ZO-1 Regulates Intercalated Disc Composition and Atrioventricular Node Conduction.

RATIONALE: ZO-1 (Zona occludens 1), encoded by the tight junction protein 1 (TJP1) gene, is a regulator of paracellular permeability in epithelia and endothelia. ZO-1 interacts with the actin cytoskeleton, gap, and adherens junction proteins and localizes to intercalated discs in cardiomyocytes. However, the contribution of ZO-1 to cardiac physiology remains poorly defined. OBJECTIVE: We aim to determine the role of ZO-1 in cardiac function. METHODS AND RESULTS: Inducible cardiomyocyte-specific Tjp1 deletion mice (Tjp1fl/fl; Myh6Cre/Esr1*) were generated by crossing the Tjp1 floxed mice and Myh6Cre/Esr1* transgenic mice. Tamoxifen-induced loss of ZO-1 led to atrioventricular (AV) block without changes in heart rate, as measured by ECG and ex vivo optical mapping. Mice with tamoxifen-induced conduction system-specific deletion of Tjp1 (Tjp1fl/fl; Hcn4CreERt2) developed AV block while tamoxifen-induced conduction system deletion of Tjp1 distal to the AV node (Tjp1fl/fl; Kcne1CreERt2) did not demonstrate conduction defects. Western blot and immunostaining analyses of AV nodes showed that ZO-1 loss decreased Cx (connexin) 40 expression and intercalated disc localization. Consistent with the mouse model study, immunohistochemical staining showed that ZO-1 is abundantly expressed in the human AV node and colocalizes with Cx40. Ventricular conduction was not altered despite decreased localization of ZO-1 and Cx43 at the ventricular intercalated disc and modestly decreased left ventricular ejection fraction, suggesting ZO-1 is differentially required for AV node and ventricular conduction. CONCLUSIONS: ZO-1 is a key protein responsible for maintaining appropriate AV node conduction through maintaining gap junction protein localization.

Electrophysiological heterogeneity of pacemaker cells in the rabbit intercaval region, including the SA node: insights from recording multiple ion currents in each cell.

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 Ca2+ pump with cyclopiazonic acid. Thereafter, in each cell, we consecutively measured the density of funny current ( If), delayed rectifier K+ current ( IK) (a surrogate of repolarization capacity), and L-type Ca2+ current ( ICa,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 If . The density of none of the currents was correlated with cell size, while ICa,L and If densities were related to baseline beating rates. If density was correlated with IK density but not with that of ICa,L. Inhibition of Ca2+ cycling had a greater beating rate slowing effect in IPCs with lower If densities. Our numerical model simulation indicated that 1) IPCs with small (or zero) If or small ICa,L can operate via a major contribution of Ca2+ clock, 2) If-Ca2+-clock interplay could be important for robust pacemaking function, and 3) coupled If- IK 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 Ca2+ cycling, which is linked to If. 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 Ca2+ current and funny current ( If) density are uncovered, along with a positive relationship between If and delayed rectifier K+ current. Links are shown between the response to Ca2+ cycling blockade and If density.

Ca(2+)/calmodulin-activated phosphodiesterase 1A is highly expressed in rabbit cardiac sinoatrial nodal cells and regulates pacemaker function.

Constitutive Ca(2+)/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP-protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca(2+)-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca(2+)/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types. We found that PDE1A protein expression was 5-fold higher in sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Additionally, total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact cells PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging), but located approximately within about 150nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca(2+) cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). We conclude that signaling via cAMP generated by Ca(2+)/CaM-activated AC in SANC lipid raft domains is limited by cAMP degradation by Ca(2+)/CaM-activated PDE1A in non-lipid raft domains. This suggests that local gradients of [Ca(2+)]-CaM or different AC and PDE1A affinity regulate both cAMP production and its degradation, and this balance determines the intensity of Ca(2+)-AC-cAMP-PKA signaling that drives SANC pacemaker function.

Real-time relationship between PKA biochemical signal network dynamics and increased action potential firing rate in heart pacemaker cells: Kinetics of PKA activation in heart pacemaker cells.

cAMP-PKA protein kinase is a key nodal signaling pathway that regulates a wide range of heart pacemaker cell functions. These functions are predicted to be involved in regulation of spontaneous action potential (AP) generation of these cells. Here we investigate if the kinetics and stoichiometry of increase in PKA activity match the increase in AP firing rate in response to ß-adrenergic receptor (ß-AR) stimulation or phosphodiesterase (PDE) inhibition, that alters the AP firing rate of heart sinoatrial pacemaker cells. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, the EC50 in response to graded increases in the intensity of ß-AR stimulation (by Isoproterenol) the magnitude of the increases in PKA activity and the spontaneous AP firing rate were similar (0.4±0.1nM vs. 0.6±0.15nM, respectively). Moreover, the kinetics (t1/2) of the increases in PKA activity and spontaneous AP firing rate in response to ß-AR stimulation or PDE inhibition were tightly linked. We characterized the system rate-limiting biochemical reactions by integrating these experimentally derived data into a mechanistic-computational model. Model simulations predicted that phospholamban phosphorylation is a potent target of the increase in PKA activity that links to increase in spontaneous AP firing rate. In summary, the kinetics and stoichiometry of increases in PKA activity in response to a physiological (ß-AR stimulation) or pharmacological (PDE inhibitor) stimuli match those of changes in the AP firing rate. Thus Ca(2+)-cAMP/PKA-dependent phosphorylation limits the rate and magnitude of increase in spontaneous AP firing rate.

Stochasticity intrinsic to coupled-clock mechanisms underlies beat-to-beat variability of spontaneous action potential firing in sinoatrial node pacemaker cells.

Recent evidence indicates that the spontaneous action potential (AP) of isolated sinoatrial node cells (SANCs) is regulated by a system of stochastic mechanisms embodied within two clocks: ryanodine receptors of the "Ca(2+) clock" within the sarcoplasmic reticulum, spontaneously activate during diastole and discharge local Ca(2+) releases (LCRs) beneath the cell surface membrane; clock crosstalk occurs as LCRs activate an inward Na(+)/Ca(2+) exchanger current (INCX), which together with If and decay of K(+) channels prompts the "M clock," the ensemble of sarcolemmal-electrogenic molecules, to generate APs. Prolongation of the average LCR period accompanies prolongation of the average AP beating interval (BI). Moreover, the prolongation of the average AP BI accompanies increased AP BI variability. We hypothesized that both the average AP BI and AP BI variability are dependent upon stochasticity of clock mechanisms reported by the variability of LCR period. We perturbed the coupled-clock system by directly inhibiting the M clock by ivabradine (IVA) or the Ca(2+) clock by cyclopiazonic acid (CPA). When either clock is perturbed by IVA (3, 10 and 30 µM), which has no direct effect on Ca(2+) cycling, or CPA (0.5 and 5 µM), which has no direct effect on the M clock ion channels, the clock system failed to achieve the basal AP BI and both AP BI and AP BI variability increased. The changes in average LCR period and its variability in response to perturbations of the coupled-clock system were correlated with changes in AP beating interval and AP beating interval variability. We conclude that the stochasticity within the coupled-clock system affects and is affected by the AP BI firing rate and rhythm via modulation of the effectiveness of clock coupling.

Endostatin and kidney fibrosis in aging: a case for antagonistic pleiotropy?

A recurring theme of a host of gerontologic studies conducted in either experimental animals or in humans is related to documenting the functional decline with age. We hypothesize that elevated circulating levels of a powerful antiangiogenic peptide, endostatin, represent one of the potent systemic causes for multiorgan microvascular rarefaction and functional decline due to fibrosis. It is possible that during the life span of an organism there is an accumulation of dormant transformed cells producing antiangiogenic substances (endostatin) that maintain the dormancy of such scattered malignant cells. The proof of this postulate cannot be obtained by physically documenting these scattered cells, and it rests exclusively on the detection of sequelae of shifted pro- and antiangiogenic balance toward the latter. Here we compared circulating levels of endostatin in young and aging mice of two different strains and showed that endostatin levels are elevated in the latter. Renal expression of endostatin increased ~5.6-fold in aging animals. This was associated with microvascular rarefaction and progressive tubulointerstitial fibrosis. In parallel, the levels of sirtuins 1 and 3 were significantly suppressed in aging mice in conjunction with the expression of markers of senescence. Treating young mice with endostatin for 28 days showed delayed recovery of circulation after femoral artery ligation and reduced patency of renal microvasculature but no fibrosis. In conclusion, the findings are consistent with the hypothesis on elevation of endostatin levels and parallel microvascular rarefaction and induction of renal fibrosis in aging mice.

New evidence for coupled clock regulation of the normal automaticity of sinoatrial nodal pacemaker cells: bradycardic effects of ivabradine are linked to suppression of intracellular Ca²âº cycling.

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.

Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand.

The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca(2+)-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca(2+) cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to ß-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca(2+) (Ca(2+)m) and an indirect effect via enhanced Ca(2+)-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca(2+) and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O2 consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O2 consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca(2+)m and cAMP increased concurrently with the increase in AP firing rate. When Ca(2+)m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca(2+)m and an increase in Ca(2+) activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level.

RGS2 overexpression or G(i) inhibition rescues the impaired PKA signaling and slow AP firing of cultured adult rabbit pacemaker cells.

Freshly isolated adult rabbit sinoatrial node cells (f-SANC) are an excellent model for studies of autonomic signaling, but are not amenable to genetic manipulation. We have developed and characterized a stable cultured rabbit SANC (c-SANC) model that is suitable for genetic manipulation to probe mechanisms of spontaneous action potential (AP) firing. After 48 h in culture, c-SANC generate stable, rhythmic APs at 34±0.5°C, at a rate that is 50% less than f-SANC. In c- vs. f-SANC: AP duration is prolonged; phosphorylation of phospholamban at Ser(16) and type2 ryanodine receptor (RyR2) at Ser(2809) are reduced; and the level of type2 regulator of G-protein signaling (RGS2), that facilitates adenylyl cyclases/cAMP/protein kinase A (PKA) via G(i) inhibition, is substantially reduced. Consistent with the interpretation that cAMP/PKA signaling becomes impaired in c-SANC, acute ß-adrenergic receptor stimulation increases phospholamban and RyR2 phosphorylation, enhances RGS2-labeling density, and accelerates the AP firing rate to the similar maximum in c- and f-SANC. Specific PKA inhibition completely inhibits all ß-adrenergic receptor effects. Adv-RGS2 infection, or pertussis toxin treatment to disable G(i)-signaling, each partially rescues the c-SANC spontaneous AP firing rate. Thus, a G(i)-dependent reduction in PKA-dependent protein phosphorylation, including that of Ca(2+) cycling proteins, reduces the spontaneous AP firing rate of c-SANC, and can be reversed by genetic or pharmacologic manipulation of PKA signaling.

The "funny" current (I(f)) inhibition by ivabradine at membrane potentials encompassing spontaneous depolarization in pacemaker cells.

Recent clinical trials have shown that ivabradine (IVA), a drug that inhibits the funny current (I(f)) in isolated sinoatrial nodal cells (SANC), decreases heart rate and reduces morbidity and mortality in patients with cardiovascular diseases. While IVA inhibits I(f), this effect has been reported at essentially unphysiological voltages, i.e., those more negative than the spontaneous diastolic depolarization (DD) between action potentials (APs). We tested the relative potency of IVA to block I(f) over a wide range of membrane potentials, including those that encompass DD governing to the SANC spontaneous firing rate. A clinically relevant IVA concentration of 3 µM to single, isolated rabbit SANC slowed the spontaneous AP firing rate by 15%. During voltage clamp the maximal I(f) was 18 ± 3 pA/pF (at -120 mV) and the maximal I(f) reduction by IVA was 60 ± 8% observed at -92 ± 4 mV. At the maximal diastolic depolarization (~-60 mV) I(f) amplitude was only -2.9 ± 0.4 pA/pF, and was reduced by only 41 ± 6% by IVA. Thus, I(f) amplitude and its inhibition by IVA at physiologically relevant membrane potentials are substantially less than that at unphysiological (hyperpolarized) membrane potentials. This novel finding more accurately describes how IVA affects SANC function and is of direct relevance to numerical modeling of SANC automaticity.

A remarkable adaptive paradigm of heart performance and protection emerges in response to marked cardiac-specific overexpression of ADCY8.

Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TGAC8, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TGAC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TGAC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TGAC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.

Beat-to-beat Ca(2+)-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm.

Whether intracellular Ca(2+) regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial. To directly test the hypothesis of beat-to-beat intracellular Ca(2+) regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca(2+) buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca(2+). Prior to introduction of the caged Ca(2+) buffer, spontaneous local Ca(2+) releases (LCRs) during diastolic depolarization were tightly coupled to rhythmic APs (r²=0.9). The buffer markedly prolonged the decay time (T50) and moderately reduced the amplitude of the AP-induced Ca(2+) transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca(2+) was acutely released from the caged compound by flash photolysis, intracellular Ca(2+) dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca(2+) dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca(2+) regulates normal SANC automaticity on a beat-to-beat basis.

A full range of mouse sinoatrial node AP firing rates requires protein kinase A-dependent calcium signaling.

Recent perspectives on sinoatrial nodal cell (SANC)(*) function indicate that spontaneous sarcoplasmic reticulum (SR) Ca(2+) cycling, i.e. an intracellular "Ca(2+) clock," driven by cAMP-mediated, PKA-dependent phosphorylation, interacts with an ensemble of surface membrane electrogenic molecules ("surface membrane clock") to drive SANC normal automaticity. The role of AC-cAMP-PKA-Ca(2+) signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca(2+) cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated local Ca(2+) releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca(2+)]. Mouse SANC also exhibits a high basal level of phospholamban (PLB) phosphorylation at the PKA-dependent site, Serine16. Inhibition of intrinsic PKA activity or inhibition of PDE in SANC, respectively: reduces or increases PLB phosphorylation, and markedly prolongs or reduces the LCR period; and markedly reduces or accelerates SAN spontaneous firing rate. Additionally, the increase in AP firing rate by PKA-dependent phosphorylation by ß-adrenergic receptor (ß-AR) stimulation requires normal intracellular Ca(2+) cycling, because the ß-AR chronotropic effect is markedly blunted when SR Ca(2+) cycling is disrupted. Thus, AC-cAMP-PKA-Ca(2+) signaling cascade is a major mechanism of normal automaticity in mouse SANC.

Ca2+ and Membrane Potential Transitions During Action Potentials Are Self-Similar to Each Other and to Variability of AP Firing Intervals Across the Broad Physiologic Range of AP Intervals During Autonomic Receptor Stimulation.

Ca2+ and V m 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 (V m 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 V m 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 ß-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 V m 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 V m 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.

cAMP-Dependent Signaling Restores AP Firing in Dormant SA Node Cells via Enhancement of Surface Membrane Currents and Calcium Coupling.

Action potential (AP) firing rate and rhythm of sinoatrial nodal cells (SANC) are controlled by synergy between intracellular rhythmic local Ca2+ releases (LCRs) ("Ca2+ clock") and sarcolemmal electrogenic mechanisms ("membrane clock"). However, some SANC do not fire APs (dormant SANC). Prior studies have shown that ß-adrenoceptor stimulation can restore AP firing in these cells. Here we tested whether this relates to improvement of synchronization of clock coupling. We characterized membrane potential, ion currents, Ca2+ dynamics, and phospholamban (PLB) phosphorylation, regulating Ca2+ pump in enzymatically isolated single guinea pig SANC prior to, during, and following ß-adrenoceptor stimulation (isoproterenol) or application of cell-permeant cAMP (CPT-cAMP). Phosphorylation of PLB (Serine 16) was quantified in the same cells following Ca2+ measurement. In dormant SANC LCRs were small and disorganized at baseline, membrane potential was depolarized (-38 ± 1 mV, n = 46), and ICaL, If, and IK densities were smaller vs SANC firing APs. ß-adrenoceptor stimulation or application of CPT-cAMP led to de novo spontaneous AP generation in 44 and 46% of dormant SANC, respectively. The initial response was an increase in size, rhythmicity and synchronization of LCRs, paralleled with membrane hyperpolarization and small amplitude APs (rate ∼1 Hz). During the transition to steady-state AP firing, LCR size further increased, while LCR period shortened. LCRs became more synchronized resulting in the growth of an ensemble LCR signal peaked in late diastole, culminating in AP ignition; the rate of diastolic depolarization, AP amplitude, and AP firing rate increased. ICaL, IK, and If amplitudes in dormant SANC increased in response to ß-adrenoceptor stimulation. During washout, all changes reversed in order. Total PLB was higher, but the ratio of phosphorylated PLB (Serine 16) to total PLB was lower in dormant SANC. ß-adrenoceptor stimulation increased this ratio in AP-firing cells. Thus, transition of dormant SANC to AP firing is linked to the increased functional coupling of membrane and Ca2+ clock proteins. The transition occurs via (i) an increase in cAMP-mediated phosphorylation of PLB accelerating Ca2+ pumping, (ii) increased spatiotemporal LCR synchronization, yielding a larger diastolic LCR ensemble signal resulting in an earlier increase in diastolic INCX; and (iii) increased current densities of If, ICaL, and IK.

Self-Similar Synchronization of Calcium and Membrane Potential Transitions During Action Potential Cycles Predict Heart Rate Across Species.

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 Ca2+ releases, whole cell action potential (AP)-induced Ca2+ transients, and APs were recorded in isolated mouse, guinea pig, rabbit, and human SAN cells. Ca2+-Vm kinetic parameters during phases of AP cycles from their ignition to recovery were quantified. RESULTS: Although both AP cycle lengths (APCLs) and Ca2+-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 Ca2+-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.

Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells.

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.

Mitochondrial health is enhanced in rats with higher vs. lower intrinsic exercise capacity and extended lifespan.

The intrinsic aerobic capacity of an organism is thought to play a role in aging and longevity. Maximal respiratory rate capacity, a metabolic performance measure, is one of the best predictors of cardiovascular- and all-cause mortality. Rats selectively bred for high-(HCR) vs. low-(LCR) intrinsic running-endurance capacity have up to 31% longer lifespan. We found that positive changes in indices of mitochondrial health in cardiomyocytes (respiratory reserve, maximal respiratory capacity, resistance to mitochondrial permeability transition, autophagy/mitophagy, and higher lipids-over-glucose utilization) are uniformly associated with the extended longevity in HCR vs. LCR female rats. Cross-sectional heart metabolomics revealed pathways from lipid metabolism in the heart, which were significantly enriched by a select group of strain-dependent metabolites, consistent with enhanced lipids utilization by HCR cardiomyocytes. Heart-liver-serum metabolomics further revealed shunting of lipidic substrates between the liver and heart via serum during aging. Thus, mitochondrial health in cardiomyocytes is associated with extended longevity in rats with higher intrinsic exercise capacity and, probably, these findings can be translated to other populations as predictors of outcomes of health and survival.

Basal Spontaneous Firing of Rabbit Sinoatrial Node Cells Is Regulated by Dual Activation of PDEs (Phosphodiesterases) 3 and 4.

BACKGROUND: Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na+/Ca2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown. METHODS: Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking. RESULTS: PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% (P<0.05) and ≈5% (P>0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% (P<0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca2+ current (ICa,L) by ≈60% (P<0.01) or ≈5% (P>0.05), respectively, and PLB phosphorylation by ≈20% (P>0.05) each, but dual PDE3+PDE4 inhibition increased ICa,L by ≈100% (P<0.01) and PLB phosphorylation by ≈110% (P<0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size (P<0.01) and reduced the SR (sarcoplasmic reticulum) Ca2+ refilling time (P<0.01) and the LCR period (time from action potential-induced Ca2+ transient to subsequent LCR; P<0.01), leading to decrease in spontaneous SANC cycle length (P<0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing. CONCLUSIONS: Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length.

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.