Frequency-Dependent Properties of the Hyperpolarization-Activated Cation Current,If, in Adult Mouse Heart Primary Pacemaker Myocytes

A number of distinct electrophysiological mechanisms that modulate the myogenic spontaneous pacemaker activity in the sinoatrial node (SAN) of the mammalian heart have been investigated extensively. There is agreement that several (3 or 4) different transmembrane ionic current changes (referred to as the voltage clock) are involved; and that the resulting net current interacts with direct and indirect effects of changes in intracellular Ca²⁺ (the calcium clock). However, significant uncertainties, and important knowledge gaps, remain concerning the functional roles in SAN spontaneous pacing of many of the individual ion channel- or exchanger-mediated transmembrane current changes. We report results from patch clamp studies and mathematical modeling of the hyperpolarization-activated current, I_f, in the generation/modulation of the diastolic depolarization, or pacemaker potential, produced by individual myocytes that were enzymatically isolated from the adult mouse sinoatrial node (SAN). Amphotericin-mediated patch microelectrode recordings at 35 °C were made under control conditions and in the presence of 5 or 10 nM isoproterenol (ISO). These sets of results were complemented and integrated with mathematical modeling of the current changes that take place in the range of membrane potentials (−70 to −50 mV), which corresponds to the ‘pacemaker depolarization’ in the adult mouse SAN. Our results reveal a very small, but functionally important, approximately steady-state or time-independent current generated by residual activation of I_f channels that are expressed in these pacemaker myocytes. Recordings of the pacemaker depolarization and action potential, combined with measurements of changes in I_f, and the well-known increases in the L-type Ca²⁺ current, I_CaL, demonstrated that I_CaL activation, is essential for myogenic pacing. Moreover, after being enhanced (approximately 3-fold) by 5 or 10 nM ISO, I_CaL contributes significantly to the positive chronotropic effect. Our mathematical model has been developed in an attempt to better understand the underlying mechanisms for the pacemaker depolarization and action potential in adult mouse SAN myocytes. After being updated with our new experimental data describing I_f, our simulations reveal a novel functional component of I_f in adult mouse SAN. Computational work carried out with this model also confirms that in the presence of ISO the residual activation of I_f and opening of I_CaL channels combine to generate a net current change during the slow diastolic depolarization phase that is essential for the observed accelerated pacemaking rate of these SAN myocytes.     

Structural Identification of the Pacemaker Cells and Expression of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in the Heart of the Wild Atlantic Cod,Gadus morhua (Linnaeus, 1758)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod (Gadus morhua), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.     

Effectiveness in Block by Dexmedetomidine of Hyperpolarization-Activated Cation Current,Independent of Its Agonistic Effect on α2-Adrenergic Receptors

Dexmedetomidine (DEX), a highly selective agonist of α₂-adrenergic receptors, has been tailored for sedation without risk of respiratory depression. Our hypothesis is that DEX produces any direct perturbations on ionic currents (e.g., hyperpolarization-activated cation current, I_h). In this study, addition of DEX to pituitary GH₃ cells caused a time- and concentration-dependent reduction in the amplitude of I_h with an IC₅₀ value of 1.21 μM and a K_D value of 1.97 μM. A hyperpolarizing shift in the activation curve of I_h by 10 mV was observed in the presence of DEX. The voltage-dependent hysteresis of I_h elicited by long-lasting triangular ramp pulse was also dose-dependently reduced during its presence. In continued presence of DEX (1 μM), further addition of OXAL (10 μM) or replacement with high K⁺ could reverse DEX-mediated inhibition of I_h, while subsequent addition of yohimbine (10 μM) did not attenuate the inhibitory effect on I_h amplitude. The addition of 3 μM DEX mildly suppressed the amplitude of erg-mediated K⁺ current. Under current-clamp potential recordings, the exposure to DEX could diminish the firing frequency of spontaneous action potentials. In pheochromocytoma PC12 cells, DEX was effective at suppressing I_h together with a slowing in activation time course of the current. Taken together, findings from this study strongly suggest that during cell exposure to DEX used at clinically relevant concentrations, the DEX-mediated block of I_h appears to be direct and would particularly be one of the ionic mechanisms underlying reduced membrane excitability in the in vivo endocrine or neuroendocrine cells.     

Physiological Roles of the Rapidly Activated Delayed Rectifier K+ Current in Adult Mouse Heart Primary Pacemaker Activity

Robust, spontaneous pacemaker activity originating in the sinoatrial node (SAN) of the heart is essential for cardiovascular function. Anatomical, electrophysiological, and molecular methods as well as mathematical modeling approaches have quite thoroughly characterized the transmembrane fluxes of Na⁺, K⁺ and Ca²⁺ that produce SAN action potentials (AP) and ‘pacemaker depolarizations’ in a number of different in vitro adult mammalian heart preparations. Possible ionic mechanisms that are responsible for SAN primary pacemaker activity are described in terms of: (i) a Ca²⁺-regulated mechanism based on a requirement for phasic release of Ca²⁺ from intracellular stores and activation of an inward current-mediated by Na⁺/Ca²⁺ exchange; (ii) time- and voltage-dependent activation of Na⁺ or Ca²⁺ currents, as well as a cyclic nucleotide-activated current, I_f; and/or (iii) a combination of (i) and (ii). Electrophysiological studies of single spontaneously active SAN myocytes in both adult mouse and rabbit hearts consistently reveal significant expression of a rapidly activating time- and voltage-dependent K⁺ current, often denoted I_Kr, that is selectively expressed in the leading or primary pacemaker region of the adult mouse SAN. The main goal of the present study was to examine by combined experimental and simulation approaches the functional or physiological roles of this K⁺ current in the pacemaker activity. Our patch clamp data of mouse SAN myocytes on the effects of a pharmacological blocker, E4031, revealed that a rapidly activating K⁺ current is essential for action potential (AP) repolarization, and its deactivation during the pacemaker potential contributes a small but significant component to the pacemaker depolarization. Mathematical simulations using a murine SAN AP model confirm that well known biophysical properties of a delayed rectifier K⁺ current can contribute to its role in generating spontaneous myogenic activity.     

Electrophysiological Effects of the Transient Receptor Potential Melastatin 4 Channel Inhibitor (4-Chloro-2-(2-chlorophenoxy)acetamido) Benzoic Acid (CBA) in Canine Left Ventricular Cardiomyocytes

Transient receptor potential melastatin 4 (TRPM4) plays an important role in many tissues, including pacemaker and conductive tissues of the heart, but much less is known about its electrophysiological role in ventricular myocytes. Our earlier results showed the lack of selectivity of 9-phenanthrol, so CBA ((4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) was chosen as a new, potentially selective inhibitor. Goal: Our aim was to elucidate the effect and selectivity of CBA in canine left ventricular cardiomyocytes and to study the expression of TRPM4 in the canine heart. Experiments were carried out in enzymatically isolated canine left ventricular cardiomyocytes. Ionic currents were recorded with an action potential (AP) voltage-clamp technique in whole-cell configuration at 37 °C. An amount of 10 mM BAPTA was used in the pipette solution to exclude the potential activation of TRPM4 channels. AP was recorded with conventional sharp microelectrodes. CBA was used in 10 µM concentrations. Expression of TRPM4 protein in the heart was studied by Western blot. TRPM4 protein was expressed in the wall of all four chambers of the canine heart as well as in samples prepared from isolated left ventricular cells. CBA induced an approximately 9% reduction in AP duration measured at 75% and 90% of repolarization and decreased the short-term variability of APD₉₀. Moreover, AP amplitude was increased and the maximal rates of phase 0 and 1 were reduced by the drug. In AP clamp measurements, CBA-sensitive current contained a short, early outward and mainly a long, inward current. Transient outward potassium current (I_to) and late sodium current (I_Na,L) were reduced by approximately 20% and 47%, respectively, in the presence of CBA, while L-type calcium and inward rectifier potassium currents were not affected. These effects of CBA were largely reversible upon washout. Based on our results, the CBA induced reduction of phase-1 slope and the slight increase of AP amplitude could have been due to the inhibition of I_to. The tendency for AP shortening can be explained by the inhibition of inward currents seen in AP-clamp recordings during the plateau phase. This inward current reduced by CBA is possibly I_Na,L, therefore, CBA is not entirely selective for TRPM4 channels. As a consequence, similarly to 9-phenanthrol, it cannot be used to test the contribution of TRPM4 channels to cardiac electrophysiology in ventricular cells, or at least caution must be applied.