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Previous observations show that β-adrenergic modulation of pacemaker current (I(f)) in sinoatrial node (SAN) cells is impaired by disruption of normal Ca(2+)-homeostasis with ryanodine or BAPTA. Recently, the presence of Ca(2+)-activated adenylyl cyclase (AC) 1 was reported in SAN, and was proposed as a possible mechanism of Ca(2+)-dependence of β-adrenergic modulation. However, direct evidence that pacemaker (HCN) channels can be regulated by Ca(2+)-activated AC and that such regulation introduces Ca(2+) dependence, is lacking. Here we co-expressed AC1 or AC6 with HCN2 in neonatal rat ventricular myocytes, which lack AC1. Although both isoforms have equivalent expression level and ability to interact with HCN2, only AC1 increases intracellular cAMP content, accelerates spontaneous beating rate and modifies HCN2 biophysics. Measured HCN2 current in the AC1 group activated ~10mV more positive than in GFP or AC6. The β-adrenergic agonist isoproterenol induced a further positive shift under control conditions, but failed to do so after pretreatment with the Ca(2+) chelator BAPTA. In the AC6 group, isoproterenol shifted the HCN2 activation relation to a similar extent in the absence and presence of BAPTA. Thus, AC1 but not AC6 over-expression introduces Ca(2+)-sensitivity to the β-adrenergic response of HCN2. These results demonstrate physical and functional interaction between AC isoforms and the HCN2 pacemaker channel and support a key role of Ca(2+) activated AC1 as a molecular mechanism in Ca(2+)-dependent modulation of β-adrenergic response of heart rate., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In rabbit, sodium current (I(Na)) contributes to newborn sinoatrial node (SAN) automaticity but is absent in adult SAN, where heart rate is slower. In contrast, heart rate is high and I(Na) is functional in adult mouse SAN. Given the slower heart rates of large mammals, we asked if I(Na) is functionally active in SAN of newborn or adult canine heart. SAN cells were isolated from newborn (6-10 days), young (40-43 days) and adult mongrels. I(Na) was observed in >80% of cells from each age. However, current density was markedly greater in newborn, decreasing with age. At all ages, I(Na) was sensitive to nanomolar tetrodotoxin (TTX); 100 nmol/L inhibited I(Na) by 46.7%, 59.9% and 90.7% in newborn, young and adult cells, respectively. While high TTX sensitivity suggested the presence of non-cardiac isoforms, steady-state inactivation was relatively negative (midpoints -89.7+/-0.7 mV, -95.1+/-1.2 mV and -93.4+/-1.9 mV from newborn to adult). Consequently, I(Na) should be unavailable at physiological potentials under normal conditions, and 100 nmol/L TTX did not change cycle length or action potential parameters of spontaneous adult SAN cells. However, computer modeling predicts the large newborn I(Na) protects against excess rate slowing from strong vagal stimulation. The results show that canine SAN cells have TTX-sensitive I(Na) which decreases with post-natal age. The current does not contribute to normal automaticity in isolated adult cells but can be recruited to sustain excitability if nodal cells are hyperpolarized. This is particularly relevant in newborn, where I(Na) is large and parasympathetic/sympathetic balance favors vagal tone., (Published by Elsevier Ltd.)

Two distinct intracellular mechanisms have been proposed to affect the firing rate of cardiac pacemaker cells: one involves modulation of the I(f) current by the second messenger cAMP, and one relies upon disruption or alteration of SR Ca2+ transients during activity. Although both mechanisms are necessary for proper automaticity and autonomic rate control, the specific contribution of each to pacemaking is still debated. We investigated if the two processes can be separated based on potentially different effects on action potential characteristics during rate modulation. To identify specific I(f)-mediated effects, we used the selective I(f) blocker ivabradine and found that ivabradine (3 microM) slows rate (-16.2%) by selectively reducing (-31.9%) the steepness of early diastolic depolarization (EDD). On the other hand ryanodine (3 microM), used to evaluate the effects of abolishment of SR Ca2+ transients, slowed rate (-31.3%) by depolarizing the take-off potential (TOP, 18.1%) without affecting EDD. We therefore used these two parameters to identify I(f)-based or SR Ca2+ transients-based processes and analyzed the effects on action potential's characteristics of Rp-cAMPs (50 microM), a membrane permeable cAMP analogue directly activating f-channels; we found that Rp-cAMPs accelerates rate by increasing EDD (+42.3%) without modifying TOP. Finally, rate modulation was achieved by muscarinic (ACh 0.01 microM) or beta-adrenergic (Iso 1 microM) stimulation; in both cases, rate changes were associated with modifications of EDD (ACh, -29.3% and Iso, +47.6%) and not of TOP. We conclude that rate-related changes in the EDD induced by autonomic agonists are mediated by I(f) and not by processes involving SR Ca2+ transients.

The expression and function of numerous cardiac ion channels change with development and disease. Whereas multiple regulatory processes and molecular mechanisms are certainly involved, one factor, sympathetic innervation, contributes to many of the developmental changes and is suggested to play a role in pathology. The onset of cardiac sympathetic innervation of the mammalian ventricle during early post-natal life has been associated with functional alterations in several ionic currents, including Na(+), L-type Ca(2+), pacemaker, inward rectifier and transient outward K(+) currents. The neural signaling molecule is not the same in each case, with evidence pointing to contributions from sustained activation of myocardial neuropeptide Y receptors, alpha-adrenergic receptors and beta-adrenergic receptors, as well as additional, but as yet unidentified, targets. Knowledge of the mechanisms by which innervation regulates ion channel expression and function during normal development may aid efforts to reverse remodel the diseased heart and to target pharmacologic agents to remodeled channels.

I(f) contributes to generation and autonomic control of spontaneous activity of cardiac pacemaker cells through a cAMP-dependent, Ca(2+)-independent mechanism of rate regulation. However, disruption of Ca(2+) release from sarcoplasmic reticulum (SR) by ryanodine (Ry) has been recently shown to slow spontaneous rate and inhibit beta-adrenergic receptor (betaAR)-induced rate acceleration, leading to the suggestion that the target of betaAR modulation of pacemaking is the intracellular Ca(2+)-regulatory process. We have investigated whether the Ry-induced decrease of betaAR rate modulation alternatively involves disruption of the betaAR-adenylate-cyclase-cAMP-I(f) mechanism. Prolonged exposure to Ry (3 microM, >2 min) slowed spontaneous rate of pacemaker cells by 29.8% via a depolarizing shift of take-off potential (TOP) without significantly changing early diastolic depolarization rate. Ry depressed rate acceleration caused by isoproterenol (Iso) (1 microM, 23.6% in control vs. 8.0%), but did not modify that caused by two membrane-permeable cAMP analogs, CPT-cAMP (300 microM, 17.7% vs. 17.3%) and Rp-cAMPs (50 microM, 18.0% vs. 20.6%). Consistent with the rate effect, exposure to Ry decreased the shift induced by Iso, but not that induced by either cAMP analog on the I(f)-activation curve. We conclude that disruption of Ry receptor function and SR Ca(2+) release depresses betaAR-induced modulation of heart rate, but does not impair cAMP-dependent rate acceleration mediated by I(f). However, abolishment of normal Ca(2+) homeostasis may result in the failure of betaAR agonists to sufficiently elevate cAMP near f-channels. The molecular basis for Ca(2+)-dependent interference in beta-adrenergic signaling remains to be determined.

Studies from our laboratory have defined alterations in Ca(i)handling in the non-dialyzed subepicardial cells that have survived in the 5 day infarcted heart (IZs). To determine whether changes in the action potential profile contributed to the observed Ca(i)changes we have used a combined voltage clamp/epifluorescent technique to determine and compare changes in fura 2 ratios in IZs compared to those of epicardial cells from the non-infarcted canine hearts (NZs). We found that Ca(i)changes in voltage clamped IZs persisted. In NZs, Ca(i)transients showed the expected voltage dependence while IZs did not. To determine whether altered NaCa exchanger activity contributed to the observed changes in Ca(i)in IZs, we measured NaCa exchanger Ca(2+)fluxes (reverse and forward mode) and ionic currents in both cell types and under different Na(i)loads (10 and 20 m m). We found that there were no significant differences in resting, peak or magnitude of fura 2 ratio changes or in outward current densities between NZs and IZs even under the different Na(i)loads. Thus, we suggest that chronic up- or downregulation of the NaCa exchanger protein does not underlie observed Ca(i)changes in IZs. Additionally, Ca(2+)released with paced voltage steps represented 79% of that released by caffeine in NZs while, in IZs, caffeine releasable Ca(2+)was equivalent to that released with step depolarization. Thus, abnormalities in Ca(i)handling in IZs appear not to arise secondarily to changes in action potential configuration nor do they appear to be due to disease-induced alteations in NaCa exchanger function., (Copyright 2000 Academic Press.)

Previously, we demonstrated that low concentrations of acetylcholine (< or = 10(-9) M) increased automaticity in neonatal but not in adult rat ventricular myocardium. In the present study, we used cultured neonatal rat ventricular myocytes grown alone or in the presence of dissociated sympathetic neurons as an experimental model to study the ontogeny of the muscarinic response. McN A343 (< or = 10(-9) M), an M1 selective agonist, increased spontaneous rate from 51 +/- 4 to 56 +/- 5 beats per minute (bpm), and this excitatory response was blocked by 10(-9) M pirenzepine, an M1 selective antagonist, but not by the M2 selective antagonist AFDX-116, nor by the alpha 1 adrenergic antagonist prazosin and the beta adrenergic antagonist propranolol (all 10(-7) M) In innervated myocytes, McN A343 also increased rate from 48 +/- 6 to 55 +/- 6 bpm. However, this effect was blocked by either 10(-9) M pirenzepine or 10(-7) M propranolol. After pretreatment with 10 ng/ml of pertussis toxin, the McN A343-induced excitatory response in non-innervated myocytes was absent, thus suggesting that this response involved a pertussis toxin-sensitive G protein dependent pathway. McN A343 failed to stimulate inositol phosphate or cAMP accumulation in non-innervated myocytes. These results demonstrate the following. (1) The muscarinic excitatory response is mediated via direct stimulation of a post-synaptic M1 receptor in non-innervated myocytes. (2) The excitatory response after innervation is related to the release of catecholamines, possibly through activation of muscarinic receptors located at the pre-synaptic sympathetic nerve terminals. (3) Sympathetic innervation prevents the functional expression of the post-synaptic myocardial M1 receptor. (4) The intracellular pathway for the post-synaptic M1 excitatory response involves a pertussis toxin-sensitive G protein, but does not depend on obvious changes in cAMP or phosphoinositide hydrolysis.

Under normal physiological conditions, the adult rat heart exhibits an alpha 1-adrenergic mediated decrease in rate. The negative chromotropic effect of alpha 1-stimulation in the adult depends upon maturation of sympathetic innervation and the presence of a pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein. We have previously used a cell culture model of neonatal myocardial cells to demonstrate more directly that sympathetic innervation is an important feature of the mature response. After alpha 1-adrenergic stimulation, neonatal rat ventricular myocytes cultured with sympathetic ganglion cells [nerve-muscle (NM) co-cultures] respond predominantly by a decrease in rate, whereas pure muscle cultures show an exclusive increase in rate. Since it has been reported that the inhibitory alpha 1-response in intact tissue is lost upon depolarization, the present study was designed to determine whether the negative chronotropic response could be reversed by potassium (K+) depolarization. We also investigated whether there might be an associated reduction in the PT-sensitive G protein linked to the negative chronotropic response. Thus, the effect of high K+ depolarization on both the alpha 1-adrenergic chronotropic response and the level of the PT-sensitive G protein was examined in NM co-cultures. Extracellular high K+ acutely and reversibly converted the phenylephrine-mediated chronotropic response from negative to positive. The positive chronotropic response in high K+ was alpha 1-mediated and not secondary to catecholamine release from adrenergic neurons. Loss of the inhibitory response in high K+ was not associated with a change in the level of the PT-sensitive G protein. Thus, the presence of a PT-sensitive G protein is necessary, but not sufficient to permit the expression of the mature alpha 1-adrenergic negative chronotropic response in innervated cardiac cells in culture.