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Cardiovascular Research 2007 75(1):99-108; doi:10.1016/j.cardiores.2007.03.018
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Copyright © 2007, European Society of Cardiology

Regulation of the Na+/Ca2+ exchanger (NCX) in the murine embryonic heart

Michael Reppela,b,*, Bernd K. Fleischmannc, Hannes Reuterd, Philipp Sassec, Heribert Schunkertb and Jürgen Heschelera

aInstitute of Neurophysiology, University of Cologne, Cologne, Germany
bMedizinische Klinik II, Universität Schleswig-Holstein, Campus Lübeck, Lübeck, Germany
cInstitute of Physiology I, Life and Brain Center, University of Bonn, Bonn, Germany
dDepartment of Internal Medicine III, University of Cologne, Germany

* Corresponding author. University of Cologne, Institute of Neurophysiology, Robert-Koch-Str. 39, D-50931 Cologne, Germany. Tel.: +49 221 478 6960; fax: +49 221 478 3834. akp72{at}uni-koeln.de

Received 17 October 2006; revised 20 March 2007; accepted 20 March 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective The Na+/Ca2+ exchanger (NCX) is involved in embryonic heart development and function demonstrated by the abnormal myofibrillar organization, defects in heartbeat, and early embryonic death of NCX-null embryos. It was therefore the aim of our study to identify key functional regulators of the embryonic NCX.

Methods NCX current (INCX) density was measured as the Ni2+ (5 mM)-sensitive current applying the whole-cell patch-clamp technique in early (EDS, E10.5V) and late developmental stage (LDS, E16.5V) mouse ventricular cardiomyocytes.

Results Compared to LDS, cardiomyocytes derived from EDS showed a significantly higher basal INCX density for the INCX forward (–120 mV: –4.1±1 pA/pF, n=15 versus –1.7±0.4, n=11, p<0.05) and reverse modes (+60 mV: 4.0±0.9 pA/pF, n=15 versus 1.8±0.4, n=11, p<0.05).

There was 2–3-fold elevation of forward and reverse current in LDS on application of ATP-{gamma}-S (2 mM) together with forskolin (1 µM) as well as intracellular application of the catalytic subunit of cAMP-dependent protein kinase (cPKA, 200 U/mL), cAMP (200 µM), phorbol 12-myristate-13-acetate (PMA), a direct activator of protein kinase C (PKC), and 8-Br-cGMP, a membrane permeable analog of cGMP. The specific PKC inhibitor Ro 31-8220 significantly reduced INCX by 70%. Co-application of 20 µM PKA inhibitor Fragment 14-22 (PKI), a specific inhibitor of PKA, and cAMP significantly reduced the exchanger activity by approx 60%. Despite these obvious effects in LDS we could not detect a significant impact of these compounds on INCX in EDS-derived cardiomyocytes. Application of the alkaline phosphatase to test for constitutive phosphorylation of NCX did not affect INCX density in LDS but led to an approx 80% reduction of INCX in EDS.

Conclusion In EDS cardiomyocytes INCX density is upregulated, at least in part by the high phosphorylation of the exchanger protein. At LDS, embryonic cardiomyocytes showed a strong increase of INCX density upon stimulation by PKC- and PKA-dependent signalling pathways.

KEYWORDS NCX; Cardiac development; Embryonic heart; Calcium; PKA; PKC; Cyclic nucleotides


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
In adult cardiomyocytes the NCX plays a key role in cardiac contractility and Ca2+ homeostasis. It is generally accepted that NCX exchanges 1 intracellular Ca2+ ion for 3 extracellular Na+ ions via the sarcolemma, resulting in a predominantly outward directed exchange current. The balance of Ca2+ influx due to voltage-dependent Ca2+ channels (VDCCs) and subsequent Ca2+-induced Ca2+ release (CICR) from intracellular stores is thereby maintained [1–3]. Minor disturbances of this equilibrium at the single cell level may lead to the clinical observation of a substantial and severe deterioration of cardiac performance [4–7].

In embryonic or neonatal cardiomyocytes the close morphological relationship between Ca2+ intrusion and extrusion mechanisms is not developed [8]. Ca2+ homeostasis is therefore believed to be critically determined by VDCCs, which are known to be expressed and to be functionally active during very early steps of cardiac differentiation, and NCX, which supports Ca2+ influx at positive membrane potentials and controls low basal Ca2+ levels during diastole [9–12]. Although the importance of VDCCs for embryonic heart development and function has been well characterized [13,14], the impact of NCX on embryonic heart function is not well understood.

Previous studies point to the elementary role of NCX especially during early stages of murine heart development. In fact targeted deletion of the NCX gene led to lethal phenotypes during early cardiac differentiation [15–20]. In line with these observations, our own group identified high intrinsic INCX density at early stages of heart development [21]. However, to date the signalling pathways being involved in alterations of embryonic NCX function have remained elusive. Some studies suggest an involvement of cyclic nucleotides and protein kinases, e.g. PKC and PKA. In adults, PKA-dependent phosphorylation of substrates, e.g. VDCCs and NCX, has been shown to be a critical determinant of contractility and Ca2+ homeostasis and to be mediated via cAMP-dependent activation of PKA [22]. Similarly, the PKC family has been implicated in a diverse array of cellular responses [23–27]. Among other proteins, e.g. troponin I and VDCCs [28–32], NCX is also discussed as serving as a substrate for activated PKC.

Our data provide evidence for a differential regulation of INCX by endogenous cAMP during fetal heart development. PKA and PKC as well as 8-Br-cGMP enhanced INCX in LDS but not in EDS. Application of alkaline phosphatase (AP), however, significantly reduced INCX only in EDS, suggesting high intrinsic phosphorylation levels as an underlying mechanism for maintenance of high INCX density in the early embryonic heart.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell preparation
Early (EDS, E9.5/10.5) and late developmental stage embryonic ventricular cardiomyocytes (LDS, E15.5–E17.5) were derived from superovulated mice (HIM:OF1) as previously described [33,34]. All experiments were carried out according to the guidelines provided by the University of Cologne animal welfare committee and conform with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Electrophysiology
Single cardiomyocytes were selected for patch-clamp experiments. [Ca2+]i was buffered to 150 nmol/L (calculated using the Maxchelator program, D. Bers, Loyola University, Chicago) by the use of BAPTA in all experiments. To inhibit all contaminating currents, the external solution contained (in mM): CsCl 10, Na+ 135, Ca2+ 2, MgCl 1, Glucose 10, Hepes 10, (in µmol/L) niflumic acid 100, Ouabain 10 and verapamil 10, adjusted to pH 7.4 (CsOH). The internal solution contained (in mmol/L): CsCl 136, NaCl 10, Aspartic acid 42, MgCl 3, Hepes 5, TEA 20, MgATP 10 and 150 nmol/L free [Ca2+]i, adjusted to pH 7.4 (CsOH). The holding potential was –30 mV to block contaminating T-type Ca2+ and Na+ current. INCX was measured as the Ni2+-sensitive current of slow-ramp pulses applied from +60 mV (reverse mode) to –120 mV (forward mode) at 0.09 V/s and 10 s intervals [35]. All experiments were performed at room temperature.

2.3 Substances
To measure INCX in response to modulation of PKA activity, we applied ATP-{gamma}-S (2 mM in pipette solution) and forskolin (2 µM, extracellular perfusion) to maximally enhance cAMP production, which subsequently stimulates endogeneous PKA [36]. Furthermore, we applied 100 µM 8-Br-cGMP [37,38], 200 µM cAMP [39,40], 200 U/mL purified catalytic subunit of PKA [38] from bovine heart and 20 µM PKA inhibitor Fragment 14-22 (PKI) [39] as a specific inhibitor of the catalytic subunit of PKA alone, or in different combinations. PKC activity was stimulated by the use of 200 nM PMA [41]. 8-Br-cGMP, a membrane permeable analog of cGMP, was applied to specifically focus on cGMP- and PKG-mediated alterations of INCX. As control measurements, we also applied 1 µM KT5720 [38], another selective inhibitor of PKA and 10 µM Ro 31-8220 [42], a specific PKC inhibitor. To determine the impact of intrinsic phosphorylation on basal INCX density, AP was applied via the patch-pipette [43]. All substances were obtained from Sigma-Aldrich Chemie GmbH, Germany and were of highest purity available.

2.4 Statistical analysis
Results are given as means±S.E.M. Unpaired t-tests were performed. Statistical significance was accepted when p<0.05. All tested substances were compared with the same control group (see also figure legends). For statistical comparison of forward and reverse modes of INCX, currents at –120 mV and +60 mV were chosen, respectively.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Regulation of embryonic INCX by cAMP and PKA
To confirm that the embryonic NCX is modulated by the adenylate cyclase (AC)/cAMP/PKA signalling cascade, 2 mM ATP-{gamma}-S and 2 µM forskolin were applied [13]. This results in maximal stimulation of AC/cAMP-dependent signalling but was without a significant effect on INCX density in EDS cardiomyocytes compared to controls (Table 1, Fig. 1a). In contrast, at the LDS ATP-{gamma}-S and forskolin led to a 3-fold increase of NCX (Table 1, Fig. 1b).


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  		Table 1
 

Figure 1
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  		Fig. 1 Regulation of INCX density due to cAMP-dependent mechanisms in embryonic cardiomyocytes. NCX activity was measured as the bidirectional Ni2+ (5 mM)-sensitive current measured from +60 mV to –120 mV (0.09 V/s) at 10 s intervals. (a) Stimulation with 2 mM ATP-{gamma}-S+1 µM forskolin in EDS cardiomyocytes. We did not observe significant differences compared to controls, whereas enhanced forward and reverse modes of NCX were observed in LDS (b). Likewise, due to application of 200 µM cAMP (intracellular solution), INCX density was unaffected in EDS (c) but significantly enhanced in LDS (d). To evaluate involvement of PKA, we applied 200 IU purified cPKA in the pipette solution. We observed no effect on INCX in EDS (e), but a strong stimulation of INCX in LDS cardiomyocytes (f). Asterisks indicate statistical significance. Effects were statistically compared to the control groups shown in (a) and (b), respectively. Note that the control group shown in this figure and in Figs. 2–5GoGoGo are identical. Original traces are shown as insets.

 
As for ATP-{gamma}-S and forskolin, intracellular administration of cAMP (200 µM) did not significantly change forward and reverse modes in EDS (Table 1, Fig. 1c). In contrast, we found a significant increase of INCX in LDS cells after cAMP administration. The forward mode of INCX was increased more than 2-fold compared to controls. For the reverse mode we found a 57% increase (Table 1, Fig. 1d). Thus, cAMP increased the INCX forward and reverse modes in cardiomyocytes derived from LDS, whereas it did not alter INCX at the EDS.

To distinguish between direct and indirect effects of cAMP, we applied cAMP, cPKA, PKI, or a combination of cAMP and PKI via the patch-pipette. As a control KT5720, another selective inhibitor of PKA was used. Fig. 1e illustrates that we did not observe significant differences in EDS cells after intracellular perfusion with cPKA. However, INCX was clearly enhanced in LDS-derived cardiomyocytes (Table 1, Fig. 1f). Subcellular compartmentation, which has been shown to occur in embryonic cardiomyocytes [21,39] may account for that finding. PKI or KT5720 did not influence basal exchanger activity in EDS (Table 1, Fig. 2a) and LDS (Table 1, Fig. 2b), whereas co-application of PKI and cAMP strongly reduced forward and reverse modes of INCX in LDS but not in EDS cardiomyocytes (Table 2, Fig. 2c and d). Likewise, application of KT5720 together with cAMP significantly reduced the forward mode of INCX. There was also a strong reduction of the reverse mode, which was, however, not statistically resolvable (Table 2).


Figure 2
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  		Fig. 2 Evaluation of direct effects of cAMP on INCX. To distinguish between direct and indirect effects of cAMP, cAMP was co-applied with the heat stable PKA inhibitor PKI in EDS and LDS-derived cardiomyocytes. For controls see Fig. 1. PKI alone did not affect INCX 4 min after intracellular perfusion in EDS (a) and LDS (b). (c) Co-application of cAMP and PKI significantly reduced INCX activity in LDS by approx 50% pointing to a direct inhibitory role of cAMP. Asterisks indicate statistical significance. (d) Overview about the effects of the cAMP signalling cascade on INCX. Effects were statistically compared to the control groups shown in Fig. 1. Original traces are shown as insets.

 

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  		Table 2
 
Taken together, these data indicate that at LDS INCX is regulated by the AC/cAMP/PKA signalling pathway and activated by cAMP due to PKA activation. cAMP itself acts as an inhibitor of INCX when PKA activity is blocked. INCX is not altered by cAMP, cPKA, PKI and KT5720 in EDS-derived cardiomyocytes.

3.2 Regulation of INCX by cGMP
To evaluate whether cGMP and PKG might be involved in INCX regulation we applied 100 µM 8-Br-cGMP, a non-hydrolyzable and cell-permeable analog of cGMP. We could not detect a modulation of INCX forward and reverse modes in cardiomyocytes derived from EDS (Table 3, Fig. 3a). In contrast, we found a 3-fold stimulation of the forward and reverse modes of INCX in response to 8-Br-cGMP at LDS (Table 3, Fig. 3b).


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  		Table 3
 

Figure 3
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  		Fig. 3 Effect of cGMP on INCX. To test the effect of cGMP on forward and reverse modes of INCX at EDS and LDS, the membrane permeable non-hydrolyzable compound 8-Br-cGMP was applied. It did not affect INCX at EDS (a), but enhanced INCX at LDS (b). For controls see Fig. 1. Effects of cPKA and 8-Br-cGMP on INCX were not additive (data not shown). Original traces are shown as insets.

 
To test for potential additive effects of PKA- and cGMP-mediated stimulation of INCX, 8-Br-cGMP and cPKA were co-applied. We could not detect additional effects as compared to cPKA or 8-Br-cGMP alone (Table 3).

3.3 Role of PKC in NCX regulation
Since Schulze et al. [44] as well as Ruknudin et al. [45] could demonstrate PKC phosphorylation sites at the NCX protein complex, we next determined the effect of PKC on INCX at the early and late embryonic stage. We applied 200 nM PMA to cardiomyocytes derived from EDS and LDS. PMA failed to alter the forward and reverse modes of INCX in EDS as compared to controls (Table 4, Fig. 4a). In LDS-derived cardiomyocytes PMA induced a more than 3-fold increase of the INCX forward mode and a 2-fold increase of the reverse mode of INCX (Table 4, Fig. 4b). Application of Ro 31-8220, a specific PKC inhibitor, led to a significant reduction of the basal forward and reverse modes of INCX in LDS. However, Ro 31-8220 was ineffective in EDS-derived cardiomyocytes (Table 4). Thus, PKC regulates INCX in cardiomyocytes derived from LDS and may be constitutatively active at LDS.


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  		Table 4
 

Figure 4
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  		Fig. 4 Effect of PKC and phosphorylation on INCX density. To evaluate the function of PKC on INCX, 200 nM PMA was applied. As for cPKA, PMA did not significantly enhance forward and reverse modes of INCX in EDS (a), but in LDS (b). Since phosphorylation processes might explain the high density of INCX, we applied alkaline phosphatase via the patch-pipette. We observed a strong and significant reduction of INCX in EDS (c), but not in LDS (d) suggesting low basal phosphorylation levels in LDS or a high intrinsic phosphorylation status and/or low phosphatase activity in EDS. Asterisks indicate statistical significance. Effects were statistically compared to the control groups shown in Fig. 1. Original traces are shown as insets.

 
3.4 Role of phosphatases in the regulation of embryonic NCX activity
To investigate whether phosphorylation processes may explain the differences between INCX density at different developmental stages, we applied AP under basal conditions via the patch-pipette. In EDS we found a nearly 80% reduction of the INCX forward mode compared to controls. Likewise, the reverse mode of INCX was decreased by approximately 83% (Table 5, Fig. 4c). No significant reduction of INCX was found in cells of LDS as compared to controls (Fig. 4d). These data indicate high intrinsic phosphorylation levels in cardiomyocytes derived from EDS.


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  		Table 5
 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
A high functional expression of NCX in early embryonic cardiomyocytes could explain why targeted deletion of NCX results in embryonic lethality after 11.0 days postcoitum in utero [15,16,19]. In accordance, we recently showed that the high functional expression of INCX of very early stages of embryonic heart development declines during development [21]. In the present study we thought to investigate signalling pathways involved in the regulation of NCX at different developmental stages. We found that the embryonic NCX is regulated by PKA, PKC and cyclic nucleotides and that phosphorylation processes are critically involved in the regulatory responsiveness of NCX.

The central role of sarcolemmal Ca2+ transport mechanisms, not only in regulation of intracellular Ca2+ concentrations but also in many downstream signalling processes like growth and differentiation, indicates that it is essential for embryonic cardiomyocytes to regulate these transport elements very precisely. Therefore the activity of the embryonic NCX might be regulated in many different ways, including protein kinases. For kinases as regulatory elements of NCX, however, there are conflicting results even in adult tissues [22,44,46–49]. The first unambiguous proof of NCX phosphorylation due to PKC was provided by Iwamoto et al. [41]. In line with their findings, further studies elaborated that cardiac NCX might not only be regulated by PKC but also by PKA-mediated phosphorylation at the intracellular loop of the adult NCX [44,50,51]. This prompted us to study PKC and PKA as potential key regulators of the embryonic NCX. A major problem in the interpretation of data may result from the fact that PKC, PKA, as well as cyclic nucleotides influence many cellular processes that also affect Ca2+ homeostasis. We therefore performed short-term studies and isolated INCX pharmacologically.

Our data confirm that PKC modulates INCX density in LDS but not in EDS-derived ventricular cardiomyocytes. Moreover, our data suggest that PKC is constitutatively active at LDS. As ATP-{gamma}-S and forskolin enhanced INCX density in LDS-derived cardiomyocytes, we suggest a regulation of the embryonic NCX by the cAMP signalling pathway [13]. This observation was confirmed by intracellular application of cAMP as well as cPKA. Quantitative differences among these groups may be explained by the experimental setup, leading to enhancement of endogeneous, compartment-related cAMP (ATP-{gamma}-S and forskolin group) and to cAMP enhancement, which may not be distributed among all, especially subsarcolemmal compartments when cAMP was directly applied via the patch-pipette. Our findings fit also well with the concept of a fuzzy space, which is supported by an earlier report from our group, where compartmentation of phosphodiesterases was found in EDS embryonic stem cell-derived cardiomyocytes [39] and by reports about subsarcolemmal microdomains described for Ca2+ and Na+ by other groups [52].

Interestingly, we demonstrate here for the first time an inhibition of the forward and reverse modes exchange activity by cAMP under conditions when the PKA is inhibited by PKI in LDS. Since PKI and KT5720 alone have no significant effect in LDS cells as compared to controls this suggests a cAMP-binding site of the NCX protein, as previously been described for f-channels [53]. Our finding that cAMP had no direct effects in EDS does not exclude the existence of this cAMP-binding site, however. Thus, the cAMP-mediated regulation of NCX seems to be complex: it may (i) lead to an activation of PKA and increase of NCX activity or (ii) inhibit NCX by direct cAMP binding. As a consequence, this may lead to a concentration-dependent fine-tuning of NCX activity by cAMP. Differences in the subsarcolemmal cAMP concentration and sarcolemmal/ion channel associated localization of PKA protein due to AKAPs [54] as well as compartmentation in the embryonic heart [39,55], which may also account for the lacking effect of PKI and cAMP in EDS, may contribute to that finding. These differences may explain the conflicting results of the role of kinases in previous studies [22,44,46–49].

As for cAMP-mediated modulation of PKA, there is also an ongoing controversial debate regarding the functional regulation of INCX by other cyclic nucleotides such as cGMP. INCX density was reported to be inhibited by dibutyryl cGMP [56], whereas it was augmented by cGMP in rat aortic myocytes [40,57]. Our present results indicate that cGMP enhances INCX density in murine embryonic cardiomyocytes, presumably because of activation of PKG.

Taken together, we conclude that the embryonic INCX is regulated by cyclic nucleotides and protein kinases. The most interesting finding was, however, that neither ATP-{gamma}-S and forskolin, nor cAMP, cPKA and PMA affected INCX density in EDS-derived embryonic cardiomyocytes. Thus, regulation of INCX occurred only at LDS. This finding might be explained either by a maximal stimulatory activation of NCX at EDS due to unknown activators [13] and/or reduced inactivation processes or compartmentalization (see also above) [39,55].

It was previously presumed that phosphorylation processes might be critically involved in regulation of NCX [58,59]. As we could clearly demonstrate that in EDS-derived cardiomyocytes phosphatases reduce the high INCX density, the level of phosphorylation seems to be a critical determinant of embryonic NCX activity. Since PKI and PMA did not alter INCX density in EDS, a maximal activation, e.g. due to phosphorylation, or a lower rate of dephosphorylation could account for that finding. In fact, in the embryonic rat heart the mRNA expression of cardiac protein phosphatase 2A, being critically involved in dephosphorylation processes of sarcolemmal ion channels in the adult heart, is expressed at significantly lower amounts [60]. Only at later stages of development, mRNA expression is also detected at high levels in myocardium suggesting a lower expression and/or activity of dephosphorylating elements in the early embryonic heart. This observation is in contrast to a previous report in embryonic stem cell-derived cardiomyocytes, where a high intrinsic activity of phosphatase type 1 and type 2A has been observed [36]. Okadaic acid was shown to increase the current density of voltage-dependent Ca2+ channels at early developmental stages by approximately 22%. Although we do not know the underlying reason for this discrepancy, differences between embryonic stem cells and native murine cardiomyocytes and/or stage-dependent compartmentation may account for that finding as suggested recently [39]. Especially for NCX a macromolecular complex was described previously in adult cells, comprising the catalytic subunit of PKA, the PKA-anchoring protein, PKC as well as phosphatase type 1 and type 2A [61]. These regulatory enzymes could be expressed in a stage-dependent manner, compartmentalized [39,55] and varyingly positioned around NCX and voltage-dependent Ca2+ channels. Other regulatory elements, however, cannot be excluded.

Our finding of high constitutive forward and reverse modes of INCX at EDS without regulatory responsiveness may also be explained by the higher resting membrane potential in embryonic cardiomyocytes [21]. Hereby INCX mediates transsarcolemmal Ca2+ in- and efflux, which may compensate for the rudimentary SR and help to initiate/coordinate cardiac contractility and to maintain low Ca2+ levels during diastole. This is of importance since ICaL as the main Ca2+ influx system and SERCA as the main Ca2+ uptake system in adult cardiomyocytes are expressed only at low levels at the embryonic stage [62]. Taken together, we hypothesize that the fine interplay of the developing proteins and regulatory systems being involved at later stages of murine heart development requires a more complex regulation, e.g. by PKA, PKC and PKG.

As a limitation we cannot distinguish between direct phosphorylation processes of NCX protein or associated proteins. Also dephosphorylation by AP might have affected NCX protein or associated regulatory elements. Due to the buffering of intracellular Ca2+, which is needed to standardarize our experiments, we cannot exclude that potential development-dependent alterations of physiological bulk Ca2+ concentrations might also contribute to changes of INCX density and regulation under physiological conditions in-vivo.

In conclusion, differences in the phosphorylation of NCX explain at least in part the striking differences between INCX densities of EDS and LDS cardiomyocytes.

Time for primary review 29 days


Figure 5
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  		Fig. 5 Summary of drug effects on INCX density. Effects of various agents in EDS (left panel) and LDS-derived cardiomyocytes (right panel). Asterisks indicate statistical significance.

 


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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A. Guo and H.-T. Yang
Ca2+ removal mechanisms in mouse embryonic stem cell-derived cardiomyocytes
Am J Physiol Cell Physiol, January 1, 2009; 297(3): C732 - C741.
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