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Rationale: Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1 (Islet-1), Tbx3 (T-box transcription factor 3), and Shox2 (short-stature homeobox protein 2), have been identified, the cis -regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified. Objective: To define the epigenetic profile of PCs using comparative ATAC-seq (assay for transposase-accessible chromatin with sequencing) and to identify novel enhancers involved in SAN gene regulation, development, and function. Methods and Results: We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched assay for transposase-accessible chromatin peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF (transcription factor) binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at embryonic day 8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations. Conclusions: (1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) cis -regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human ISL1 enhancer may regulate human SAN function.

Pacemaking is a basic physiological process, and the cellular mechanisms involved in this function have always attracted the keen attention of investigators. The “funny” ( I f) current, originally described in sinoatrial node myocytes as an inward current activated on hyperpolarization to the diastolic range of voltages, has properties suitable for generating repetitive activity and for modulating spontaneous rate. The degree of activation of the funny current determines, at the end of an action potential, the steepness of phase 4 depolarization; hence, the frequency of action potential firing. Because I f is controlled by intracellular cAMP and is thus activated and inhibited by β-adrenergic and muscarinic M2 receptor stimulation, respectively, it represents a basic physiological mechanism mediating autonomic regulation of heart rate. Given the complexity of the cellular processes involved in rhythmic activity, an exact quantification of the extent to which I f and other mechanisms contribute to pacemaking is still a debated issue; nonetheless, a wealth of information collected since the current was first described more than 30 years ago clearly agrees to identify I f as a major player in both generation of spontaneous activity and rate control. I f- dependent pacemaking has recently advanced from a basic, physiologically relevant concept, as originally described, to a practical concept that has several potentially useful clinical applications and can be valuable in therapeutically relevant conditions. Typically, given their exclusive role in pacemaking, f-channels are ideal targets of drugs aiming to pharmacological control of cardiac rate. Molecules able to bind specifically to and block f-channels can thus be used as pharmacological tools for heart rate reduction with little or no adverse cardiovascular side effects. Indeed a selective f-channel inhibitor, ivabradine, is today commercially available as a tool in the treatment of stable chronic angina. Also, several loss-of-function mutations of HCN4 (hyperpolarization-activated, cyclic-nucleotide gated 4), the major constitutive subunit of f-channels in pacemaker cells, are known today to cause rhythm disturbances, such as for example inherited sinus bradycardia. Finally, gene- or cell-based methods for in situ delivery of f-channels to silent or defective cardiac muscle represent novel approaches for the development of biological pacemakers eventually able to replace electronic devices. This article is the introduction of a new thematic series on Mechanisms of Pacemaking in the Heart , which includes the following articles: Be Still, My Beating Heart – Never! [2010;106:238–239] Development of the Pacemaker Tissues of The Heart [2010;106:240–254] Mapping Cardiac Pacemaker Circuits: Methodological Puzzles of the Sinoatrial Node Optical Mapping [2010;106:255–271] The Role of The Funny Current in Pacemaker Activity Ca2+ Cycling in the Mechanism of Pacemaking Cardiac Pacemaking: Historical Overview and Future Directions Dennis Noble Guest Editor and Brian O'Rourke Editor

Abstract : Historically, milestones in science are usually associated with methodological breakthroughs. Likewise, the advent of electrocardiography, microelectrode recordings and more recently optical mapping have ushered in new periods of significance of advancement in elucidating basic mechanisms in cardiac electrophysiology. As with any novel technique, however, data interpretation is challenging and should be approached with caution, as it cannot be simply extrapolated from previously used methodologies and with experience and time eventually becomes validated. A good example of this is the use of optical mapping in the sinoatrial node (SAN): when microelectrode and optical recordings are obtained from the same site in myocardium, significantly different results may be noted with respect to signal morphology and as a result have to be interpreted by a different set of principles. Given the rapid spread of the use of optical mapping, careful evaluation must be made in terms of methodology with respect to interpretation of data gathered by optical sensors from fluorescent potential-sensitive dyes. Different interpretations of experimental data may lead to different mechanistic conclusions. This review attempts to address the origin and interpretation of the “double component” morphology in the optical action potentials obtained from the SAN region. One view is that these 2 components represent distinctive signals from the SAN and atrial cells and can be fully separated with signal processing. A second view is that the first component preceding the phase 0 activation represents the membrane currents and intracellular calcium transients induced diastolic depolarization from the SAN. Although the consensus from both groups is that ionic mechanisms, namely the joint action of the membrane and calcium automaticity, are important in the SAN function, it is unresolved whether the double-component originates from the recording methodology or represents the underlying physiology. This overview aims to advance a common understanding of the basic principles of optical mapping in complex 3D anatomic structures.

Background: Bradyarrhythmias occur more frequently at night. On the basis of heart rate variability this is attributed to high vagal tone. Here we tested the alternative hypothesis that an intrinsic circadian clock-driven remodelling of pacemaking ion channels underlies fluctuations in heart rate (HR). Methods and results: The occurrence of a circadian rhythm in HR was tested by placing nocturnal C57BL6/J mice under a strict 12/12h light-dark cycle and telemetry-based ECG intervals measured every 2 h for 48 h. Under these conditions, the R-R interval was rhythmic (n=10). To test whether this is caused by circadian rhythms in the expression of ion channels controlling HR, sinus node (SAN) biopsies were collected at time points corresponding to the minima (ZT0, subjective day) and maxima (ZT12, subjective night) of HR, as determined by ECG recordings. Real-time PCR normalised to 28s demonstrated an elevated expression of the key pacemaking ion channel HCN4 that carries the pacemaker current I f and genes encoding the Ca 2+ -handling proteins SERCA2a and RYR2 at ZT12 ( P mPer1 Luc mice which carry the 5’ upstream region of the mPer1 gene (a key core clock component) fused to a luciferase gene. mPer1-luc bioluminescence was recorded in the isolated SAN using a light-tight photomultiplier tube assembly to reveal a circadian rhythm with a periodicity of 24 h (n=3). Disruption of the molecular clock by global knockout of core clock components Cry1 and Cry2 abrogated the circadian cycling of mPer1-luc in the SN of Cry1 -/- /Cry2 -/- double knockout mice (n=3). Examination of 10[[Unable to Display Character: ]]kb of the Hcn4 promoter revealed a conserved consensus binding site for CLOCK and its heterodimer BMAL1, other essential transcription factors involved in setting intrinsic circadian rhythms. Conclusions: This is the first demonstration of a peripheral circadian clock in the cardiac pacemaker and circadian oscillations in key pacemaker mechanisms. Data reveal a novel regulator of SN function and the occurrence of bradyarrythmias at night.

Abstract —Cardiac beating arises from the spontaneous rhythmic excitation of sinoatrial (SA) node cells. Here we report that SA node pacemaker activity is critically dependent on Ca 2+ /calmodulin-dependent protein kinase II (CaMKII). In freshly dissociated rabbit single SA node cells, inhibition of CaMKII by a specific peptide inhibitor, autocamtide-2 inhibitory peptide (AIP, 10 μmol/L), or by KN-93 (0.1 to 3.0 μmol/L), but not its inactive analog, KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 10 μmol/L AIP and 3 μmol/L KN-93 completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP. To understand the ionic mechanisms of the CaMKII effects, we measured L-type Ca 2+ current ( I Ca, L ), which contributes both to AP upstroke and to pacemaker depolarization. KN-93 (1 μmol/L), but not its inactive analog, KN-92, decreased I Ca, L amplitude from 12±2 to 6±1 pA/pF without altering the shape of the current-voltage relationship. Both AIP and KN-93 shifted the midpoint of the steady-state inactivation curve leftward and markedly slowed the recovery of I Ca, L from inactivation. Similar results were observed using the fast Ca 2+ chelator BAPTA, whereas the slow Ca 2+ chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca 2+ transients. Indeed, confocal immunocytochemical imaging showed that active CaMKII is highly localized beneath the surface membrane in the vicinity of L-type channels and that AIP and KN-93 significantly reduced CaMKII activity. Thus, we conclude that CaMKII plays a vital role in regulating cardiac pacemaker activity mainly via modulating I Ca, L inactivation and reactivation, and local Ca 2+ is critically involved in these processes.

Abstract —Several time- and voltage-dependent ionic currents have been identified in cardiac pacemaker cells, including Na + current, L- and T-type Ca 2+ currents, hyperpolarization-activated cation current, and various types of delayed rectifier K + currents. Mathematical models have demonstrated that spontaneous action potentials can be reconstructed by incorporating these currents, but relative contributions of individual currents vary widely between different models. In 1995, the presence of a novel inward current that was activated by depolarization to the potential range of the slow diastolic depolarization in rabbit sinoatrial (SA) node cells was reported. Because the current showed little inactivation during depolarizing pulses, it was called the sustained inward current ( I st ). A similar current is also found in SA node cells of the guinea pig and rat and in subsidiary pacemaker atrioventricular node cells. Recently, single-channel analysis has revealed a nicardipine-sensitive, 13-pS Na + current, which is activated by depolarization to the diastolic potential range in guinea pig SA node cells. This channel differs from rapid voltage-gated Na + or L-type Ca 2+ channels both in unitary conductance and gating kinetics. Because I st was observed only in spontaneously beating SA node cells, ie, it was absent in quiescent cells dissociated from the same SA or atrioventricular node, an important role of I st for generation of intrinsic cardiac automaticity was suggested. (Circ Res. 2000;87:88-91.)

Rationale: A recent study published in Circulation Research by Gao et al used sinoatrial node (SAN)–targeted, incomplete Ncx1 knockout in mice to explore the role of the Na + /Ca 2+ exchanger (NCX) in cardiac pacemaker. The authors concluded that NCX is required for increasing sinus rates, but not for maintaining resting heart rate. This conclusion was based, in part, on numeric model simulations performed by Gao et al that reproduced their experimental results of unchanged action potentials in the knockout SAN cells. The authors, however, did not simulate the NCX current (I NCX ), that is, the subject of the study. Objective: We extended numeric examinations to simulate I NCX in their incomplete knockout SAN cells that is crucial to interpret the study results. Methods and Results: I NCX and Ca 2+ dynamics were simulated using different contemporary numeric models of SAN cells. We found that minimum diastolic Ca 2+ levels and I NCX amplitudes generated by remaining NCX molecules (only 20% of control) remained almost unchanged. Simulations using a new local Ca 2+ control model indicate that these powerful compensatory mechanisms involve complex local cross-talk of Ca 2+ cycling proteins and NCX. Specifically, lower NCX expression facilitates Ca 2+ -induced Ca 2+ release and larger local Ca 2+ releases that stabilize diastolic I NCX . Further reduction of NCX expression results in arrhythmia and halt of automaticity. Conclusions: Remaining NCX molecules in the incomplete knockout model likely produce almost the same diastolic I NCX as in wild-type cells. I NCX contribution is crucially important for both basal automaticity of SAN cells and during the fight-or-flight reflex.

Abstract : Interaction between a membrane oscillator generated by voltage-dependent ion channels and an intracellular calcium signal oscillator was present in the earliest models (1984 to 1985) using representations of the sarcoplasmic reticulum. Oscillatory release of calcium is inherent in the calcium-induced calcium release process. Those historical results fully support the synthesis proposed in the articles in this review series. The oscillator mechanisms do not primarily compete with each; they entrain each other. However, there is some asymmetry: the membrane oscillator can continue indefinitely in the absence of the calcium oscillator. The reverse seems to be true only in pathological conditions. Studies from tissue-level work and on the development of the heart also provide valuable insights into the integrative action of the cardiac pacemaker.

Abstract: Ion channels on the surface membrane of sinoatrial nodal pacemaker cells (SANCs) are the proximal cause of an action potential. Each individual channel type has been thoroughly characterized under voltage clamp, and the ensemble of the ion channel currents reconstructed in silico generates rhythmic action potentials. Thus, this ensemble can be envisioned as a surface “membrane clock” (M clock). Localized subsarcolemmal Ca 2+ releases are generated by the sarcoplasmic reticulum via ryanodine receptors during late diastolic depolarization and are referred to as an intracellular “Ca 2+ clock,” because their spontaneous occurrence is periodic during voltage clamp or in detergent-permeabilized SANCs, and in silico as well. In spontaneously firing SANCs, the M and Ca 2+ clocks do not operate in isolation but work together via numerous interactions modulated by membrane voltage, subsarcolemmal Ca 2+ , and protein kinase A and CaMKII-dependent protein phosphorylation. Through these interactions, the 2 subsystem clocks become mutually entrained to form a robust, stable, coupled-clock system that drives normal cardiac pacemaker cell automaticity. G protein–coupled receptors signaling creates pacemaker flexibility, ie, effects changes in the rhythmic action potential firing rate, by impacting on these very same factors that regulate robust basal coupled-clock system function. This review examines evidence that forms the basis of this coupled-clock system concept in cardiac SANCs.

While a diversity of cell types and distribution within the sinoatrial node and cell-cell interactions add complexity to a complete elucidation of the heart’s pacemaker function, it has become clear that cyclic variation of submembrane [Ca 2+ ] and activation of the Na + -Ca 2+ exchanger during diastolic depolarization (DD) act in concert with ion channels to confer on sinoatrial node cells (SANCs) their status of dominance with respect to pacemaker function. Studies using confocal microscopy indicate that subsarcolemmal Ca 2+ release via ryanodine receptors occurs not only in response to the action potential (AP) upstroke, but also during the DD, and this is augmented by β-adrenergic receptor (β-AR) stimulation. Spontaneous APs simulated by mathematical SANC models beat at a faster rate when this subsarcolemmal Ca 2+ waveform measured under β-AR stimulation is introduced into the modeling scheme. Thus, in future investigation of pacemaker functioning in health, disease, and disease therapies the “bar ought to be raised” to embrace the impact of cyclic variation in submembrane [Ca 2+ ] on pacemaker function. The full text of this article is available at http://www.circresaha.org.

Abstract —The HCN family of ion channel subunits underlies the currents I f in heart and I h and I q in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a β subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a β subunit for the cardiac delayed rectifier I Kr , these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.

This article is the introduction of a new thematic series on Mechanisms of Pacemaking in the Heart , which includes the following articles: Be Still, My Beating Heart—Never! Development of the Pacemaker Tissues of the Heart [2010;106:240–254] Mapping Cardiac Pacemaker Circuits: Methodological Puzzles of the Sinoatrial Node Optical Mapping [2010;106:255–271] The Role of the Funny Current in Pacemaker Activity Ca2+ Cycling in the Mechanism of Pacemaking Cardiac Pacemaking: Historical Overview and Future Directions Denis Noble Guest Editor and Brian O'Rourke Editor For me, it takes only a short bout of insomnia to trigger an acute awareness and reflection on the pacemaker mechanisms of the heart. In my quiet wakefulness, attempting to suppress vexing thoughts of grant deadlines, perplexing experimental results, or various other items, I often focus on counting heartbeats rather than sheep. How can the …

Intracellular microelectrodes and small unipolar leads applied to endocardial surface of the right atrium in vitro were used to study the complex extracellular wave patterns recorded from the neighborhood of the cardiac pacemaker during spontaneous activity. Sinus activity propagated slowly toward the site of atrial invasion on the venous border of the crista terminalis. Atrial activation was marked by a primary negative wave that appeared 20-40 msec after pacemaker firing. Two sources of complex multiphasic waves were found. First, potentials from transitional sinus tissue propagated toward the atrium and caused low-voltage waves that preceded and slurred the onset of the atrial initial negativity. Second, bundles and layers of the crista terminalis muscle were excited asynchronously around the invasion region, as if cross-connections were infrequent. Waves originating from this source occurred after the firing of the invasion site. No extracellular wave could be associated with the firing of the true pacemaker cells. The sinoatrial ring bundle (SARB) yielded a discrete biphasic deflection along most of its way toward the coronary sinus. This potential appeared most frequently after that of the adjoining cristal muscle, raising questions about the functional role of the SARB as an internodal preferential pathway.

The effect of the timing of discrete bursts of efferent vagal impulses on heart rate was determined in anesthized dogs. Two modes of stimulation were employed. In the first mode, one stimulus burst was delivered per cardiac cycle. As the time from the beginning of the P wave to the vagal stimulus (P-St interval) was progessively increased, there was first a progressive lengthening of the cardiac cycle (P-P interval), then a rapid decrease in P-P interval, and finally a gradual augmentation of the P-P interval. The amplitudes of curves of P-P interval as a function of P-St interval increased as the number of stimuli per burst was augmented, with 10 stimuli/burst yielding nearly maximal effects. Vagal stimuli applied at P-St intervals which coincided with the negative-slope (d[P-P]/d[P-St]) region of such curves tended to evoke sinus arrhythmias. With 5 stimuli/burst or more, these arrhythmias were pronounced and consisted of alternate short and long P-P intervals. In the second mode of stimulation, bursts of stimuli were delivered to the vagus nerve at a frequency independent of heart rate. The cardiac pacemaker tended to become synchronized in some fixed ratio of vagal stimuli to P waves, and this tendency became greater the larger the number of stimuli per burst of impulses. Within any range of synchronization, a paradoxical effect was manifest--increasing frequencies of vagal stimulation produced increasing rather than decreasing heart rates.

In anesthetized open-chest dogs, the right carotid sinus nerve was stimulated with brief bursts of electrical impulses. When a single burst was given with each heart beat, the reflexly induced change in heart rate depended on the time within the cardiac cycle that the stimulus was applied. The maximum response was obtained when the P-St interval (i.e., the time from the beginning of the P wave to the beginning of the stimulus burst) was approximately zero. At this P-St interval the cardiac cycle (P-P interval) was prolonged to a mean value of 583 ± 145 (SD) msec from a prestimulation control value of 408 ± 80 msec. The minimum response, a mean P-P interval 45 ± 20 msec less than the maximum, was obtained at a mean P-St interval of 322 ± 48 msec. As a consequence of this phase dependency, over a definite but small range of stimulation frequencies, the discharge of the cardiac pacemaker became synchronized with the neural activity in the baroreceptor reflex arc. Within this critical range of stimulation frequencies, the cardiac pacemaker responded paradoxically to carotid sinus nerve stimulation: a change in the frequency of stimulation evoked a parallel, rather than the well-known inverse, change in heart rate.

Dynamic heart rate control by parasympathetic nervous input involves feedback mechanisms and reflex bursting of efferent cardiac vagal fibers. Periodic vagal bursting induces phasic changes in sinoatrial cycle length and can entrain the pacemaker to beat at periods that may be identical to those of the vagal burst. We investigated the electrophysiological basis of these phenomena in isolated sinus node preparations (rabbit, cat, and sheep). In the presence of propranolol (3.9 X 10(-6)M), relatively brief (50-150 msec) trains of stimuli, applied onto the endocardial surface of the preparation, activated postganglionic vagal terminals and induced a brief hyperpolarization of sinoatrial pacemaker cells. This vagally mediated hyperpolarization could alter the pacemaker rhythm by an amount that depended on its duration and its position in the cycle, as well as on the duration of the free-running pacemaker period. When the free-running period was sufficiently long and the hyperpolarization was induced sufficiently early in the spontaneous cycle, a "paradoxical" acceleration of the pacemaker rhythm ensued. Phasic changes were plotted on phase-response curves, constructed by scanning systematically the sinoatrial pacemaker period with single or repetitive vagal trains. These phase-response curves enabled us to predict the entrainment characteristics and the levels of synchronization of the pacemaker to the vagal periodicity. The overall data explain the cellular mechanisms involved in the phasic effects of brief vagal discharges on sinoatrial periodicity, and provide conclusive evidence for the prediction that repetitive vagal input is capable of forcing the cardiac pacemaker to beat at rates that can be faster or slower than the intrinsic pacemaker rate. These data should improve our knowledge of the dynamic control of heart rate by neural reflexes and aid in our understanding of rhythm disturbances generated by the interaction of the cardiac pacemaker with vagal activity.

Experiments were performed on open-chest anesthetized dogs to determine the quantitative effects of autonomic nerve stimulation on pacemaker activity and conduction. The lead II electrocardiogram together with bipolar electrograms were recorded from the atria, the His bundles, and the ventricles. The vagi or the stellate ganglia were stimulated in dogs which exhibited either sinus rhythm, ectopic atrial rhythm, junctional rhythm, or ectopic ventricular rhythm. The time courses of the change in heart rate in response to vagal or stellate stimulation were characteristic for each type of rhythm. The characteristic responses of different cardiac pacemaker sites to autonomic influence were demonstrated to be important factors in the production of wandering pacemakers and in the emergence of ectopic beats. Sinus pacemaker activity was more sensitive to modification by autonomic stimulation than was atrioventricular (AV) conduction. However, subliminal autonomic effects on AV transmission were brought out during conduction of premature atrial beats, thereby demonstrating a coupling interval dependency of autonomic influences on AV conduction. The present experiments also showed how fluctuations in autonomic activity could result in Mobitz type II second-degree heart block, pseudosupernormal conduction, and the concertina effect observed in the preexcitation syndrome.

The effects of variations in external K + and Ca 2+ concentrations on electrical and mechanical activity of the isolated rat portal vein were studied using a sucrose-gap technique. Spontaneous activity in normal Krebs solution consists of bursts of action potentials accompanied by phasic contractions. Moderate reduction in [K+] o results in slight hyperpolarization with decreased discharge frequency. Greater reduction or total removal of extracellular K causes a temporary increase in burst frequency. Graded increases in [K+] o from 6 up to 128 mM cause depolarization with an increase in discharge frequency and in contraction amplitude until [K+] o reaches a level of 6 to 7 times normal when spike potentials are abolished and a contracture develops. The relationship between membrance potential and log [K + ] o indicates a relatively low permeability to K + . Increasing [Ca 2+ ] o causes slight hyperpolarization, decreased burst frequency, increased burst duration and increased contraction amplitude. Reducing [Ca 2+ ] o causes increased frequency of discharge and shortening of bursts, until single spikes appear, each preceded by a prepotential; thus the activity resembles that in cardiac pacemaker cells. In Ca 2+ -free solution, electrical and mechanical activity ceases within 10 to 15 min. Calcium is essential for activation of the contractile mechanism and for maintenance of spontaneous spike discharge.

The interactions between pacemakers, and the effects on pacemakers, of terminating imposed driving were studied in the in situ heart of anesthetized dogs. Following atrial fibrillation or termination of a fast drive imposed through an artificial pacemaker, pacemaker action in intrinsic pacemakers is suppressed. Pacemakers tend to accelerate and compete with imposed drives which exceed control rates by only a small percentage (10 to 15%). Arrhythmias may result if imposed drive is slower than or identical with intrinsic pacemaker rate. Post-drive depression of pacemakers and the resulting deceleration of the heart is followed normally by an overshoot or supranormal acceleration. The magnitudes and durations of depression and late acceleration are proportional, within limits, to the rate and duration of drive. Atrioventricular and ectopic atrial pacemakers are much more readily depressed than is the sinoatrial pacemaker. Furthermore, beats of ectopic origin are much more likely to occur while subsidiary pacemakers are recovering from post-drive depression. Augmentation of depression by Prostigmin, its diminution by atropine, and the potentiation of late acceleration by cocaine and its absence after reserpine or guanethidine pretreatment, indicate that acetylcholine and catecholamines are liberated by driving stimuli. Placement of the pacemaker over the sinoatrial node, or near to regions where nerve terminals are concentrated, results in the greatest post-drive effects. The fact that propagated action potentials cause depressions and accelerations subject to drug block or potentiation indicates that mediators are also released in the course of propagated activity. Since atropine does not completely block post-drive depression, it is thought that a potassium ion shift may be involved.