Long term regulation of cardiac L-type calcium channel by small G proteins.

Calcium ions are crucial elements of excitation-contraction coupling in cardiac myocytes. The intracellular Ca(2+ ) concentration changes continously during the cardiac cycle, but the Ca(2+ ) entering to the cell serves as an intracellular second messenger, as well. The Ca(2+ ) as a second messenger influences the activity of many intracellular signalling pathways and regulates gene expression. In cardiac myocytes the major pathway for Ca(2+ ) entry into cells is L-type calcium channel (LTCC). The precise control of LTCC function is essential for maintaining the calcium homeostasis of cardiac myocytes. Dysregulation of LTCC may result in different diseases like cardiac hypertrophy, arrhytmias, heart failure. The physiological and pathological structural changes in the heart are induced in part by small G proteins. These proteins are involved in wide spectrum of cell biological functions including protein transport, regulation of cell proliferation, migration, apoptosis, and cytoskeletal rearrangement. Understanding the crosstalk between small G proteins and LTCC may help to understand the pathomechanism of different cardiac diseases and to develop a new generation of genetically-encoded Ca(2+ ) channel inhibitors.

Monovalent cations contribute to T-type calcium channel (Cav3.1 and Cav3.2) selectivity.

Low voltage-activated (LVA) Ca2+ channels regulate chemical signaling by their ability to select for Ca2+. Whereas Ca2+ is the main permeating species through Ca2+ channels, Ca2+ permeation may be modified by abundant intra- and extracellular monovalent cations. Therefore, we explored monovalent cation regulation of LVA Ca2+ permeation in the cloned T-type Ca2+ channels alpha1G (Cav3.1) and alpha1H (Cav3.2). In physiological [Ca2+], the reversal potential in symmetrical Li+ was 19 mV in alpha1G and 18 mV in alpha1H, in symmetrical Cs+ the reversal potential was 36 mV in alpha1G and 37 mV in alpha1H, and in the bi-ionic condition with Li+ in the bath and Cs+ in the pipette, the reversal potential was 46 mV in both alpha1G and alpha1H. When Cs+ was used in the pipette, replacement of external Cs+ with Li+ (or Na+) shifted the reversal potential positive by 5-6 mV and increased the net inward current in alpha1G. Taken together the data indicate that in physiological [Ca2+], external Li+ (or Na+) permeates more readily than external Cs+, resulting in a positive shift of the reversal potential. We conclude that external monovalent cations dictate T-type Ca2+ channel selectivity by permeating through the channel. Similar to Li+, we previously reported that external [H+] can regulate T-type Ca2+ channel selectivity. Alpha1H's selectivity was more sensitive to external pH changes compared to alpha1G. When Cs+ was used in the pipette and Li+ was used in the bath external acidification from pHo 7.4 to 6.0 caused a negative shift of the reversal by 8 mV in alpha1H. Replacement of internal Cs+ with Li+ reduced the pH-induced shift of the reversal potential to 2 mV. We conclude that, similar to other external monovalent cations, H+ can modify T-type Ca2+ channel selectivity. However, in contrast to external monovalent ions that readily permeate, H+ regulate T-type Ca2+ channel selectivity by increasing the relative permeability of the internal monovalent cation.

Identification of the t-type calcium channel (Ca(v)3.1d) in developing mouse heart.

During cardiac development, there is a reciprocal relationship between cardiac morphogenesis and force production (contractility). In the early embryonic myocardium, the sarcoplasmic reticulum is poorly developed, and plasma membrane calcium (Ca(2+)) channels are critical for maintaining both contractility and excitability. In the present study, we identified the Ca(V)3.1d mRNA expressed in embryonic day 14 (E14) mouse heart. Ca(V)3.1d is a splice variant of the alpha1G, T-type Ca(2+) channel. Immunohistochemical localization showed expression of alpha1G Ca(2+) channels in E14 myocardium, and staining of isolated ventricular myocytes revealed membrane localization of the alpha1G channels. Dihydropyridine-resistant inward Ba(2+) or Ca(2+) currents were present in all fetal ventricular myocytes tested. Regardless of charge carrier, inward current inactivated with sustained depolarization and mirrored steady-state inactivation voltage dependence of the alpha1G channel expressed in human embryonic kidney-293 cells. Ni(2+) blockade discriminates among T-type Ca(2+) channel isoforms and is a relatively selective blocker of T-type channels over other cardiac plasma membrane Ca(2+) handling proteins. We demonstrate that 100 micromol/L Ni(2+) partially blocked alpha1G currents under physiological external Ca(2+). We conclude that alpha1G T-type Ca(2+) channels are functional in midgestational fetal myocardium.

Identification of a T-type Ca(2+) channel isoform in murine atrial myocytes (AT-1 cells)

Calcium channels are important targets for therapeutics, but their molecular diversity complicates characterization of these channels in native heart cells. In this study, we identify a new splice variant of a low-voltage activated, or T-type Ca(2+), channel in murine atrial myocytes. To date, alpha1G and alpha1H are the only 2 T-type Ca(2+) channel isoforms found in cardiovascular tissue. We compared alpha1G and alpha1H channel current heterologously expressed in HEK 293 cells with T-type current from the murine atrial tumor cell, AT-1. AT-1 cell T-type current (I(T)) has the same voltage dependence of activation and inactivation as alpha1G and alpha1H. The cloned T-type channels and AT-1 T-type current share similar kinetics of macroscopic inactivation and deactivation. The kinetics of recovery from inactivation of T-type currents serves as an electrophysiological signature for T-channel isoform. alpha1G and AT-1 I(T) have a similar recovery from inactivation time course that is faster than that for alpha1H. In all cases, T-type current recovers with a biexponential time course, and the relative amplitude of fast and slow time courses explains the slower alpha1H recovery kinetics, rather than differences in the time constants of the individual transitions. Thus, the T-type channels may be an important contributor to automaticity in heart cells, and molecular diversity is reflected in the pathway of recovery from inactivation.

pH modification of human T-type calcium channel gating.

External pH (pH(o)) modifies T-type calcium channel gating and permeation properties. The mechanisms of T-type channel modulation by pH remain unclear because native currents are small and are contaminated with L-type calcium currents. Heterologous expression of the human cloned T-type channel, alpha1H, enables us to determine the effect of changing pH on isolated T-type calcium currents. External acidification from pH(o) 8.2 to pH(o) 5.5 shifts the midpoint potential (V(1/2)) for steady-state inactivation by 11 mV, shifts the V(1/2) for maximal activation by 40 mV, and reduces the voltage dependence of channel activation. The alpha1H reversal potential (E(rev)) shifts from +49 mV at pH(o) 8.2 to +36 mV at pH(o) 5.5. The maximal macroscopic conductance (G(max)) of alpha1H increases at pH(o) 5.5 compared to pH(o) 8.2. The E(rev) and G(max) data taken together suggest that external protons decrease calcium/monovalent ion relative permeability. In response to a sustained depolarization alpha1H currents inactivate with a single exponential function. The macroscopic inactivation time constant is a steep function of voltage for potentials < -30 mV at pH(o) 8.2. At pH(o) 5.5 the voltage dependence of tau(inact) shifts more depolarized, and is also a more gradual function of voltage. The macroscopic deactivation time constant (tau(deact)) is a function of voltage at the potentials tested. At pH(o) 5.5 the voltage dependence of tau(deact) is simply transposed by approximately 40 mV, without a concomitant change in the voltage dependence. Similarly, the delay in recovery from inactivation at V(rec) of -80 mV in pH(o) 5.5 is similar to that with a V(rec) of -120 mV at pH(o) 8.2. We conclude that alpha1H is uniquely modified by pH(o) compared to other calcium channels. Protons do not block alpha1H current. Rather, a proton-induced change in activation gating accounts for most of the change in current magnitude with acidification.

Arachidonic acid modulation of alpha1H, a cloned human T-type calcium channel.

Arachidonic acid (AA) and the products of its metabolism are central mediators of changes in cellular excitability. We show that the recently cloned and expressed T-type or low-voltage-activated Ca channel, alpha1H, is modulated by external AA. AA (10 microM) causes a slow, time-dependent attenuation of alpha1H current. At a holding potential of -80 mV, 10 microM AA reduces peak inward alpha1H current by 15% in 15 min and 70% in 30 min and shifts the steady-state inactivation curve -25 mV. AA inhibition was not affected by applying the cyclooxygenase inhibitor indomethacin or the lipoxygenase inhibitor nordihydroguaiaretic acid. The epoxygenase inhibitor octadecynoic acid partially antagonized AA attenuation of alpha1H. The epoxygenase metabolite epoxyeicosatrienoic acid (8,9-EET) mimicked the inhibitory effect of AA on alpha1H peak current. A protein kinase C (PKC)-specific inhibitor (peptide fragment 19-36) only partially antagonized the AA-induced reduction of peak alpha1H current and the shift of the steady-state inactivation curve but had no effect on 8,9-EET-induced attenuation of current. In contrast, PKA has no role in the modulation of alpha1H. These results suggest that AA attenuation and shift of alpha1H may be mediated directly by AA. The heterologous expression of T-type Ca channels allows us to study for the first time properties of this important class of ion channel in isolation. There is a significant overlap of the steady-state activation and inactivation curves, which implies a substantial window current. The selective shift of the steady-state inactivation curve by AA reduces peak Ca current and eliminates the window current. We conclude that AA may partly mediate physiological effects such as vasodilatation via the attenuation of T-type Ca channel current and the elimination of a T-type channel steady window current.

Glycosylation influences voltage-dependent gating of cardiac and skeletal muscle sodium channels.

The role of glycosylation on voltage-dependent channel gating for the cloned human cardiac sodium channel (hH1a) and the adult rat skeletal muscle isoform (microl) was investigated in HEK293 cells transiently transfected with either hH1a or microl cDNA. The contribution of sugar residues to channel gating was examined in transfected cells pretreated with various glycosidase and enzyme inhibitors to deglycosylate channel proteins. Pretreating transfected cells with enzyme inhibitors castanospermine and swainsonine, or exo-glycosidase neuroaminidase caused 7 to 9 mV depolarizing shifts of V(1/2) for steady-state activation of hH1a, while deglycosylation with corresponding drugs elicited about the same amount of depolarizing shifts (8 to 9 mV) of V(1/2) for steady-state activation of microl. Elevated concentrations of extracellular Mg(2+) significantly masked the castanospermine-elicited depolarizing shifts of V(1/2) for steady-state activation in both transfected hH1a and microl. For steady-state activation, deglycosylation induced depolarizing shifts of V(1/2) for hH1a (10.6 to 12 mV), but hyperpolarizing shifts for microl (3.6 to 4.4 mV). Pretreatment with neuraminidase had no significant effects on single-channel conductance, the mean open time, and the open probability. These data suggest that glycosylation differentially regulates Na channel function in heart and skeletal muscle myocytes.

Cloning and characterization of alpha1H from human heart, a member of the T-type Ca2+ channel gene family.

Voltage-activated Ca2+ channels exist as multigene families that share common structural features. Different Ca2+ channels are distinguished by their electrophysiology and pharmacology and can be classified as either low or high voltage-activated channels. Six alpha1 subunit genes cloned previously code for high voltage-activated Ca2+ channels; therefore, we have used a database search strategy to identify new Ca2+ channel genes, possibly including low voltage-activated (T-type) channels. A novel expressed sequence-tagged cDNA clone of alpha1G was used to screen a cDNA library, and in the present study, we report the cloning of alpha1H (or CavT.2), a low voltage-activated Ca2+ channel from human heart. Northern blots of human mRNA detected more alpha1H expression in peripheral tissues, such as kidney and heart, than in brain. We mapped the gene, CACNA1H, to human chromosome 16p13.3 and mouse chromosome 17. Expression of alpha1H in HEK-293 cells resulted in Ca2+ channel currents displaying voltage dependence, kinetics, and unitary conductance characteristic of native T-type Ca2+ channels. The alpha1H channel is sensitive to mibefradil, a nondihydropyridine Ca2+ channel blocker, with an IC50 of 1.4 micromol/L, consistent with the reported potency of mibefradil for T-type Ca2+ channels. Together with alpha1G, a rat brain T-type Ca2+ channel also cloned in our laboratory, these genes define a unique family of Ca2+ channels.

Modal behavior of the mu 1 Na+ channel and effects of coexpression of the beta 1-subunit.

The adult rat skeletal muscle Na+ channel alpha-subunit (mu 1) appears to gate modally with two kinetic schemes when the channel is expressed in Xenopus oocytes. In the fast mode mu 1 single channels open only once or twice per depolarizing pulse, but in the slow mode the channels demonstrate bursting behavior. Slow-mode gating was favored by hyperpolarized holding potentials and slow depolarizing rates, whereas fast-mode gating was favored by depolarized holding potentials and rapid depolarizations. Single-channel studies showed that coexpression of beta 1 reduces slow-mode gating, so that channels gate almost exclusively in the fast mode. Analysis of open-time histograms showed that mu 1 and mu 1 + beta 1 both have two open-time populations with the same mean open times (MOTs). The difference lies in the relative sizes of the long and short MOT components. When beta 1 was coexpressed with mu 1 in oocytes, the long MOT fraction was greatly reduced. It appears that although mu 1 and mu 1 + beta 1 share the same two open states, the beta 1-subunit favors the mode with the shorter open state. Examination of first latencies showed that it is likely that the rate of activation is increased upon coexpression with beta 1. Experiments also showed that the rate of activation for the fast mode of mu 1 is identical to that for mu 1 + beta 1 and is thus more rapid than the rate of activation for the slow mode. It can be concluded that beta 1 restores native-like kinetics in mu 1 by favoring the fast-gating mode.

The saxitoxin/tetrodotoxin binding site on cloned rat brain IIa Na channels is in the transmembrane electric field.

The rat brain IIa (BrIIa) Na channel alpha-subunit and the brain beta 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (INa) was measured at a test potential of -10 mV. Hyperpolarization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of BrIIa Na channels. The apparent half-block concentration (ED50) for STX of BrIIa current decreased with hyperpolarizing holding potentials (Vhold). At Vhold of -100 mV, the ED50 was 2.1 +/- 0.4 nM, and the affinity increased to a ED50 of 1.2 +/- 0.2 nM with Vhold of -140 mV. In the absence of toxin, the peak current amplitude was the same for all potentials negative to -90 mV, demonstrating that all of the channels were in a closed conformation and maximally available to open in this range of holding potentials. The Woodhull model (1973) was used to describe the increase of the STX ED50 as a function of holding potential. The equivalent electrical distance of block (delta) by STX was 0.18 from the extracellular milieu when the valence of STX was fixed to +2. Analysis of the holding potential dependence of TTX block yielded a similar delta when the valence of TTX was fixed to +1. We conclude that the guanidinium toxin site is located partially within the transmembrane electric field. Previous site-directed mutagenesis studies demonstrated that an isoform-specific phenylalanine in the BrIIa channel is critical for high affinity toxin block. Therefore, we propose that amino acids at positions corresponding to this Phe in the BrIIa channel, which lie in the outer vestibule of the channel adjacent to the pore entrance,are partially in the transmembrane potential drop.

Post-repolarization block of cloned sodium channels by saxitoxin: the contribution of pore-region amino acids.

Sodium channels expressed in oocytes exhibited isoform differences in phasic block by saxitoxin (STX). Neuronal channels (rat IIa co-expressed with beta 1 subunit, Br2a + beta 1) had slower kinetics of phasic block for pulse trains than cardiac channels (RHI). After the membrane was repolarized from a single brief depolarizing step, a test pulse at increasing intervals showed first a decrease in current (post-repolarization block) then eventual recovery in the presence of STX. This block/unblock process for Br2a + beta 1 was 10-fold slower than that for RHI. A model accounting for these results predicts a faster toxin dissociation rate and a slower association rate for the cardiac isoform, and it also predicts a shorter dwell time in a putative high STX affinity conformation for the cardiac isoform. The RHI mutation (Cys374-->Phe), which was previously shown to be neuronal-like with respect to high affinity tonic toxin block, was also neuronal-like with respect to the kinetics of post-repolarization block, suggesting that this single amino acid is important for conferring isoform-specific transition rates determining post-repolarization block. Because the same mutation determines both sensitivity for tonic STX block and the kinetics of phasic STX block, the mechanisms accounting for tonic block and phasic block share the same toxin binding site. We conclude that the residue at position 374, in the putative pore-forming region, confers isoform-specific channel kinetics that underlie phasic toxin block.

Post-repolarization block of cardiac sodium channels by saxitoxin.

Phasic block of rat cardiac Na+ current by saxitoxin was assessed using pulse trains and two-pulse voltage clamp protocols, and the results were fit to several kinetic models. For brief depolarizations (5 to 50 ms) the depolarization duration did not affect the rate of development or the amplitude of phasic block for pulse trains. The pulse train data were well described by a recurrence relation based upon the guarded receptor model, and it provided rate constants that accurately predicted first-pulse block as well as recovery time constants in response to two-pulse protocols. However, the amplitudes and rates of phasic block development at rapid rates (> 5 Hz) were less than the model predicted. For two pulse protocols with a short (10 ms) conditioning step to -30 mV, block developed only after repolarization to -150 mV and then recovered as the interpulse interval was increased. This suggested that phasic block under these conditions was caused by binding with increased affinity to a state that exists transiently after repolarization to -150 mV. This "post-repolarization block" was fit to a three-state model consisting of a transient state with high affinity for the toxin, the toxin bound state, and the ultimate resting state of the channel. This model accounted for the biphasic post-repolarization block development and recovery observed in two-pulse protocols, and it more accurately described phasic block in pulse trains. The transient state after repolarization was predicted to have a dwell time of 570 ms, an on rate for saxitoxin of 16 s-1 micro M-1, and an off rate of 0.2 s-1 (KD = 12 nM). These results and the proposed model suggest a novel variation on phasic block mechanisms and suggest a long-lived transient Na+ channel conformation during recovery.

Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin-resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction.

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to -1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)

The cloned cardiac Na channel alpha-subunit expressed in Xenopus oocytes show gating and blocking properties of native channels.

The neonatal rat cardiac Na channel alpha-subunit directed currents in oocytes show characteristic cardiac relative resistance to tetrodotoxin (TTX) block. TTX-sensitive currents obtained by expression in Xenopus oocytes of the alpha-subunits of the rat brain (BrnIIa) and adult skeletal muscle (microI) Na channels show abnormally slow decay kinetics. In order to determine if currents directed by the cardiac alpha-subunit (RHI) exhibit kinetics in oocytes like native currents, we compared RHI-directed currents in oocytes to Na currents in freshly isolated neonatal rat myocytes. The decay rate of RHI currents approached that of neonatal myocytes and was faster than BrnIIa and microI currents in oocytes. The voltage dependence of availability and activation was the same as that in the rat myocytes except for a 12-19 mV shift in the depolarizing direction. The RHI Na currents were sensitive to Cd2+ block, and they showed use dependence of TTX and lidocaine block similar to native currents. The current expressed in oocytes following injection of the cRNA encoding for the alpha-subunit of the cardiac Na channel possesses most of the characteristic kinetic and pharmacological properties of the native cardiac Na current.

A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties.

The cardiac sodium channel alpha subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxitoxin (STX) and more sensitive to cadmium than brain and skeletal muscle (microliter) isoforms. An RHI mutant, with Tyr substituted for Cys at position 374 (as in microliter) confers three properties of TTX-sensitive channels: (i) greater sensitivity to TTX (730-fold); (ii) lower sensitivity to cadmium (28-fold); and (iii) altered additional block by toxin upon repetitive stimulation. Thus, the primary determinant of high-affinity TTX-STX binding is a critical aromatic residue at position 374, and the interaction may take place possibly through an ionized hydrogen bond. This finding requires revision of the sodium channel pore structure that has been previously suggested by homology with the potassium channel.

Functional expression of the rat heart I Na+ channel isoform. Demonstration of properties characteristic of native cardiac Na+ channels.

We describe the expression of functional Na+ channels in Xenopus oocytes injected with cRNA transcribed from the rat heart I cDNA clone. The expressed rat heart I Na+ currents show kinetic properties and resistance to tetrodotoxin and saxitoxin which are characteristic of native cardiac Na+ currents. The primary amino acid sequence of the rat heart I alpha-subunit is therefore sufficient for expression of tetrodotoxin resistance, and the rat heart I clone is likely to account for the tetrodotoxin-resistant phenotype of cardiac and denervated skeletal muscle.

Development of cardiac beat rate in early chick embryos is regulated by regional cues.

The mesoderm of each of the paired lateral heart-forming regions (HFRs) in the stage 5-7 chick embryo includes prospective conus (pre-C), ventricle (pre-V), and sinoatrial (pre-SA) cells, arranged in a rostrocaudal sequence (C-V-SA). With microsurgery we divided each HFR into three rostrocaudally arranged segments. After 24 hr of further incubation, each segment differentiated into a spontaneously beating vesicle of heart tissue to form a multiheart embryo. The cardiac vesicles in these embryos expressed left-right and rostrocaudal beat rate gradients: the left caudal pre-SA mesoderm produced tissue with the fastest beat rate of the six while the rostral vesicle formed from right pre-C was the slowest. In another operation, we prevented the HFRs from fusing in the midline by cutting through the anterior intestinal portal at stage 8, to produce cardia bifida (CB) embryos with an independently beating half-heart on each side. In these cases, the left half-heart of 87.2% of CB embryos beat faster than the right, confirming the left-right difference in intrinsic beat rate. To assess whether the future beat rate of each region is already determined in the st 5-7 HFR, we exchanged rectangular fragments of left pre-SA mesoderm and attached endoderm with right pre-C fragments to yield a left HFR with the sequence C-V-C and a right HFR with the sequence SA-V-SA. A CB operation was subsequently performed on these exchange embryos to prevent fusion of the lateral HFRs. Preconus mesoderm, transplanted to the pre-SA region, differentiated into tissue with a rapid beat rate, while pre-SA mesoderm relocated to the preconus region formed heart tissue with a slow spontaneous rate typical of the conus. In 73% of the exchange CB embryos, the left half-heart beat faster than the right, despite the origins of its mesoderm. The exchanged mesoderm developed a rate that was appropriate for its new location rather than the site of origin of the mesodermal fragment. In a third set of operations, we implanted a fragment of st 15 differentiated conus tissue into a site lateral to the left caudal HFR in st 5, 6, and 7 embryos, and subsequently performed CB operations on them. The implant caused the adjacent half-heart to develop with a slower beat rate than in unoperated or sham-operated controls.(ABSTRACT TRUNCATED AT 400 WORDS)