synchronized Ca release from sarcoplasmic reticulum (SR), a major intracellular Ca store. However, under abnormal conditions Ca leaks from the SR, decreasing heart contraction amplitude and increasing risk of life-threatening arrhythmia. The mechanisms and regimes of SR operation generating the abnormal Ca leak remain unclear. Here we employed both numerical and analytical modeling to get mechanistic insights into the emergent Ca leak phenomenon. Our numerical simulations using a detailed realistic model of Ca release unit (CRU) reveal sharp transitions resulting in Ca leak. The emergence of leak is closely mapped mathematically to the Ising model from statistical mechanics. The system steady-state behavior is determined by two aggregate parameters: the analogues of magnetic field ($h$) and the inverse temperature ($\beta$) in the Ising model, for which we have explicit formulas in terms of SR Ca and release channel opening/closing rates. The classification of leak regimes takes the shape of a phase $\beta$-$h$ diagram, with the regime boundaries occurring at $h$=0 and a critical value of $\beta$ ($\beta*$) which we estimate using a classical Ising model and mean field theory. Our theory predicts that a synchronized Ca leak will occur when $h$>0 and $\beta>\beta*$ and a disordered leak occurs when $\beta, Comment: 20 pages, 6 figures, supplemental material

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.

Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (I CaL ). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to I CaL activation threshold. During the ignition phase, I CaL -mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and I CaL , as diastolic LCRs begin to occur below and continue within the voltage range of I CaL activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (∼0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.

This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na(+) channel function and regulation, Na(+) channel structure and function, and Na(+) channel trafficking, sequestration and complexing.

Cardiac pacemaker cells, including cells of the sinoatrial node, are heterogeneous in size, morphology, and electrophysiological characteristics. The exact extent to which these cells differ electrophysiologically is unclear yet is critical to understanding their functioning. We examined major ionic currents in individual intercaval pacemaker cells (IPCs) sampled from the paracristal, intercaval region (including the sinoatrial node) that were spontaneously beating after enzymatic isolation from rabbit hearts. The beating rate was measured at baseline and after inhibition of the Ca(2+) pump with cyclopiazonic acid. Thereafter, in each cell, we consecutively measured the density of funny current (I(f)), delayed rectifier K(+) current (I(K)) (a surrogate of repolarization capacity), and L-type Ca(2+) current (I(Ca,L)) using whole cell patch clamp(.) The ionic current densities varied to a greater extent than previously appreciated, with some IPCs demonstrating very small or zero I(f) . The density of none of the currents was correlated with cell size, while I(Ca,L) and I(f) densities were related to baseline beating rates. I(f) density was correlated with I(K) density but not with that of I(Ca,L). Inhibition of Ca(2+) cycling had a greater beating rate slowing effect in IPCs with lower I(f) densities. Our numerical model simulation indicated that 1) IPCs with small (or zero) I(f) or small I(Ca,L) can operate via a major contribution of Ca(2+) clock, 2) I(f)-Ca(2+)-clock interplay could be important for robust pacemaking function, and 3) coupled I(f)-I(K) function could regulate maximum diastolic potential. Thus, we have demonstrated marked electrophysiological heterogeneity of IPCs. This heterogeneity is manifested in basal beating rate and response to interference of Ca(2+) cycling, which is linked to I(f). NEW & NOTEWORTHY In the present study, a hitherto unrecognized range of heterogeneity of ion currents in pacemaker cells from the intercaval region is demonstrated. Relationships between basal beating rate and L-type Ca(2+) current and funny current (I(f)) density are uncovered, along with a positive relationship between I(f) and delayed rectifier K(+) current. Links are shown between the response to Ca(2+) cycling blockade and I(f) density.

Intracellular Ca signals represent a universal mechanism of cell function. Messages carried by Ca are local, rapid, and powerful enough to be delivered over the thermal noise. A higher signal to noise ratio is achieved by a cooperative action of Ca release channels such as IP3 receptors or ryanodine receptors arranged in clusters or release units containing a few to several hundred release channels. The release channels synchronize their openings via Ca-induced-Ca-release, generating high-amplitude local Ca signals known as puffs in neurons or sparks in muscle cells. Despite the high release amplitude and positive feedback nature of the activation, Ca signals are strictly confined in time and space by an unexplained termination mechanism. Here we show that the collective transition of release channels from an open to a closed state is identical to the phase transition associated with the reversal of magnetic field in an Ising ferromagnet. We demonstrate this mechanism using numerical model simulations of Ca sparks over a wide range of cluster sizes from 25 to 169 release channels. While prior studies suggested contributions of stochastic attrition and Ca store depletion, our new simple quantitative criterion closely predicts the depletion level required for spark termination for each cluster size. We further formulate exact requirements for a cluster of release channels to follow the Ising model in any cell type. Thus we describe deterministically the behaviour of a system on a coarser scale (release unit) which is random on a finer scale (release channels), bridging the gap between scales. Our results provide the first exact mapping of a nanoscale biological signalling model to an interacting particle system in statistical physics, making the extensive mathematical apparatus available to quantitative biology., Comment: 22 pages, 4 figures, 4 videos. Published in PNAS 2017

In our previous study, we analyzed heart rate variability and heart rate from a large variety of cardiac preparations (including humans, living animals, Langendorff-perfused isolated hearts, and single sinoatrial nodal cells) in diverse species, combining our data with those of previously published articles. The analysis revealed that regardless of conditions, heart rate variability (for the purposes of the study assessed as standard deviation) of beat-to-beat intervals heart rate follows a universal exponential decay-like relationship. Numerical simulations of DI variability by adding a randomly fluctuating term to net current revealed a similar relationship. In the present study, using a Taylor series, we found that this relationship is, in fact, inverse square, and we derive an explicit formula for sd(CL) vs. heart rate with biophysically meaningful parameters.

Late Na+ current (INaL) contributes to action potential (AP) duration and Ca2+ handling in cardiac cells. Augmented INaL was implicated in delayed repolarization and impaired Ca2+ handling in heart failure (HF). We tested if Na+ channel (Nav) neuronal isoforms contribute to INaL and Ca2+ cycling defects in HF in 17 dogs in which HF was achieved via sequential coronary artery embolizations. Six normal dogs served as control. Transient Na+ current (INaT) and INaL in left ventricular cardiomyocytes (VCMs) were recorded by patch clamp while Ca2+ dynamics was monitored using Fluo-4. Virally delivered short interfering RNA (siRNA) ensured Nav1.1 and Nav1.5 post-transcriptional silencing. The expression of six Navs was observed in failing VCMs as follows: Nav1.5 (57.3%) > Nav1.2 (15.3%) > Nav1.1 (11.6%) > Nav2.1 (10.7%) > Nav1.3 (4.6%) > Nav1.6 (0.5%). Failing VCMs showed up-regulation of Nav1.1 expression, but reduction of Nav1.6 mRNA. A similar Nav expression pattern was found in samples from human hearts with ischaemic HF. VCMs with silenced Nav1.5 exhibited residual INaT and INaL (∼30% of control) with rightwardly shifted steady-state activation and inactivation. These currents were tetrodotoxin sensitive but resistant to MTSEA, a specific Nav1.5 blocker. The amplitude of the tetrodotoxin-sensitive INaL was 0.1709 ± 0.0299 pA pF–1 (n = 7 cells) and the decay time constant was τ = 790 ± 76 ms (n = 5). This INaL component was lacking in VCMs with a silenced Nav1.1 gene, indicating that, among neuronal isoforms, Nav1.1 provides the largest contribution to INaL. At –10 mV this contribution is ∼60% of total INaL. Our further experimental and in silico examinations showed that this new Nav1.1 INaL component contributes to Ca2+ accumulation in failing VCMs and modulates AP shape and duration. In conclusion, we have discovered an Nav1.1-originated INaL component in dog heart ventricular cells. This component is physiologically relevant to controlling AP shape and duration, as well as to cell Ca2+ dynamics. Key points Late Na+ current (INaL) contributes to action potential remodelling and Ca2+/Na+ changes in heart failure. The molecular identity of INaL remains unclear. The contributions of different Na+ channel isoforms, apart from the cardiac isoform, remain unknown. We discovered and characterized a substantial contribution of neuronal isoform Nav1.1 to INaL. This new component is physiologically relevant to the control of action potential shape and duration, as well as to cell Ca2+ dynamics, especially in heart failure. Introduction A physiological role of neuronal isoforms of voltage-sensitive Na+ channels (Navs) in heart has been suggested many years ago by Corabeouf (Coraboeuf et al. 1979) based on their result that low concentrations (33 nm) of tetrodotoxin (TTX) significantly shortened action potential (AP) duration recorded in cardiac Purkinje fibres. At this low concentration TTX blocks only so-called ‘TTX-sensitive’ Navs which are neuronal isoforms (Haufe et al. 2005a2005a). Indeed, during the past decade, besides the dominating cardiac isoform main α-subunit, Nav1.5, different transcripts of highly TTX-sensitive neuronal Nav isoforms have been found in mouse (Nav1.1, 1.3, 1.6) and dog (Nav1.1, 1.2, 1.3) heart (Maier et al. 2004; Haufe et al. 2005a2005a,b). These neuronal Navs are responsible for 10% and 20% of the peak transient Na+ current, INaT, in dog myocardial and Purkinje cells, respectively (Haufe et al. 2005b2005b). The contribution of neuronal Navs to the late Na+ current, INaL, has not been studied in cardiac cells, therefore the molecular identity of INaL needs further careful consideration. This problem is especially important in HF where the gene expression profile changes dramatically. Thus an intriguing possibility could be that the differential expression of neuronal Navs can contribute, at least in part, to INaL alterations (augmentation) (Valdivia et al. 2005; Maltsev et al. 2007) and to INaL-related electrophysiological abnormalities observed in HF (for review see Maltsev & Undrovinas, 2008). In the present study we used a combination of molecular and pharmacological approaches, as well as numerical modelling, to test a hypothesis that neuronal Nav isoforms contribute to INaL in a physiologically significant manner in ventricular myocytes (VCMs) isolated from failing adult dog hearts. We performed post-transcriptional silencing of Nav1.1 and Nav1.5 genes by sequence-directed RNA using virally delivered short interfering RNA (siRNA). We found the expression of six Navs in both human heart and adult dog VCMs. Analysis of the expression revealed up-regulation of Nav1.1 and down-regulation of Nav1.6 in failing heart compared to normal heart. We took advantage of the method of culturing adult dog VCMs, established by our group, that preserves INaT and INaL over 5 days, i.e. sufficient time for gene silencing and membrane protein turnover (Maltsev et al. 2008a2008a; Mishra et al. 2011). In VCMs with silenced Nav1.5 we found a substantial residual INaT and INaL. Our biophysical and pharmacological examinations confirmed its neuronal isoform origin. Diastolic Ca2+ accumulation was reduced via blockade of the neuronal INaL component, indicating its physiological importance. Our numerical model simulations also demonstrated the physiological importance of the novel INaL component: the Nav1.1- associated INaL effectively modulates AP shape and duration, as well as intracellular Ca2+ homeostasis.

Numerical modeling indicates that hierarchical clustering of ryanodine receptors in cells of the sinoatrial node is crucial to the calcium clock and thereby to regulation of heart rate., The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This “calcium clock” takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic–mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in beating rate in response to β-adrenergic stimulation. The model indicates that the hierarchical clustering of surface RyRs in SANCs may be a crucial adaptive mechanism. Pathological desynchronization of the clocks may explain sinus node dysfunction in heart failure and RyR mutations.

Beneficial clinical bradycardic effects of ivabradine (IVA) have been interpreted solely on the basis of If inhibition, because IVA specifically inhibits If in sinoatrial nodal pacemaker cells (SANC). However, it has been recently hypothesized that SANC normal automaticity is regulated by crosstalk between an "M clock," the ensemble of surface membrane ion channels, and a "Ca(2+) clock," the sarcoplasmic reticulum (SR). We tested the hypothesis that crosstalk between the two clocks regulates SANC automaticity, and that indirect suppression of the Ca(2+) clock further contributes to IVA-induced bradycardia. IVA (3 μM) not only reduced If amplitude by 45 ± 6% in isolated rabbit SANC, but the IVA-induced slowing of the action potential (AP) firing rate was accompanied by reduced SR Ca(2+) load, slowed intracellular Ca(2+) cycling kinetics, and prolonged the period of spontaneous local Ca(2+) releases (LCRs) occurring during diastolic depolarization. Direct and specific inhibition of SERCA2 by cyclopiazonic acid (CPA) had effects similar to IVA on LCR period and AP cycle length. Specifically, the LCR period and AP cycle length shift toward longer times almost equally by either direct perturbations of the M clock (IVA) or the Ca(2+) clock (CPA), indicating that the LCR period reports the crosstalk between the clocks. Our numerical model simulations predict that entrainment between the two clocks that involves a reduction in INCX during diastolic depolarization is required to explain the experimentally AP firing rate reduction by IVA. In summary, our study provides new evidence that a coupled-clock system regulates normal cardiac pacemaker cell automaticity. Thus, IVA-induced bradycardia includes a suppression of both clocks within this system.

Life Emerges From Functions of a System of Synchronized Clocks A hierarchical system of clocks within cells throughout the body creates and synchronizes rhythmic functions from which life emerges. Biological circadian rhythms, linked to light and darkness in an organism's environment, are

Physiological processes governing the heart beat have been under investigation for several hundred years. Major advances have been made in the recent past. A review of the present paradigm is presented here, including a look back at important steps that led us to where we are today, alongside a glimpse into the exciting future of pacemaker research.

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)

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition–a phenomenon we dub “spark metastability.” Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.