Ca2+-stimulated Basal Adenylyl Cyclase Activity Localization in Membrane Lipid Microdomains of Cardiac Sinoatrial Nodal Pacemaker Cells

In sinoatrial node (SAN) cells, electrogenic sodium-calcium exchange (NCX) is the dominant calcium (Ca) efflux mechanism. However, the role of NCX in the generation of SAN automaticity is controversial. To investigate the contribution of NCX to pacemaking in the SAN, we performed optical voltage mapping and high-speed 2D laser scanning confocal microscopy (LSCM) of Ca dynamics in an ex vivo intact SAN/atrial tissue preparation from atrial-specific NCX knockout (KO) mice. These mice lack P waves on electrocardiograms, and isolated NCX KO SAN cells are quiescent. Voltage mapping revealed disorganized and arrhythmic depolarizations within the NCX KO SAN that failed to propagate into the atria. LSCM revealed intermittent bursts of Ca transients. Bursts were accompanied by rising diastolic Ca, culminating in long pauses dominated by Ca waves. The L-type Ca channel agonist BayK8644 reduced the rate of Ca transients and inhibited burst generation in the NCX KO SAN whereas the Ca buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl ester) (BAPTA AM) did the opposite. These results suggest that cellular Ca accumulation hinders spontaneous depolarization in the NCX KO SAN, possibly by inhibiting L-type Ca currents. The funny current (I f ) blocker ivabradine also suppressed NCX KO SAN automaticity. We conclude that pacemaker activity is present in the NCX KO SAN, generated by a mechanism that depends upon I f . However, the absence of NCX-mediated depolarization in combination with impaired Ca efflux results in intermittent bursts of pacemaker activity, reminiscent of human sinus node dysfunction and "tachy-brady" syndrome.sinoatrial node | sodium-calcium exchange | pacemaker activity | arrhythmia | intracellular calcium P hysiological heart rhythm originates in the sinoatrial node (SAN), a cluster of specialized pacemaker cells located on the endocardial surface of the right atrium (RA). SAN dysfunction (SND) leads to serious arrhythmias characterized by pathological pauses, often alternating with rapid heart rates or atrial fibrillation (1). Each year in the United States, close to 200,000 patients affected with SAN disease require surgical implantation of an electronic pacemaker (2). Therefore, advances in our understanding of SAN pacemaker activity are essential for developing new therapies to avoid this costly procedure and its related morbidity.In SAN pacemaker cells, action potentials (APs) are thought to be triggered by spontaneous diastolic depolarization (SDD) produced by a coupled system of cellular "clocks" (3). The first clock, known as the "membrane clock," initiates SDD in response to inward funny current (I f ) carried mostly by HCN4 channels (4) although other ion channels, like voltage-dependent Ca channels, have also been implicated (5). The second (and more controversial) clock is referred to as the "Ca clock." This clock produces a depolarizing current in late diastole when local Ca released by ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) is extruded by the e...

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“…5A). We speculate that this increased rate may be a consequence of activation of the adenylate/cAMP pathway by Ca (35).…”

Section: Discussionmentioning

confidence: 99%

Burst pacemaker activity of the sinoatrial node in sodium–calcium exchanger knockout mice

Torrente¹,

Zhang²,

Zaini³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

109

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“…1), i.e., surface membrane electrogenic proteins, functioning as a voltage oscillator ("membrane clock"), and sarcoplasmic reticulum (SR) ( (2,19). This high-throughput Ca 2ϩ -calmodulin-activated AC/PKA-CaMKII-Ca 2ϩ release signaling cascade in pacemaker cells is regulated in a feed-forward manner (27,43) and requires physiological "brakes" to maintain the basal spontaneous action potential (AP) firing rate well below its maximum (Fig. 1).…”

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confidence: 99%

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

Yaniv

Spurgeon

Ziman

et al. 2013

American Journal of Physiology-Heart and Circulatory Physiology

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EG. Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand. Am J Physiol Heart Circ Physiol 304: H1428 -H1438, 2013. First published April 19, 2013 doi:10.1152/ajpheart.00969.2012.-The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca 2ϩ -calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca 2ϩ / calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca 2ϩ cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to ␤-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 2ϩ (Ca 2ϩ m) and an indirect effect via enhanced Ca 2ϩ -cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 Ϯ 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca 2ϩ 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 2ϩ m and cAMP increased concurrently with the increase in AP firing rate. When Ca 2ϩ 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 2ϩ m and an increase in Ca 2ϩ activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level.calcium-activated adenylyl cyclase; bioenergetics; pacemaker automaticity REGULATION OF SINOATRIAL NODE cells (SANCs) pacemaker function involves a coupled-clock system (Fig. 1) (2,19). This high-throughput Ca 2ϩ -calmodulin-activated AC/PKA-CaMKII-Ca 2ϩ release signaling cascade in pacemaker cells is regulated in a feed-forward manner (27, 43) and requires physiological "brakes" to maintain the basal spontaneous action potential (AP) firing rate well below its maximum (Fig. 1). This regulation is accomplished by basal phosphodiesterase (PDE) (15, 27, 37) and protein phosphatase activity (1, 44) (Fig. 1). ATP is required to activate AC to produce cAMP, and a significant part of ATP is consumed to pump Ca 2ϩ into the SR, to maintain Na ϩ /K ϩ pump function and to maintain cell ionic homeostasis. Our prior studies indicate that the ATP supply required to support the basal SANC firing rate is also controlled by the Ca 2ϩ -AC/PKA-CaMKII cascade (39, 41) (Fig. 1). When the AP firing rate increases, e.g., in response to ␤-adrenergic receptor (␤-AR) stimulation (see Fig. 1), ATP production must increase to...

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“…In brief, cDNA was synthesized in a 22 μL reaction per sample with 2 μg total RNA, 0.5 μg/μL oligo (dT) 12-18 , 0.5 μL 20 U RNAsin (Promega, Madison, WI, USA), 4 μL of 5× RT buffer, 5 μL of 2.5 mM dNTPs, and 20 U Moloney murine leukemia virus RT enzyme (Invitrogen). Semi-quantitative polymerase chain reaction (PCR) was performed with the TPersonal Thermocycler System from Biometra (Göttingen, Germany), all primer sequences were adopted from (Younes et al 2008) and shown in Table 1. Each reaction contained 1 μL cDNA, MgCl 2 of 3 mM final concentration in case of AC1, otherwise 2.5 mM, 2.5 mM dNTP, 5 μL 10× RT buffer, 1 pmol of each primer, and 0.25 μL 5 U A-Taq polymerase (Promega, Madison, WI, USA).…”

Section: Methodsmentioning

confidence: 99%

Pharmacological characterization of adenylyl cyclase isoforms in rabbit kidney membranes

Erdorf

Seifert

2011

Naunyn-Schmied Arch Pharmacol

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Polycystic kidney disease (PKD) is the most common life-threatening genetic disorder with bilateral cysts caused by increased level of cyclic adenosine 3',5'-monophosphate (cAMP). Since adenylyl cyclases (ACs) catalyze cAMP formation, pharmacological characterization of renal AC isoforms is essential. Therefore, we analyzed differences in activation, inhibition, and regulation of AC isoforms in rabbit cortex and medulla membranes. Glucagon, [8-arginine]vasopressin (AVP) and catecholamines significantly activated cortical AC. However, in medulla only glucagon and AVP activated AC. Under Mg(2+) conditions the profile of cortical membrane AC enzyme kinetics and the inhibitory profile of 2'(3')-O-(N-methylanthraniloyl) (MANT) nucleotides resembled recombinant AC5. In contrast, the K (i) values of MANT nucleotides for medullary membrane AC and its kinetic properties were similar to those of recombinant AC1. Reverse-transcriptase PCR confirmed the presence of AC1 and AC5 in medulla and cortex, respectively. Cortical AC was sensitive to inhibition by Ca(2+), corroborating the importance of AC5. However, Ca(2+)/CaM dependency specific for AC1 was not found in medulla. In conclusion, according to expression, kinetics and inhibition by MANT nucleotides both parts of the kidney differ in their AC isoforms. Whereas Ca(2+)-inhibitable AC5 was confirmed in renal cortex, the initially assumed AC1 activation in medulla could not be confirmed, pointing to the involvement of another AC isoform with some similarity to AC1. Since PKD is characterized by predominant involvement of the collecting duct and the distal nephrons located in renal cortex, AC5 may be the major AC isoform in this part of the kidney where cAMP increases cyst growth. Thus, potent and selective AC5 inhibitors could constitute a novel approach to treat PKD.

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Ca2+-stimulated Basal Adenylyl Cyclase Activity Localization in Membrane Lipid Microdomains of Cardiac Sinoatrial Nodal Pacemaker Cells

Cited by 97 publications

References 43 publications

Burst pacemaker activity of the sinoatrial node in sodium–calcium exchanger knockout mice

Burst pacemaker activity of the sinoatrial node in sodium–calcium exchanger knockout mice

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

Pharmacological characterization of adenylyl cyclase isoforms in rabbit kidney membranes

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