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 Ca/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca 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).

Ca 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.

Ca and 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 (Ca domain) and limit-cycle ( 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 Ca and 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 -species AP firing intervals (APFIs) of these cells , to heart rate , and to body mass.

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.

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 Ca releases (LCRs) ("Ca clock") and sarcolemmal electrogenic mechanisms ("membrane clock"). However, some SANC do not fire APs (dormant SANC). Prior studies have shown that β-adrenoceptor stimulation can restore AP firing in these cells.

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

Rationale: ZO-1 (Zona occludens 1), encoded by the tight junction protein 1 () 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.

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.

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

The spontaneous rhythmic action potentials generated by the sinoatrial node (SAN), the primary pacemaker in the heart, dictate the regular and optimal cardiac contractions that pump blood around the body. Although the heart rate of humans is substantially slower than that of smaller experimental animals, current perspectives on the biophysical mechanisms underlying the automaticity of sinoatrial nodal pacemaker cells (SANCs) have been gleaned largely from studies of animal hearts. Using human SANCs, we demonstrated that spontaneous rhythmic local Ca releases generated by a Ca clock were coupled to electrogenic surface membrane molecules (the M clock) to trigger rhythmic action potentials, and that Ca-cAMP-protein kinase A (PKA) signaling regulated clock coupling.

Mammalian γ2 AMPK regulates intrinsic heart rate.

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αβγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia).

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 β-adrenergic receptor (β-AR) stimulation or phosphodiesterase (PDE) inhibition, that alters the AP firing rate of heart sinoatrial pacemaker cells.

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.

Synchronization of sinoatrial node pacemaker cell clocks and its autonomic modulation impart complexity to heart beating intervals.

Background: A reduction of complexity of heart beating interval variability that is associated with an increased morbidity and mortality in cardiovascular disease states is thought to derive from the balance of sympathetic and parasympathetic neural impulses to the heart. However, rhythmic clocklike behavior intrinsic to pacemaker cells in the sinoatrial node (SAN) drives their beating, even in the absence of autonomic neural input.

Objective: To test how this rhythmic clocklike behavior intrinsic to pacemaker cells interacts with autonomic impulses to the heart beating interval variability in vivo.

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 Ca²⁺ 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.

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.

Ca²+/calmodulin-dependent protein kinase II (CaMKII) activity and sinoatrial nodal pacemaker cell energetics.

Unlabelled: : Ca(2+)-activated basal adenylate cyclase (AC) in rabbit sinoatrial node cells (SANC) guarantees, via basal cAMP/PKA-calmodulin/CaMKII-dependent protein phosphorylation, the occurrence of rhythmic, sarcoplasmic-reticulum generated, sub-membrane Ca(2+) releases that prompt rhythmic, spontaneous action potentials (APs). This high-throughput signaling consumes ATP.

Aims: We have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca(2+) indirectly, regulate mitochondrial ATP production.

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.

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.

Crosstalk between mitochondrial and sarcoplasmic reticulum Ca2+ cycling modulates cardiac pacemaker cell automaticity.

Background: Mitochondria dynamically buffer cytosolic Ca(2+) in cardiac ventricular cells and this affects the Ca(2+) load of the sarcoplasmic reticulum (SR). In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca(2+) releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+)-Ca(2+) exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP).

Objective: To determine if mitochondrial Ca(2+) (Ca(2+) (m)), cytosolic Ca(2+) (Ca(2+) (c))-SR-Ca(2+) crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.

Glycogen synthase kinase-3beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore.

Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria.