Rabbit Sino-atrial Node Myocytes Research Articles

Beta-1 adrenergic receptors modulate pacemaker activity of mouse sino-atrial myocytes through L-type Cav1.3 channels

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Doctoral program in Biology and Biotechnologies b1- and b2- adrenergic receptors (ARs) are co-expressed in different regions of the heart. The b2/ b1 expression ratio is higher in the sino-atrial node (SAN) than in atria and ventricles, but the specific contribution of either type of receptor to modulation of pacemaker activity is still not well established. Specific stimulation of b2-ARs in rabbit SAN myocytes is associated with a positive shift of the pacemaker "funny" current (If) activation curve. However, previous studies showed that L-type Cav1.3 channels play an important role in the generation of cardiac pacemaker activity by contributing to the diastolic depolarization (DD) in SAN myocytes. Since Cav1.3 channels are positively regulated by b-ARs activation2, we investigated which is the main b-ARs isoform that modulates Cav1.3-mediated ICaL andthe pacemaker activity of SAN myocytes. To address this point, we recorded spontaneous activity and Cav1.3-mediated ICaL from mouse SAN myocytes. We found that the positive chronotropic effect of the non-selective b-AR agonist isoproterenol (ISO, 0.1mM) was decreased by the b1-ARs antagonist CGP-20712 (0.3mM) and the b2-ARs antagonist ICI-118,551 (1mM) by -18% and -9%, respectively. Perfusion of CGP-20712 strongly reduced the positive chronotropic effect induced by ISO. Finally, we recorded Cav1.3-mediated L-type currents in presence of the b1-ARs antagonist. CGP-20712 reduced the basal Cav1.3-mediated ICaL. Furthermore, the increase in Cav1.3-mediated ICaL by isoproterenol was abolished during b1-ARs inhibition by CGP-20712. In conclusion, these preliminary data show that b1- and b2-ARs differently modulate the spontaneous activity of mouse SAN myocytes. In addition, b1-ARs play a predominant role in the adrenergic regulation of L-type Cav1.3 channels to increase pacemaker activity. Future studies will be performed to clarify the role of b2-ARs antagonist on Cav1.3-mediated ICaL and the functional relationships between b-ARs and Cav1.3 channels.

Multiple effects of 4-aminopyridine on feline and rabbit sinoatrial node myocytes and multicellular preparations

4-aminopyridine (4-AP) is commonly used to block the transient outward potassium current, I(to), in cardiac and noncardiac tissues. In the present work, we found that 4-AP inhibited the rapid component of the delayed rectifier potassium current, I(Kr), in rabbit-isolated sinoatrial node myocytes by 25% (1 mM) and 51% (5 mM) and inhibited the slow component of the delayed rectifier potassium current, I(Ks), in cat- isolated sinoatrial node myocytes by 39% (1 mM) and 62% (5 mM). In cat- and rabbit-isolated sinoatrial node myocytes, 4-AP activated muscarinic receptors in a voltage-dependent manner to increase the acetylcholine-activated potassium current, I(KACh). In multicellular preparations of the central region of the sinoatrial node from nonreserpinized rabbits, 4-AP produced an increase in action potential overshoot, frequency, and rate of diastolic depolarization. In the presence of the beta-adrenergic antagonist propranolol, 4-AP produced a marked increase in duration and a marked decrease in maximum diastolic potential and eventually, cessation of the spontaneous activity in preparations from the sinoatrial central region. In multicellular preparations from reserpinized rabbits, 4-AP produced similar effects to those observed in the presence of propranolol. We conclude that 4-AP inhibits multiple cardiac K(+) currents, including I(to), I(Kr), and I(Ks), and that these activities mask I(KACh) activation. In addition, in multicellular preparations, 4-AP produces neurotransmitter release from the autonomic nerve terminals. These multiple effects need to be considered when using 4-AP as a "specific" I(to) blocker.

Two distinct pathways of muscarinic current responses in rabbit sino-atrial node myocytes.

Acetycholine (ACh) slows the heart rate by acting on sino-atrial node currents. Low ACh concentrations act on muscarinic receptors to inhibit the hyperpolarization-activated current (if) by a adenosine 3',5'-cyclic monophosphate (cAMP)-dependent cytoplasmic pathway. ACh also activates a muscarinic potassium conductance (iK,ACh) via a pertussis toxin-sensitive guanine nucleotide-binding protein (G-protein) that gates the channel directly. This pathway has been called membrane-delimited or "fast" because cytoplasmic components are not required and hence activation is relatively rapid. Such a pathway has also been proposed for the muscarinic inhibition of if. Here we show that, under steady-state current conditions, 0.1-1 microM ACh activates iK,ACh with a time constant of 1 s or less that is inversely proportional to ACh concentration, consistent with a fast, membrane-delimited pathway. ACh also causes a significantly slower inhibition of if which is not proportional to ACh binding. The changes in if are consistent with muscarinic effects mediated exclusively through the cAMP pathway.

Selective cardiodepressant activity of fluodipine, a fluorenone-1,4-dihydropyridine derivative

The effect of the dihydropyridine derivative, 1,4-dihydro-2,6-dimethyl-4-(fluorenon-4-yl)pyridine-3,5-dicarboxylic acid diallyl ester (fluodipine) was studied in vitro in different rabbit, rat and guinea pig preparations and in vivo in the rabbit in order to characterize its pharmacological profile at cardiac and at vascular sites. Compared to nifedipine, fluodipine showed a similar cardiodepressant activity, and a much lower inhibitory activity on vascular contraction. The highest tissue selectivity was observed in guinea pig preparations: fluodipine was about 2–3 times more effective than nifedipine on chronotropism and inotropism in isolated atria, and about 150 times less effective on aortic strip contraction. Accordingly, fluodipine (i) showed high-affinity binding to guinea pig ventricular L-type cardiac Ca2+ channels (Ki=2.57 nM), (ii) was about 80 times less effective than nifedipine to inhibit Ca2+ influx in vascular smooth muscle cells and (iii) induced a significant reduction of heart rate in the anesthetized rabbit (ID25=8.5 mg kg−1, i.v.) without affecting the blood pressure up to 20 mg kg−1, whereas nifedipine showed a significant hypotensive effect at very low doses (ID25=0.18 mg kg−1, i.v.). The pacemaker current If of rabbit sino-atrial node myocytes was not affected by fluodipine. These findings demonstrate that fluodipine exerts selective cardiodepressant activity, likely due to a higher affinity for cardiac than for vascular Ca2+ channels.

Tyrosine kinase inhibition reduces i(f) in rabbit sinoatrial node myocytes.

We studied pacemaker current (i(f)), the inward current activated by hyperpolarization in rabbit sinoatrial (SA) node myocytes, with the permeabilized-patch-clamp technique. The tyrosine kinase inhibitors genistein (50 microM) or herbimycin A (35 microM) reduced the amplitude of i(f) in response to step hyperpolarizations in the diastolic range of potentials. A two-step voltage-clamp protocol revealed that the reduction in i(f) is due to a decrease in maximal i(f) conductance. The observed effects are due to tyrosine kinase inhibition since an inactive analog of genistein did not reduce i(f). To further examine the mechanism of action, we added 2 mM chlorophenylthio cAMP (CPTcAMP, a membrane-permeant cAMP analog) to the bathing Tyrode, which increased i(f). Genistein still reduced i(f) in the presence of CPTcAMP. This suggests that the pathway mediating the actions of tyrosine kinase inhibition on i(f) is independent of cAMP- or protein-kinase-A-mediated phosphorylation.

Mechanism of muscarinic control of the high-threshold calcium current in rabbit sino-atrial node myocytes

The mechanism of the action of acetylcholine (ACh) on the L-type calcium current (ICa,L) was examined using a whole-cell voltage-clamp technique in single sino-atrial myocytes from the rabbit heart. ACh depressed basal ICa,L at concentrations in the range 0.05-10 microM, without previous beta-adrenergic stimulation. The ACh-induced reduction of ICa,L was reversed by addition of atropine, indicating that muscarinic receptors mediate it. Incubation of cells with a solution containing pertussis toxin led to abolition of the ACh effect, suggesting that this effect is mediated by G proteins activated by muscarinic receptors. Dialysis of cells with protein kinase inhibitor or 5'-adenylyl imidodiphosphate, inhibitors of the cAMP-dependent protein kinase, decreased basal ICa,L by about 85% and suppressed the effect of ACh. The ACh effect was also absent in cells dialysed with a non-hydrolysable analogue of cAMP, 8-bromo-cAMP. The results suggest that, in basal conditions, a large part of the L-type calcium channels should be phosphorylated by protein kinase A stimulated by a high cAMP level correlated with a high adenylate cyclase activity. The depressing effect of ACh on ICa,L may occur via inhibition of the high basal adenylate cyclase activity leading to a decrease of cAMP-dependent protein kinase stimulation and thus to a dephosphorylation of calcium channels.

The effects of manganese and barium on the cardiac pacemaker current, i f , in rabbit sino-atrial node myocytes

The isolation of ionic fluxes contributing to electric currents through cell membranes often requires block of other undesired components which can be achieved, among others, by divalent cations. Mn2+ and Ba2+ are often used, for example, to block Ca and K currents. Here we have investigated the effects of these two cations on the properties of the hyperpolarization-activated pacemaker current if, in rabbit sino-atrial node myocytes, as obtained by voltage clamp analysis. We find that 2 mM Mn2+ shifts the if activation curve by 3.2 +/- 0.3 mV towards more positive values. However, when 1 mM Ba2+ is also added, the positive shift is more than halved (1.3 +/- 0.2 mV). We find, too, that in the absence of blocking cations the ACh-induced if inhibition is slightly higher than in their presence. These results indicate that the alteration of if kinetic properties by Ba2+ plus Mn(2+)-containing solutions is minimal.

Block of the cardiac pacemaker current (If) in the rabbit sino-atrial node and in canine Purkinje fibres by 9-amino-1,2,3,4-tetrahydroacridine

We have investigated the action of 9-amino-1, 2, 3, 4-tetrahydroacridine (THA) on the pacemaker current If in rabbit sino-atrial node myocytes and in canine Purkinje fibers. THA at concentrations in the range 3-300 microM blocked If in a voltage-independent manner, as revealed by measurements on the fully activated I/V relation for If. The dose/response relationship of the If maximal slope conductance (Gf) can be fitted by assuming a cooperative binding reaction where two THA molecules are required to block one If channel. Half-maximal block occurred at 18.2 microM in the sino-atrial node and 36.6 microM in Purkinje fibers. THA also affected the If kinetic properties. This was examined in the sino-atrial node where the current activation curve was shifted in the negative direction on the voltage axis (- 21 mV at 30 microM THA). The delayed rectifier current, IK, was also reduced by THA in sino-atrial node myocytes: at - 40 mV the IK fully activated value was decreased to 37% of its control value by 30 microM THA, with only a minor modification of the position of the activation curve at the same potential. Thus, although THA blocks If at a lower concentration than other known If-channel blockers [DiFrancesco (1982) J Physiol (Lond) 329:485-507], its action on the pacemaker current is not specific.