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The purpose of the present study was to examine the role of G(i2)alpha in Ca(2+) channel regulation using G(i2)alpha gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the beta-adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca(2+) currents in both 129Sv wild-type (WT) and G(i2)alpha gene knockout (G(i2)alpha-/-) mice. Perfusion with CCh significantly inhibited the Ca(2+) current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca(2+) currents in G(i2)alpha-/- ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca(2+) current in WT cardiomyocytes but not in G(i2)alpha-/- cells. These findings demonstrate that, whereas the Iso-G(s)alpha signal pathway is intact in G(i2)alpha gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca(2+) channels by CCh. Therefore, G(i2)alpha is necessary for the muscarinic regulation of Ca(2+) channels in the mouse heart. Further studies are needed to delineate the possible interaction of G(i) and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca(2+) current in the heart.

The purpose of the present study was to determine whether age-related changes in the expression and function of the cardiac isoform of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) play a role in SR Ca(2+)release and cell contraction. SERCA2a protein levels and subcellular localization were compared between fetal, neonatal, juvenile and adult New Zealand White rabbits. Studies of SERCA function in isolated myocytes were performed in situ by examining the rate of reloading of the SR Ca(2+)stores following caffeine-induced depletion. We found that significant quantities of SERCA2a were present early in immature heart and that SERCA2a expression reached adult levels within 15-30 days after birth. Furthermore, SERCA2a protein is present as a series of transverse striations within the cell as early as 1 day of age. In contrast to previous studies of SERCA in vitro, the SERCA protein function in situ was found to be comparable between neonatal and adult myocytes in maintaining SR Ca(2+)stores. These results indicate that the paucity of SR Ca(2+)release in immature ventricular cardiac myocytes is not the result of immaturity in SERCA2a expression., (Copyright 2000 Academic Press.)

Canine junctin is a 26-kDa transmembrane protein found in the sarcoplasmic reticulum (SR) membrane in cardiac and skeletal muscle. Junctin has recently been shown to bind directly to calsequestrin, the ryanodine receptor, and triadin. Junctin is thought to play a role in facilitating (and perhaps regulating) Ca(2+) release from the SR. Immature heart exhibits decreased utilization of SR Ca(2+) stores for cell contraction. We have cloned human and rabbit cardiac junctin and investigated the expression of junctin in developing rabbit heart. Human junctin was cloned from an adult cardiac cDNA library. Rabbit junctin was cloned by RT-PCR. Northern blot analysis demonstrates a single primary mRNA transcript of approximately 2.8 kb in hearts from both species. Sequence analysis demonstrates greater than 97% homology between the predicted amino acid sequences of human, rabbit, and canine junctin in the putative transmembrane domain and subsequent initial 61 amino acid portion of the putative luminal domain. These domains also exhibit sequence homology with triadin. The C-terminal region shows much lower (72 to 75%) sequence homology among the three species. In addition, Northern blot analysis demonstrates that the expression of junctin increases markedly in postnatal rabbit myocardium. These findings suggest that the putative transmembrane domain and subsequent initial portion of the putative luminal domain of junctin play an important role in the binding of junctin to calsequestrin, the ryanodine receptor, and triadin in the postnatal heart. Furthermore, the previously described increase in SR Ca(2+) release with development is associated with the increased expression of junctin., (Copyright 2000 Academic Press.)

Radiofrequency (RF) ablation is a nonsurgical technique using catheter-directed RF energy for treating cardiac arrhythmias in children and adults. Previous reports have suggested that sequestration of calcium (Ca2+) by the sarcoplasmic reticulum may partially protect mature cardiac myocytes from the effects of RF energy. The purposes of this study were to determine whether differences exist between neonatal and adult myocyte responses to RF energy and if myocyte damage is a Ca2+-dependent process. Because immature myocardium is functionally deficient in sarcoplasmic reticulum, we hypothesized that immature myocytes would be more susceptible to damage induced by RF energy. Isolated ventricular myocytes were obtained from neonatal and adult New Zealand White rabbits by enzymatic dissociation, then placed in a perfusion chamber designed to deliver RF energy or a heated perfusate solution. Measurements of bath temperature, cell morphology, and contractile response to electrical stimuli were recorded. RF energy application associated with increased perfusate temperature resulted in cell death, but not when the temperature rise was inhibited. Thus, the acute damage to cells exposed to RF energy appears to be mediated by thermal energy. After exposure to thermal energy, neonatal cells underwent contracture at lower temperatures than did adult cells. Perfusion with solutions containing low Ca2+ concentrations, comparable to intracellular diastolic Ca2+ levels, had a protective effect for both neonatal and adult myocytes. These findings indicate that acute cell damage after exposure to RF energy is mediated by a Ca2+-dependent process. Furthermore, immature myocardium is particularly susceptible to RF-mediated cell damage, possibly secondary to reduced Ca2+ sequestration by the sarcoplasmic reticulum.

Background: Amiodarone is an effective antiarrhythmic drug used to treat a wide variety of ventricular and supraventricular tachyarrhythmias. Recent voltage clamp studies indicate that amiodarone may possess a variety of antiarrhythmic effects., Methods: The tight-seal, whole-cell voltage clamp technique was used to investigate the acute effects of amiodarone on L-type Ca2+ channel kinetics in isolated neonatal ventricular myocytes., Results: We found that acute perfusion with 1 mumol/L amiodarone inhibited peak inward Ca2+ current by 39.1% (4.85 +/- 0.42 to 2.95 +/- 0.6 pA/pF, n = 10, p < 0.001) without changing the shape of the current-voltage relation. In addition, amiodarone shifted Ca2+ channel steady-state inactivation to more negative membrane potentials. In the absence of amiodarone, half inactivation of the Ca2+ current occurred at a membrane potential of -23.8 +/- 0.2 mV compared to -34.2 +/- 0.6 mV after addition of amiodarone (n = 11, p < 0.01). Furthermore, amiodarone significantly delayed Ca2+ current recovery from previous inactivation., Conclusions: These results provide evidence that amiodarone blocks voltage-dependent Ca2+ current in isolated neonatal rabbit ventricular myocytes by a variety of different mechanisms. The inhibitory effect of amiodarone on L-type Ca2+ current may represent an important facet of amiodarone's acute antiarrhythmic activity in the immature heart.

Cardiac responsiveness to beta-adrenergic stimulation changes with age. Developmental changes in expression of guanine nucleotide-binding coupling protein (G protein) subunits may account for these physiologic changes. We measured steady-state levels of mRNA encoding the alpha-subunit of the specific G protein that stimulates adenylyl cyclase (Gs alpha) and three isoforms of beta-subunit of G proteins (G beta) in developing myocardium. Total RNA prepared from the right and left ventricles of fetal, neonatal, juvenile, and adult rabbits was size-fractionated, blotted, and probed with 32P-labeled cDNAs encoding rat Gs alpha, bovine G beta-1, human G beta-2, and human G beta-3. For standardization, these blots were subsequently hybridized with a 32P-labeled cDNA encoding glyceraldehyde 3-phosphate dehydrogenase (GAPD). Two-dimensional densitometric analysis of autoradiographs was used to quantify relative hybridization intensities. An age-dependent decrease in mRNAs encoding Gs alpha, G beta-1, and G beta-2 relative to mRNA encoding GAPD was observed in both ventricles, while G beta-3 mRNA was not detected. At all ages studied, levels of Gs alpha and G beta-1 mRNA were similar in the two ventricles. However, G beta-2 mRNA declined more in the left ventricle than in the right ventricle during maturation. Our results demonstrate developmental control in heart for mRNAs encoding several G protein subunits. In addition, differential declines in G beta-1 and G beta-2 mRNA in the right ventricle suggest that these G beta isoforms are regulated uniquely and may reflect functional roles for these G beta isoforms in different signaling cascades.

We investigated the effects of added beta gamma subunits of G proteins (G beta gamma) on beta-adrenergic responsiveness of transmembrane Ca2+ currents (ICa) in ventricular myocytes from neonatal rabbits. G beta 1 gamma 1 purified from retinal rods was dialyzed into cells via the voltage clamp micro-electrode. Stimulation of ICa by isoproterenol was not affected by added intracellular G beta 1 gamma 1 or by carbachol alone but was completely blocked by combined G beta 1 gamma 1 and carbachol. Pretreatment of cells with pertussis toxin or temporal separation of carbachol and isoproterenol allowed stimulation of ICa by isoproterenol in cells dialyzed with G beta 1 gamma 1. Carbachol and G beta 1 gamma 1 together also did not prevent stimulation of ICa by dibutyryl-cyclic AMP. Thus, rather than simply inactivating Gs alpha by mass action, G beta 1 gamma 1 acts in concert with carbachol to inhibit isoproterenol stimulation of ICa.

Recent studies have demonstrated a relative deficiency in voltage-gated Ca2+ currents (ICa) in immature myocardium. We hypothesized that contraction in developing heart results in part from Ca2+ influx via "reverse" Na+/Ca2+ exchange current (INa/Ca). Accordingly, INa/Ca and cell contraction amplitude were measured in single neonatal and adult rabbit ventricular myocytes. INa/Ca was dependent on Ca2+ concentration, Na+ concentration, and membrane potential and was blocked by 5 mM Ni2+ but not by the Ca(2+)-channel blocker nifedipine. In neonatal cells, contraction amplitude reached a plateau for depolarizations positive to 0 mV. In adult myocytes, contraction amplitude was maximal at 0 mV and decreased at positive membrane potentials. Inhibition of ICa with nifedipine did not affect maximal contraction amplitude in neonatal myocytes but almost completely suppressed contraction of adult cells. These data suggest that Ca2+ influx via ICa is not required for contraction of neonatal rabbit cardiac myocytes. Moreover, Ca2+ influx via reversal of the Na+/Ca2+ exchange mechanism may provide a significant portion of the Ca2+ regulating cell contraction, especially during depolarization to positive membrane potentials.

Neonatal myocardium is distinctly more sensitive to extracellular calcium levels than is mature myocardium. This has been ascribed to the poorly developed sarcoplasmic reticulum of neonatal myocardium. Recent evidence has suggested that there is an increased dependence of neonatal myocardium on the sodium-calcium exchange current, and that sodium-calcium exchange may be a major source of calcium influx in neonatal myocardial cells. We determined the effect of halothane on the sodium-calcium exchange current on single neonatal (2- to 5-day-old) rabbit ventricular myocytes by means of the whole cell voltage clamp. Lower (1.5%) halothane decreased sodium-calcium exchange current by 49%, from 29 +/- 3 to 15 +/- 6 pA. Higher (3%) halothane decreased this current by 66%, from 50 +/- 9 to 17 +/- 9 pA. Thus halothane has a reversible inhibition of sodium-calcium exchange current in neonatal myocardium. Inhibition of sodium-calcium exchange current would be expected to have a magnified effect on contractility in neonatal as opposed to adult myocardium, and could theoretically ameliorate reperfusion injury due to influx of calcium via the sodium-calcium exchanger.

We evaluated the effects of clinically relevant concentrations of halothane (1%) and ketamine (10(-4) M) on activation, inactivation, and recovery from inactivation of voltage-gated sarcolemmal calcium current (ICa) in single guinea pig ventricular myocytes, using the whole cell voltage clamp. Both anesthetics had qualitatively similar effects. The potential at half-activation was shifted from -18 to -23 mV for halothane (p < 0.03) and from -17 to -21 mV for ketamine (p = 0.005). There was no change in the slope of the activation curve for either anesthetic. The potential at half-inactivation was shifted from -29 to -40 mV with exposure to halothane (p < 0.001) and from -27 to -33 mV (p < 0.001) with exposure to ketamine. There was no change in the slope of the inactivation curve with either agent. The changes in time constant of recovery from steady-state inactivation with halothane did not reach statistical significance (178 vs. 207 ms, p = 0.20) and was significantly prolonged with exposure to ketamine (106 vs. 157 ms, p = 0.005). The two anesthetics show parallel shifts in activation, inactivation, and recovery from inactivation of ICa in ventricular myocardial cells. These findings in normal ventricular myocytes may help interpret the interactions of these anesthetics with other types of heart muscle, such as ischemic and immature myocardium.

Measurement of transsarcolemmal voltage-gated Ca2+ current (ICa) in myocytes isolated from immature rabbit heart has demonstrated an unexpectedly low level of Ca2+ channel activity. We have characterized the kinetic properties of ICa in acutely isolated 21-day fetal, 1-5-day-old neonatal, and adult cardiac myocytes by the whole-cell voltage-clamp technique. The membrane potential for half-maximal steady-state inactivation became less negative with maturation (-24 +/- 3 [mean +/- SEM] mV, n = 5; -19 +/- 2 mV, n = 5; and -11 +/- 2 mV, n = 6 for fetal, neonatal, and adult myocytes, respectively; p < 0.005). In contrast, the membrane potential for half-maximal steady-state activation was not statistically different among the age groups studied. These parameters accurately predicted the voltage dependence of the sustained ICa present at the end of a 400-msec depolarization. This "window" current was significantly smaller in immature cells than in adult cells and occurred at a more negative membrane potential in the younger age groups. The time course of inactivation of ICa was not significantly different between age groups. However, ICa was inhibited by increasing the frequency of stimulation. This effect was most prominent in immature cells, particularly at more positive holding potentials. This developmental alteration in the frequency dependence of ICa was due in part to a prolonged time constant of recovery from inactivation in the younger age groups. In summary, the kinetic properties of ICa in immature cardiac cells place them at a relative disadvantage in terms of the total Ca2+ influx during a depolarization. Thus, the role of ICa in the control of cell contraction may change with development.

The properties of the ATP-sensitive potassium (KATP) current were studied in freshly isolated rabbit ventricular myocytes using the patch clamp technique. Removing ATP from the bath (intracellular) solution activated a large K+ conductance in patches from neonatal cells with properties similar to those of KATP channels in other preparations. In membrane patches from neonatal ventricular myocytes, the density of KATP channels was higher than the density of inwardly rectifying K+ channels and the mean patch KATP current was approximately 10 times that of the inwardly rectifying K+ current, at a patch membrane potential of -60 mV. Glibenclamide (10 microM) in the bath solution decreased the number of functional KATP channels, the open-state probability, and the mean patch membrane current. The single-channel conductance of the KATP channel was dependent on the external K+ concentration, and the relationship between channel conductance and external K+ concentration was fit by an exponential equation. In addition, the voltage dependence, channel density, and open-state probability of this channel were compared between neonatal and adult isolated ventricular myocytes. The single-channel conductance and channel density of the KATP channel in neonatal myocytes were significantly smaller than in adult cells. These results suggest that age-related changes occur in the properties of KATP channels.

Action potentials and voltage clamp-induced ionic currents were recorded in acutely isolated neonatal rabbit cardiac myocytes using the whole-cell voltage clamp technique. Time- and voltage-dependent Ca2+ currents in neonatal myocytes were elicited by depolarizations from a holding potential of -80 mV to various clamp potentials. The maximal measured inward Ca2+ current was 206 +/- 10 pA (mean +/- SEM, n = 51). The peak current occurred at a mean membrane potential of 7.8 +/- 1.3 mV (n = 51). The Ca2+ current voltage relation was shifted 26 mV in the positive direction when the external Ca2+ concentration was increased 10-fold. Ca2+ current rundown was observed with a half-time of approximately 20 min. Cells dialyzed with solution containing the Ca2+ chelating agent, EGTA (0.04 mM), had action potential durations similar to those previously reported in papillary muscle. In contrast, a higher concentration of EGTA (14 mM) prolonged the action potential duration. Control of the cell internal ionic composition was achieved by dialysis of the cell with a time constant for Na+ ions of 1.2 to 2.6 min. Tetrodotoxin (10 microM), included in some experiments to block Ca2+ entry via Na+ channels, was shown to be more than 98% effective. These results characterize the whole-cell voltage clamp technique as applied to immature heart cells.

We have compared transsarcolemmal Ca2+ currents in acutely isolated neonatal (1- to 5-d-old) and adult rabbit cardiac myocytes prepared using similar enzymatic techniques. Time- and voltage-dependent inward Ca2+ currents were measured using the whole-cell voltage clamp technique. In neonatal myocytes, peak Ca2+ currents measured 114 +/- 10 pA (mean +/- SEM, n = 18) as compared with 2014 +/- 403 pA in adult myocytes (n = 5, p less than 0.001). Although adult myocytes had a larger surface area (estimated from cell capacitance) than neonatal cells (113 +/- 15 x 10(-6) versus 28 +/- 2 x 10(-6) cm2, p less than 0.001), the calculated peak current density was also significantly larger in adult cells (17.9 +/- 2.5 compared to 4.3 +/- 0.4 microA/cm2 for neonatal cells, p less than 0.001). The voltage dependence of the peak Ca2+ current was similar in neonatal and adult myocytes. Early transient (T-type) Ca2+ currents were also studied by comparing the current induced by depolarization to -20 mV from holding potentials of -40 and -80 mV. T-type Ca2+ channels were present in 91% of the adult cells but were evident in only 39% of the neonatal cells. In summary, voltage-gated Ca2+ current amplitude, current density, and T-type Ca2+ channel prevalence all increase with maturation. These data suggest that neonatal myocytes may be relatively deficient in Ca2+ channel activity when compared to adult myocytes.

Properties of the inwardly rectifying K+ channel, which contributes to the maintenance of the resting membrane potential, were studied in neonatal rabbit ventricular myocytes using the patch-clamp technique. Inward rectification was evident in single-channel current-voltage (I-V) relations at potentials positive to the potassium equilibrium potential (Ek = 0 mV with [K+]o = [K+]i = 150 mM, [Mg2+]i = 2 mM). The single-channel conductance was 3.2 +/- 0.1 pS in physiological (5.4 mM) [K+]o. The zero-current potential shifted 48.4 +/- 2.4 mV for a ten-fold change in [K+]o in neonatal cells. External Ba2+ blocked the current in a dose-dependent manner. The voltage dependence, open-state probability and channel density of this channel were compared between neonatal and adult ventricular myocytes isolated by similar techniques. The open-state probability of the channel was approximately the same in neonatal (0.39 +/- 0.06, n = 13) as in adult cells (0.4 +/- 0.05, n = 11). However, in symmetrical transmembrane K+ concentration [( K+]o = [K+]i = 150 mM), the single channel conductance was significantly smaller in neonatal (25 +/- 0.3 pS, n = 25) as compared with adult cells (31 +/- 0.4 pS, n = 12). In addition, the relationship between resting membrane potential and [K+]o was measured in neonatal and adult myocytes. The resting membrane potential in the neonate was less dependent on [K+]o than in the adult. These results are consistent with an age-related change in resting membrane K+ permeability which may result from a developmental change in the single-channel conductance properties of the inwardly rectifying K+ channel.

We have reported previously that norepinephrine (NE) and epinephrine reduce acetylcholine (ACh) overflow from superfused rat atria apparently through interaction with a presynaptic alpha-1 receptor. To characterize further this novel alpha-1-mediated effect, we tested the ability of a series of alpha antagonists and agonists to modulate ACh release in this preparation. The alpha-1 selective antagonists YM 12617 and WB 4101 blocked the inhibitory action of NE with IC50 values of about 0.1 and 1 nM, respectively, whereas the alpha-2 selective antagonists Wy 26703 and rauwolscine were much less potent. These data are consistent with the involvement of an alpha-1 receptor in the response to NE. ACh release was diminished by (-)-alpha-methyl-NE but similar concentrations of the alpha-2 selective agonists B-HT 920 and UK 14304 had no effect on ACh release. A number of alpha-1 selective agonists including amidephrine, cirazoline, St 587 and SK&F 89748 failed to inhibit [3H]ACh release or had only a small effect (phenylephrine). When tested as antagonists, however, phenylephrine, cirazoline and SK&F 89748 could block the inhibitory effect of NE at concentrations consistent with their affinities at alpha-1 receptors in other systems. These compounds thus bind to but do not activate the alpha receptor regulating ACh release, apparently due to their low efficacies compared to NE. Experiments carried out after alpha receptor inactivation with phenoxybenzamine demonstrate that there is little receptor reserve for the inhibition of ACh release by NE.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated rat atria take up [3H]choline and synthesize [3H]acetylcholine (ACh). The uptake of [3H]choline has a high-affinity component with a Km of approximately 0.2 microM and a Vmax of approximately 6 fmol/min/mg wet wt. This high-affinity component of choline uptake is difficult to measure directly because it represents only a small portion of total [3H]choline uptake. However, the rate of synthesis of [3H] ACh from [3H]choline appears to reflect the activity of the high-affinity choline uptake system. Thus, [3H]ACh synthesis is most efficient at low choline concentrations and is inhibited in the presence of hemicholinium-3 and low NaCl medium. The neuronal localization of the [3H]Ach synthesized from [3H]choline is demonstrated by the finding that [3H]ACh is released from the atria by depolarization with 57 mM K+ medium. The release is Ca++ -dependent and there is a compensatory increase in the synthesis of [3H]ACh after depolarization-induced ACh release. These data suggest that [3H]choline can be specifically incorporated into a releasable pool of [3H]ACh localized in cardiac parasympathetic neurons. The synthesis of [3H]ACh is inhibited by blockade of high-affinity choline uptake and is regulated in response to neuronal activity. The application of these methods will provide a means for directly examining the physiological and pharmacological control of ACh synthesis and release from cardiac parasympathetic neurons.