The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by "funny" current (If) through HCN4 channels (the "Membrane ...Clock" hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two ("Coupled Clock"). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of If in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.
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 (If) 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 If. 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.
The topology of the plasma membrane Na+/Ca2+ exchanger of cardiac muscle, NCX1, is uncertain. Biochemical analyses have indicated the presence of 9 transmembrane segments (TMSs) whereas the recent ...crystal structure of a prokaryotic homologue has 10 TMSs. The discrepancy is towards the C-terminus of the proteins where the prokaryotic homologue has an additional TMS8. To resolve this apparent disagreement, we re-assessed the topology of the C-terminal TMSs of NCX1. We examined the ability of internal or external cysteine residues in the N-terminal portion of NCX1 to crosslink with cysteine residues, of uncertain orientation, in the C-terminal portion of the protein. The results strongly support a model of NCX1 with 10 TMSs as found in the prokaryotic homologue.
Na/Ca exchange and contraction of the heart Ottolia, Michela; Torres, Natalia; Bridge, John H.B ...
Journal of molecular and cellular cardiology,
08/2013, Letnik:
61
Journal Article
Recenzirano
Odprti dostop
Abstract Sodium–calcium exchange (NCX) is the major calcium (Ca) efflux mechanism of ventricular cardiomyocytes. Consequently the exchanger plays a critical role in the regulation of cellular Ca ...content and hence contractility. Reductions in Ca efflux by the exchanger, such as those produced by elevated intracellular sodium (Na) in response to cardiac glycosides, raise sarcoplasmic reticulum (SR) Ca stores. The result is an increased Ca transient and cardiac contractility. Enhanced Ca efflux activity by the exchanger, for example during heart failure, may reduce diadic cleft Ca and excitation–contraction (EC) coupling gain. This aggravates the impaired contractility associated with SR Ca ATPase dysfunction and reduced SR Ca load in failing heart muscle. Recent data from our laboratories indicate that NCX can also impact the efficiency of EC coupling and contractility independent of SR Ca load through diadic cleft priming with Ca during the upstroke of the action potential. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”.
Plasma membrane Na(+)-Ca2+ exchange is an essential component of Ca2+ signaling pathways in several tissues. Activity is especially high in the heart where the exchanger is an important regulator of ...contractility. An expanding exchanger superfamily includes three mammalian Na(+)-Ca2+ exchanger genes and a number of alternative splicing products. New information indicates that the exchanger protein has nine transmembrane segments. The exchanger, which transports Na+ and Ca2+, is also regulated by these substrates. Some molecular information is available on regulation by Na+ and Ca2+ and by PIP2 and phosphorylation. Altered expression of the exchanger in pathophysiological states may contribute to various cardiac phenotypes. Use of transgenic approaches is beginning to improve our knowledge of exchanger function.
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•We created an atrial-specific NCX1 KO mouse to study the role of NCX1 in SAN automaticity and EC coupling.•NCX1 KO SAN cells lack spontaneous action potentials, indicating that NCX1 ...is essential for operation of the calcium clock.•In NCX1 KO SAN tissue, Ca-activated SK channels cause abnormal sinus pauses.•Similar to humans, mouse atrial myocytes exhibit transverse axial tubules (TATs), but these are lost in the NCX1 KO.•In the absence of TATs, triggered Ca release occurs only near the sarcolemma in NCX1 KO atrial myocytes.
Na/Ca exchange is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. Although our knowledge of exchanger function (NCX1 in the heart) was originally established using biochemical and electrophysiological tools such as cardiac sarcolemmal vesicles and the giant patch technique 1–4, many advances in our understanding of the physiological/pathophysiological roles of NCX1 in the heart have been obtained using a suite of genetically modified mice. Early mouse studies focused on modification of expression levels of NCX1 in the ventricles, with transgenic overexpressors, global NCX1 knockout (KO) mice (which were embryonic lethal if homozygous), and finally ventricular-specific NCX1 KO 5–12. We found, to our surprise, that ventricular cardiomyocytes lacking NCX1 can survive and function by engaging a clever set of adaptations to minimize Ca entry, while maintaining contractile function through an increase in excitation-contraction (EC) coupling gain 5,6,13. Having studied ventricular NCX1 ablation in detail, we more recently focused on elucidating the role of NCX1 in the atria through altering NCX1 expression. Using a novel atrial-specific NCX1 KO mouse, we found unexpected changes in atrial cell morphology and calcium handling, together with dramatic alterations in the function of sinoatrial node (SAN) pacemaker activity. In this review, we will discuss these findings and their implications for cardiac disease.
Key points
Repolarizing currents through K+ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca2+ on repolarization in the SAN is uncertain.
We ...identified all three isoforms of Ca2+‐activated small conductance K+ (SK) channels in the murine SAN.
SK channel blockade slows repolarization and subsequent depolarization of SAN cells.
In the atrial‐specific Na+/Ca2+ exchanger (NCX) knockout mouse, cellular Ca2+ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing.
These findings suggest that Ca2+‐sensitive SK channels can translate changes in cellular Ca2+ into a repolarizing current capable of modulating pacemaking.
SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca2+.
Small conductance K+ (SK) channels have been implicated as modulators of spontaneous depolarization and electrical conduction that may be involved in cardiac arrhythmia. However, neither their presence nor their contribution to sinoatrial node (SAN) pacemaker activity has been investigated. Using quantitative PCR (q‐PCR), immunostaining and patch clamp recordings of membrane current and voltage, we identified all three SK isoforms (SK1, SK2 and SK3) in mouse SAN. Inhibition of SK channels with the specific blocker apamin prolonged action potentials (APs) in isolated SAN cells. Apamin also slowed diastolic depolarization and reduced pacemaker rate in isolated SAN cells and intact tissue. We investigated whether the Ca2+‐sensitive nature of SK channels could explain arrhythmic SAN pacemaker activity in the atrial‐specific Na+/Ca2+ exchange (NCX) knockout (KO) mouse, a model of cellular Ca2+ overload. SAN cells isolated from the NCX KO exhibited higher SK current than wildtype (WT) and apamin prolonged their APs. SK blockade partially suppressed the arrhythmic burst pacing pattern of intact NCX KO SAN tissue. We conclude that SK channels have demonstrable effects on SAN pacemaking in the mouse. Their Ca2+‐dependent activation translates changes in cellular Ca2+ into a repolarizing current capable of modulating regular pacemaking. This Ca2+ dependence also promotes abnormal automaticity when these channels are hyperactivated by elevated Ca2+. We propose SK channels as a potential target for modulating SAN rate, and for treating patients affected by SAN dysfunction, particularly in the setting of Ca2+ overload.
Key points
Repolarizing currents through K+ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca2+ on repolarization in the SAN is uncertain.
We identified all three isoforms of Ca2+‐activated small conductance K+ (SK) channels in the murine SAN.
SK channel blockade slows repolarization and subsequent depolarization of SAN cells.
In the atrial‐specific Na+/Ca2+ exchanger (NCX) knockout mouse, cellular Ca2+ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing.
These findings suggest that Ca2+‐sensitive SK channels can translate changes in cellular Ca2+ into a repolarizing current capable of modulating pacemaking.
SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca2+.
Cardiovascular disease is a leading cause of death worldwide, with ischemic heart disease alone accounting for >12% of all deaths, more than HIV/AIDS, tuberculosis, lung, and breast cancer combined. ...Heart disease has been the leading cause of death in the United States for the past 85 years and is a major cause of disability and health-care expenditures. The cardiac conditions most likely to result in death include heart failure and arrhythmias, both a consequence of ischemic coronary disease and myocardial infarction, though chronic hypertension and valvular diseases are also important causes of heart failure. Sodium-calcium exchange (NCX) is the dominant calcium (Ca2+) efflux mechanism in cardiac cells. Using ventricular-specific NCX knockout mice, we have found that NCX is also an essential regulator of cardiac contractility independent of sarcoplasmic reticulum Ca2+ load. During the upstroke of the action potential, sodium (Na+) ions enter the diadic cleft space between the sarcolemma and the sarcoplasmic reticulum. The rise in cleft Na+, in conjunction with depolarization, causes NCX to transiently reverse. Ca2+ entry by this mechanism then "primes" the diadic cleft so that subsequent Ca2+ entry through Ca2+ channels can more efficiently trigger Ca2+ release from the sarcoplasmic reticulum. In NCX knockout mice, this mechanism is inoperative (Na+ current has no effect on the Ca2+ transient), and excitation-contraction coupling relies upon the elevated diadic cleft Ca2+ that arises from the slow extrusion of cytoplasmic Ca2+ by the ATP-dependent sarcolemmal Ca2+ pump. Thus, our data support the conclusion that NCX is an important regulator of cardiac contractility. These findings suggest that manipulation of NCX may be beneficial in the treatment of heart failure.
Transverse-axial tubules (TATs) are commonly assumed to be sparse or absent in atrial myocytes from small animals. Atrial myocytes from rats, cats and rabbits lack TATs, which results in a ...characteristic “V”-shaped Ca release pattern in confocal line-scan recordings due to the delayed rise of Ca in the center of the cell. To examine TAT expression in isolated mouse atrial myocytes, we loaded them with the membrane dye Di-4-ANEPPS to label TATs. We found that >80% of atrial myocytes had identifiable TATs. Atria from male mice had a higher TAT density than female mice, and TAT density correlated with cell width. Using the fluorescent Ca indicator Fluo-4-AM and confocal imaging, we found that wild type (WT) mouse atrial myocytes generate near-synchronous Ca transients, in contrast to the “V”-shaped pattern typically reported in other small animals such as rat. In atrial-specific Na–Ca exchanger (NCX) knockout (KO) mice, which develop sinus node dysfunction and atrial hypertrophy with dilation, we found a substantial loss of atrial TATs in isolated atrial myocytes. There was a greater loss of transverse tubules compared to axial tubules, resulting in a dominance of axial tubules. Consistent with the overall loss of TATs, NCX KO atrial myocytes displayed a “V”-shaped Ca transient with slower and reduced central (CT) Ca release and uptake in comparison to subsarcolemmal (SS) Ca release. We compared chemically detubulated (DT) WT cells to KO, and found similar slowing of CT Ca release and uptake. However, SS Ca transients in the WT DT cells had faster uptake kinetics than KO cells, consistent with the presence of NCX and normal sarcolemmal Ca efflux in the WT DT cells. We conclude that the remodeling of NCX KO atrial myocytes is accompanied by a loss of TATs leading to abnormal Ca release and uptake that could impact atrial contractility and rhythm.
•Mouse atria possess an extensive transverse-axial tubule (TAT) system.•Abundant TATs in mouse atria synchronize electrically-stimulated Ca transients.•Atrial-specific Na–Ca exchanger (NCX) knockout (KO) mice lack atrial TATs.•Low TAT density in NCX KO mice leads to non-synchronized “V”-shaped Ca transients.•Pathological changes in TAT density could alter contractility and rhythm in atrium.
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