Regulation of the L-type Ca21 channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition G. J. JI,*,1 B. K. FLEISCHMANN,*,1 W. BLOCH,† M. FEELISCH,‡ C. ANDRESSEN,† K. ADDICKS,† AND J. HESCHELER*,2 *Institute of Neurophysiology, University of Cologne, Cologne, Germany; †Institute of Anatomy I, University of Cologne, Cologne, Germany; and ‡Wolfson Institute for Biomedical Research, University College London, London, U.K. ABSTRACT In adult mammalian cardiomyocytes, stimulation of muscarinic receptors counterbalances the b-adrenoceptor-mediated increase in myocardial contractility and heart rate by decreasing the L-type Ca21 current (ICa) (1, 2). This effect is mediated via inhibition of adenylyl cyclase and subsequent reduction of cAMP-dependent phosphorylation of voltagedependent L-type Ca21 channels (3). Little is known, however, about the nature and origin of this pivotal inhibitory pathway. Using embryonic stem cells as an in vitro model of cardiomyogenesis, we found that muscarinic agonists depress ICa by 58 63% (n534) in early stage cardiomyocytes lacking functional b-adrenoceptors. The cholinergic inhibition is mediated by the nitric oxide (NO)/cGMP system since it was abolished by application of NOS inhibitors (L-NMA, L-NAME), an inhibitor of the soluble guanylyl cyclase (ODQ), and a selective phosphodiesterase type II antagonist (EHNA). The NO/cGMP-mediated ICa depression was dependent on a reduction of cAMP/ protein kinase A (PKA) levels since application of the catalytic subunit of PKA or of the PKA inhibitor PK) prevented the carbachol effect. In late development stage cells, as reported for ventricular cardiomyocytes (2, 4), muscarinic agonists had no effect on basal ICa but antagonized b-adrenoceptor-stimulated ICa by 43 64% (n516). This switch in signaling pathways during development is associated with distinct changes in expression of the two NO-producing isoenzymes, eNOS and iNOS, respectively. These findings indicate a fundamental role for NO as a signaling molecule during early embryonic development and demonstrate a switch in the signaling cascades governing ICa regulation.—Ji, G. J., Fleischmann, B. K., Bloch, W., Feelisch, M., Andressen, C., Addicks, K., Hescheler, J. Regulation of the L-type Ca21 channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition. FASEB J. 13, 313–324 (1999)
Key Words: ES cell-derived cardiomyocytes z patch-clamp z nitric oxide z switch in the regulation of ICa during development 0892-6638/99/0013-0313/$02.25 © FASEB
In the heart, the force of contraction is critically dependent on the influx of Ca21 ions through voltage-dependent L-type Ca21 channels (VDCC)3 (5). Under conditions of b-adrenoceptor stimulation, the amplitude of the L-type Ca21 current (ICa) is augmented and the force of contraction increased (6). Subsequent muscarinic receptor activation decreases ICa and results in negative inotropy (2, 7). Even though the regulation of ICa by the sympathetic and parasympathetic nervous systems has been thoroughly investigated (3, 8), it is currently under debate whether changes in the involved signaling cascades may underlie or contribute to certain myocardial disorders. Recent evidence suggests that nitric oxide (NO) may be involved in the decreased responsiveness to b-adrenergic stimulation under conditions of left ventricular dysfunction and immunotherapy-induced cardiomyopathy (9 –12) by exerting a negative inotropic and chronotropic action (13). This works possibly via modulation of isoprenaline (Iso) prestimulated ICa secondary to an activa1
These authors contributed equally to the manuscript. Correspondence: Institut fu¨r Neurophysiologie. Universita¨t zu Ko¨ln. Robert-Koch-Str. 39, D-50931 Ko¨ln, Germany. E-mail:
[email protected] 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, erythro9-(2-hydroxyl-3-nonyl)adenine; ES, embryonic stem; IBMX, isobutylmethylxanthine; If, hyperpolarization-activated nonselective cation current; IK,Ach, acetylcholine-induced K1 current; ICa, L-type Ca21 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, cAMPdependent 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 Ca21 channels. 2
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tion of soluble guanylyl cyclase (sGC) and cGMPdependent phosphodiesterases (PDEs) (14 –16). However, the origin and implication of these findings remain obscure. As dysfunctions in adult cells may stem from activation of a dormant pathway developed earlier during cellular differentiation, we sought to investigate the regulation of ICa during cardiac ontogenesis. For this purpose we have used embryonic stem (ES) cell-derived (line D3) (17) cardiomyocytes (18), which provide a unique tool for electrophysiological and functional studies of developing mammalian cells (19, 20). 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).
MATERIALS AND METHODS ES cell preparation Murine embryonic stem cells of the line D3 were cultured and differentiated into spontaneously beating cardiomyocytes as described previously (19). Briefly, cells were grown as spheroidal aggregates (embryoid bodies; EBs) in hanging drops for 2 days, then transferred into suspension for 5 days, and finally plated for different periods (3– 4 and 9 –12 days, respectively) in 24-microwell plates. Single cardiomyocytes were isolated from clusters of spontaneously beating areas by a modified procedure of Isenberg and Klo¨ckner (21). Beating areas of 15 to 20 EBs were isolated with a sterile microscalpel and collected in low Ca21 solution containing (in mM): 120 NaCl, 5.4 KCl, 5 MgSO4, 5 Na pyruvate, 20 glucose, 20 taurine, 10 HEPES (pH 6.9 with NaOH). The tissue was then incubated in enzyme medium (1 mg/ml collagenase B; Boehringer, Mannheim, Germany; 30 mM CaCl2) for 20 min at 37°C. Tissue fragments were transferred into a medium containing (in mM): 85 KCl, 30 K2HPO4, 5 MgSO4, 1 EGTA, 2 Na2ATP, 5 Na pyruvate, 5 creatine, 20 taurine, 20 glucose, pH 7.2, where they were kept at room temperature for 1 h and then resuspended in Dulbecco’s modified Eagle’s medium (Gibco, Eggenstein, Germany) complemented with 20% fetal calf serum. Isolated cells were plated on sterile, gelatine-coated glass coverslips and kept in the incubator for 24 – 48 h. Spontaneously contracting myocytes could be observed within 12 h after cell preparation. Murine ventricular cardiomyocytes were isolated from adult mice by collagenase treatment, as reported elsewhere (21). 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 250 mV, and trains of depolarizing pulses lasting 20 ms were 314
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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 240 mV to 40 mV in 10 mV steps (HP 250 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 6sem. 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 ICa was 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 MV 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 voltageclamp 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 (Gru¨nberg, 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 220°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 714 and 719 day-old EBs were used for immunocytochemical investigation of NOS isoform distribution and a-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 (b-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 a-actinin antibody (Sigma) for 1 h at 37°C. Peroxidase rabbit IgG kit (Vector Labs., Burlingame, Calif.) was then used as recommended, with 3,39 diaminobenzidine as the chromogen. Cell preparations were indirectly immunolabeled with a dilution of 1:600 a-actinin
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Figure 1. A, B) Expression of NADPH-diaphorase at different developmental stages in cardiomyocytes. A) EDS cells showing strong NADPH-diaphorase activity. B) LDS cardiomyocyte with very weak NADPH-diaphorase activity. C) Upper panel: EDS cell was identified as cardiomyocyte by a-actinin staining; lower panel: this cell showed strong eNOS expression. D) Upper panel: LDS cell was identified as cardiomyocyte by a-actinin staining, with clearly visible sarcomeric structures. Lower panel: only weak expression of eNOS was visible. Horizontal calibration bar equals 10 mm. E ) Action potentials (AP) recorded from a spontaneously contracting EDS cell in the current clamp mode. The cell had a relatively depolarized resting membrane potential and only a small overshoot. Application of carbachol (CCh) produced a prominent negative chronotropic effect, but no membrane hyperpolarization. F ) APs recorded from a spontaneously contracting LDS cell. Application of CCh did not alter AP frequency.
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.
RESULTS Early development stage (EDS) cardiomyocytes (713– 4 days, i.e., differentiated for 7 days within EBs L-TYPE Ca21 CHANNEL REGULATION
in suspension and kept for another 3– 4 days after plating on glass coverslips; Fig. 1A) predominantly express L-type Ca21 channels and transiently inactivating K1 channels (19). In contrast, late development stage (LDS) cardiomyocytes (719 –12 days, Fig. 1B) show a diversity in phenotype and express most of the ionic currents typical for adult ventricular, atrial, or sinusnodal cardiomyocytes (19). Both EDS and LDS in culture contained a small percentage (20 –25%) of LDS or EDS cells, respectively. Superfusion of spontaneously contracting EDS cells with carbachol (CCh, 1 mM) strongly suppressed their action potential (AP) frequency (n533), 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 K1 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 (n528) had a more hyperpolarized resting membrane potential (Fig. 1F) with a longer AP duration, resembling adult 315
Figure 2. Effect of carbachol (CCh, 1 mM) superfusion on basal ICa in ES cell-derived EDS and LDS cardiomyocytes. Aa) Representative recordings from an EDS cell. The cell was voltage clamped (HP 250 mV) and 20 ms-lasting depolarizing pulses to 0 mV evoked ICa. The control ICa (1) was clearly depressed upon CCh application (2), and this effect was reversed at washout (3). Ab) Time course of the peak ICa in the same cell. Depolarizing pulses to 0 mV were applied repetitively at a frequency of 0.2 Hz; the upper trace shows the holding current. The numbers indicate the currents displayed in panel Aa. Ba, Bb) Current traces and ICa time course for an LDS cell. No depression of ICa upon CCh application was observed (note typical current rundown). Ac) ICa density in EDS cells with CCh response. The first control pulse was taken after rupturing the membrane. The second control pulse was recorded just before CCh application; note the absence of a prominent ICa run-up. *Statistical significant difference between current densities during CCh application and after washout (P,0.05). C) Percentage of EDS and LDS cardiomyocytes displaying a CCh-induced depression of basal ICa.
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 63%, 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 316
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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 (n55, data not shown) unless prestimulated with b-adrenergic agonists. As reported for murine embryonic cardiomyocytes (25), only 15% of EDS cells revealed a b-adrenergic increase of ICa (n516, data not shown). 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 mg/ml for 12 h), CCh no longer decreased basal ICa (Fig. 3Aa–Ac). Intracellular perfusion via the patch pipette of the cAMPdependent PKA inhibitor PKI (fragment 6-22 amide, 2 mM) caused complete abolishment of the muscarinic depression of ICa in EDS cells (n58, 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 mM) resulted in an irreversible stimulation of ICa density by 115 624% 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 mM intracellular application, 100 mM 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 PDEdependent 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 (n56, J. G. Ji and B. K. Fleischmann, unpublished results). 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 mM for 10 –15 min) (Fig. 4Aa–Ac) and N-nitro-L-arginine methylester (L-NAME, 1 mM for 4 h, n529; 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 mM; shown for L-NMA in Fig. 4Ba–Bc). In agreement with these findings, application of the NO-generating compounds SNAP (150 mM) and
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(Fig. 4Ca–Cc). In contrast, 89% of all LDS cells tested failed to increase the basal ICa density upon application of AG (n59, 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 NOdependent effect was investigated by application of a selective blocker of sGC ODQ (10 mM via pipette, 10
Figure 3. Effect of various agents on CCh-induced depression of ICa in EDS cells. Column a: Representative time course of peak ICa in individual cells in response to application of the test agents. Column b: Percentage of cells responding (filled bars) or nonresponding (shaded bars) to CCh. Column c: Changes in ICa density upon different interventions. Aa) Cell preincubated with PTX (1 mg/ml, 12 h). In this and most other cells tested, the CCh effect on basal ICa was absent. Ab) Almost no cells responded to CCh. Ac) Lack of effect of CCh on ICa density in nonresponding cells (decline in ICa density due to current rundown). Ba) Intracellular perfusion via the patch pipette of the catalytic subunit of PKA (7 mM) led to a strong stimulation of the basal ICa. The CCh effect on ICa was abolished. Bb) All EDS cells lack the CCh response. Bc) Stimulation of ICa density by the catalytic subunit of PKA and lack of the CCh-induced depression of basal ICa. Ca) Combined application of IBMX (10 mM via pipette, 100 mM via superfusion) increased ICa amplitude and prevented the CCh-induced depression of ICa. Cb) Practically no cells responded to CCh. Cc) ICa density in cells responding to IBMX, but not to CCh.
spermine-NONOate (200 mM) reversibly decreased ICa density by 38 67% and 51 65%, respectively, and mimicked the effect of CCh in 83% (n56) and 77% (n522), respectively, of EDS cells tested (data not shown). To investigate which NOS isoform is involved in the endogenous production of NO and modulation of ICa aminoguanidine (AG; 100 mM), 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 44 610% L-TYPE Ca21 CHANNEL REGULATION
Figure 4. Effect of NOS inhibitors and the selective iNOS inhibitor aminoguanidine (AG) on CCh-induced depression of ICa in EDS cells. Column a: Representative time course of peak ICa in individual cells in response to application of various test agents. Column b: Percentage of cells responding (filled bars) or not (shaded bars) to CCh. Column c: Changes in ICa density upon different interventions. Aa) Cell preincubated and superfused with L-NMA (200 mM). In this and most of the other cells tested, the CCh effect on basal ICa was essentially absent. Ab) The majority of cells do not respond to CCh. Ac) Lack of effect of CCh on ICa density in nonresponding cells (decline in ICa density due to current rundown). Ba) Cell preincubated and superfused with L-NMA (200 mM) and L-arginine (L-Arg; 400 mM) showing clear CCh-induced depression of ICa. Bb) Most EDS cells respond to CCh as under control conditions. Bc) CCh-induced depression of the basal ICa in responder cells. Ca) AG (100 mM) increased ICa amplitude, but preserved the CCh-induced depression of ICa. Cb) Most cells responded with an increase in ICa to application of AG (middle bar). Almost all cells with an AG response did also respond to CCh (right column). Cc) ICa density in cells responding to AG and CCh. *Statistical significant differences between current densities after washout and CCh application (P,0.05). 317
rinic modulation, however, preincubation in L-NMA (n510, Fig. 6Ba, Bb) or ODQ (n520, Fig. 6Ca, Cb) did not affect ANP-induced inhibition of ICa in most cardiomyocytes examined. In LDS cells, Iso (0.1 mM) stimulated basal ICa by 42 610% whereas the CCh-mediated inhibition of the prestimulated ICa density amounted to 43 64% (Fig. 7Aa–Ac). In clear contrast to the findings in EDS cells, the muscarinic receptor-mediated inhibi-
Figure 5. Effect of inhibitors of the sGC (ODQ) and the PDE type-II (EHNA) on CCh-induced depression of ICa in EDS cells. Column a: Representative time course of peak ICa in individual cells in response to application of the test agents. Column b: Percentage of cells responding (filled bars) or nonresponding (shaded bars) to CCh. Column c: Changes in ICa density upon different interventions. Aa) Prominent disinhibition of basal ICa by addition of ODQ to pipette solution (10 mm) and bath (10 mm), but lack of effect of CCh. Ab) In most of the cells, no change in ICa was seen after CCh application, whereas a few cells either displayed a depression (middle bar) or a small increase in ICa (right bar). Ac) Cells with no CCh response. Ba) Increase in basal ICa amplitude but no effect of CCh in a cardiomyocyte superfused with EHNA (30 mM). Bb) Most of the cardiomyocytes incubated with EHNA did not respond to CCh. Bc) Cardiomyocytes not responding to CCh.
mM 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-3nonyl)adenine (EHNA) (28) (30 mM) increased the basal ICa of EDS cells by 45 67% (Fig. 5Ba–Bc) and completely blocked the effect of CCh on basal ICa (Fig. 5Ba–Bc). Conversely, application of milrinone (10 mM), a blocker of the cGMP inhibited PDE isoform, PDE-III, increased basal ICa but did not prevent the CCh effect in EDS cardiomyocytes (n517, data not shown). 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 (n524, Fig. 6Aa, Ab). Opposite to the musca318
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Figure 6. Effect of atrial natriuretic peptide (ANP, rat, fragment 3-28, 20 nM) superfusion on basal ICa in EDS cardiomyocytes. Aa) Representative recordings from an EDS cell. The cell was voltage clamped (HP ms50 mV) and 20 ms-lasting depolarizing pulses to 0 mV evoked ICa. The control ICa (1) was clearly depressed upon ANP application (2), and this effect was partially reversed at washout (3). Ab) Time course of the peak ICa in the same cell. Depolarizing pulses to 0 mV were applied repeatedly at a frequency of 0.2 Hz; upper trace shows the holding current. Ba) Representative recordings of time course (Bb) of an EDS cell preincubated and superfused with L-NMA (200 mM) (experimental protocol identical to panel Aa, numbers correspond to current traces in panel Ba). In the presence of the NOS inhibitor, ANP still depressed basal ICa; the effect was reversed upon washout, suggesting that the ANP effect on ICa was independent on NO generation. Ca) Representative recordings and time course (Cb) of an EDS cell after superfusion with ODQ (10 mM) and CCh (experimental protocol identical to panel Aa). ODQ disinhibited strongly basal ICa, but ANP still reversibly depressed ICa in the presence of ODQ, indicating that the ANP effect was not mediated through activation of sGC.
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Figure 7. Effects of inhibitors of NOS, NO donors, and sGC on isoprenaline (0.1 mM) -induced stimulation and CCh (1 mM) -induced depression of ICa in LDS cardiomyocytes. Column a: Representative time course of peak ICa in individual cells in response to application of various test agents. Column b: Percentage of cells responding to b-adrenergic stimulation with Iso (shaded bars) and to muscarinic depression of Iso stimulated ICa with CCh (filled bars). Column c: Changes in ICa density upon different interventions. Aa) Cell showing Iso-induced stimulation and CCh-induced depression of ICa. Ab) Almost all cardiomyocytes responding to Iso subsequently respond to CCh. Ac) Summarized data on changes in ICa density by Iso and CCh. Ba) Iso stimulated and CCh depressed the prestimulated ICa after preincubation and superfusion with L-NMA (200 mM). Bb) Almost all cardiomyocytes responding to Iso also respond to CCh in presence of L-NMA. Bc) Responses to Iso and CCh in the presence of L-NMA. Ca) ICa stimulation by Iso and depression by CCh in the presence of ODQ (10 mM in pipette and bath solution). 4Cb) Almost all cells responding to Iso with an ICa increase displayed subsequent ICa depression by CCh in the presence of ODQ. Cc) Responses to Iso and CCh in the presence of ODQ. Da) Cardiomyocytes prestimulated by Iso responding to application of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP; 200 mM) with a depression of ICa. Db) All cells responding to Iso stimulation display ICa decrease on SNAP application. Dc) Stimulation of ICa density by Iso and reversal by SNAP. *Statistical significant differences between current densities after washout and during CCh application (P,0.05).
tion of ICa after b-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), b-adrenoceptorprestimulated 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 L-TYPE Ca21 CHANNEL REGULATION
setting of ICa in LDS cells. However, when the availability of NO was enhanced by addition of the NO donor SNAP (150 mM) to Iso-prestimulated cells, ICa density was depressed by 37 66% in six out of six cells (Fig. 7Da–Dc). To identify which NOS isoform(s) may be responsible for the NO production in early stage cardiomyocytes, single EDS cells were isolated from beating 319
areas of EBs and investigated immunocytochemically using specific antibodies against the three known NOS isoforms and a-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 a-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). 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).
DISCUSSION Although it is well known that NO plays a fundamental role in the regulation of numerous physiological and pathophysiological functions (reviewed by Moncada et al., ref 31), its importance for normal cellular development has only recently emerged (32–34). Using ES cells as an in vitro model of cardiomyogenesis, we describe a crucial role for NO as a functional modulator of L-type Ca21 channels in the early embryonic heart. The most important findings of our study are that 1) muscarinic inhibition of ICa already occurs in early stage cardiomyocytes, which lack functional b-adrenoceptors; 2) this inhibition is mediated entirely by the NO/cGMP system; 3) the hormonal regulation of ICa changes during cardiomyogenesis, the switch in signaling pathways during development being associated with distinct changes in expression of eNOS and iNOS; and 4) although the rather primitive NO-triggered pathway is dormant at later developmental stages, it can dominate the system to such an extent that the 320
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Table 1. Extent of expression of different NOS-isoforms in EDS and LDS cardiomyocytesa
EDS LDS
NADPH-d
eNOS
iNOS
nNOS
111 1
111 1
111 2
2 2
a Scoring: no staining (2), partial staining (1), complete but weak staining (11), complete and distinct staining of cardiomyocytes (111) See Fig. 1.
more elaborate b-adrenoceptor-mediated control over ICa no longer functions when NO availability is increased. 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 downregulation in EDS cells already occurred in the absence of b-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, b-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 b-adrenergic stimulation of ICa, probably as a result of dysfunctional b-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 Ca21 channel (36, 37) or cGMPindependent 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 ventric-
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Figure 8. Expression of iNOS and cGMP content in the left ventricular myocardium of the mouse during different stages of ontogenesis. Left panels: The myocardium of an early developmental stage (E11) embryo shows distinct iNOS expression (upper part) as well as high cGMP content (lower part). Right panels: In later developmental stages (E16), the iNOS expression is switched off (upper part) and the cGMP content (lower part) is significantly reduced.
ular 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 Ca21 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 (E9E13), 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 reL-TYPE Ca21 CHANNEL REGULATION
ported 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 Gprotein. 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 321
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 b-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 Ca21 channel, and the reduction of Ca21 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 b-adrenergic prestimulation. At this develop-
mental 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. 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
Figure 9. Simplified scheme of pathways involved in the regulation of ICa in EDS and LDS cardiomyocytes (see text for details).
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exert negative inotropic effects (10), possibly through a depression of ICa. This may at least partly explain b-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). We thank Dr. A. M. Wobus for providing ES cells of the cell line D3, M. Faulhaber and B. Hops for assistance in cell culture work, and O. Cremanns, C. Hoffmann, and J. Siodlaczek for their help in the immunocytochemistry. The support of the machine and electronic shop is gratefully acknowledged.
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Received for publication March 30, 1998. Revised for publication July 31, 1998. Accepted for publication September 29, 1998.
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