Rhythmic Ca2 Research Articles

Electrically Dormant Sinoatrial Nodal Cells (SANC) are Awakened by Increased Camp-Dependent Phosphorylation of Coupled-Clock Proteins

SANC firing rate and rhythm is controlled via the degree of coupling between rhythmic local Ca2+ releases (LCRs) emerging beneath the cell membrane (‘Ca2+clock’) and sarcolemmal electrogenic mechanisms (‘membrane clock’). Dormant SANC (ones that don’t fire spontaneous action potentials (APs)) may therefore represent the extreme of clock uncoupling. We hypothesized that increased cAMP-mediated, PKA-dependent phosphorylation of the coupled-clock system proteins would restore rhythmic AP firing in dormant SANC. We recorded membrane potential (Vm) and intracellular Ca2+ kinetics in enzymatically isolated single, dormant guinea pig SANC prior to and during β-adrenoceptor stimulation (BARs, isoproterenol 0.1-1 µM) or during exposure to cell-permeant cAMP (CPT-cAMP, 300 µM). Dormant SANC that did not fire still maintained a resting membrane potential of −35+/-3mV (n=17), and exhibited small, random LCRs. ∼40% responded to BARs/CPT-cAMP. This initial response involved increases in LCR size and rhythmicity, and hyperpolarization of Vm, accompanied by small amplitude, spontaneous APs (rate ∼1Hz) triggering whole-cell Ca2+ transients. During the subsequent transition to steady-state AP firing, average LCR size increased while average LCR period shortened, partially synchronizing LCRs to late diastole, (rate ∼2Hz); this was paralleled by MDP hyperpolarization to ∼-60 mV. AP amplitude increased, and average AP cycle length shortened similar to LCR period. When BARs/CPT-cAMP was washed out, all changes reversed in order, and SANC again exhibited small random LCRs, with Vm ∼-40 mV. In conclusion, at least in some SANC, dormancy represents membrane & Ca2+ clock uncoupling that is rescuable through increased cAMP and PKA-dependent phosphorylation of both membrane and Ca2+ clock proteins. Specifically, rescuing impaired clock coupling occurs through: (1) increased spatial LCR synchronization, yielding larger LCRs; (2) increased temporal LCR synchronization, decreasing average LCR period and shifting larger LCR occurrence to late diastole.

Loss of Caveolin-3 Causes Heart Rhythm Abnormalities by Affecting Ca2+-Voltage Coupling in the Mouse Sinoatrial Node

Caveolin-3 (Cav3) is a myocyte-specific scaffolding protein within caveolar membranes that compartmentalize a number of ion channels and transporters involved in sinoatrial node (SAN) pacemaking. These channels and transporters are coupled with intracellular Ca2+ cycling (Ca2+-voltage coupling) through rhythmic local Ca2+ releases (LCRs) from ryanodine receptors to initiate an action potential. We hypothesize that disruption of caveolae-associated macromolecular signaling complexes alters SAN automaticity leading to SAN dysfunction. In vivo ECG telemetry, in vitro high-resolution optical mapping, and confocal imaging of intracellular Ca2+ cycling were performed in wild type (WT, n=8) and conditional tamoxifen-induced α-MHC-controlled Cav3 knockout (Cav3-/-, n=8) mice and isolated SAN myocytes. Both in vivo and in vitro,Cav3-/- mice exhibited SAN pacemaking abnormalities characterized by alternating periods of tachycardia-bradycardia rhythm (cycle length varied from 123.6±2.6 ms to 464.4±130 ms), significant beat-to-beat cycle length variability (3.8±1.1 ms in WT vs. 19.5±3.6 ms in Cav3-/-, p<0.01) and shift of the leading pacemaker from the SAN to ectopic foci. In SAN myocytes, Cav3 deficiency led to irregular spontaneous rate with significant cycle length variability (30.9±5.2 ms in WT vs. 644.7±256.4 ms in Cav3-/-, p<0.01). Because LCRs are critically important for the Na+/Ca2+ exchanger mediated component of slow diastolic depolarization of transmembrane potential, we measured the period from LCRs to Ca2+ transient initiation in order to evaluate Ca2+-voltage coupling. Cav3-/- SAN myocytes showed significant prolongation (73.9±6.7 ms in WT vs. 130.6±15.0 ms in Cav3-/-, p<0.01) of this period with large beat-to-beat variability (15.5±2.6 ms in WT vs. 71.6±17.4 ms in Cav3-/-, p<0.01). Our findings demonstrate that Cav3 plays a crucial role in supporting functional integrity of the SAN by synchronizing Ca2+-voltage coupling and regulating rhythm of LCRs.

TRPC3-GEF-H1 axis mediates pressure overload-induced cardiac fibrosis.

Structural cardiac remodeling, accompanying cytoskeletal reorganization of cardiac cells, is a major clinical outcome of diastolic heart failure. A highly local Ca2+ influx across the plasma membrane has been suggested to code signals to induce Rho GTPase-mediated fibrosis, but it is obscure how the heart specifically decodes the local Ca2+ influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca2+ handling required for pump function. We found that an inhibition of transient receptor potential canonical 3 (TRPC3) channel activity exhibited resistance to Rho-mediated maladaptive fibrosis in pressure-overloaded mouse hearts. Proteomic analysis revealed that microtubule-associated Rho guanine nucleotide exchange factor, GEF-H1, participates in TRPC3-mediated RhoA activation induced by mechanical stress in cardiomyocytes and transforming growth factor (TGF) β stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubule-associated NADPH oxidase (Nox) 2, and inhibition of Nox2 attenuated mechanical stretch-induced GEF-H1 activation in cardiomyocytes. Finally, pharmacological TRPC3 inhibition significantly suppressed fibrotic responses in human cardiomyocytes and cardiac fibroblasts. These results strongly suggest that microtubule-localized TRPC3-GEF-H1 axis mediates fibrotic responses commonly in cardiac myocytes and fibroblasts induced by physico-chemical stimulation.

Electrophysiological characterization of voltage-dependent calcium currents and TRPV4 currents in human pulmonary fibroblasts

We have presented indirect evidence of a key role for voltage-dependent Ca(2+) currents in TGFβ-induced synthetic function in human pulmonary fibroblast (HPF), as well as in bleomycin-induced pulmonary fibrosis in mice. Others, however, have provided indirect evidence for transient receptor potential vanilloid 4 (TRPV4) channels in both of those effects. Unfortunately, definitive electrophysiological descriptions of both currents in HPFs have been entirely lacking. In this study, we provide the first direct electrophysiological and pharmacological evidence of the currents in HPFs at rest and during overnight stimulation with TGFβ. These currents include a Ca(2+)-dependent K(+) current, a TRPV4 current, a chloride current, and an L-type voltage-dependent Ca(2+) current. Evidence for the TRPV4 current include activation of a large-conductance change by two putatively TRPV4-selective agonists (4α-phorbol-12,13-didecanoate; GSK1016790A), with a reversal potential near 0 mV, partial sensitivity to two different TRPV4-selective blockers (RN1734; HC067047), and partial reduction following removal of external Na(+) Substantial reduction of the evoked current was seen following the coapplication of RN1734, DIDS, and niflumic acid, suggesting that a chloride current is also involved. The voltage-dependent Ca(2+) current is found to be "L-type" in nature, as indicated by the voltage and time dependence of its activation, deactivation, and inactivation properties, and by its pharmacology (sensitivity to replacement with barium and inhibition by nifedipine, verapamil, or mibefradil). We also found that overnight treatment with TGFβ evoked a periodic current (inward at negative holding potentials, with reversal potential near 0 mV), which is sufficient to trigger the voltage-dependent Ca(2+) currents and, thereby, account for the rhythmic Ca(2+) oscillations, which we have described previously in these cells.

Synchronization of Local Calcium Releases (LCRs) in Guinea Pig Single, Isolated SA Node Cells Contributes to Generation of Rhythmic Action Potential-Induced Ca2+ Transients

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

The end effector of circadian heart rate variation: the sinoatrial node pacemaker cell.

Cardiovascular function is regulated by the rhythmicity of circadian, infradian and ultradian clocks. Specific time scales of different cell types drive their functions: circadian gene regulation at hours scale, activation-inactivation cycles of ion channels at millisecond scales, the heart's beating rate at hundreds of millisecond scales, and low frequency autonomic signaling at cycles of tens of seconds. Heart rate and rhythm are modulated by a hierarchical clock system: autonomic signaling from the brain releases neurotransmitters from the vagus and sympathetic nerves to the heart’s pacemaker cells and activate receptors on the cell. These receptors activating ultradian clock functions embedded within pacemaker cells include sarcoplasmic reticulum rhythmic spontaneous Ca2+ cycling, rhythmic ion channel current activation and inactivation, and rhythmic oscillatory mitochondria ATP production. Here we summarize the evidence that intrinsic pacemaker cell mechanisms are the end effector of the hierarchical brain-heart circadian clock system. [BMB Reports 2015; 48(12): 677-684]

STIM1–Ca2+ signaling modulates automaticity of the mouse sinoatrial node

Cardiac pacemaking is governed by specialized cardiomyocytes located in the sinoatrial node (SAN). SAN cells (SANCs) integrate voltage-gated currents from channels on the membrane surface (membrane clock) with rhythmic Ca(2+) release from internal Ca(2+) stores (Ca(2+) clock) to adjust heart rate to meet hemodynamic demand. Here, we report that stromal interaction molecule 1 (STIM1) and Orai1 channels, key components of store-operated Ca(2+) entry, are selectively expressed in SANCs. Cardiac-specific deletion of STIM1 in mice resulted in depletion of sarcoplasmic reticulum (SR) Ca(2+) stores of SANCs and led to SAN dysfunction, as was evident by a reduction in heart rate, sinus arrest, and an exaggerated autonomic response to cholinergic signaling. Moreover, STIM1 influenced SAN function by regulating ionic fluxes in SANCs, including activation of a store-operated Ca(2+) current, a reduction in L-type Ca(2+) current, and enhancing the activities of Na(+)/Ca(2+) exchanger. In conclusion, these studies reveal that STIM1 is a multifunctional regulator of Ca(2+) dynamics in SANCs that links SR Ca(2+) store content with electrical events occurring in the plasma membrane, thereby contributing to automaticity of the SAN.

Sarcoplasmic reticulum Ca2 + cycling protein phosphorylation in a physiologic Ca2 + milieu unleashes a high-power, rhythmic Ca2 + clock in ventricular myocytes: Relevance to arrhythmias and bio-pacemaker design

Basal phosphorylation of sarcoplasmic reticulum (SR) Ca2+ proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca2+ releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca2+ sparks. We tested the hypothesis that in a physiologic Ca2+ milieu, and independent of increased Ca2+ influx, an increase in basal phosphorylation of SR Ca2+ cycling proteins will convert stochastic Ca2+ sparks into periodic, high-power Ca2+ signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca2+ release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca2+], prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca2+ releases of low power and low amplitude, as assessed by confocal Ca2+ imaging and spectral analysis. When the kinetics of Ca2+ pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, “clock-like” local Ca2+ releases, partially synchronized in space and time (Ca2+ wavelets), emerged, and the ensemble of these rhythmic local Ca2+ wavelets generated a periodic high-amplitude Ca2+ signal. Thus, a Ca2+ clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca2+ cycling increases and spontaneous local Ca2+ release becomes partially synchronized. This unleashed Ca2+ clock that emerges in a physiological Ca2+ milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature of novel bio-pacemaker designs.

Rhythmic Ca2+ Signaling: Keeping Time with MicroRNAs

Pacemaker cells are specialized cell types that drive biological rhythms like the heartbeat and intestinal peristalsis. What determines whether a cell functions as a pacemaker? Studies in Caenorhabditis elegans suggest that pacemaking activity may be controlled in part by microRNAs.

Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts

Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell origins are similar. However, MSC-derived hiPSCs revealed a higher cardiac differentiation efficiency than keratinocyte- and fibroblast-derived hiPSCs.

Rhythmic dendritic Ca2+ oscillations in thalamocortical neurons during slow non‐REM sleep‐related activity in vitro

The distribution of T-type Ca2+ channels along the entire somatodendritic axis of sensory thalamocortical (TC) neurons permits regenerative propagation of low threshold spikes (LTS) accompanied by global dendritic Ca2+ influx. Furthermore, T-type Ca2+ channels play an integral role in low frequency oscillatory activity (<1–4 Hz) that is a defining feature of TC neurons. Nonetheless, the dynamics of T-type Ca2+ channel-dependent dendritic Ca2+ signalling during slow sleep-associated oscillations remains unknown. Here we demonstrate using patch clamp recording and two-photon Ca2+ imaging of dendrites from cat TC neurons undergoing spontaneous slow oscillatory activity that somatically recorded δ (1–4 Hz) and slow (<1 Hz) oscillations are associated with rhythmic and sustained global oscillations in dendritic Ca2+. In addition, our data reveal the presence of LTS-dependent Ca2+ transients (Δ[Ca2+]) in dendritic spine-like structures on proximal TC neuron dendrites during slow (<1 Hz) oscillations whose amplitudes are similar to those observed in the dendritic shaft. We find that the amplitude of oscillation associated Δ[Ca2+] do not vary significantly with distance from the soma whereas the decay time constant (τdecay) of Δ[Ca2+] decreases significantly in more distal dendrites. Furthermore, τdecay of dendritic Δ[Ca2+] increases significantly as oscillation frequency decreases from δ to slow frequencies where pronounced depolarised UP states are observed. Such rhythmic dendritic Ca2+ entry in TC neurons during sleep-related firing patterns could be an important factor in maintaining the oscillatory activity and associated biochemical signalling processes, such as synaptic downscaling, that occur in non-REM sleep.

An Inherent Ability of Ventricular Myocytes to Self-Organize Spontaneous RyRs Activation that Generate Synchronized Local Ca2+ Releases is Physiologically Suppressed by Constitutive Phosphodiesterase and Protein Phosphatase Activity

Stochastic, spontaneous, local RyRs activation underlies basal “Ca2+ sparks” in ventricular myocytes (VM). We have previously shown that addition of exogenous cAMP to saponin-permeabilized rabbit VM synchronizes stochastic Ca2+ sparks into rhythmic local Ca2+ releases. We hypothesized that in the basal state VM have an intrinsic ability to self-organize spontaneous RyRs activation and synchronize Ca2+ releases but this self-organization is suppressed by endogenous phosphodiesterase (PDE) and protein phosphatase (PP) activity, permitting only the rare and stochastic occurrence of Ca2+ sparks. We found that incubation of saponin-permeabilized VM (confocal imaging of Fluo-4 saltfluorescence) with a PP inhibitor (CyA 0.5 μM) or/and PDE inhibitor (IBMX 10μM) converts stochastic sparks into periodic, spatially synchronized local Ca2+ releases (Table). Moreover, the combination PP and PDE inhibitors produced rhythmic, local Ca2+ releases with a shorter period than that produced by either of inhibitors alone. Thus, VM have an inherent capacity to self-organize spontaneous basal RyR activation and synchronize Ca2+ release, but this is physiologically suppressed by constitutive PDE and PP activity.

Synchronization of Local Calcium Releases by Beta-Adrenergic Stimulation in Cardiac Pacemaker Cells

Sympathetic heart rate acceleration is a complex orchestration of intercellular, subcellular and molecular events. According to a modern concepts, rhythmic local Ca2+releases (LCRs), generated by a subcellular “Ca-clock”, interact with a “membrane-voltage-clock”, forming a coupled-clock system that produces rhythmic action potentials (AP). We imaged LCRs using a fast-2D-camera with simultaneous AP recording via perforated-patch-clamping to explore how β-AR stimulation modulates the phase and synchronization of the ensemble of individual LCRs within a given diastole to accelerate spontaneous beating rate. In single rabbit SA-node cells (36oC) isoproterenol (100nM): (i) synchronized LCR occurrence [the standard deviation of individual LCR periods (time from Ca2+transient peak to LCR occurrence) decreased (−30%; baseline,71.5ms vs. ISO,50ms)], (ii) shifted the LCR phases to earlier time-point, (iii) shifted “Ca-nadir”, i.e. time-point when AP-induced Ca2+-transient decay ceases and [Ca2+] begins to increase due to the occurrence of LCRs (time from maximum-diastolic-potential to Ca-nadir: baseline, 103ms vs. ISO,86ms); (iv) accelerated diastolic depolarization (baseline,0.09mV/ms vs. ISO,0.15mV/ms); (v) shortened spontaneous cycle length (baseline,420ms vs. ISO,345ms). We previously had demonstrated a close relationship between the decay of the global AP-induced Ca2+ transient and the LCR phase (within a given cycle) and the AP cycle length; we now found that the decays of each LOCAL Ca2+Transient (LCaT) induced by the prior AP vary in different subcellular locations/neighborhoods and that isoproterenol not only accelerated LCaT-decays (average time constant τ: baseline, 104 vs. ISO,87ms) but also synchronized LCaT-decays among neighborhoods (τ's SD/mean: baseline,40.5% vs. ISO,33.7%). More synchronous LCaT-decay mirrors more synchronous local SR Ca2+-pumping and therefore more synchronous achievement of the threshold SR Ca load required for spontaneous LCR occurrence. Thus, earlier and more synchronized LCR occurrence under β-AR stimulation insures an earlier and larger Na+/Ca2+-exchanger current that ultimately accelerates diastolic depolarization rate.

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity Modulates Sinoatrial Nodal Pacemaker Cell Energetics

In rabbit sinoatrial node cells (SANC), high basal (i.e., without β-adrenergic receptor stimulation) Ca2+ activated adenylate cyclase (AC) via cAMP/PKA-CaMKII-dependent protein phosphorylation guarantees the occurrence of rhythmic intracellular Ca2+ release from sarcoplasmic-reticulum which drives spontaneous action potentials (APs). This high-throughput signaling consumes ATP. We have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca2+ indirectly, not only consume ATP, but also regulate mitochondrial ATP production. A role for CaMKII in regulation of SANC ATP supply has not been explored. Based upon its involvement in the aforementioned phosphorylation signaling cascade we tested the hypotheses that, basal CaMKII activity not only regulates ATP consumption, but also ATP production. We superfused single, isolated rabbit SANC or SANC suspensions with CaMKII inhibitors (KN-93 or autocamtide-2 Related Inhibitory Peptide (AIP)) and measured cytosolic Ca2+, cAMP, energetic indices and spontaneous AP firing rate. A partial reduction in basal CaMKII activity by KN-93 (0.5 μmol/L) or AIP (2 μmol/L) markedly slowed the kinetics of intracellular Ca2+ cycling and decreased the spontaneous AP firing rate. Further suppression of CaMKII activity (KN-93 3 μmol/L; AIP 10 μmol/L) to a level that eliminates all spontaneous APs, decreased the cAMP level and reduced O2 consumption and flavoprotein fluorescence. ATP was depleted,even though the ATP demand decreased. The structural inactive analog of KN-93, KN-92, neither decreased ATP demand (no effect on spontaneous AP firing rate) nor ATP turnover. Therefore,CaMKII signaling is required not only to drive normal automaticity in rabbit SANC, but is also tightly linked to SANC bioenergetics. Future studies are required to determine whether this link between CaMKII signaling and mitochondrial energetics occurs indirectly, via changes in Ca2+ release that affects activation of Ca2+-AC-cAMP/PKA signaling, or directly, via phosphorylation of mitochondrial proteins.

How Ca2+-Activated, cAMP/PKA-Dependent Phosphorylation Signaling Mediates Pacemaker Cell Activity: Experimental and In Silico Biochemical and Biophysics Perspectives

Ca2+-activated, adenylate cyclase (AC)-cAMP/PKA-dependent phosphorylation of both surface membrane electrogenic proteins of (“Membrane-clock”), and of intracellular proteins that generate rhythmic Ca2+ oscillations (“Ca2+-clock”), regulate the periodicity of each clock, and couple the two-clocks to regulate sinoatrial node cells (SANCs) normal automaticity.We developed a novel numerical model to simulate the coupling of SANC Ca2+-AC-cAMP/PKA signaling to functions of surface membrane and Ca2+ cycling molecules. The model incorporates experimentally measured a term for Ca2+-dependent AC-activity, and when Ca2+ changes predicts resulting changes in downstream cAMP/PKA phosphorylation-dependent signaling that produce changes in ion channels conductance and intracellular Ca2+ kinetics that ultimately change the spontaneous action potential (AP) firing rate.Model predictions of Ca2+-dependent changes in the cAMP/PKA phosphorylation cascade, of spontaneous AP firing rate and of the stoichiometry-relationships between cAMP, phospholamban phosphorylation and AP firing rate (line in the figure) faithfully reproduced the experimentally measured variables and their stoichiometry (points in the figure).The simulations of this novel integrative model of biochemical and biophysical signaling within the coupled-clock model further support the importance of high throughput signaling via Ca2+-AC-cAMP-PKA phosphorylation cascade in normal SANC automaticity.View Large Image | View Hi-Res Image | Download PowerPoint Slide

High density cultures of embryoid bodies enhanced cardiac differentiation of murine embryonic stem cells

Murine embryonic stem cell (mESC)-derived cardiomyocytes represent a promising source of cells for use in the development of models for studying early cardiac development as well as cell-based therapies in postnatal pathologies. Here, we report a highly efficient cardiac differentiation system in which high density embryoid body (EB) cultures leads to a marked increase of cardiomyocytes production from multiple mESC lines without the addition of any cardiogenic growth factors. Our results show that high density EB cultures significantly increase the yield of functional cardiomyocytes, which express typical cardiac markers, exhibit normal rhythmic Ca 2+ transients, and respond to both β-adrenergic and electric stimulations. During the differentiation period, the inhibition of bone morphogenetic protein (BMP) signaling significantly attenuates the increase of cardiac differentiation as well as the increased expression of cardiac-specific genes, NK2 transcription factor related 5 ( Nkx2. 5) and myosin light chain 2v ( Mlc2v) by high density EB cultures. Therefore, we believe that we offer a novel and efficient means of cardiomyocyte production for practical use of mESCs in cardiac regenerative medicine.

Ca 2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand

Rationale In sinoatrial node cells (SANC), Ca 2+ activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca 2+ cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca 2+ oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca 2+ to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. Objective Since feedback mechanisms link ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca 2+-cAMP/PKA dependent; and (2) due to its feed-forward nature, a decrease in flux through the Ca 2+-cAMP/PKA signaling axis will reduce the basal ATP production rate. Methods and results O 2 consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca 2+-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r 2 = 0.96), and reduced O 2 consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca 2+ flux reduced the ATP level. Conclusions Feed-forward basal Ca 2+-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca 2+-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes.

Catecholaminergic-induced arrhythmias in failing cardiomyocytes associated with human HRCS96A variant overexpression

The histidine-rich calcium binding protein (HRC) Ser96Ala polymorphism was shown to correlate with ventricular arrhythmias and sudden death only in dilated cardiomyopathy patients but not in healthy human carriers. In the present study, we assessed the molecular and cellular mechanisms underlying human arrhythmias by adenoviral expression of the human wild-type (HRC(WT)) or mutant HRC (HRC(S96A)) in adult rat ventricular cardiomyocytes. Total HRC protein was increased by ∼50% in both HRC(WT)- and HRC(S96A)-infected cells. The HRC(S96A) mutant exacerbated the inhibitory effects of HRC(WT) on the amplitude of Ca(2+) transients, prolongation of Ca(2+) decay time, and caffeine-induced sarcoplasmic reticulum Ca(2+) release. Consistent with these findings, HRC(S96A) reduced maximal sarcoplasmic reticulum calcium uptake rate to a higher extent than HRC(WT). Furthermore, the frequency of spontaneous Ca(2+) sparks, which was reduced by HRC(WT), was increased by mutant HRC(S96A) under resting conditions although there were no spontaneous Ca(2+) waves under stress conditions. However, expression of the HRC(S96A) genetic variant in cardiomyocytes from a rat model of postmyocardial infarction heart failure induced dramatic disturbances of rhythmic Ca(2+) transients. These findings indicate that the HRC Ser96Ala variant increases the propensity of arrhythmogenic Ca(2+) waves in the stressed failing heart, suggesting a link between this genetic variant and life-threatening ventricular arrhythmias in human carriers.

Small molecule Wnt inhibitors enhance the efficiency of BMP-4-directed cardiac differentiation of human pluripotent stem cells

Human induced pluripotent stem (iPS) cells potentially provide a unique resource for generating patient-specific cardiomyocytes to study cardiac disease mechanisms and treatments. However, existing approaches to cardiomyocyte production from human iPS cells are inefficient, limiting the application of iPS cells in basic and translational cardiac research. Furthermore, strategies to accurately record changes in iPS cell-derived cardiomyocyte action potential duration (APD) are needed to monitor APD-related cardiac disease and for rapid drug screening. We examined whether modulation of the bone morphogenetic protein 4 (BMP-4) and Wnt/β-catenin signaling pathways could induce efficient cardiac differentiation of human iPS cells. We found that early treatment of human iPS cells with BMP-4 followed by late treatment with small molecule Wnt inhibitors led to a marked increase in production of cardiomyocytes compared to existing differentiation strategies. Using immunocytochemical staining and real-time intracellular calcium imaging, we showed that these induced cardiomyocytes expressed typical sarcomeric markers, exhibited normal rhythmic Ca 2+ transients, and responded to both β-adrenergic and electric stimulation. Furthermore, human iPS cell-derived cardiomyocytes demonstrated characteristic changes in action potential duration in response to cardioactive drugs procainamide and verapamil using voltage-sensitive dye-based optical recording. Thus, modulation of the BMP-4 and Wnt signaling pathways in human iPS cells leads to highly efficient production of cardiomyocytes with typical electrophysiological function and pharmacologic responsiveness. The use of human iPS cell-derived cardiomyocytes and the application of calcium- and voltage-sensitive dyes for the direct, rapid measurement of iPS cell-derived cardiomyocyte activity promise to offer attractive platforms for studying cardiac disease mechanisms and therapeutics.

Identification of a Chemical Supressor of Cardiac Arrhythmia Induced by Aberrant Calcium Homeostasis

Ca2+ homeostasis is essential for rhythmic contractions of the adult myocardium. In the developing embryo, a role of Ca2+ in establishing cardiac rhythmicity has been proposed, but the underlying mechanism has yet to be fully explored. We have previously shown that zebrafish tremblor mutant embryos develop a fibrillating heart and that the tremblor locus encodes a cardiac-specific isoform of the Na+/Ca2+ exchanger, NCX1h. While rhythmic Ca2+ waves were detected with each cardiac contraction in wild type hearts, only local, unsynchronized Ca2+ signals together with sporadic contractions were observed in tremblor hearts. These data suggest that loss of function of NCX1h induces cardiac fibrillation in tremblor and that tremblor mutants can serve as a model for cardiac arrhythmia induced by aberrant calcium homeostasis.From a collection of small molecules, we found a novel compound, OK-F7, that could restore rhythmic cardiac contractions in tremblor embryos at low micromolar concentrations in a reversible and dosage-dependent manner. We tested the effect of OK-F7 on isolated cardiomyocytes under Ca2+ overload conditions and found that treatment with 1 µM OK-F7 reduces the frequency of spontaneously propagating Ca2+ waves by approximately half, while 25 µM OK-F7 almost entirely abolishes Ca2+ waves. A biochemical pull-down assay using an OK-F7 affinity column isolated a mitochondrial membrane protein that selectively bound to OK-F7. These findings suggest that OK-F7 suppresses cardiac fibrillation by reducing the frequency of arrhythmogenic Ca2+ waves in Ca2+-overloaded cardiomyocytes and indicate a novel role of mitochondria in cardiac Ca2+ handling and control of rhythmicity. Further validation of the target protein and biophysical experiments confirming a role of mitochondria in the mechanism of OK-F7 action are underway and will be discussed.Supported by NIH-1R01HL096980-01A1 and FWF-J3065-B11.