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RESEARCH COMMUNICATION |
a Institute of Neurophysiology, University of Cologne, Cologne, Germany;
b Institute of Anatomy I, University of Cologne, Cologne, Germany; and
c Wolfson Institute for Biomedical Research, University College London, London, U.K.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ES cell-derived cardiomyocytes • patch-clamp • nitric oxide • switch in the regulation of ICa during development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have found that muscarinic modulation of ICa during the early stages of cardiomyogenesis is regulated entirely through NO. Moreover, we demonstrate that, in later stages of ES cell-derived cardiomyocytes, the signaling cascades involved in the regulation of ICa switch to the phenotype described for adult ventricular guinea pig cardiomyocytes (2) .
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Electrophysiology
Only spontaneously beating, single cardiomyocytes were selected
for patch-clamp recordings, using the whole-cell variation of the
patch-clamp technique (22)
. The cells were held in the
voltage-clamp or current-clamp mode using an Axopatch 200-A amplifier
(Axon Instruments, Foster City, Calif.). Time `0' in the figures
displaying the time course of ICa indicates the start of
recording after establishment of the whole cell configuration.
Voltage-clamped cells were held at -50 mV, and trains of depolarizing
pulses lasting 20 ms were applied to a test potential of 0 mV at a
frequency of 0.2 Hz. Current-voltage (I/V) relationships were
determined by applying 150 ms-lasting depolarizing voltage steps from
test potentials of -40 mV to 40 mV in 10 mV steps (HP -50 mV).
Membrane capacity was determined on line using the ISO 2 acquisition
software program. Data were acquired at a sampling rate of 10 kHz,
filtered at 1 kHz, stored on hard disk, and analyzed off-line using the
ISO2 analysis software package (MFK, Frankfurt, Germany). Averaged data
are expressed as means ±SEM. Statistical analysis was
performed using paired Student's t test; a
P value of <0.05 after Bonferroni correction was
considered significant. Substances were applied only after
establishment of stable ICa. The linear approximation
between control amplitude of peak ICa prior to carbachol
(CCh) application and after washout of CCh was used as an estimate for
ICa rundown. For calculation of the CCh-dependent
ICa depression, the washout value was defined as the peak
current taken on the linear regression in the same time point as the
maximal inhibition of ICa by CCh. For calculation of basal
ICa stimulation, the control ICawas taken as the reference value. Experiments where the drug
effect was less than 10% were considered without effect.
Glass coverslips containing the cells were placed in a
temperature-controlled (37°C) recording chamber and perfused
continuously with extracellular solution by gravity at a rate of 1
ml/min. Substances were applied by exchanging the solution in the
chamber; a 90% volume exchange was achieved within approximately
20 s. Patch pipettes (2–4 M
resistance) from borosilicate
glass were pulled from Hilgenberg (Malsfeld, Germany) or Clark
(Electromedical Instruments, Reading, U.K.) using a Zeitz puller (DMZ,
Munich, Germany). For current-clamp recordings, the solutions used
contained the following (in mM): internal solution: KCl 50, K aspartate
80, MgCl2 1, MgATP 3, EGTA 10, HEPES 10, pH 7.4 (KOH);
external solution: NaCl 140, KCl 5.4, CaCl2 3.6,
MgCl2 1, HEPES 10, glucose 10 pH 7.4 (NaOH). For
voltage-clamp recordings, internal solution: CsCl 120,
MgCl2 3, MgATP 5, EGTA 10, HEPES 5, pH 7.4 (CsOH); external
solution: NaCl 120, KCl 5, CaCl2 3.6, TEA-Cl 20,
MgCl2 1, HEPES 10, pH 7.4 (TEAOH).
The catalytic subunit of PKA was purchased from Promega (Heidelberg, Germany), ODQ, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), and spermine-NONOate were purchased from Alexis (Grünberg, Germany). ODQ was dissolved in DMSO (100 mM, final DMSO concentration 0.01%), all other substances were purchased from Sigma (Deisenhofen, Germany), dissolved in extracellular solution, and either prepared freshly (Isoprenaline, SNAP) or stored frozen at -20°C. Aliquots were thawed immediately before use and diluted in the bath solution to the concentration desired.
Immunocytochemistry in ES cell-derived cardiomyocytes
Single cell preparations of 7+4 and 7+9 day-old EBs were used
for immunocytochemical investigation of NOS isoform distribution and
-actinin staining. Histochemical estimation of NOS activity was
performed by applying NADPH-diaphorase staining. Single cell
preparations were fixed in 4% paraformaldehyde in 0.1 M
phosphate-buffered saline for 20 min. NADPH-diaphorase staining was
applied with a Tris buffer solution (pH 8.0) containing 83 mg
nicotinamide adenine dinucleotide phosphate (ß-NADPH), 40 mg nitro
blue tetrazolium, 125 mg monosodium maleate, and 0.1% triton X-100 at
100 ml for 2 h, followed by incubation with a 1:600 dilution of
mouse anti-rat -actinin antibody (Sigma) for 1 h at 37°C.
Peroxidase rabbit IgG kit (Vector Labs., Burlingame, Calif.) was then
used as recommended, with 3,3' diaminobenzidine as the chromogen. Cell
preparations were indirectly immunolabeled with a dilution of 1:600
-actinin mouse anti-rat antibody and a rabbit anti-mouse antibody
for iNOS, eNOS (Biomol, Hamburg, Germany), or nNOS (Alexis) in a
dilution of 1:1000 for 1 h at 37°C, followed by a TRITC-labeled
IgG goat anti-rabbit antibody (Sigma) and a biotin-labeled IgG goat
anti-mouse antibody (Vector Labs.). Thereafter, cells were treated with
extravidin FITC (Sigma). Double immunostaining for eNOS and iNOS was
performed with an eNOS rabbit anti-mouse antibody (Biomol) and a 1:1000
dilution of iNOS antibody from mouse (Affinitti, Nottingham, U.K.),
followed by the same treatment as described above.
Immunocytochemistry in murine embryonic cardiomyocytes
Murine cardiomyocytes were prepared as described before
(23)
. Briefly, mouse embryos from E9-E20 were fixed by
immersion with either Bouin's fixative or 4% paraformaldehyde.
Embryos were embedded in paraffin and sectioned. Immunocytochemistry
was performed using the same primary and secondary antibodies as
described for the ES cell-derived cardiomyocytes. Content of cGMP was
evaluated using a cGMP rabbit antibody (Quartett, Hamburg, Germany) at
a dilution of 1:600.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Superfusion of spontaneously contracting EDS cells with carbachol (CCh, 1 µM) strongly suppressed their action potential (AP) frequency (n=33), and this effect was fully reversible after washout (Fig. 1E ). In contrast to sinusnodal and atrial cardiomyocytes (24) , the majority of EDS cells did not show concomitant hyperpolarization of the resting membrane potential (Fig. 1E ), indicating that an acetylcholine-induced K+ current (IK,ACh) was not involved. This effect could, however, be explained by a CCh-mediated depression of ICa. About 20% of the LDS cell population (n=28) had a more hyperpolarized resting membrane potential (Fig. 1F ) with a longer AP duration, resembling adult ventricular cardiomyocytes. In these cells, application of CCh had no effect (Fig. 1F ).
The effect of CCh on the peak amplitude of basal ICa was studied under voltage-clamp conditions. A depression by CCh of ICa (58 ±3%, Fig. 2Aa–Ac )was observed in 72% of EDS cells (Fig. 2C ), and this effect was rapidly reversed after washout of the agonist (Fig. 2Aa–Ac ). In EDS cells, I/V curves demonstrated that the CCh-mediated decrease of basal ICa was linear at all potentials tested. By contrast, CCh did not affect basal ICa in most LDS cells (Fig. 2Ba, Bb, C ) or in murine ventricular cardiomyocytes (n=5, data not shown) unless prestimulated with ß-adrenergic agonists. As reported for murine embryonic cardiomyocytes (25) , only 15% of EDS cells revealed a ß-adrenergic increase of ICa (n=16, data not shown).
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To understand the signaling cascades involved in the muscarinic depression of basal ICa in EDS cells, we tested whether this effect was mediated through pertussis toxin (PTX) -sensitive G-proteins and cAMP/protein kinase A (PKA), respectively. Indeed, in cells pretreated with PTX (1 µg/ml for 12 h), CCh no longer decreased basal ICa (Fig. 3Aa–Ac ).Intracellular perfusion via the patch pipette of the cAMP-dependent PKA inhibitor PKI (fragment 6-22 amide, 2 µM) caused complete abolishment of the muscarinic depression of ICa in EDS cells (n=8, data not shown). In addition, PKI inclusion in the pipette solution resulted in an enhanced rundown of basal ICa as compared to control measurements in the absence of PKI indicating a high intrinsic PKA activity. Intracellular application of the catalytic subunit of PKA (7 µM) resulted in an irreversible stimulation of ICa density by 115 ±24% over basal levels in all cells tested. Under these conditions, CCh was no longer able to depress ICa (Fig. 3Ba–Bc ). Similarly, inhibition of cAMP breakdown by the PDE inhibitor isobutylmethylxanthine (IBMX; 10 µM intracellular application, 100 µM bath application) increased basal ICa and blocked the CCh effect in EDS cells (Fig. 3Ca–Cc ). These data suggest that the muscarinic inhibition of basal ICa is mediated through PTX-sensitive Gi/o proteins and a PDE-dependent reduction of the cAMP-PKA activity. The muscarinic depression of cAMP in EDS cells was further confirmed by investigations on the regulation of the hyperpolarization-activated nonselective cation current (If), where the application of CCh resulted in a reduction of If and ICa in the same cell (n=6, J. G. Ji and B. K. Fleischmann, unpublished results).
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To determine the role of endogenous NO production in the regulation of ICa in ES cell-derived cardiomyocytes, EDS cells were preincubated with the NO synthase (NOS) inhibitors N-methyl-L-arginine (L-NMA, 200 µM for 10–15 min) (Fig. 4Aa–Ac )and N-nitro-L-arginine methylester (L-NAME, 1 mM for 4 h, n=29; data not shown), respectively, before addition of CCh. Both inhibitors completely prevented the CCh-mediated inhibition of ICa. This effect was reversed on coapplication of an excess of the endogenous NOS substrate, L-arginine (L-Arg, 400 µM; shown for L-NMA in Fig. 4Ba–Bc ). In agreement with these findings, application of the NO-generating compounds SNAP (150 µM) and spermine-NONOate (200 µM) reversibly decreased ICa density by 38 ±7% and 51 ±5%, respectively, and mimicked the effect of CCh in 83% (n=6) and 77% (n=22), respectively, of EDS cells tested (data not shown).
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To investigate which NOS isoform is involved in the endogenous production of NO and modulation of ICa aminoguanidine (AG; 100 µM), a compound with relative selectivity for inhibition of iNOS (26) was applied via the patch pipette. AG was found to increase basal ICa density of EDS cells by 44 ±10% (Fig. 4Ca–Cc ). In contrast, 89% of all LDS cells tested failed to increase the basal ICa density upon application of AG (n=9, data not shown). Despite the inhibition of iNOS by AG, CCh, probably via eNOS (see below), decreased ICa density in EDS cells to much the same extent as under control conditions (Fig. 4Ca–Cc ).
The possible involvement of cGMP in the NO-dependent effect was investigated by application of a selective blocker of sGC ODQ (10 µM via pipette, 10 µM superfusion) (27) . ODQ completely prevented the CCh-mediated inhibition of ICa (Fig. 5Aa–Ac ).Concurrent with a blockade of the CCh effect, ODQ markedly and consistently increased basal ICa in EDS cells. In agreement with the notion that an increase in cGMP may stimulate the activity of type II phosphodiesterase (PDE-II), which in turn may result in a decrease in cAMP levels (14) , application of the selective PDE-II inhibitor erythro-9-(2-hydroxyl-3-nonyl)adenine (EHNA) (28) (30 µM) increased the basal ICa of EDS cells by 45 ±7% (Fig. 5Ba–Bc ) and completely blocked the effect of CCh on basal ICa (Fig. 5Ba–Bc ). Conversely, application of milrinone (10 µM), a blocker of the cGMP inhibited PDE isoform, PDE-III, increased basal ICa but did not prevent the CCh effect in EDS cardiomyocytes (n=17, data not shown).
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Since the ANF receptor is coupled directly to the particulate isoform of guanylyl cyclase (29) , we tested the effect of atrial natriuretic peptide (ANP, rat fragment 3-28) on basal ICa in EDS cells. ANP (20 nM) strongly depressed basal ICa in 71% of the cells tested (n=24, Fig. 6Aa, Ab ).Opposite to the muscarinic modulation, however, preincubation in L-NMA (n=10, Fig. 6Ba, Bb ) or ODQ (n=20, Fig. 6Ca, Cb ) did not affect ANP-induced inhibition of ICa in most cardiomyocytes examined.
In LDS cells, Iso (0.1 µM) stimulated basal ICa by 42 ±10% whereas the CCh-mediated inhibition of the prestimulated ICa density amounted to 43 ±4% (Fig. 7Aa–Ac ).In clear contrast to the findings in EDS cells, the muscarinic receptor-mediated inhibition of ICa after ß-adrenergic stimulation in LDS cardiomyocytes was neither affected by blockade of NOS nor by inhibition of sGC. Even in the presence of maximally effective concentrations of L-NMA (Fig. 7Ba–Bc ) and ODQ (Fig. 7Ca–Cc ), ß-adrenoceptor-prestimulated ICa was consistently down-regulated by CCh to a similar extent as in control cells, suggesting that basal NO production does not contribute to the setting of ICa in LDS cells. However, when the availability of NO was enhanced by addition of the NO donor SNAP (150 µM) to Iso-prestimulated cells, ICa density was depressed by 37 ±6% in six out of six cells (Fig. 7Da–Dc ).
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To identify which NOS isoform(s) may be responsible for the NO
production in early stage cardiomyocytes, single EDS cells were
isolated from beating areas of EBs and investigated
immunocytochemically using specific antibodies against the three known
NOS isoforms and -actinin for myofilament staining. The
histochemical NADPH-diaphorase stain, which reacts with the reductase
domain of all NOS isoforms, revealed distinct differences in enzyme
expression between EDS and LDS cardiomyocytes. EDS cells expressed
-actinin with no or only discrete myofilament organization and
displayed strong NADPH-diaphorase staining, predominantly in
perinuclear areas (Fig. 1A
). In contrast, LDS cells
contained well-organized myofilaments (Fig. 1B
) and, in
agreement with findings in adult hearts (30)
, revealed
only relatively weak NADPH-diaphorase activity (Fig. 1B
).
EDS cells expressed both eNOS (Fig. 1C
) and iNOS, whereas
LDS cells expressed only eNOS at low levels (Fig. 1D
). The
neuronal isoform nNOS was found in neither early nor late stage
cardiomyocytes (see Table 1
).
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To confirm the pattern of NOS-expression detected in ES cell-derived cardiomyocytes, immunocytochemistry was performed in the developing murine embryonic heart. Similar to EDS cells, cardiomyocytes of E9–E13-old embryos displayed strong expression of iNOS (Fig. 8 )and eNOS. In contrast, in E16-old murine cardiomyocytes, a relatively weak antibody staining for eNOS was detected. As reported for LDS cells, no iNOS expression was observed (Fig. 8) . Moreover, the cGMP content determined by antibody staining was found to be high in young murine embryonic cardiomyocytes, but very low in old embryonic cardiomyocytes (Fig. 8) .
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We demonstrate here that in EDS cardiomyocytes, as in adult ventricular cardiomyocytes, ICa is regulated by changes in the intracellular concentration of cAMP (2) . However, in contrast to LDS cells and adult murine ventricular cardiomyocytes, ICa down-regulation in EDS cells already occurred in the absence of ß-adrenergic prestimulation of ICa, probably as a consequence of high basal AC activity and, thus, high cAMP-PKA levels. This is supported by the observation that application of PKI caused a fast rundown of basal ICa, whereas the nonselective PDE inhibitor IBMX and the PDE-II inhibitor EHNA resulted in marked disinhibition of basal ICa. Although at present it remains unclear what mechanism accounts for the stimulation of AC activity in EDS cells, resting ICa was found to be markedly decreased upon challenge with the muscarinic receptor agonist CCh. Unlike in terminally differentiated cardiomyocytes, ß-adrenoceptors in EDS cells are not yet functionally coupled to their target enzyme, AC, which explains the lack of effect of Iso on basal ICa in these cells. A lack of ß-adrenergic stimulation of ICa, probably as a result of dysfunctional ß-adrenoceptor/G-protein coupling (35) , has also been reported for embryonic murine cardiomyocytes (25) .
The inhibitory effect of CCh on ICa was fully dependent on the activity of NOS, as demonstrated by the complete blockade of CCh action by L-NMA and L-NAME, respectively, and prevention of this blockade with excess L-Arg. The inhibitory action of CCh was mimicked by application of the NO donors SNAP and spermine-NONOate. These NO-dependent decreases in ICa appeared to be mediated entirely by the sGC/cGMP system, as evidenced by the complete inhibition of CCh effect by the sGC inhibitor ODQ, ruling out a direct modulatory action of NO on the Ca2+ channel (36 , 37 ) or cGMP-independent NO effects. Consistent with the functional role for NO, in EDS cells and early murine embryonic cardiomyocytes both eNOS and iNOS expression and activity were high and decreased dramatically in LDS cells and late embryonic ventricular cardiomyocytes, underscoring the similarity in cardiomyogenesis between ES cell-derived and murine embryonic cardiomyocytes (19 , 20 ).
Activation of the ANF receptor, known to be expressed early during murine cardiac development (38) and directly coupled to the particulate pGC (29 , 39 ), also resulted in depression of basal ICa in EDS cells. As expected, this effect was not sensitive to NOS inhibition or sGC blockade, further underscoring the specificity of the muscarinic regulation during early and later stages of cardiomyogenesis.
Together, these results suggest that cGMP( via its action on cGMP-stimulated PDE-II) represents the muscarinic inhibitory messenger of cardiac Ca2+ entry, which may indicate an early regulatory motive during ontogenesis (40) . cGMP levels elevated by four- to fivefold have been detected in rat embryos and neonates (41) . This is consistent with our findings in early embryonic murine cardiomyocytes (E9-E13), where very high cGMP concentrations were found. In contrast to rat ventricular myocytes (42) and embryonic chick cardiomyocytes (43 , 44 ), where the cGMP-dependent protein kinase PKG has been suggested to be involved in the regulation of ICa, in EDS cells the cGMP effect appears to be mediated entirely through the PDE II-cAMP pathway, as reported for rabbit sinusnodal cells (16) . This is supported by the observations that maximal stimulation of PKA activity by the catalytic subunit of PKA, inhibition by PKI, or blockade of PDE II by EHNA abolished the muscarinic effect on ICa. Accordingly, If, an ion channel known to be stimulated directly by cAMP (45) , was also inhibited by muscarinic receptor activation. The effect of CCh was completely blocked by PTX, suggesting an involvement of Gi or Go proteins in the muscarinic generation of NO in EDS cells. These data agree with a recent report demonstrating a PTX-dependent increase in eNOS activity and protein expression upon muscarinic receptor activation (46) .
We envisage the following scenario (see Fig. 9 ):In EDS cardiomyocytes, muscarinic down-regulation of ICa is mediated entirely via the NO/cGMP pathway. Both eNOS and iNOS are constitutively expressed at relatively high levels, whereas the former appears to be coupled to the muscarinic receptor via a G-protein. Basal (iNOS-mediated, AG-sensitive) and stimulated (eNOS-mediated) formation of NO activates sGC, resulting in an increase in intracellular cGMP concentration. Although basal levels of endogenously produced NO are already high, an enhancement in NO availability through application of NO donors or, physiologically, additional release from neighboring cells or nitrergic nerves may further stimulate cGMP production. AC is not yet functionally coupled to the ß-adrenoceptor and presumably has a high constitutive turnover rate for ATP. Thus, increased NO formation secondary to stimulation of muscarinic receptors translates, via activation of PDE-II, into a decreased level of cAMP, which in turn results in a diminished PKA activity, reduced phosphorylation of the L-type Ca2+ channel, and the reduction of Ca2+ entry into the cardiomyocyte (1 , 6 ). In LDS cells as in adult ventricular cardiomyocytes of mouse and other mammals (2 , 4 ), muscarinic receptor activation depresses ICa only after ß-adrenergic prestimulation. At this developmental stage, the muscarinic receptor is coupled via Gi proteins to AC (3) . As this allows for control of cAMP concentrations at the level of its production rather than its degradation, NO formation does not need to be as high as in EDS cells, offering an explanation for the lower expression of NOS in LDS cells.
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We suggest that the developmentally early NO/cGMP-related pathway of ICa regulation, which is dormant in LDS cells, can be activated upon an increase in NO availability, e.g., after induction of iNOS. Consistent with this view, adult cardiomyocytes do not express iNOS unless pathologically altered (47) . When iNOS expression is induced (48) , however, the increased concentration of NO can exert negative inotropic effects (10) , possibly through a depression of ICa. This may at least partly explain ß-adrenergic hyporesponsiveness and myocardial dysfunction in inflammatory heart disease. If so, then therapeutic interventions should aim at either intercepting at the level of the increased NO and cGMP formation (by inhibiting iNOS and/or sGC) or elevating cAMP levels (e.g., by inhibiting cAMP-degrading PDE-II).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 These authors contributed equally to the
manuscript.
3 Abbreviations: AC, adenylylcyclase; AG, aminoguanidine; ANP, atrial natriuretic peptide; AP, action potential; CCh, carbachol; E, embryonic day (E9, embryonic day 9); EB, embryoid body; EDS, early development stage; EHNA, erythro-9-(2-hydroxyl-3-nonyl)adenine; ES, embryonic stem; IBMX, isobutylmethylxanthine; If, hyperpolarization-activated nonselective cation current; IK,Ach, acetylcholine-induced K+ current; ICa, L-type Ca2+ current; iNOS, inducible NOS; Iso, isoprenaline; I/V, current-voltage; LDS, late development stage; L-Arg, L-arginine; L-NAME, N-nitro-L-arginine methylester; L-NMA, N-methyl-L-arginine; nNOS, neuronal NOS; NO, nitric oxide;NOS, nitric oxide synthase; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PDE, phosphodiesterases; PDE-II, type II phosphodiesterase; PKA, cAMP-dependent protein kinase A; PKI, protein kinase inhibitor; PTX, pertussis toxin; sGC, soluble guanylyl cyclase; SNAP, S-nitroso-N-acetyl-D,L-penicillamine; VDCC, voltage-0 dependent L-type Ca2+ channels.
Received for publication March 30, 1998.
Revision received July 31, 1998.
Accepted for publication September 29, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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