Effect of transverse gap-junction channels on transverse propagation in an enlarged PSpice model of cardiac muscle

Background  

In previous PSpice modeling studies of simulated action potentials (APs) in parallel chains of cardiac muscle, it was found that transverse propagation could occur between adjacent chains in the absence of gap-junction (gj) channels, presumably by the electric field (EF) generated in the narrow interstitial space between the chains. Transverse propagation was sometimes erratic, the more distal chains firing out of order.     

Transverse propagation in an expanded PSpice model for cardiac muscle with gap-junction ion channels

Transverse propagation was previously found to occur in a two-dimensional model of cardiac muscle using the PSpice software program for electronic circuit design and analysis. Longitudinal propagation within each chain, and transverse propagation between parallel chains, occurred even when there were no gap-junction (g-j) channels inserted between the simulated myocardial cells either longitudinally or transversely. In those studies, there were pronounced edge (boundary) effects and end-effects even within single chains. Transverse velocity increased with increase in model size. The present study was performed to examine boundary effects on transverse propagation velocity when the length of the chains was held constant at 10 cells and the number of parallel chains was varied from 3 to 5, to 7, to 10, and to 20. The number of g-j channels was either zero, both longitudinally and transversely (0/0), or 100/100. Some experiments were also made at 100/0, 1/1, and 10/10. Transverse velocity and overall velocity (both longitudinal and transverse components) was calculated from the measured total propagation time (TPT), i.e., the elapsed time between when the first action potential (AP) and the last AP crossed the zero potential level. The transverse g-j channels were placed only at the ends of each chain, such that propagation would occur in a zigzag pattern. Electrical stimulation was applied intracellularly between cells A1 and A2. It was found that, with no g-j channels (0/0), overall velocity increased almost linearly when more and more chains were placed in parallel. In contrast, with many g-j channels (100/100), there was a much flatter relationship between overall velocity and number of parallel chains. The difference in velocities with 0/0 channels and 100/100 channels was reduced as the number of chains was increased. In conclusion, edges have important effects on propagation velocity (overall and transverse) in cardiac muscle simulations.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.     

Boundary effects influence velocity of transverse propagation of simulated cardiac action potentials

Background  

We previously demonstrated that transverse propagation of excitation (cardiac action potentials simulated with PSpice) could occur in the absence of low-resistance connections (gap – junction channels) between parallel chains of myocardial cells. The transverse transmission of excitation between the chains was strongly dependent on the longitudinal resistance of the interstitial fluid space between the chains: the higher this resistance, the closer the packing of the parallel chains within the bundle. The earlier experiments were carried out with 2-dimensional sheets of cells: 2 × 3, 3 × 4, and 5 × 5 models (where the first number is the number of parallel chains and the second is the number of cells in each chain). The purpose of the present study was to enlarge the model size to 7 × 7, thus enabling the transverse velocities to be compared in models of different sizes (where all circuit parameters are identical in all models). This procedure should enable the significance of the role of edge (boundary) effects in transverse propagation to be determined.     

Vasoconstrictors inhibit ATP-sensitive K+ channels in arterial smooth muscle through protein kinase C

The effects of vasoconstrictor-receptor (neuropeptide Y, alpha- adrenergic, serotonergic, histaminergic) stimulation on currents through ATP-sensitive potassium (KATP) channels in arterial smooth muscle cells were examined. Whole-cell KATP currents, activated by the synthetic KATP channel opener pinacidil or by the endogenous vasodilator, calcitonin gene-related peptide, which acts through protein kinase A, were measured in smooth muscle cells isolated from mesenteric arteries of rabbit. Stimulation of NPY-, alpha 1-, serotonin (5-HT2)-, and histamine (H1)-receptors inhibited KATP currents by 40- 56%. The signal transduction pathway that links these receptors to KATP channels was investigated. An inhibitor of phospholipase C (D609) and of protein kinase C (GF 109203X) reduced the inhibitory effect of these vasoconstrictors on KATP currents from 40-56% to 11-23%. Activators of protein kinase C, a diacylglycerol analogue and phorbol 12-myristate 13- acetate (PMA), inhibited KATP currents by 87.3 and 84.2%, respectively. KATP currents, activated by calcitonin gene-related peptide, were also inhibited (47-87%) by serotonin, phenylephrine, and PMA. We propose that KATP channels in these arterial myocytes are subject to dual modulation by protein kinase C (inhibition) and protein kinase A (activation).     

Monovalent ion current through single calcium channels of skeletal muscle transverse tubules.

Akali monovalents, Li, Na, K, Cs, and organic monovalents of molecular cross section less than 20 A2, ammonium, methylammonium, hydrazinium, guanidinium, are shown to have a measurable conductance through Ca channels of muscle transverse tubules reconstituted into planar bilayers. For the alkali series, single channel conductances follow the sequence Cs approximately equal to K greater than Na greater than Li with a conductance ratio [g(Cs)/g(Li)] = 1.7. For permeability ratios, the sequence is Li greater than Na greater than K approximately equal to Cs with [P(Li)/P(Cs)] = 1.5. Monovalent current is only unmasked when Ba ions are not present. In mixtures of Cs and Ba, single channel current reverses close to the Ba equilibrium potential and more than 100 mV away from the Cs equilibrium potential. A cutoff in conduction is found for organic cations larger than trimethylammonium; this suggests an apparent pore aperture of about 5 X 5 A. Even in such a large pore, the fact that the alkali cation permeability sequence and conductance sequence are inverted rules out molecular sieving as the mechanism of selection among monovalents.     

Biochemical analysis of L-type calcium channels from skeletal and cardiac muscle

The development of specific pharmacological agents that modulate different types of ion channels has prompted an extensive effort to elucidate the molecular structure of these important molecules. The calcium channel blockers that specifically modulate the L-type calcium channel activity have aided in the purification and reconstitution of this channel from skeletal muscle transverse tubules. The L-type calcium channel from skeletal muscle is composed of five subunits designated alpha 1, alpha 2, beta, gamma, and sigma. The alpha 1-subunit is the pore-forming polypeptide and contains the ligand binding and phosphorylation sites through which channel activity can be modulated. The role of the other subunits in channel function remains to be studied. The calcium channel components have also been partially purified from cardiac muscle. The channel consists of at least three subunits that have properties related to the subunits of the calcium channel from skeletal muscle. A core polypeptide that can form a channel and contains ligand binding and phosphorylation sites has been identified in cardiac preparations. Here we summarize recent biochemical and molecular studies describing the structural features of these important ion channels.     

Regulation of cation channels in cardiac and smooth muscle cells by intracellular magnesium

Magnesium regulates various ion channels in many tissues, including those of the cardiovascular system. General mechanisms by which intracellular Mg(2+) (Mg(i)(2+)) regulates channels are presented. These involve either a direct interaction with the channel, or an indirect modification of channel function via other proteins, such as enzymes or G proteins, or via membrane surface charges and phospholipids. To provide an insight into the role of Mg(i)(2+) in the cardiovascular system, effects of Mg(i)(2+) on major channels in cardiac and smooth muscle cells and the underlying mechanisms are then reviewed. Although Mg(i)(2+) concentrations are known to be stable, conditions under which they may change exist, such as following stimulation of beta-adrenergic receptors and of insulin receptors, or during pathophysiological conditions such as ischemia, heart failure or hypertension. Modifications of cardiovascular electrical or mechanical function, possibly resulting in arrhythmias or hypertension, may result from such changes of Mg(i)(2+) and their effects on cation channels.     

Transverse propagation of action potentials between parallel chains of cardiac muscle and smooth muscle cells in PSpice simulations

Background  

We previously examined transverse propagation of action potentials between 2 and 3 parallel chain of cardiac muscle cells (CMC) simulated using the PSpice program. The present study was done to examine transverse propagation between 5 parallel chains in an expanded model of CMC and smooth muscle cells (SMC).     

Transverse propagation of action potentials between parallel chains of cardiac muscle and smooth muscle cells in PSpice simulations

Background  

The goal of our study is to examine the effect of stimulating a two-dimensional sheet of myocardial cells. We assume that the stimulating electrode is located in a bath perfusing the tissue.     

Comparison of heterologously expressed human cardiac and skeletal muscle sodium channels.

In this study we have expressed and characterized recombinant cardiac and skeletal muscle sodium channel alpha subunits in tsA-201 cells under identical experimental conditions. Unlike the Xenopus oocyte expression system, in tsA-201 cells (transformed human embryonic kidney) both channels seem to gate rapidly, as in native tissue. In general, hSkM1 gating seemed faster than hH1 both in terms of rate of inactivation and rate of recovery from inactivation as well as time to peak current. The midpoint of the steady-state inactivation curve was approximately 25 mV more negative for hH1 compared with hSkM1. In both isoforms, the steady-state channel availability relationships ("inactivation curves") shifted toward more negative membrane potentials with time. The cardiac isoform showed a minimal shift in the activation curve as a function of time after whole-cell dialysis, whereas hSkM1 showed a continued and marked negative shift in the activation voltage dependence of channel gating. This observation suggests that the mechanism underlying the shift in inactivation voltage dependence may be similar to the one that is causing the shift in the activation voltage dependence in hSkM1 but that this is uncoupled in the cardiac isoform. These results demonstrate the utility and limitations of measuring cardiac and skeletal muscle recombinant Na+ channels in tsA-201 cells. This baseline characterization will be useful for future investigations on channel mutants and pharmacology.     

Chimeric study of sodium channels from rat skeletal and cardiac muscle.

Two isoforms of voltage-dependent Na channels, cloned from rat skeletal muscle, were expressed in Xenopus oocytes. The currents of rSkM1 and rSkM2 differ functionally in 4 properties: (i) tetrodotoxin (TTX) sensitivity, (ii) mu-conotoxin (mu-CTX) sensitivity, (iii) amplitude of single channel currents, and (iv) rate of inactivation. rSkM1 is sensitive to both TTX and mu-CTX. rSkM2 is resistant to both toxins. Currents of rSkM1 have a higher single channel conductance and a slower rate of inactivation than those of rSkM2. We constructed (i) chimeras by interchanging domain 1 (D1) between the two isoforms, (ii) block mutations of 22 amino acids in length that interchanged parts of the loop between transmembrane segments S5 and S6 in both D1 and D4, and (iii) point mutations in the SS2 region of this loop in D1. The TTX sensitivity could be switched between the two isoforms by the exchange of a single amino acid, tyrosine-401 in rSkM1 and cysteine-374 in rSkM2 in SS2 of D1. By contrast most chimeras and point mutants had an intermediate sensitivity to mu-CTX when compared with the wild-type channels. The point mutant rSkM1 (Y401C) had an intermediate single-channel conductance between those of the wild-type isoforms, whereas rSkM2 (C374Y) had a slightly lower conductance than rSkM2. The rate of inactivation was found to be determined by multiple regions of the protein, since chimeras in which D1 was swapped had intermediate rates of inactivation compared with the wild-type isoforms.     

Agonists Bay-K8644 and CGP-28392 open calcium channels reconstituted from skeletal muscle transverse tubules.

The recently described calcium channel agonists Bay-K8644 and CGP-28392 have been used to induce long-term opening of calcium channels from purified rat muscle transverse tubules (t-tubules) incorporated into planar phospholipid bilayers. Agonist-open channels are selective for divalent cations (except Mg++), display voltage-dependent kinetics, and are blocked by the calcium channel antagonist, nitrendipine. The sensitivity to dihydropyridine agonists and antagonists indicate that a pool of t-tubule calcium channels remain functional after membrane fractionation and purification.     

Structural determinants of slow inactivation in human cardiac and skeletal muscle sodium channels.

Skeletal and heart muscle excitability is based upon the pool of available sodium channels as determined by both fast and slow inactivation. Slow inactivation in hH1 sodium channels significantly differs from slow inactivation in hSkM1. The beta(1)-subunit modulates fast inactivation in human skeletal sodium channels (hSkM1) but has little effect on fast inactivation in human cardiac sodium channels (hH1). The role of the beta(1)-subunit in sodium channel slow inactivation is still unknown. We used the macropatch technique on Xenopus oocytes to study hSkM1 and hH1 slow inactivation with and without beta(1)-subunit coexpression. Our results indicate that the beta(1)-subunit is partly responsible for differences in steady-state slow inactivation between hSkM1 and hH1 channels. We also studied a sodium channel chimera, in which P-loops from each domain in hSkM1 sodium channels were replaced with corresponding regions from hH1. Our results show that these chimeras exhibit hH1-like properties of steady-state slow inactivation. These data suggest that P-loops are structural determinants of sodium channel slow inactivation, and that the beta(1)-subunit modulates slow inactivation in hSkM1 but not hH1. Changes in slow inactivation time constants in sodium channels coexpressed with the beta(1)-subunit indicate possible interactions among the beta(1)-subunit, P-loops, and the slow inactivation gate in sodium channels.     

Antipsychotics inhibit TREK but not TRAAK channels

Schizophrenia is a chronic mental illness affecting 0.4% of the population. Existing antipsychotic drugs are mainly used to treat positive symptoms such as hallucinations but have only poor effects on negative symptoms such as cognitive deficits or depression. TREK and TRAAK channels are two P domain background potassium channels activated by polyunsaturated fatty acids and mechanical stress. TREK but not TRAAK channels are regulated by Gs- and Gq-coupled pathways. The inactivation of the TREK-1 but not the TRAAK channel in mice results in a depression-resistant phenotype. In addition, it has been shown that antidepressants such as fluoxetine or paroxetine directly inhibit TREK channel activity. Here we show that different antipsychotic drugs directly inhibit TREK currents with IC(50) values of approximately 1 to approximately 20 microM. No effect is seen on TRAAK channel activity. We conclude that TREK channels might be involved in the therapeutic action of antipsychotics or in their secondary effects. Furthermore, TREK channels could play a role in the pathophysiology of psychiatric disorders such as depression and schizophrenia.     

Spike propagation in dendrites with stochastic ion channels

We investigate the effects of the stochastic nature of ion channels on the faithfulness, precision and reproducibility of electrical signal transmission in weakly active, dendritic membrane under in vitro conditions. The properties of forward and backpropagating action potentials (BPAPs) in the dendritic tree of pyramidal cells are the subject of intense empirical work and theoretical speculation (Larkum et al., 1999; Zhu, 2000; Larkum et al., 2001; Larkum and Zhu, 2002; Schaefer et al., 2003; Williams, 2004; Waters et al., 2005). We numerically simulate the effects of stochastic ion channels on the forward and backward propagation of dendritic spikes in Monte-Carlo simulations on a reconstructed layer 5 pyramidal neuron. We report that in most instances there is little variation in timing or amplitude for a single BPAP, while variable backpropagation can occur for trains of action potentials. Additionally, we find that the generation and forward propagation of dendritic Ca²⁺ spikes are susceptible to channel variability. This indicates limitations on computations that depend on the precise timing of Ca²⁺ spikes. Action Editor : Alain Destexhe     

Insulation of the conduction pathway of muscle transverse tubule calcium channels from the surface charge of bilayer phospholipid

Functional calcium channels present in purified skeletal muscle transverse tubules were inserted into planar phospholipid bilayers composed of the neutral lipid phosphatidylethanolamine (PE), the negatively charged lipid phosphatidylserine (PS), and mixtures of both. The lengthening of the mean open time and stabilization of single channel fluctuations under constant holding potentials was accomplished by the use of the agonist Bay K8644. It was found that the barium current carried through the channel saturates as a function of the BaCl2 concentration at a maximum current of 0.6 pA (at a holding potential of 0 mV) and a half-saturation value of 40 mM. Under saturation, the slope conductance of the channel is 20 pS at voltages more negative than -50 mV and 13 pS at a holding potential of 0 mV. At barium concentrations above and below the half-saturation point, the open channel currents were independent of the bilayer mole fraction of PS from XPS = 0 (pure PE) to XPS = 1.0 (pure PS). It is shown that in the absence of barium, the calcium channel transports sodium or potassium ions (P Na/PK = 1.4) at saturating rates higher than those for barium alone. The sodium conductance in pure PE bilayers saturates as a function of NaCl concentration, following a curve that can be described as a rectangular hyperbola with a half-saturation value of 200 mM and a maximum conductance of 68 pS (slope conductance at a holding potential of 0 mV). In pure PS bilayers, the sodium conductance is about twice that measured in PE at concentrations below 100 mM NaCl. The maximum channel conductance at high ionic strength is unaffected by the lipid charge. This effect at low ionic strength was analyzed according to J. Bell and C. Miller (1984. Biophysical Journal. 45:279-287) and interpreted as if the conduction pathway of the calcium channel were separated from the bilayer lipid by approximately 20 A. This distance thereby effectively insulates the ion entry to the channel from the bulk of the bilayer lipid surface charge. Current vs. voltage curves measured in NaCl in pure PE and pure PS show that similarly small surface charge effects are present in both inward and outward currents. This suggests that the same conduction insulation is present at both ends of the calcium channel.     

Hormonal and neurotransmitter regulation of Ca++ influx through voltage-dependent slow channels in cardiac muscle membrane

The voltage- and time-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca++ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. These slow channels behave kinetically as if their gates open, close, and recover more slowly than those of the fast Na+ channels; in addition, the slow channel gates operate over a less negative (more depolarized) voltage range. Tetrodotoxin does not block the slow channels, whereas the calcium antagonistic drugs, Mn++, Co++, and La ions do. The slow channels have some special properties, including their functional dependence on metabolic energy, their selective blockade by acidosis, and their regulation by cyclic AMP level. Because of their regulation by cyclic AMP, it is proposed that either the slow channel protein or an associated regulatory protein must be phosphorylated in order for the channel to be made available for voltage activation during excitation. That is, the dephosphorylated channel would be electrically silent. The requirement for phosphorylation allows the extrinsic control of the slow channels and Ca++ influx by neurotransmitters, hormones, and autacoids that affect the cyclic nucleotide levels.