Yevgeniya O. Lukyanenko

Ca2+-stimulated basal adenylyl cyclase activity localization in membrane lipid microdomains of cardiac sinoatrial nodal pacemaker cells

Spontaneous, rhythmic subsarcolemmal local Ca2+ releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca2+ releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca2+ and cAMP/PKA regulatory components of automaticity are coupled via Ca2+ activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca2+-activated AC isoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca2+ over the entire physiological Ca2+ range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca2+ cycling proteins drive sarcoplasmic reticulum Ca2+ releases, which, in turn, activate ACs. This feed forward “fail safe” system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells.

Ca2+-Dependent Phosphorylation of Ca2+ Cycling Proteins Generates Robust Rhythmic Local Ca2+ Releases in Cardiac Pacemaker Cells

Distinctive features in calcium cycling may explain the pacemaker activity of sinoatrial node cells in the heart. Setting the Beat A heartbeat requires the spontaneous firing of sinoatrial node cells followed by the contraction of ventricular myocytes. Sirenko et al. analyzed why sinoatrial node cells, rather than ventricular myocytes, are the pacemakers in the heart. Rhythmic spontaneous firing requires the spontaneous release of Ca2+ through ryanodine receptors from the sarcoplasmic reticulum, and sinoatrial node cells released more Ca2+ through ryanodine receptors in a rhythmic manner compared to ventricular myocytes, which released Ca2+ in a more random manner. The abundance and phosphorylation status of various proteins involved in Ca2+ cycling by the sarcoplasmic reticulum differed between sinoatrial node cells and ventricular myocytes, and these differences would be expected to contribute to the ability of sinoatrial node cells to release large amounts of Ca2+ from the sarcoplasmic reticulum in a rhythmic fashion. These insights may help in the design of gene- or cell-based biological pacemakers that could be used instead of electronic devices in individuals with abnormal heart rates caused by sinoatrial node dysfunctions. The spontaneous beating of the heart is governed by spontaneous firing of sinoatrial node cells, which generate action potentials due to spontaneous depolarization of the membrane potential, or diastolic depolarization. The spontaneous diastolic depolarization rate is determined by spontaneous local submembrane Ca2+ releases through ryanodine receptors (RyRs). We sought to identify specific mechanisms of intrinsic Ca2+ cycling by which sinoatrial node cells, but not ventricular myocytes, generate robust, rhythmic local Ca2+ releases. At similar physiological intracellular Ca2+ concentrations, local Ca2+ releases were large and rhythmic in permeabilized sinoatrial node cells but small and random in permeabilized ventricular myocytes. Furthermore, sinoatrial node cells spontaneously released more Ca2+ from the sarcoplasmic reticulum than did ventricular myocytes, despite comparable sarcoplasmic reticulum Ca2+ content in both cell types. This ability of sinoatrial node cells to generate larger and rhythmic local Ca2+ releases was associated with increased abundance of sarcoplasmic reticulum Ca2+-ATPase (SERCA), reduced abundance of the SERCA inhibitor phospholamban, and increased Ca2+-dependent phosphorylation of phospholamban and RyR. The increased phosphorylation of RyR in sinoatrial node cells may facilitate Ca2+ release from the sarcoplasmic reticulum, whereas Ca2+-dependent increase in phosphorylation of phospholamban relieves its inhibition of SERCA, augmenting the pumping rate of Ca2+ required to support robust, rhythmic local Ca2+ releases. The differences in Ca2+ cycling between sinoatrial node cells and ventricular myocytes provide insights into the regulation of intracellular Ca2+ cycling that drives the automaticity of sinoatrial node cells.

Ca2 +/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.

A bioluminescence method for direct measurement of phosphodiesterase activity.

We have adapted bioluminescence methods to be able to measure phosphodiesterase (PDE) activity in a one-step technique. The method employs a four-enzyme system (PDE, adenylate kinase (AK) using excess CTP instead of ATP as substrate, pyruvate kinase (PK), and firefly luciferase) to generate ATP, with measurement of the concomitant luciferase-light emission. Since AK, PK, and luciferase reactions are coupled to recur in a cyclic manner, AMP recycling maintains a constant rate of ATP formation, proportional to the steady-state AMP concentration. The cycle can be initiated by the PDE reaction that yields AMP. As long as the PDE reaction is rate limiting, the system is effectively at steady state and the bioluminescence kinetics progresses at a constant rate proportional to the PDE activity. In the absence of cAMP and PDE, low concentrations of AMP trigger the AMP cycling, which allows standardizing the system. The sensitivity of the method enables detection of <1 μU (pmol/min) of PDE activity in cell extracts containing 0.25-10 μg protein. Assays utilizing pure enzyme showed that 0.2 mM IBMX completely inhibited PDE activity. This single-step enzyme- and substrate-coupled cyclic-reaction system yields a simplified, sensitive, reproducible, and accurate method for quantifying PDE activities in small biological samples.

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 &agr;-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.

Concurrent PDE3 and PDE4 Activation Suppresses Local Ca2+ Releases (LCR) to Regulate Normal Spontaneous Firing of Sinoatrial Node Cells (SANC)

The spontaneous SANC beating rate is controlled by cAMP-mediated, PKA-dependent (cAMP/PKA) LCRs from ryanodine receptors (RyR). LCRs activate an inward Na+/Ca2+ exchange current increasing diastolic depolarization (DD) rate and spontaneous SANC firing. High basal level of cAMP in SANC is regulated by high phosphodiesterase (PDE) activity. In rat ventricular myocytes (VM) dual inhibition of both PDE3 and PDE4 synergistically increased the contraction amplitude. We studied whether PDE3 alone or concurrent (PDE3+PDE4) activation regulated spontaneous firing of rabbit SANC. Specific PDE3 inhibitor (cilostomide) or PDE4 inhibitor (rolipram) alone increased spontaneous firing (perforated patch-clamp) by only∼20% and ∼5%, respectively. But concurrent (PDE3+PDE4) inhibition increased spontaneous firing by∼48% accompanied by increases in LCR number, size and decrease in the LCR period that predicted concomitant reduction in SANC cycle length. In permeabilized SANC inhibition of PDE3 or PDE4 alone produced minor changes in LCR characteristics, while concurrent (PDE3+PDE4) inhibition markedly increased LCR size and number. Inhibition of PDE3 or PDE4 alone increased phospholamban (PLB) phosphorylation at Ser16site (marker of cAMP/PKA-dependent phosphorylation) by∼20%; inhibition of (PDE3+PDE4), however, increased PLB phosphorylation by∼110%. L-type Ca2+ current (ICa,L) provides Ca2+available for pumping into sarcoplasmic reticulum. PDE3 or PDE4 inhibition alone increased ICa,L amplitude by∼60% and∼5%, respectively, while concurrent (PDE3+PDE4) inhibition increased ICa,L by∼100%. When RyR were disabled by ryanodine (PDE3+PDE4) inhibition failed to increase SANC firing rate. Western blots revealed diverse expression of PDE subtypes in SA node and ventricle: more PDE4D was expressed in former, but more PDE3A in the latter; PDE4B was similarly expressed in both tissues. Thus, concurrent (PDE3+PDE4) activation regulates spontaneous SANC firing in a synergistic manner, by suppressing basal cAMP/PKA-dependent phosphorylation, reducing RyR Ca2+release and prolonging the LCR period and spontaneous cycle length.

Phosphodiesterase Expression Pattern and Activity Partitioning in Lipid Raft and in Non-Lipid-Raft Microenvironment in Cardiac Sinoatrial Nodal Cells

Rationale: Rhythmic, action potential firing of sinoatrial nodal cells (SANC) in the basal state requires constitutively active Ca2+/calmodulin (CaM)-activated adenylyl cyclase (AC) -generated cAMP signaling, originating within lipid-raft-rich microdomains. Neither the phosphodiesterase (PDE) expression profile, nor the relative contributions of PDE types to total PDE activity, and its partitioning in lipid-raft microdomains are known.Objective: To measure the PDE mRNA expression pattern in isolated rabbit SANC and in left ventricular cardiomyocytes (LVC), the total PDE activity in SANC and LVC lysates and in SANC lysate sucrose density gradient (SDG) fractions; and measure response of PDE activity to PDE inhibitors.Methods and results: PDE3 and PDE4 expression (RT-QPCR) was high in all cell types. The Ca2+/CaM-activated PDE1A, PDE4B, and PDE3B, respectively, were 12, 6, and 4 - fold greater in SANC than in LVC; in contrast, PDE1C, PDE3A and PDE4D, respectively, in SANC were only 14, 13 and 9% of that in LVC. Total cell lysate PDE activity did not differ between SANC and LVC. The nimodipine sensitive PDE1 accounted for 43% of the total PDE activity in SANC lysates compared to 5% in LVC. PDE activity was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor SANC microdomains.Conclusion: That Ca2+/CaM -activated PDE1A is the dominant functional PDE type in SANC suggests that the local [Ca2+] not only regulates cAMP production by AC within lipid rafts, but also regulates its degradation via PDE1A activation. The markedly higher PDE activity in lipid-raft-poor microdomains of SANC may limit cAMP diffusion away from its production sites in lipid-raft-rich microdomains, localizing a large component of cAMP signaling to lipid rafts.