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Cardiovascular Research 2007 73(1):111-119; doi:10.1016/j.cardiores.2006.10.016
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Copyright © 2006, European Society of Cardiology

Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex

P. Edera, D. Probsta, C. Roskera, M. Potesera, H. Wolinskib, S.D. Kohlweinb, C. Romaninc and K. Groschnera,*

aInstitute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University, Graz, Austria
bInstitute of Molecular Biosciences, Karl-Franzens-University, Graz, Austria
cInstitute of Biophysics, University of Linz, Austria

* Corresponding author. Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Universitaetsplatz 2, 8010 Graz, Austria. Tel.: +43 316 380 5570; fax: +43 316 380 9890. Email address: klaus.groschner{at}uni-graz.at

Received 28 July 2006; revised 12 October 2006; accepted 17 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na+/Ca2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes.

Methods: Protein expression and cellular distribution were determined by Western blot and immunocytochemistry. Protein–protein interactions were investigated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments. Recruitment of protein complexes into the plasma membrane was assayed by surface biotinylation. The functional role of TRPC3 was investigated by fluorimetric recording of angiotensin II-induced calcium signals employing a dominant negative knockdown strategy.

Results: TRPC3 immunoreactivity was observed in surface plasma membrane regions and in an intracellular membrane system. Co-immunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GST-pulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca2+ entry, which was dependent on TRPC3-mediated Na+ loading of myocytes. This NCX-mediated Ca2+ signaling was significantly suppressed by expression of a dominant negative fragment of TRPC3. PLC stimulation was associated with increased membrane presentation of both TRPC3 and NCX1.

Conclusion: These results suggest a PLC-dependent recruitment of a TRPC3-NCX1 complex into the plasma membrane as a pivotal mechanism for the control of cardiac Ca2+ homeostasis.

KEYWORDS Transient receptor potential protein; TRPC3; Na/Ca-exchanger; Cardiac myocytes; Calcium signaling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Sodium calcium exchanger (NCX) proteins play a crucial role in calcium (Ca2+) homeostasis of a variety of cell types [1]. These proteins control Ca2+ flux across cell membranes by transporting Ca2+ in exchange for Na+ in either direction (1 Ca2+/3–4 Na+) depending on the electrochemical gradient for Na+ and Ca2+. In cardiomyocytes, NCX1, the cardiac isoform of NCX, primarily extrudes cytoplasmic Ca2+ (forward mode NCX) during cardiac relaxation [2]. Nonetheless, NCX1 mediated Ca2+ influx by reverse mode NCX has been suggested for pathophysiological states and may contribute to Ca2+ transients during the action potential [3,4]. Ca2+ entry via reverse mode NCX increases substantially when intracellular Na+ rises. This may occur as a result of Na+/K+ ATPase inhibition [5] or sodium hydrogen exchanger (NHE) mediated Na+ increase [6]. Promotion of reverse mode NCX has been suggested for pathophysiological situations such as heart failure [7] or ischemia which are typically associated with increases in intracellular Na+ levels [8].

Recently, TRPC3, a non selective cation channel that is activated by receptor-mediated stimulation of phospholipase C (PLC), has been implicated in cellular control of NCX [9,10]. TRPC3 displays a Na+/Ca2+ permeability ratio (PCa/PNa) of 1/1.5 [11,12] and consequently allows significant Na+ loading upon activation. This Na+ entry mechanism has been shown to enable local accumulation of intracellular Na+, together with membrane depolarization, sufficient to drive Ca2+ entry via reverse mode NCX in HEK293 cells [9,10]. This functional link was confirmed by a tight physical interaction between these ion transport systems. A functional interaction between NCX (CalX) and TRP channels has also been found in Drosophila retinal cells [13]. TRPC3-NCX1 complexes may be part of a macromolecular signal complex because for both TRPC3 [14–16] and NCX1, targeting to specific membrane domains has been postulated [17–20].

Since NCX1 is an important regulator of cardiac function and TRPC3 expression has also been suggested for the myocardium [21] we were prompted to investigate expression of TRPC3 and its possible interaction with NCX1 in this tissue. Our findings provide evidence for an NCX-mediated Ca2+ influx in cardiac myocytes, which is dependent on an Na+ entry mechanism triggered by PLC-dependent activation of TRPC3 channels. Physical interaction between TRPC3 and NCX1 and an increase of cell membrane expression upon PLC stimulation provided further evidence for these ion transport systems to be associated in a cardiac signaling complex.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Chemicals
Tissue culture medium and TRIZOL reagent were from Gibco BRL (Vienna, Austria); fura-2/AM was from Molecular Probes (Leiden, Netherlands), GeneJuice Transfection reagent from Novagen (Darmstadt, Germany), Magnetofection reagent from chemicell GmbH (Berlin, Germany), protease inhibitors and N-Glycosidase F, recombinant from Roche (Vienna, Austria), EZ-Link Sulfo-NHS-SS-Biotin and streptavidin–agarose-beads from Pierce (Rockford, USA), collagenase (CLS2) from Worthington Biochemical Corporation (New Jersey, USA). All other chemicals were purchased from Sigma Chemical Co (Vienna, Austria).

2.2. Antibodies
A polyclonal TRPC3 antibody was raised against an N-terminal fragment of TRPC3 (aa 1-302) which was purified as a GST (glutathione-S-transferase) fusion protein. The selectivity of the TRPC3-antibody was tested in Western blots. Anti-NCX (R3F1) was purchased from swant (Switzerland). All other antibodies were from Sigma Chemical Co (Vienna, Austria).

2.3. Primary ventricular cardiomyocyte cultures
Ventricular cardiomyocytes were isolated from hearts of adult Sprague-Dawley rats under the guide lines for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). Rats were injected with heparin sulfate (1000 U/kg. ip) and anesthetized with diethyl ether. Hearts were removed and perfused retrogradely in a Langendorff set-up (4 ml/min) with a Krebs–Henseleit bicarbonate buffer containing (in mmol/L) 118 NaCl, 4.7 KCl, 1.25 CaCl, 1.2 MgSO47H2O, 1.2 KH2PO4, 25 NaHCO3 and 11 glucose (pH 7.4) at 37 °C, 95% O2/5% CO2 for 5 min. The solution was switched to a Ca2+ free Tyrode buffer containing (in mmol/L) 128 NaCl, 6 KCl, 1 Na2HPO4, 0.2 NaH2PO4, 1.4 MgSO4, 9 HEPES, 2.7 glucose and 1 pyruvate (pH 7.4) to stop spontaneous cardiac contraction. Heart digestion was initiated by adding 0.11% collagenase. Atria and aorta were removed, the ventricles minced and filtered through a nylon mesh. Cells were centrifuged, re-suspended in Tyrode containing 2% bovine serum albumin (BSA) and the Ca2+ concentration was increased step wise to 2 mmol/L. The cell pellet was re-suspended in M-199 culture medium containing 0.2% BSA, 10–7 mol/L insulin, 5 mmol/L creatine, 2 mmol/L carnitine, 5 mmol/L taurine, 100 U/ml penicillin/streptomycin, 1.25 µg/ml amphotericin and plated on culture dishes coated with laminin. After at least 1 h of incubation in a humidified incubator with 95% air/5% CO2, medium was changed in order to remove rounded cells (damaged cells) and debris from adherent cells that displayed the typical rod-shaped morphology of native, differentiated cardiomyocytes. Myocytes were used within a time window of 5–48 h after isolation. Within this time, the cells did not show any morphological signs of dedifferentiation.

2.4. DNA constructs and cardiomyocyte transfection
EYFP-NTRPC3, an N-terminal fragment of hTRPC3 (aa 1-302 of TRPC3), subcloned into pEYFP-N1, and pEYFP-N1 as control, were used to transfect adult rat cardiomyocytes. Two transfection methods were combined: GeneJuice Transfection reagent composed of a cellular protein and polymine and Magnetofection. 100 µL of Optimem-Medium was mixed with 3 µL of GeneJuice reagent and incubated for 5 min. 1 µg of plasmid was added and after 15 min of incubation 2 µL of magnetic particles (CombiMag) was added followed by further 15 min of incubation. This mixture was added to cardiomyocytes plated in a 6 well culture dish which was transferred to a magnetic plate for 15 min. The cells were kept in culture for further 24–48 h until expression of the YFP fusion protein was detectable in the cells. Transfection efficiency was typically about 5%.

2.5. Immunocytochemistry and microscopy
HEK 293 (human embryonic kidney)-WT (wild type) cells, T3-9 cells (HEK 293 cells stably transfected with TRPC3) and isolated adult rat cardiomyocytes were fixed with 2% paraformaldehyde for 30 min. After fixation, the cells were rinsed with phosphate-buffered saline (PBS), pH 7.4. The samples were incubated with a blocking solution (5% goat serum) for 1 h at room temperature (RT). Afterwards, the cells were incubated with the primary antibody against NCX or TRPC3 for 1 h at RT and then washed with PBS. Subsequently, the samples were incubated with the secondary FITC or TRITC conjugated antibody for 1 h at RT and washed in PBS. Primary and secondary antibodies were diluted 1:300 in PBS/5% goat serum (v/v). After mounting the samples, microscopy was performed using a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany) with spectral detection and a Leica 63x oil immersion objective (HCX PL APO OIL CS, 1.32 NA).

2.6. Crude membrane preparation
Isolated ventricular cardiomyocytes were washed with ice cold PBS and sonicated (3x15 s) in 400 µL of buffer I containing (in mmol/L): 10 Tris–HCL (pH 7.5), 1 sodium vanadate, 1 phenylmethylsulfonyl fluoride (PMSF), 100 NaF, 1 EGTA, and protease inhibitors. 400 µL of buffer II containing (in mmol/L) 10 Tris (pH 7.5), 300 KCl, 1 Na+ vanadate, 1 PMSF, 100 NaF, 1 EGTA, 20% sucrose and protease inhibitors were added and centrifugation at 10,000 g for 10 min at 4 °C followed. The supernatant was subjected to ultracentrifugation at 100,000 g for 1 h, 4 °C. The pellet was washed and re-suspended in buffer III containing (in mmol/L) 50 Tris–HCL (pH 7.5), 150 NaCl, 60 OG (Octyl β-D-glucopyranoside), 1% Triton-X, 0.5% deoxycholate, 1 PMSF and protease inhibitors and incubated for 30 min at 4 °C. After ultracentrifugation the supernatant was used for immunoprecipitation experiments.

2.7. Co-immunoprecipitation of NCX1 and TRPC3 and Western blotting
Solubilized proteins were pre-incubated with protein A-sepharose beads. 400 µg of precleared proteins was incubated either with anti-NCX or anti-TRPC3 overnight. Protein A-sepharose beads were added to the immuno-complex for 2 h. Control was performed by incubation with rabbit IgG (Sigma). The beads were centrifuged, washed and boiled in Laemmli buffer to release the bound proteins. After SDS-PAGE and Western blotting, nitrocellulose membranes were blocked and treated with the first antibody against NCX (1:1000) or TRPC3 (1:1000). The secondary horseradish peroxidase conjugated antibody (1:5000) was added and the blots were developed using a chemiluminescence detection system. Band intensities were calculated by densitometric analysis (HEROLAB, E.A.S.Y win 32 system).

2.8. Glutathione S-Transferase (GST) pulldown
The N-terminus (NT; aa 1-302) and the C-terminus (CT; aa 670-848) of TRPC3 were cloned into the pGEX4T-1 plasmid. GST pulldown experiments were performed as described previously [10]. 200 µg of lysed protein of ventricular myocytes was incubated with glutathione-Sepharose 4B beads charged either with GST (control), GST-T3N, or GST-T3C for 2 h. Bound proteins were analyzed by SDS page and immunoblotting. Blots were probed with anti-NCX (1:5000).

2.9. Whole cell lysates
T3-9-, HEK 293-, T4-60-cells and rat cardiomyocytes were lysed using buffer III (‘see crude membrane preparation’), sonicated and centrifuged. 100 µg of the supernatant was used for SDS-page and Western blotting. Blots were probed with anti-TRPC3.

2.10. Measurement of intracellular calcium
Rat cardiomyocytes seeded on coverslips were loaded with the fluorescence dye fura 2-AM (5 µmol/l) for 30 min in a humidified incubator with 95% air/5% CO2 and treated with ryanodine (4 µmol/L; 2 min), thapsigargin (150 nmol/L; 8 min), and nifedipine (10 µmol/L). During experiments the cells were constantly perfused with a buffer containing either (in mmol/L) 137 NaCl, 5.4 KCl, 10 HEPES (20 µmol/L Ca2+ or with 2 mmol/L Ca2+); or (in mmol/L) 5 NaCl, 5.4 KCl, 150 NMDG, 10 HEPES (20 µmol/L Ca2+ or with 2 mmol/L Ca2+). Recording of Ca2+ sensitive fluorescence ratios was performed in single cells as described previously [10].

2.11. Biotinylation of cell surface proteins
Ventricular cardiomyocytes were stimulated with Angiotensin II (Ang II) in Tyrode buffer containing 20 µmol/L Ca2+ for 1 min. Control was performed without stimulation. The following procedures are described elsewhere [22]. In short, cells were incubated with 1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin, centrifuged and lysed in buffer III (see ‘crude membrane preparation’). After centrifugation, 400 µg of solubilized proteins was incubated with 60 µL of streptavidin–agarose-beads. Eluted from the beads, proteins were subjected to SDS-PAGE and Western blotting. Membranes were probed with anti-TRPC3 and anti-NCX. Band intensities were quantified by densitometric analysis.

2.12. Statistical analysis
Results are expressed as mean value±S.E.M. Differences were considered statistically significant at p<0.05 using Student's t-test for unpaired values.

For analysis of overlap between red and green fluorescence signals within cardiac myocytes, nonlinear correlations of single intensity linescans for the red and green channel (r=E((V1–mw1)*(V2–mw2))/(s1*s2); r=correlation coefficient, E=average, V=data value, mw=curve average, s=standard deviation) were calculated and correlation quality was estimated using a z-test (SISA Statistical Analysis, Hilversum, NL).


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Expression of TRPC3 in rat cardiomyocytes
TRPC3 protein expression in rat cardiomyocytes was analyzed using a polyclonal antibody raised against the N-terminus of TRPC3. The specificity of the antibody was demonstrated using cell lines expressing different TRPC isoforms. As shown in Fig. 1A, the antibody recognizes TRPC3 as a protein with a molecular mass of ~97 kDa in HEK293 cells (T3-9 cells), and does not cross react with endogenous proteins in HEK 293 cells or TRPC4 (T4-60 cells). Using this antibody, TRPC3 immunoreactivity was clearly detectable at ~97 kDa (Fig. 1A; RCM).


Figure 1
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  		Fig. 1 A. TRPC3 protein expression in adult rat cardiomyocytes. The TRPC3 antibody (1:1000) recognizes a protein with a molecular mass of ~97 kDa in T3-9 and cardiomyocyte lysates (RCM) but not in HEK 293 or T4-60 cell lysates. B. Fluorescence images of T3-9 cells, HEK 293 cells and of a cardiomyocyte (RCM) stained with the TRPC3 antibody. Arrows indicate distinct TRPC3 immunoreactivity in the plasma membrane. Bar indicates 40 µm.

 
Localization of TRPC3 in single cardiomyocytes was examined in immunocytochemistry and confocal fluorescence microscopy. Immunostaining of T3-9 cells showed a distinct localization of TRPC3 in the plasma membrane while no immunoreactivity was detected in wild type HEK 293 cells (Fig. 1B) or when the first antibody was omitted (not shown). In cardiomyocytes (RCM), TRPC3 immunoreactivity was found in distinct regions of the plasma membrane and intracellular compartments most likely in a longitudinally oriented intracellular membrane system. Co-immunolabeling of cardiomyocytes with anti-TRPC3 and anti-NCX revealed an overlap of immunoreactivity (Fig. 2). For quantification of cellular co-localization, linescans of fluorescence intensity through the cells were analyzed for correlation of fluorescence intensity of the two marker fluorophors. As shown in Fig. 2 (lower panel), overlap of fluorescence intensity peaks was clearly evident and a nonlinear correlation of r=0.43 was calculated with p<0.001.


Figure 2
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  		Fig. 2 Co-localization of TRPC3 and NCX in adult rat cardiomyocytes. Confocal fluorescence images of cells stained using anti-TRPC3 (red) and anti-NCX (green). Merged images are shown on the right displaying fluorescence overlap in yellow. Bar indicates 10 µm. Extent of overlap is illustrated by displaying fluorescence intensity profiles (lower panel) along a linescan, as indicated in image (sc). A non-linear correlation coefficient of r=0.43 with p<0.001 was calculated for fluorescence intensity overlap. Well correlated intensity peaks are marked by arrows.

 
3.2. Association of cardiac TRPC3 with NCX1
To test for association of cardiac TRPC3 and NCX1 we performed reciprocal co-immunoprecipitation experiments (IP). Solubilized proteins of crude membrane fractions were immunoprecipitated with either anti-TRPC3 or anti-NCX in order to pull down the respective signalplexes. Interaction between TRPC3 and NCX1 was clearly detectable. Non-specific binding was determined using anti-rabbit IgG which failed to pull down any TRPC3/NCX1 immunoreactivity (Fig. 3A).


Figure 3
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  		Fig. 3 TRPC3 associates with NCX1 in adult rat cardiomyocytes. A. Proteins solubilized from crude membrane preparations of cardiomyocytes were immunoprecipitated (IP) with anti-NCX (left panel) or anti-TRPC3 (right panel) and immunoblotted to detect TRPC3 and NCX, respectively. Input: solubilized crude membrane proteins; IP: immunoprecipitated with the antibody indicated. B. GST-fusion proteins of the N-terminus (T3N) or of the C-terminus (T3C) were incubated with solubilized proteins of cardiomyocytes. Input: proteins from total cell lysate; Lane 2–3: proteins retained by GST (control), T3C and T3N.

 
The TRPC3-NCX1 interaction was further confirmed in GST pulldown-experiments. An N-terminal (aa 1-302; T3N) and a C-terminal region (aa 670-848; T3C) of TRPC3 were used as baits to test for binding to NCX1 from cardiomyocyte lysates. As illustrated in Fig. 3B, NCX1 is efficiently retained by the C-terminus, but barely by the N-terminus of TRPC3 and not in control experiments.

3.3. PLC-dependent activation of cardiac TRPC3 promotes reverse mode NCX
The functional consequences of the physical coupling between TRPC3 and NCX1 were examined in fura-2 experiments measuring intracellular Ca2+ signals. As TRPC3 channels are typically activated in response to receptor-mediated stimulation of phospholipase C (PLC), we first set out to investigate the effects of PLC stimulation on NCX-mediated Ca2+ signals and obtained evidence for promotion of reverse mode NCX as a consequence of angiotensin-induced activation of the G protein Gq- (Gq) PLC pathway in cardiomyocytes. Reverse mode Ca2+ entry was initiated by reducing the extracellular Na+ concentration [Na+]e [23]. In order to eliminate or minimize Ca2+ signals via mechanisms other than reverse mode NCX and TRPC activation, cells were pre-incubated with thapsigargin and ryanodine to block the SR and the Ca2+ channel antagonist nifedipine. As illustrated in Fig. 4A, reduction of [Na+]e from 137 mmol/L to 5 mmol/L at low [Ca2+]e of about 20 µmol/L resulted in a distinct, albeit small elevation of intracellular Ca2+ ([Ca2+]i). This Ca2+ increase was clearly attributed to NCX1 reverse mode activity, as KB-R7943, a specific inhibitor of NCX [5], diminished this signal (Fig. 4A). Angiotensin II (Ang II), which activates Gq-coupled receptors (AT1R) and initiates the phosphatidyl inositol (PI)-PLC-inositol-tris-phosphate (InsP3)-Ca2+-transduction pathway [24–26] strongly promoted this Ca2+ signal. As illustrated in Fig. 4A, Ang II (1 µmol/L) triggered a small rise in [Ca2+]i, at low [Ca2+]e conditions. This Ca2+ signal was substantially increased by Na+ reduction. Consistent with the concept, which we have recently proposed for a signaling partnership between TRPC3 and NCX1 in HEK293 cells [10], Ang II may initiate TRPC3-mediated Na+ entry to generate a substantial shift in the equilibrium for Na+/Ca2+ exchange allowing for operation of NCX in reverse mode. To test this hypothesis, we performed classical Ca2+ re-addition experiments. Cardiomyocytes were stimulated with Ang II (1 µmol/L) in low [Ca2+]e, and extracellular Ca2+ was subsequently elevated to 2 mmol/L to initiate Ca2+ entry via reverse mode NCX operation. In the presence of 137 mmol/L, Na+e, which allows for substantial Na+ entry via TRPC3 channels, Ang II stimulation triggered a weak Ca2+ influx that was increased upon addition of 2 mmol/L Ca2+e, and drastically reduced when [Na+]e was lowered to 5 mmol/L during Ang II stimulation. Moreover, KB-R7943 significantly diminished the Ca2+ signal initiated by Ca2+ re-addition (Fig. 4B).


Figure 4
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  		Fig. 4 Angiotensin II (Ang II) promotes reverse mode NCX in adult rat cardiomyocytes due to a TRPC3-mediated Na+ entry. A. Left: Representative time courses of Ca2+-sensitive fura-2 fluorescence ratios (F340/380) recorded during an Na+ reduction protocol. Cardiomyocytes were kept in a low Ca2+ solution (20 µmol/L Ca2+) containing 137 mmol/L NaCl which was reduced to 5 mmol/L after 300 s (–Na+e). After 100 s, a set of cells was stimulated with Ang II (1 µmol/L; +Ang II) and compared to un-stimulated cells (–Ang II) treated with/without KB-R9743. Right: Statistical comparison of intracellular Ca2+ signals induced by Na+ reduction in cardiomyocytes stimulated with Ang II (+Ang II; n=30), un-stimulated cells (–Ang II, n=28) and un-stimulated cells treated with KB-R9743 (5 µmol/L; –Ang II/+KB-R, n=32). Asterisks denote statistically significant differences versus the response in cells stimulated with Ang II. B. Time course of Ca2+-sensitive fura-2 fluorescence ratios (F340/380) recorded during a Ca2+ re-addition protocol. Left: Cardiomyocytes were stimulated with Ang II (1 µM) in a low Ca2+ solution (20 µmol/L Ca2+) either containing 137 mmol/L NaCl, 5 mmol/L NaCl or 137 mmol/L NaCl plus KB-R9743 (5 µmol/L). Extracellular Ca2+ (Ca2+e) was elevated to 2 mmol/L after 200 s. Right: Statistical comparison between intracellular Ca2+ signals induced by Ca2+ re-addition at the indicated experimental conditions. 137 mmol/L Na+: cardiomyocytes stimulated with Ang II in the presence of 137 mmol/L Na+ (n=28); 5 mmol/L Na+: stimulation in 5 mmol/L Na+ (n=32); KB-R: stimulation in the presence of KB-R7943 (n=32). Asterisks denote statistically significant differences versus the response in cells stimulated in the presence of 137 mmol/L Na+. C. Time course of Ca2+-sensitive fura-2 fluorescence ratios (F340/380) recorded during a Ca2+ re-addition protocol. Left: Cardiomyocytes transiently transfected with EYFP (YFP) or EYFP-NTRPC3 (NTRPC) were stimulated with 1 µmol/L Ang II in a low Ca2+ solution and Ca2+e was elevated to 2 mmol/L after 200 s. Right: Statistical comparison between intracellular Ca2+ signals induced by Ca2+ re-addition under different experimental conditions. NTRPC3: cells transfected with NTRPC3 (n=18); YFP: cells transfected with YFP (n=21). Asterisks denote statistically significant differences versus the response in control cells transfected with YFP.

 
These results indicate that reverse mode NCX operation is promoted by a PLC-dependent Na+ entry mechanisms. Since TRPC3 and NCX1 have been shown to be assembled to a signalplex in cardiomyocytes (Fig. 3) we expected TRPC3 to mediate Na+ loading responsible for promotion of reverse mode NCX. To test for a role of TRPC3 function in the observed promotion of reverse mode NCX, we transiently transfected cardiomyocytes to express an N-terminal fragment of TRPC3, which exerts dominant negative effects on TRPC3 [27]. Cardiomyocytes transfected with either YFP-NTRPC3 or YFP (control) were selected and subjected to measurement of NCX-mediated cellular Ca2+ signals (Fig. 4C). Expression of YFP-NTRPC3 significantly suppressed Ca2+ signals initiated by Ca2+ re-addition, supporting the concept of TRPC3-dependent Na+ loading as a key determinant of cardiac NCX operation. The results suggest expression of a functionally relevant signaling complex containing NCX1 and TRPC3 in rat cardiomyocytes.

3.4. PLC-dependent recruitment of TRPC3-NCX1 complexes to the plasma membrane of cardiomyocytes
Recently, recruitment of TRPC3 to the plasma membrane via rapid vesicular trafficking has been proposed as a key event of TRPC3 conductance regulation [28]. We therefore tested if such exocytotic mechanism may occur in native cardiomyocytes and if plasma membrane recruitment of TRPC3 is associated with changes in NCX1 surface presentation. Thus, we examined the effect of PLC stimulation on cell surface expression of TRPC3 and NCX1 in biotinylation experiments. Cardiomyocytes were stimulated with Ang II (1 µmol/L) and biotinylated together with un-stimulated cells. Equal protein amounts (400 µg; input) from treated (+Ang II) and untreated cells (–Ang II) were pulled down with streptavidin–agarose beads and immunoblotted with anti-NCX (Fig. 5A) and anti-TRPC3 (Fig. 5B), respectively. TRPC3, as well as NCX1 immunoreactivity in the biotinylated protein fraction was significantly increased in Ang II-stimulated cells as compared to un-stimulated controls (n=6 for NCX1; n=5 for TRPC3; Fig. 5, bar graphs). These data indicate a substantial PLC-dependent membrane presentation of both TRPC3 and NCX1.


Figure 5
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  		Fig. 5 Angiotensin II (Ang II) increases plasma membrane presentation of NCX1 and TRPC3 in adult rat cardiomyocytes. NCX1 (A) and TRPC3 (B)-immunoreactivity in the biotinylated protein fraction representing plasma membrane expressed proteins. Comparison of surface protein expression of un-stimulated cells (–Ang II) and cells stimulated with 1 µmol/L Ang II (+Ang II). Representative experiments are displayed. Bar graphs show quantification and statistical analysis (n=6 for NCX1; n=5 for TRPC3). Ang II-induced membrane expression was quantified relative to basal expression.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
With the present study we provide evidence for cardiac expression of TRPC3 and for cross talk of this cation channel protein with NCX1 forming a signalplex that controls cardiac Ca2+ homeostasis. Expression of TRPC3 in cardiomyocytes was evident from both immunoblotting and immunocytochemistry.

As we have recently obtained evidence for a signaling partnership between TRPC3 and NCX1 [10], a key player in cardiac Ca2+ homeostasis, we tested whether native NCX1 would physically interact with the TRPC3 proteins expressed in rat cardiomyocytes. Interestingly, co-immunoprecipitation experiments revealed an interaction of NCX1 with TRPC3. GST pulldown-experiments revealed only a weak association of NCX with an N-terminal fragment of TRPC3, while a C-terminal domain efficiently retained NCX1 which is consistent with previous results obtained in the HEK293 expression system [10].

The present study demonstrates that activation of the cardiac Gq-phospholipase C (PLC)-pathway by Ang II promotes Ca2+ influx initiated by either Ca2+ re-addition (2 mmol/L) or Na+ removal (from 137 mmol/L to 5 mmol/L) consistent with an involvement of reverse mode NCX-mediated Ca2+ entry. This Ca2+ signal was suppressed by 5 µmol/L KB-R7943, which fairly selectively inhibits reverse mode NCX at this concentration [5]. These experiments required the inhibition of voltage-gated Ca2+ channels, as potential additional targets of PLC stimulation [29] with nifedipine. Thapsigargin and ryanodine were used to eliminate a contribution of intracellular stores to Ca2+ signaling. At this point we cannot exclude that a Ca2+-mediated Ca2+ release takes place in native cells as a consequence of the TRPC3-NCX1 mediated Ca2+ entry. Notably, Ang II failed to promote Ca2+ entry when cells were challenged by the receptor agonist at low [Na+]e, indicating that this Ca2+ entry does not behave like a store operated pathway but is rather dependent on the preceding Na+ entry associated with PLC stimulation. Expression of NTRPC3, an N-terminal dominant negative fragment of TRPC3, which has been effectively employed to suppress TRPC3 function [10,27], eliminated this Ca2+ signal, substantiating the concept of TRPC3 as crucial determinant of cardiac NCX1 function.

PLC-dependent regulation of the cardiac NCX has previously been reported and attributed to Na+/H+ exchanger (NHE1) activity [6]. Indeed, we observed a modest inhibitory effect of the NHE inhibitor Hoechst 943 on reverse mode NCX-mediated Ca2+ signals (data not shown). Our present results do not exclude that NCX1 activity may in certain conditions be subject to fine-tuning by sodium transporters, such as NHE1 activity or Na+/K+-ATPase. However, functional coupling between Na+–K+–ATPase and NCX1 appears to be restricted to middle stages of differentiation of cardiomyocytes [30] and is therefore unlikely to contribute to the effects reported here. Moreover, our data unequivocally demonstrate the prominent role of TRPC3-mediated Na+ entry as a determinant of NCX function and Ca2+ signaling in cardiac myocytes during PLC stimulation. As previously calculated for the HEK 293 expression system [10], TRPC3-mediated local rises in Na+i might exceed 10 mM, thereby shifting ENCX to values negative of –30 mV and, thus, enable reverse mode action at physiologically relevant membrane potentials. It is of note that in particular cellular situations, functional significance of TRPC3/NCX1 coupling may arise from NCX forward mode action.

TRPC3-dependent, NCX1-mediated Ca2+ influx into cardiomyocytes was associated with an increased cell surface presentation of the two ion transport proteins. Stimulation with Ang II increased surface expression of both TRPC3 and NCX1 in the cardiomyocytes. It is tempting to speculate that TRPC3 and NCX1 are co-transported as a preformed protein complex via vesicular trafficking to the plasma membrane. Such receptor/PLC-dependent membrane recruitment has been observed for TRPC channel proteins in other cell systems, and rapid vesicular trafficking has been suggested as mechanism regulating membrane insertion and thereby activation of cellular TRPC3 conductances. Singh et al. [28] demonstrated agonist-stimulated insertion of TRPC3 from a mobile vesicle fraction into the plasma membrane. Thus, exocytotic membrane insertion is considered as potential mechanism underlying PLC-dependent promotion of surface expression of cardiac TRPC3/NCX1 complexes.

The novel TRPC3-NCX1 signaling complexes described here, are likely to play a significant role in cardiac physiology or/and pathophysiology. Cardiac heart failure and hypertrophy are typically associated with an increased PLC activation due to an enhanced input via angiotensin (AT-1R)- or {alpha}-adrenergic receptors. Tonic elevation of cellular PLC activity in such pathophysiological states may generate local Ca2+ signals and enhanced cellular Ca2+ loading due to the here described TRPC3-NCX1 interaction. Excess Ca2+ influx via reverse mode NCX during the terminal phase of the action potential plateau may contribute to abnormally slow relaxation in cardiomyocytes [7] and to abnormal excitability. Alternatively, increased Ca2+ entry via reverse mode NCX may as well be considered as an inotropic support in conditions in which SR function is impaired. Future studies are required to investigate the role of TRPC3-NCX1 in cardiac physio/pathophysiology.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the Fonds zur Foerderung der Wissenschaftlichen Forschung," P18280 [GenBank] (to K.G.), P18475 [GenBank] (to M.P), P18169 [GenBank] and P1538 (to C.R.). We thank Mrs. Renate Schmidt, Kerstin Geckl and Mr. Gerald Woelkart for their excellent technical assistance.


   Notes
 
Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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