Rate- and voltage-dependent effects of m-nisoldipine on action potential of partially depolarized guinea pig papillary muscle.

The rate- and voltage-dependent effects of m-Nis were studied using standard microelectrode technique and real-time microcomputer analyzing system. The onset rate for rate-dependent inhibition (RDI) on action potentials of partially depolarized papillary muscle of guinea pig was accelerated as the concentration of m-Nis was increased from 0.5 to 2 mumol.L-1 or the driving frequency decreased from 0.8 to 0.2 Hz. The steady-state values of Vmax and APA were markedly decreased by elevating the concentration of m-Nis or increasing the driving frequency. The recovery time constants of Vmax, APA, and latency period from RDI were all increased by m-Nis (1 mumol.L-1). The inhibitory effects of m-Nis on Vmax and APA were more pronounced as the resting membrane potential was decreased by elevating K+ concentration in the perfusate.

Electrophysiological effects of m-nisoldipine and nisoldipine on pacemaker cells in sinoatrial node of rabbits.

UNLABELLED: The effects of m-nisoldipine (m-Nis) and nisoldipine (Nis) on the electrical activity of pacemaker cells in sinoatrial node (SAN) of rabbit were studied using intracellular microelectrodes. THE RESULTS: APA and Vmax in SAN pacemaker cells were markedly reduced by m-Nis and Nis. The inhibitory effects of Nis on APA and Vmax were greater than those of the m-Nis. Elevation of Ca2+ concentration in the perfusate partially antagonized the inhibitory actions of m-Nis and Nis. VDD of SAN pacemaker cells were reduced by m-Nis. The change of RPF was not consistent with that of VDD. CSRT was prolonged by m-Nis and Nis in a dose-dependent manner. The inhibitory effects of Nis on SAN pacemaker cells were greater than that of the m-Nis. Both MDP and APD50 were not affected by m-Nis and Nis.

Electrophysiological effects of m-nisoldipine and nisoldipine on papillary muscles of guinea pig.

UNLABELLED: The effects of m-nisoldipine (m-Nis) and nisoldipine (Nis) on action potentials of papillary muscles in guinea pigs were studied using intracellular microelectrodes. THE RESULTS: (1) APD and Vmaxs in normal papillary muscles were reduced by m-Nis and Nis. However, the APA, Vmaxf and overshoot were not affected. (2) In the partially depolarized papillary muscles, the APA, overshoot, Vmax and APD were depressed in a dose-dependent manner. The inhibitory effects of Nis on APA, APD50 and PPD were greater than those of the m-Nis. (3) There was a good correlation between APD50 and PPD derived from a linear regression. By the linear equation, PPD was easily calculated from APD50.

Effects of m-nisoldipine on delayed afterdepolarization of canine Purkinje fibers.

Effects of m-nisoldipine (m-Nis) on delayed after depolarization (DAD) and triggered activity (TA) were studied using standard microelectrode technique. The development of DAD and TA were markedly inhibited by pre-treatment with m-Nis (1-4 mumol.L-1) as well as m-Nis treatment after DAD and TA had been elicited. The amplitude of DAD was reduced from 15.3 +/- 2.7 to 2.3 +/- 2.0 mV, and the duration from 980 +/- 45 to 130 +/- 27 ms. The occurrence of TA was also reduced or prevented by m-Nis. These effects of m-Nis might be attributed to its blocking effects on voltage-dependent calcium channel and the resultant alleviation of intracellular calcium overload.

Effects of m-nisoldipine on transmembrane currents of guinea pig papillary muscles.

Effects of m-Nis on the transmembrane currents were studied using single sucrose gap voltage clamp technique. The amplitude of slow inward current (I(si)) was 10.6 +/- 4.1 microA. Maximal inward current was induced at a membrane potential range between -20 to -25 mV. The amplitude of I(si) were significantly decreased by m-Nis (0.2 mumol.L-1) with a reduction of 47.3%. The transient inward current (I(ti)) induced by ouabain was also greatly depressed or prevented by m-Nis, resulting in the inhibition of DAD.

[Effects of m-nisoldipine on ischemic arrhythmia in conscious rats].

m-Nisoldipine iv 3, 5 and 10 micrograms/kg before coronary artery ligation markedly reduced the early ischemic arrhythmia in conscious rats. Nifedipine iv 5 and 10 micrograms/kg had the same effect on preventing ischemic arrhythmia. Nisoldipine was less effective than m-nisoldipine at equal doses. Myocardial infarct size was reduced and electrical stability of the myocardium was markedly improved as a result of the increment in VDT, VFT and VERP. These effects may be related to the mechanisms of their respective antiarrhythmic actions. It is unlikely that these calcium antagonists prevent ischemic arrhythmia by improving the blood supply to the ischemic myocardium.

The utility of fluorescent in vivo reporter genes in molecular cardiology.

In vivo reporter genes can be used in different ways in molecular cardiology. In this paper studies are presented using the green fluorescent protein and one of its mutants, S65T-GFP, as in vivo reporter genes. With this new molecular tool we studied cell type specificity of the murine ventricular myosin light chain 2 promoter, positive cell identification prior to patch clamp procedures, and the use of fluorescence activated cell sorting of transiently transfected mammalian cells.

Lidocaine block of LQT-3 mutant human Na+ channels.

In transiently transfected mammalian cells we have identified pharmacological consequences of a naturally occurring deletion mutation, delta KPQ, of the human heart Na+ channel alpha subunit that previously has been linked to one form of the long QT syndrome, an inherited heart disease. Our results show that the Class IB antiarrhythmic agent lidocaine blocks maintained inward current through and slows recovery from inactivation of delta KPQ-encoded Na+ channels. Block is greater for maintained than for peak current. Because incomplete inactivation of mutant Na+ channels is now thought to underlie the prolonged ventricular action potential, which is the phenotype of this disease, and we find that the delta KPQ mutation speeds the recovery from inactivation of drug-free mutant channels, our results provide evidence, for the first time, that clinically relevant dysfunctional properties of an ion channel can be selectively targeted on the basis of the molecular properties conferred on the channel by an inherited genetic disorder.

Effects of m-nisoldipine and nisoldipine on electric activity of human atrial tissue.

Using intracellular microelectrode technique and microcomputer analyzing system, the effects of m-nisoldipine (m-Nis) and nisoldipine (Nis) on spontaneous electric activity of human atrial tissue were studied. APA and Vmax were remarkably decreased by m-Nis (0.25 and 1.25 mumol.L-1) and Nis (0.25 mumol.L-1). RPF was also greatly decreased as a result of inhibition in VDD. The inhibitory effects of m-Nis on transmembrane potentials were less than those of Nis at equal concentration (0.25 mumol.L-1). Neither MDP nor APD90 was affected by m-Nis and Nis.

Novel LQT-3 mutation affects Na+ channel activity through interactions between alpha- and beta1-subunits.

The congenital long-QT syndrome (LQT), an inherited cardiac arrhythmia characterized in part by prolonged ventricular repolarization, has been linked to 5 loci, 4 of which have been shown to harbor genes that encode ion channels. Previously studied LQT-3 mutations of SCN5A (or hH1), the gene that encodes the human Na+ channel alpha-subunit, have been shown to encode voltage-gated Na+ channels that reopen during prolonged depolarization and hence directly contribute to the disease phenotype: delayed repolarization. Here, we report the functional consequences of a novel SCN5A mutation discovered in an extended LQT family. The mutation, a single A-->G base substitution at nucleotide 5519 of the SCN5A cDNA, is expected to cause a nonconservative change from an aspartate to a glycine at position 1790 (D1790G) of the SCN5A gene product. We investigated ion channel activity in human embryonic kidney (HEK 293) cells transiently transfected with wild-type (hH1) or mutant (D1790G) cDNA alone or in combination with cDNA encoding the human Na+ channel beta1-subunit (hbeta1) using whole-cell patch-clamp procedures. Heteromeric channels formed by coexpression of alpha- and beta1-subunits are affected: steady-state inactivation is shifted by -16 mV, but there is no D1790G-induced sustained inward current. This effect is independent of the beta1-subunit isoform. We find no significant effect of D1790G on the biophysical properties of monomeric alpha- (hH1) channels. We conclude that the effects of the novel LQT-3 mutation on inactivation of heteromeric channels are due to D1790G-induced changes in alpha- and beta1-interactions.

Distinctions in the molecular determinants of charged and neutral dihydropyridine block of L-type calcium channels.

We investigated block of the alpha1Cb subunit of L-type calcium channels by dihydropyridines (DHPs) in which a permanently charged or neutral head group was linked to the active DHP moiety by a spacer chain containing ten methylene (-CH2) groups. We compared the sensitivity of channel modulation by the charged (DHPch) and neutral (DHPn) forms to specific alpha1Cb mutations in domains IIIS5, IIIS6, and IVS6, which had previously been shown to reduce channel modulation by the neutral DHP (+)-isradipine. The effects of these mutations were studied on channel block recorded from polarized (-80 mV) and depolarized (-40 mV) holding potentials (HPs). We found that channel block by DHPn was markedly reduced at both HPs by each mutation studied. In contrast, channel block by DHPch was only modestly reduced by mutations in IIIS6 and IVS6 for block from either -40 mV or -80 mV. Replacement of IIIS5 Thr1061 by Tyr, which abolished block by DHPn in an HP-independent manner, had little effect on channel block by DHPch recorded from -40 mV. However, this mutation markedly reduced DHPch block of currents recorded from a -80 mV HP. Inhibition of current by DHPch was not markedly use-dependent, in contrast with block by verapamil, another charged calcium channel blocker. These results suggest that the presence of a permanently charged head group restricts the access of the attached DHP moiety to a subset of interaction residues on the alpha1C subunit in a voltage-dependent manner. Furthermore, these restricted interactions confer distinct functional properties upon the charged DHP molecules.

Differentiation of cardiomyocytes in floating embryoid bodies is comparable to fetal cardiomyocytes.

Embryonic stem cells will cluster and differentiate into embryoid bodies, which can develop spontaneous rhythmic contractions. From these embryoid bodies, cardiomyocytes can be isolated based on density by a discontinuous Percoll gradient. These cardiomyocytes differentiate into ventricular myocytes, which is demonstrated by the expression of the ventricular specific isoform of the myosin light chain 2 gene. In this study the functional expression of ion channels was compared between fetal cardiomyocytes (in vivo) and stem cell derived cardiomyocytes (in vitro). Sodium and calcium currents together with transient potassium currents could be detected in early developmental stages (

Developmental changes in ionic channel activity in the embryonic murine heart.

We have isolated murine embryonic atrial and ventricular cells derived from timed-pregnant females at different periods and used patch-clamp procedures to investigate age- and chamber-specific expression of ionic channels in the developing fetal mouse. Our data indicate that L-type Ca2+ channels play a dominant role in excitation during early murine cardiac embryogenesis and that Na+ channel expression increases dramatically just before birth. K+ channel expression is particularly sensitive to changes during development. Neither atrial nor ventricular cells express a slowly activating component of delayed rectification (IKs) until just before birth, and inwardly rectifying channel activity, associated with determination of cellular resting potential, is not markedly apparent until late stages of embryogenesis. Instead, we find robust expression of the ATP-regulated K+ channel at early and late states of embryonic development, which may indicate a novel functional role for this channel during morphogenesis of the heart. These results have important implications for the physiology and development of the murine cardiac conduction system and will also serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in the myocardium of both wild-type and genetically modified mice.

Overexpression of nerve growth factor in the heart alters ion channel activity and beta-adrenergic signalling in an adult transgenic mouse.

1. The electrophysiological and pharmacological properties of cardiac myocytes from the hearts of adult transgenic mice engineered to overexpress nerve growth factor (NGF) in the heart were studied. 2. There was a 12% increase in the ventricular myocyte capacitance in NGF myocytes consistent with cardiac hypertrophy, and action potential duration at 90% repolarization (APD90) was prolonged by 142 % compared with wild-type (WT) myocytes. This was due, at least in part, to a decrease in the density of two K+ currents, Ito and IK(ur), which were significantly reduced in NGF mice with no change in their electrophysiological characteristics. We found no change in the current density or electrophysiological properties of the L-type Ca2+ current. 3. The effect on Ito and IK(ur) of TEA and 4-aminopyridine (4-AP) was not different in cells isolated from WT and NGF mice. The prolongation of APD observed in NGF cells was mimicked in WT cells by exposure to 1 mM 4-AP, which partially blocked Ito, completely blocked IK(ur) and increased APD90 by 157%. 4. The isoprenaline-induced increase in ICa was significantly smaller in NGF myocytes than in WT myocytes. This was not due to a decrease in beta-adrenergic receptor (beta-AR) density, as this was increased in NGF tissue by 55%. Analysis of beta-AR subtypes showed that this increase was entirely due to an increase in beta2-AR density with no change in beta1-ARs. 5. The response of the beta-AR-coupled adenylyl cyclase system to isoprenaline, Gpp(NH)p and forskolin was studied by measuring cAMP production. In NGF tissue, isoprenaline elicited a significantly smaller response than in WT myoyctes and this was not due to reduced adenylyl cyclase activity as the responses of NGF tissue to guanylylimidodiphosphate (Gpp(NH)p) and forskolin were unaffected. 6. In conclusion, the overexpression of NGF in the mouse heart resulted in a decrease in the current density of two K+ channels, which contributed to the prolongation of the cardiac action potential. Despite an increase in beta2-AR density in the hearts of the NGF mice, the response to isoprenaline was diminished, and this was due to an uncoupling of the beta-ARs from the intracellular signalling cascade. These potentially pathological changes may be involved in the occurrence of ventricular arrhythmias in cardiac hypertrophy and failure, and this mouse provides a novel model in which to study such changes.

Developmental changes in beta-adrenergic modulation of L-type Ca2+ channels in embryonic mouse heart.

In the adult mammalian myocardium, cellular Ca2+ entry is regulated by the sympathetic nervous system. L-type Ca2+ channel currents are markedly increased by beta-adrenergic (beta-A) agonists, which contribute to changes in pacing and contractile activity of the heart. In the developing mammalian heart, the regulation of Ca2+ entry by this enzyme cascade has not been clearly established, because changes in receptor density and coupling to downstream elements of the signaling cascade are known to occur during embryogenesis. In this study, we systematically investigated the regulation of L-type Ca2+ channel currents during development of the murine embryonic heart. We used conventional whole-cell and perforated-patch-clamp procedures to study modulation of L- type Ca2+ channel currents and to assay functional activity of distinct steps in the beta-A signaling cascade in murine embryonic myocytes at different stages of gestation. Our data indicate that the L-type Ca2+ channels in early-stage (day-11 to -13) myocytes are unresponsive to either isoproterenol or cAMP. L-type Ca2+ channels in late-stage (day-17 to -19) murine myocytes, however, exhibit responses to isoproterenol and cAMP similar to responses in adult cells, providing evidence that the beta-A cascade becomes functionally active during this period of embryonic development. We found that L-type Ca2+ channel activity in early-stage cells is increased by cell dialysis with the catalytic subunit of cAMP-dependent protein kinase (cA-PK) and that dialysis of early-stage cells with the holoenzyme of cA-PK restores functional responses to forskolin and cAMP, but not to isoproterenol. Our results provide strong evidence that a key factor in the early-stage insensitivity of L-type Ca2+ channels to cAMP is the absence, or low expression level, of the holoenzyme of cA-PK but that in addition, another element in the signaling cascade upstream from adenylate cyclase is expressed at a nonfunctional level or is uncoupled from the cascade and thus contributes to L-type Ca2+ channel insensitivity to beta-A agonists in early stages of the developing murine heart.