Intrinsic Beating Rate Research Articles

Human Sinoatrial Node Pacemaker Activity: Role of the Slow Component of the Delayed Rectifier K+ Current, IKs.

The pacemaker activity of the sinoatrial node (SAN) has been studied extensively in animal species but is virtually unexplored in humans. Here we assess the role of the slowly activating component of the delayed rectifier K+ current (IKs) in human SAN pacemaker activity and its dependence on heart rate and β-adrenergic stimulation. HEK-293 cells were transiently transfected with wild-type KCNQ1 and KCNE1 cDNA, encoding the α- and β-subunits of the IKs channel, respectively. KCNQ1/KCNE1 currents were recorded both during a traditional voltage clamp and during an action potential (AP) clamp with human SAN-like APs. Forskolin (10 µmol/L) was used to increase the intracellular cAMP level, thus mimicking β-adrenergic stimulation. The experimentally observed effects were evaluated in the Fabbri-Severi computer model of an isolated human SAN cell. Transfected HEK-293 cells displayed large IKs-like outward currents in response to depolarizing voltage clamp steps. Forskolin significantly increased the current density and significantly shifted the half-maximal activation voltage towards more negative potentials. Furthermore, forskolin significantly accelerated activation without affecting the rate of deactivation. During an AP clamp, the KCNQ1/KCNE1 current was substantial during the AP phase, but relatively small during diastolic depolarization. In the presence of forskolin, the KCNQ1/KCNE1 current during both the AP phase and diastolic depolarization increased, resulting in a clearly active KCNQ1/KCNE1 current during diastolic depolarization, particularly at shorter cycle lengths. Computer simulations demonstrated that IKs reduces the intrinsic beating rate through its slowing effect on diastolic depolarization at all levels of autonomic tone and that gain-of-function mutations in KCNQ1 may exert a marked bradycardic effect during vagal tone. In conclusion, IKs is active during human SAN pacemaker activity and has a strong dependence on heart rate and cAMP level, with a prominent role at all levels of autonomic tone.

Chronic Optogenetic Pacing of Human-Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissues.

The delivery of cells into damaged myocardium induces limited cardiac regeneration due to extensive cell death. In an effort to limit cell death, our lab formulates three-dimensional matrices as a delivery system for cell therapy. Our primary work has been focused on the formation of engineered cardiac tissues (ECTs) from human-induced pluripotent stem cell-derived engineered cardiac cells. However, ECT immaturity hinders ability to fully recover damaged myocardium. Various conditioning regimens such as mechanical stretch and/or electric pacing have been used to activate maturation pathways. To improve ECT maturity, we use non-contacting chronic light stimulation using heterologously expressed light-sensitive channelrhodopsin ion channels. We transduce ECTs with an AAV packaged channelrhodopsin and chronically optically pace (C-OP) ECTs for 1week above the intrinsic beat rate, resulting in increased ECT electrophysiological properties.

Dynamics of beating cardiac tissue under slow periodic drives.

Effects of mechanical coupling on cardiac dynamics are studied by monitoring the beating dynamics of a cardiac tissue which is being pulled periodically at a pace slower than its intrinsic beating rate. The tissue is taken from the heart of a bullfrog that includes pacemaker cells. The cardiac tissue beats spontaneously with an almost constant interbeat interval (IBI) when there is no external forcing. On the other hand, the IBI is observed to vary significantly under an external periodic drive. Interestingly, when the period of the external drive is about two times the intrinsic IBI of the tissue without pulling, the IBI as a function of time exhibits a wave packet structure. Our experimental results can be understood theoretically by a phase-coupled model under external driving. In particular, the theoretical prediction of the wave-packet period as a function of the normalized driving period agrees excellently with the observations. Furthermore, the cardiac mechanical coupling constant can be extracted from the experimental data from our model and is found to be insensitive to the external driving period. Implications of our results on cardiac physiology are also discussed.

Cavin-1 deficiency modifies myocardial and coronary function, stretch responses and ischaemic tolerance: roles of NOS over-activity.

Caveolae and associated cavin and caveolins may govern myocardial function, together with responses to mechanical and ischaemic stresses. Abnormalities in these proteins are also implicated in different cardiovascular disorders. However, specific roles of the cavin-1 protein in cardiac and coronary responses to mechanical/metabolic perturbation remain unclear. We characterised cardiovascular impacts of cavin-1 deficiency, comparing myocardial and coronary phenotypes and responses to stretch and ischaemia-reperfusion in hearts from cavin-1 +/+ and cavin-1 -/- mice. Caveolae and caveolins 1 and 3 were depleted in cavin-1 -/- hearts. Cardiac ejection properties in situ were modestly reduced in cavin-1 -/- mice. While peak contractile performance in ex vivo myocardium from cavin-1 -/- and cavin-1 +/+ mice was comparable, intrinsic beating rate, diastolic stiffness and Frank-Starling behaviour (stretch-dependent diastolic and systolic forces) were exaggerated in cavin-1 -/- hearts. Increases in stretch-dependent forces were countered by NOS inhibition (100µM L-NAME), which exposed negative inotropy in cavin-1 -/- hearts, and were mimicked by 100µM nitroprusside. In contrast, chronotropic differences appeared largely NOS-independent. Cavin-1 deletion also induced NOS-dependent coronary dilatation, ≥3-fold prolongation of reactive hyperaemic responses, and exaggerated pressure-dependence of coronary flow. Stretch-dependent efflux of lactate dehydrogenase and cardiac troponin I was increased and induction of brain natriuretic peptide and c-Fos inhibited in cavin-1 -/- hearts, while ERK1/2 phospho-activation was preserved. Post-ischaemic dysfunction and damage was also exaggerated in cavin-1 -/- hearts. Diverse effects of cavin-1 deletion reveal important roles in both NOS-dependent and -independent control of cardiac and coronary functions, together with governing sarcolemmal fragility and myocardial responses to stretch and ischaemia.

Patient-Specific Induced Pluripotent Stem Cell Cardiac Myocytes as Predictors of Sudep Risk

Dravet syndrome (DS) is a severe and intractable pediatric epileptic encephalopathy that is largely caused by de novo loss-of-function mutations in the voltage-gated sodium channel SCN1A. Approximately 15% of DS patients undergo Sudden Unexpected Death in Epilepsy (SUDEP). A growing body of evidence suggests that cardiac arrhythmia may precede SUDEP in DS. Previous work in DS mouse cardiomyocytes (CMs) demonstrated increased transient and persistent TTX-resistent sodium current (INa), action potential excitability, and incidence of early afterdepolarizations.To test whether cardiac excitability is altered in DS patients, we differentiated induced pluripotent stem cells (iPSCs) from two control and four SCN1A-linked DS patients into CMs. iPSC neurons tested from one of these DS patients was found to be hyperexcitable with increased INa density.Confluent iPSC-CMs from all DS patients had an increased intrinsic beating rate compared to controls (P<0.05, N≥8 per group). Multi-electrode array analysis of iPSC-CMs from DS4 confirmed that beating rate was increased in DS. Consistent with our previous studies in DS mice, we observed a ∼two-fold increase in peak INa in iPSC-CMs from 3 of the 4 DS patients (P<0.05, N≥9 per group). INa density from the remaining patient (DS5) was ∼3 fold higher than control. Due to the high level of INa density, the patient was recommended for a cardiac consultation. The patient presented with sinus tachycardia and repolarization abnormalities that are now being closely monitored.Taken together, our data suggest that alterations in cardiac excitability contribute to the mechanism of SUDEP in DS via increased INa density, which may underlie arrhythmia in DS patients. DS patient-derived iPSC-CMs provide a valuable model for advancing our knowledge of how cardiac arrhythmias contribute to the mechanism of SUDEP and may provide novel biomarkers for SUDEP risk.

Low dose fish oil increases the omega-3 index improving cycling efficiency and heart rate recovery

Background: Dietary fish oil provides long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The heart (Charnock et al., 1986: Annals of Nutrition and Metabolism, 30, 393-406) and skeletal muscle (Peoples & McLennan, 2010: British Journal of Nutrition, 104(12), 1771-1779) preferentially incorporate DHA and modulates both myocardial (Pepe & McLennan, 2002: Circulation, 105, 2303-2308) and skeletal muscle oxygen consumption translated as improved muscle fatigue resistance (Peoples & McLennan, 2014: British Journal of Nutrition, 111, 996-1004). However, studies of well-trained endurance athletes have used excessive doses of DHA (Peoples et al., 2008: Journal of Cardiovascular Pharmacology, 52, 540-547;  Buckley et al., 2009: Journal of Science and Medicine in Sport, 12(4), 503-507). Given that even a very low fish oil supplement maximises membrane incorporation of DHA (Slee et al., 2010: Journal of Lipid Research, 51, 1841-1847) there is strong physiological basis for cyclists to improve oxygen efficiency through a nutritional approach. Purpose: The current study primarily aimed to establish if provision of a low dose of dietary fish oil in cyclists could convey improved oxygen efficiency in heart and skeletal muscle under conditions of established cycling fatigue and acute recovery. Methods: Using a double-blind design, trained males (N=26) were supplemented with (2x1g.d-1) soy oil (control n=13) or tuna fish oil (FO n=13) providing LCn-3PUFA, DHA: 560mg and EPA: 140mg (Nu-Mega, Australia) for 8 weeks. Erythrocyte omega-3 index (% EPA+DHA) was measured at baseline at following supplementation. At both time points participants underwent a repeated Wingate cycling protocol (6x30sec / 150seconds recovery) to establish cycling fatigue, immediately followed by a 5min cycling time trial (Wattbike, UK). Upon completion, participants were place in a supine recovery position for 10mins. Oxygen consumption (O2), heart rate (HR) and power (W) were continuously monitored.Results: Erythrocyte omega-3 index at baseline (Control: 4.2±0.2; FO: 4.7±0.2%) was not different between groups. After 8weeks the omega-3 index was unchanged in control (3.9±0.2%) but increased in FO group (6.3±0.3%, P< 0.01). There was no effect of FO supplementation on; peak HR (Control: 174±1 FO: 176±1beats.min-1), mean power during Wingate cycling fatigue protocol (Control: 545±28; FO: 511±33W), or cycling time trial performance under established fatigue (Control: 267±19; FO: 253±16W). However, FO supplementation improved oxygen efficiency (delta change) during the cycling time trial (Control: -23±26; FO: -154±59ml O2/min/100W P<0.05) and accelerated HR recovery (logit half-time) in the supine position immediately afterwards (Control: -0.4±1.2s; FO: -8.0±1.7s, P<0.05). The later was independent of a change in parasympathetic tone and reflective of reduced myocardial oxygen consumption.Discussion: A low dose fish oil supplement providing DHA increased the omega-3 index in trained cyclists. The improved cycling efficiency is reflective of the oxygen modulation previously demonstrated in rodent skeletal muscle (Peoples & McLennan, 2014), while the faster heart rate recovery without alteration in autonomic tone is supportive of the a reduction in intrinsic beat rate of the SA node following DHA administration (Verkerk, et al., 2009: Heart Rhythm, 6(10), 1485-1492). Conclusion: Provision of LCn-3 PUFA, DHA, in the form of a low dose fish oil supplement can have profound effects on heart and skeletal muscle incorporation, and as consequence improve cycling efficiency and heart rate recovery, indicative of reduced heart load. As a consequence, the concentrations used in the current study can be modeled for nutritional recommendations of DHA in elite cyclists.

Intrinsic heart rate recovery after dynamic exercise is improved with an increased omega-3 index in healthy males.

Dietary fish consumption contributes to a reduced risk of cardiac mortality. In the present study, the effect of low-dose fish oil (FO) supplementation on heart rate (HR) response to intense exercise and recovery was investigated in physically fit males. The subjects (n 26) were supplemented (double-blind, parallel design) with (2 × 1 g/d) soya bean oil (control) or tuna FO providing the long-chain n-3 PUFA DHA (560 mg) and EPA (140 mg). Erythrocyte omega-3 index (%EPA+DHA), HR, HR variability and HR recovery were analysed during rest, intense exercise and recovery at baseline and after 8 weeks of supplementation. The mean erythrocyte omega-3 index, which did not differ between the groups at baseline (control 4.2 (sem 0.2), n 13; FO 4.7 (sem 0.2), n 13), remained unchanged in the control group (3.9 (sem 0.2)), but increased in the FO group (6.3 (sem 0.3); P< 0.01). The mean HR during supine resting conditions (control 56 (sem 10); FO 59 (sem 9)) was not affected by FO supplementation. Poincaré analysis of HR variability at rest exhibited a decreasing trend in parasympathetic activity in the FO group (SD1 (standard deviation of points perpendicular to the axis of line of identity)/SD2 (standard deviation of points along the axis of line of identity): control 0.02 (sem 0.01); FO - 0.05 (sem 0.02); P= 0.18). Peak HR was not affected by supplementation. However, during submaximal exercise over 5 min, fewer total heart beats were recorded in the FO group (-22 (sem 6) ( = -4.5 beats/min)), but not in the control group (+1 (sem 4)) (P< 0.05). Supine HR recovery (half-time) after cycling was significantly faster after FO supplementation (control - 0.4 (sem 1.2) s; FO - 8.0 (sem 1.7) s; P< 0.05). A low intake of FO increased the omega-3 index and reduced the mean exercise HR and improved HR recovery without compromising the peak HR. A direct influence of DHA via reductions in the cardiac intrinsic beat rate was balanced by a reciprocal decrease in vagal tone.

Human Embryonic and Induced Pluripotent Stem Cell–Derived Cardiomyocytes Exhibit Beat Rate Variability and Power-Law Behavior

The sinoatrial node is the main impulse-generating tissue in the heart. Atrioventricular conduction block and arrhythmias caused by sinoatrial node dysfunction are clinically important and generally treated with electronic pacemakers. Although an excellent solution, electronic pacemakers incorporate limitations that have stimulated research on biological pacing. To assess the suitability of potential biological pacemakers, we tested the hypothesis that the spontaneous electric activity of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) exhibit beat rate variability and power-law behavior comparable to those of human sinoatrial node. We recorded extracellular electrograms from hESC-CMs and iPSC-CMs under stable conditions for up to 15 days. The beat rate time series of the spontaneous activity were examined in terms of their power spectral density and additional methods derived from nonlinear dynamics. The major findings were that the mean beat rate of hESC-CMs and iPSC-CMs was stable throughout the 15-day follow-up period and was similar in both cell types, that hESC-CMs and iPSC-CMs exhibited intrinsic beat rate variability and fractal behavior, and that isoproterenol increased and carbamylcholine decreased the beating rate in both hESC-CMs and iPSC-CMs. This is the first study demonstrating that hESC-CMs and iPSC-CMs exhibit beat rate variability and power-law behavior as in humans, thus supporting the potential capability of these cell sources to serve as biological pacemakers. Our ability to generate sinoatrial-compatible spontaneous cardiomyocytes from the patient's own hair (via keratinocyte-derived iPSCs), thus eliminating the critical need for immunosuppression, renders these myocytes an attractive cell source as biological pacemakers.

Specific inhibition of HCN channels slows rhythm differently in atria, ventricle and outflow tract and stabilizes conduction in the anoxic-reoxygenated embryonic heart model

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in pacemaker cells very early during cardiogenesis. This work aimed at determining to what extent these channels are implicated in the electromechanical disturbances induced by a transient oxygen lack which may occur in utero. Spontaneously beating hearts or isolated ventricles and outflow tracts dissected from 4-day-old chick embryos were exposed to a selective inhibitor of HCN channels (ivabradine 0.1–10 μM) to establish a dose–response relationship. The effects of ivabradine on electrocardiogram, excitation–contraction coupling and contractility of hearts submitted to anoxia (30 min) and reoxygenation (60 min) were also determined. The distribution of the predominant channel isoform, HCN4, was established in atria, ventricle and outflow tract by immunoblotting. Intrinsic beating rate of atria, ventricle and outflow tract was 164 ± 22 ( n = 10), 78 ± 24 ( n = 8) and 40 ± 12 bpm ( n = 23, mean ± SD), respectively. In the whole heart, ivabradine (0.3 μM) slowed the firing rate of atria by 16% and stabilized PR interval. These effects persisted throughout anoxia-reoxygenation, whereas the variations of QT duration, excitation–contraction coupling and contractility, as well as the types and duration of arrhythmias were not altered. Ivabradine (10 μM) reduced the intrinsic rate of atria and isolated ventricle by 27% and 52%, respectively, whereas it abolished activity of the isolated outflow tract. Protein expression of HCN4 channels was higher in atria and ventricle than in the outflow tract. Thus, HCN channels are specifically distributed and control finely atrial, ventricular and outflow tract pacemakers as well as conduction in the embryonic heart under normoxia and throughout anoxia-reoxygenation.

Temperature acclimation modifies sinoatrial pacemaker mechanism of the rainbow trout heart

The hypothesis of pacemaker level origin of thermal compensation in heart rate was tested by recording action potentials (AP) in intact sinoatrial tissue and enzymatically isolated pacemaker cells of rainbow trout acclimated at 4 degrees C (cold) and 18 degrees C (warm). With electrophysiological recordings, the primary pacemaker was located at the base of the sinoatrial valve, where a morphologically distinct ring of tissue comprising myocytes and neural elements was found by histological examination. Intrinsic beating rate of this pacemaker was higher in cold-acclimated (46 +/- 6 APs/min) than warm-acclimated trout (38 +/- 3 APs/min; P < 0.05), and a similar difference was seen in beating rate of isolated pacemaker cells (44 +/- 6 vs. 38 +/- 6 APs/min; P < 0.05), supporting the hypothesis that thermal acclimation modifies the intrinsic pacemaker mechanism of fish heart. Inhibition of sarcoplasmic reticulum (SR) with 10 microM ryanodine and 1 microM thapsigargin did not affect heart rate in either warm- or cold-acclimated trout at 11 degrees C but reduced heart rate in warm-acclimated trout from 74 +/- 2 to 42 +/- 6 APs/min (P < 0.05) at 18 degrees C. At 11 degrees C, a half-maximal blockade of the delayed rectifier K+ current (I(Kr)) with 0.1 microM E-4031 reduced heart rate more in warm-acclimated (from 45 +/- 1 to 24 +/- 5 APs/min) than cold-acclimated trout (56 +/- 3 vs. 48 +/- 2 APs/min), whereas I(Kr) density was higher and AP duration less in cold-acclimated trout (P > 0.05). Collectively, these findings suggest that a cold-induced increase in AP discharge frequency is at least partly due to higher density of the I(Kr) in the cold-acclimated trout, whereas contribution of SR Ca2+ release to thermal compensation of heart rate is negligible.

Phase resetting and dynamics in isolated atrioventricular nodal cell clusters.

In the heart, the AV node is the primary conduction pathway between the atria and ventricles and subserves an important function by virtue of its rate-dependent properties. Cell clusters isolated from the rabbit atrioventricular (AV) node beat with a stable rhythm (cycle length: 300-520 ms) and are characterized by slow action potential upstroke velocities (7 to 30 V/s). The goal of this study is to better characterize the phase resetting and the rhythms during periodic stimulation of this slow inward current system. Single or periodic depolarizing pulses (20 ms in duration) were injected into AV nodal cell clusters using glass microelectrodes. Phase resetting curves of both strong, weak as well as discontinuous types were obtained by applying single current pulses of different intensities and latencies following every ten action potentials. Graded responses were elicited in a wide range of stimulus phases and amplitudes. A single premature stimulus caused a transient prolongation of the cycle length. Sustained periodic stimulation, at rates faster than the intrinsic beat rate, resulted in various N:M (stimulus frequency: action potential frequency) entrainment rhythms as well as periodic or irregular changes in action potential morphology. The changes in action potential characteristics were evaluated by computing the area under the action potential trace and above a fixed threshold (-45 mV). We show that the variations in action potential morphology play a major role in the onset of complicated dynamics observed in this experimental preparation. In this context, the prediction of entrainment rhythms using techniques based on the iteration of phase resetting curves (PRCs) is inadequate since the PRC does not carry information directly related to the changes in action potential morphology. This study demonstrates the need to consider graded events which, though not propagated, have important implications in the understanding of dynamical diseases of the heart. (c) 1995 American Institute of Physics.

Role of Sympathetic Innervation in Cardiac Development in Oculoa

Embryonic heart cultured in oculo differentiates into adult-like heart tissue, increases in size, and maintains spontaneous activity driven by a defined pacemaker. The growth and beating rate of embryonic heart maturing in oculo are modulated by its neural milieu. Culture in a sympathetically denervated eye chamber compromised growth and increased intrinsic beating rate. Exposure to an additional source of sympathetic neurons increased parasympathetic control of heart rate but did not alter growth or intrinsic beating rate. Because eye chamber sympathectomy alters neurotransmitter expression in parasympathetic and sensory neurons, it is possible that the growth inhibition observed in heart grafts is not a direct consequence of preventing sympathetic innervation. This possibility is being tested in additional experiments.

Genetic, neurohumoral, and hemodynamic influences on spontaneously hypertensive rat heart development in oculo.

To distinguish among genetic, neurohumoral, and hemodynamic explanations for structural and functional differences in the hearts of young spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) control rats, embryonic SHR and WKY rat heart tissue was cultured in the anterior eye chamber of adult SHR and WKY rats. In study 1, atria from E-12 WKY rat embryos grafted into anterior eye chambers of either SHR or WKY host rats achieved a larger size than did SHR grafts by 8 weeks in oculo (2.98 +/- 0.75 and 2.55 +/- 0.32 mm2 vs. 1.80 +/- 0.20 and 2.04 +/- 0.44 mm2). Beating rates did not differ between SHR and WKY rat atria implanted into SHR or WKY host rats. In study 2, ventricles from E-13 embryonic SHR and WKY rat hearts grew to similar size and weight when implanted into SHR or WKY host rats (e.g., SHR hearts, 1.81 +/- 0.32 vs. 1.74 +/- 0.33 mm2; WKY rat hearts, 1.75 +/- 0.29 vs. 2.29 +/- 0.32 mm2). Ventricle grafts from SHR embryos into SHR host rats beat more rapidly (165 +/- 19 beats/min) during weekly measurements than either WKY rat ventricles (92 +/- 9 beats/min in SHR hosts and 99 +/- 9 beats/min in WKY host rats) or SHR ventricles grafted into WKY host rats (109 +/- 7 beats/min, p less than 0.001). In study 3, atria from E-13 SHR and WKY rat embryos were grafted into sympathectomized and intact eye chambers of SHR or WKY host rats. Sympathectomy of the eye chamber compromised growth of grafts into WKY host rats (1.54 +/- 0.24 vs. 0.90 +/- 0.14 mm2) but not SHR hosts (1.54 +/- 0.25 vs. 1.73 +/- 0.24 mm2). Grafts into sympathectomized eye chambers of WKY host rats beat more slowly than grafts into eye chambers with sympathetic innervation intact (282 +/- 14 vs. 202 +/- 14 beats/min); sympathectomy did not alter beating rate of grafts in SHR hosts (266 +/- 14 vs. 255 +/- 18 beats/min). These results suggest that the growth and beating rate of SHR atrial grafts may be less sensitive to sympathetic innervation than WKY rat atrial grafts. In these studies, SHR grafts did not grow larger than WKY heart grafts and did not show an increased intrinsic beating rate, suggesting that the cardiac hypertrophy and increased intrinsic beating rate observed in intact SHR are unlikely to result from direct genetic programming.

Development of cardiac beat rate in early chick embryos is regulated by regional cues

The mesoderm of each of the paired lateral heart-forming regions (HFRs) in the stage 5–7 chick embryo includes prospective conus (pre-C), ventricle (pre-V), and sinoatrial (pre-SA) cells, arranged in a rostrocaudal sequence (C-V-SA). With microsurgery we divided each HFR into three rostrocaudally arranged segments. After 24 hr of further incubation, each segment differentiated into a spontaneously beating vesicle of heart tissue to form a multiheart embryo. The cardiac vesicles in these embryos expressed left-right and rostrocaudal beat rate gradients: the left caudal pre-SA mesoderm produced tissue with the fastest beat rate of the six while the rostral vesicle formed from right pre-C was the slowest. In another operation, we prevented the HFRs from fusing in the midline by cutting through the anterior intestinal portal at stage 8, to produce cardia bifida (CB) embryos with an independently beating half-heart on each side. In these cases, the left half-heart of 87.2% of CB embryos beat faster than the right, confirming the left-right difference in intrinsic beat rate. To assess whether the future beat rate of each region is already determined in the st 5–7 HFR, we exchanged rectangular fragments of left pre-SA mesoderm and attached endoderm with right pre-C fragments to yield a left HFR with the sequence C-V-C and a right HFR with the sequence SA-V-SA. A CB operation was subsequently performed on these exchange embryos to prevent fusion of the lateral HFRs. Preconus mesoderm, transplanted to the pre-SA region, differentiated into tissue with a rapid beat rate, while pre-SA mesoderm relocated to the preconus region formed heart tissue with a slow spontaneous rate typical of the conus. In 73% of the exchange CB embryos, the left half-heart beat faster than the right, despite the origins of its mesoderm. The exchanged mesoderm developed a rate that was appropriate for its new location rather than the site of origin of the mesodermal fragment. In a third set of operations, we implanted a fragment of st 15 differentiated conus tissue into a site lateral to the left caudal HFR in st 5, 6, and 7 embryos, and subsequently performed CB operations on them. The implant caused the adjacent half-heart to develop with a slower beat rate than in unoperated or sham-operated controls. We conclude that (i) at st 5–7 the precardiac mesoderm is not yet stably coded for its future intrinsic beat rate; and (ii) local cues from the tissues surrounding the preconus mesoderm can alter the intrinsic beat rate of the differentiating precardiac cells.

The effect of β-adrenergic blocking drugs on the intrinsic beating rate of cultured rat myocytes

Cultured myocytes from neonate rats were subject to increasing doses of dl-, d- or l-propranolol or dl-atenolol, and the intrinsic heart rates were measured. Both propranolol isomers and racemate stopped beating activity at very high concentrations (10 −3 m) while atenolol did not. The results are interpreted to indicate that constant β-adrenoceptor stimulation is not an obligatory requirement for maintenance of spontaneous beating in this system as has been suggested.