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Cardiovascular Research 2007 73(2):376-385; doi:10.1016/j.cardiores.2006.10.018
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Copyright © 2006, European Society of Cardiology

Insulin potentiates TRPC3-mediated cation currents in normal but not in insulin-resistant mouse cardiomyocytes

Jérémy Fauconniera, Johanna T. Lannera, Ariane Sultanb, Shi-Jin Zhanga, Abram Katza, Joseph D. Brutona and Håkan Westerblada,*

aDepartment of Physiology and Pharmacology, Karolinska Institutet, SE-171 77 Stockholm, Sweden
bCenter for Molecular Medicine, Department of Medicine, Karolinska Institutet, SE-171 76 Stockholm, Sweden

* Corresponding author. Tel.: +46 8 524 872 53; fax: +46 8 32 70 26. Email address: hakan.westerblad{at}ki.se

Received 24 April 2006; revised 6 October 2006; accepted 24 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Recent studies show that bioactive lipids alter intracellular Ca2+ handling of cardiac cells differently in normal and insulin-resistant cardiomyocytes. In the present study we measured non-selective cation currents (NSCC) focusing on the interaction between insulin, the bioactive lipid diacylglycerol (DAG) and canonical transient receptor potential 3 (TRPC3) channels.

Methods: Whole cell patch-clamp was used to measure NSCC in ventricular cardiomyocytes isolated from adult wild-type (WT) and insulin resistant, obese ob/ob mice. Western blot, immunoprecipitation and immunofluorescence staining were used to study the concentration and cellular distribution of TRPC3 channels.

Results: Application of the membrane permeable DAG analogue (OAG, 30 µM) induced an NSCC, which was ~40% smaller in ob/ob than in WT cardiomyocytes. Insulin induced a small NSCC with similar amplitude in ob/ob and WT cells. Pretreatment with insulin (60 nM) increased the OAG-induced NSCC in WT (by ~50%) but not in ob/ob cells. OAG-induced currents were inhibited by adding anti-TRPC3 antibodies to the patch pipette solution. The expression of TRPC3 was lower in ob/ob than in WT cardiomyocytes. TRPC3 was detected in glucose transporter 4 (GLUT4) immunoprecipitates. Insulin exposure resulted in a translocation of TRPC3 to the plasma membrane in WT but not in ob/ob cardiomyocytes.

Conclusions: Insulin-resistant ob/ob cardiomyocytes showed decreases in DAG-mediated NSCC, which were accompanied by decreased TRPC3 expression and defective insulin-mediated trafficking of this protein.

KEYWORDS Insulin resistance; Lipid signaling; TRPC channels; Cellular calcium; Type 2 diabetes; Obesity; Patch clamp


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Obesity, impaired insulin signalling and type 2 diabetes are major causes of coronary heart disease and heart failure [1,2]. Insulin signalling occurs via interaction between the insulin receptor, which belongs to the tyrosine kinase receptor family, and a complex array of downstream proteins [3,4]. In the heart, insulin regulates various physiological processes including energy metabolism, contractility, protein expression, and ion transport [5]. Translocation of membrane proteins (such as transporters, ion channels and receptors) from intracellular stores to the cell surface is often a key step in their activation [6]. Insulin facilitates glucose uptake by inducing translocation of the glucose transporter 4 (GLUT4) from an intracellular pool to the plasma membrane via a phosphoinositide 3-kinase (PI3K)-dependent mechanism [2,7]; this process is defective in insulin resistant conditions and type 2 diabetes [8–10]. The mechanisms underlying the translocation and fusion of GLUT4 vesicles with the plasma membrane are unclear, but several proteins have been identified in association with the GLUT4 vesicles and appear to move with GLUT4 to the plasma membrane in an insulin-dependent manner [2,11]. These proteins include VAMP2 (vesicle-associated membrane protein 2), which is a vesicle SNAP receptor (v-SNARE) protein that interacts with SNARE proteins on the sarcolemma (t-SNARE:s).

Insulin perfusion has been shown to increase the concentration of the lipid second messenger diacylglycerol (DAG) in isolated rat hearts [12]. Furthermore, increased DAG concentrations have been observed in diabetic and in failing hearts [13,14]. The classical action of DAG is via activation of protein kinase C (PKC) [15]. More recent studies have shown that DAG also activates the canonical transient receptor potential (TRPC) 3/6/7 channel subgroup in a PKC-independent manner [16–20]. In mammals, TRP homologs represent a family of ion channels that generally conduct mono- and divalent cations with relatively poor discrimination, i.e. they generate non-selective cation currents (NSCC) [21]. Interestingly, TRPC3 was recently found to co-localize with, and bind to, SNARE proteins including VAMP2 [22]. Furthermore, agonist stimulation caused a VAMP2-dependent increase in surface expression of TRPC3, presumably arising from translocation of TRPC3 containing vesicles localized in the vicinity of the plasma membrane [22].

In the present study we measured plasma membrane ion currents in freshly isolated, adult cardiomyocytes focusing on the interaction between insulin, the bioactive lipid DAG and TRPC3 channels. We used freshly isolated cardiomyocytes to minimize dedifferentiation and changes in cellular function that occur when cells are cultured [23] and applied a saturating concentration of the membrane permeable DAG analogue 1-oleyl-2-acetyl-sn-glycerol (OAG). The specific aims were to determine whether in cardiomyocytes: 1) DAG activates an NSCC; 2) insulin affects the DAG-induced NSCC; 3) insulin induces translocation of TRPC3 to the plasma membrane; 4) these processes are altered in cardiomyocytes from insulin resistant, obese ob/ob mice. The results show a DAG-mediated NSCC that was smaller in ob/ob than in wildtype (WT) cardiomyocytes. Furthermore, insulin increased the DAG-mediated NSCC and translocated TRPC3 to the plasma membrane in WT but not in ob/ob cardiomyocytes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Animal model, cell isolation and materials
Young (3–5 months) C57BL genetically obese male mice (ob/ob) and their WT counterparts were killed by rapid neck disarticulation and the heart was excised. Single cardiomyocytes were isolated from the ventricles following the protocols developed by the Alliance for Cellular Signalling (AfCS Procedure Protocol ID PP00000 125) [24,25]. All experiments were approved by the Stockholm North local ethical committee. The investigation conforms 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).

Human insulin (Actrapid) was from Novo-Nordisk. OAG, 2-aminoethoxydiphenyl borate (2-APB), wortmannin, SK&F96365, Gd3+, and tetanus toxin (TeNT) were from Sigma. All compounds were prepared as stock solutions in appropriate solvents. On the day of the experiment, stock solutions were diluted to the desired final concentration in the bath solution and the same volume of solvent was added to the control solution.

2.2. Measurement of non-selective cation currents
After isolation, myocytes were placed in a perfusion chamber on the stage of a Nikon Diaphot 200 inverted microscope and continuously perfused with standard Tyrode solution of the following composition (mM): 121 NaCl; 5.0 KCl; 1.8 CaCl2; 0.5 MgCl2; 0.4 NaH2PO4; 24 NaHCO3; 0.1 EDTA; 5.5 glucose. The solution was bubbled with 5% CO2/95% O2, which gives a bath pH of 7.4. Whole-cell patch-clamp experiments were performed at room temperature (~24 °C) with an Axopatch 200B amplifier (Axon Instruments, Burlingham, CA, USA). Patch pipettes had a resistance of 2–3 M{Omega}. Currents were normalized to the cell membrane capacitance and presented as current densities (pA/pF). Series resistances were electronically compensated before voltage-clamp recordings. Leak current was not compensated and all cells exhibiting a current leak larger than 100 pA were excluded. The patch pipette solution contained (mM): 120 CsCl, 6.8 MgCl2, 5 Na2ATP, 5 sodium creatine phosphate, 0.4 Na2GTP, 11 EGTA, 4.7 CaCl2 (120 nM free [Ca2+]), and 20 HEPES; pH was adjusted with CsOH to 7.2. In some experiments we added TeNT (10 nM) or anti-TRPC3 antibodies (1:200 dilution)+/–blocking peptide (1:100 dilution) (Alomone Labs, see below) to the patch pipette solution. In order to record an NSCC induced by OAG and/or insulin, the bathing solution was switched to a modified Tyrode solution where: Na+ was replaced by Li+ to inhibit the Na+–Ca2+ exchanger; 200 µM ouabain was added to inhibit Na+–K+ ATPase; 1 mM BaCl2 was added to block residual K+ and background conductance. In some experiments, 0.5 µM of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Na+–HCO3– co-transport and Cl– conductance, was added but this had no effect on NSCC characteristics (data not shown). Quasi steady-state I–V relations of the NSCC were obtained by applying a descending voltage ramp that covered the physiological range of membrane potentials. The voltage ramp went from +50 mV to –120 mV with a slope of 0.057 V/s (see Fig. 1A) and was repeated at 0.03 Hz, which inactivates both rapid Na+ currents and L-type Ca2+ currents. OAG- and insulin-mediated NSCCs were determined as the difference between steady state currents recorded after and before application of the respective compound.


Figure 1
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  		Fig. 1 The OAG-induced NSCC was smaller in ob/ob than in WT cardiomyocytes. (A) Representative I–V curves obtained after application of OAG (30 µM) in WT (black line) and ob/ob (grey line) cells. The stimulation protocol is displayed at the top left. (B) Mean data (± SEM; n=10 WT and 9 ob/ob cells) of the OAG-induced NSCC density at +50 mV and the effect of pharmacological agents (n=7 in each conditions). *indicates significant difference from OAG alone within each group (P<0.05); {dagger} shows significant difference between WT and ob/ob cells (P<0.05).

 
2.3. Western blot analyses
Left ventricles of WT and ob/ob hearts were isolated and homogenized in lysis buffer consisting of (mM) 20 HEPES (pH 7.6), 150 NaCl, 5 EDTA, 1 Na3V04, 25 KF, 20% glycerol (v/v), 0.5% Triton X-100 (v/v) and protease inhibitor cocktail (Roche). The protein content of the crude lysate was determined using the Bradford assay (BioRad). Equal amounts of total protein (30 µg) were loaded into each well and separated by electrophoresis using NuPAGE Novex 4–12% Bis–Tris Gels (Invitrogen) and transferred onto a polyvinylidine fluoride (PVDF) membrane (BioRad). Membranes were blocked in 5% (w/v) non-fat milk in Tris-buffered saline containing 0.05% Tween 20 followed by incubation with primary antibody (anti-TRPC3, 1:200 dilution, Alomone Labs). Membranes were then incubated with horseradish peroxidase-conjugated antibody (HRP-conjugated anti-rabbit Ig 1:30000 dilution, Amersham) and immunoreactive bands were visualised using enhanced chemiluminescence (Super Signal, Pierce).

The specificity of commercially available TRPC antibodies has been questioned [26]. However, when endogenous TRPC3 expression was knocked down in muscle cells, there was a specific decrease of both TRPC3 mRNA and protein, the latter detected with the Alomone anti-TRPC3 antibody [27]. Thus, endogenous expression of TRPC3 in muscle cells can be specifically detected by the anti-TRPC3 antibody used in the present experiments.

2.4. Immunoprecipitation
Freshly isolated cardiomyocytes were allowed to rest for 1 h in standard Tyrode solution. Cells were exposed to Tyrode with or without insulin (60 nM) for for 20 min. They were then centrifuged for 45 s at 500 rpm, the supernatant was removed and the pellet was suspended in lysis buffer (same as for Western blots). Cells were homogenized and lysates cleared by centrifugation at 10,000 g for 10 min at 4 °C. The protein content was determined using the Bradford assay (BioRad). Equal amounts of protein (1 mg) were incubated with 10 µl goat anti-GLUT4 antibody (Chemicon) for 5 min at room temperature, followed by addition of 30 µl of protein G agarose suspension (Santa Cruz Biotechnology, CA) and overnight incubation at 4°C with rotation. Samples were washed three times with lysis buffer, and eluted with 40 µl LDS sample buffer when heated for 10 min at 70 °C. Western blots for TRPC3 were then performed as described above. Finally membranes were stripped (Pierce), blocked and re-blotted with mouse anti-GLUT4 antibody (5ug/ml, Acris) and anti-mouse Ig (1:1000 dilution, Pierce).

2.5. Immunofluorescence staining
Freshly isolated cardiomyocytes were placed on laminin-coated dishes in normal Tyrode solution. After 1 h, the Tyrode solution was replaced by a Tyrode solution with or without insulin (60 nM=10 mU/ml) and cells were exposed to this solution for 20 min. In some experiments phosphoinositide 3-kinase (PI3K) was inhibited by exposing cells to wortmannin (0.5 µM) [28] for 20 min before insulin was added to the medium. Cells were fixed in PBS with 0.2% paraformaldehyde for 20 min and then permeabilized by 0.3% Triton X-100 in PBS solution. After rinsing, cardiomyocytes were preincubated for 30 min in 10% normal goat serum. Cells were then incubated with anti-TRPC3 antibody (1:50 dilution in 1% BSA) at 4 °C overnight and then washed and incubated with biotinylated goat anti-rabbit antibody (1:400, Vector) followed by incubation with Avidin Texas Red (1:400, Invitrogen-Molecular Probes). To assess the specificity of the anti-TRPC3 antibody, cells were exposed to the primary antibody and a 10-fold excess of TRPC3 or TRPC6 blocking peptide (Alomone Labs) at 4 °C overnight. Stained cells were mounted on cover slips using fluorescence mounting medium (DakoCytomation). Images of longitudinal thin sections of stained cells were obtained with laser confocal microscopy using a BioRad MRC 1024 unit (BioRad Microscopy Division, Hertfordshire, England) attached to a Nikon Diaphot 200 inverted microscope with a Nikon Plan Apo 60x oil immersion objective (N.A. 1.3). Focus was set at the height where the cell diameter was maximal. Excitation was at 568 nm and the emitted light was collected through a 585 nm narrow band filter.

2.6. Analyses and statistics
Confocal images of TRPC3 immunostained cell sections were analysed with ImageJ (NIH, USA; http://rsb.info.nih.gov/ij/). TRPC3 staining occurred mainly at the cell surface (see Fig. 6) and to enable comparisons between TRPC3 stained cells, the fluorescence profile was in each cell measured along at least three lines drawn perpendicular to its long axis and maximal fluorescence in each line was set to 1.0. These measurements were performed at representative sites in each cell where both edges of the cell were stained. An average transverse fluorescence profile for each cell was constructed from these measurements and the normalized fluorescence was then measured at 5% intervals relative to the distance from the edge of the cell. The diameter of WT and ob/ob cells was 25.9±1.3 and 26.3±1.0 µm, respectively, and hence the 5% intervals represented ~1.3 µm. Electrophysiological analyses were performed using Pclamp (version 8.1, Axon Instruments). All averaged data are presented as mean±SEM and the number of cells (n) is given. For each experimental condition, cells were isolated from at least three animals. Statistical analyses were performed using Student's t test (for paired or unpaired samples) or when three or more groups were compared, one-way analysis of variance (ANOVA) followed by a Newman–Keuls post hoc test. Differences were considered significant when the P value was less than 0.05.


Figure 6
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  		Fig. 6 Insulin induced TRPC3 translocation to the plasma membrane in WT but not in ob/ob cardiomyocytes. (A) Negative controls were produced by incubating cells with anti-TRPC3 antibody together with an excess of TRPC3 antipeptide. (B) Representative confocal images of isolated WT cardiomyocytes stained for TRPC3 under control conditions, after exposure to insulin (60 nM), and after insulin exposure following pretreatment with the PI3K inhibitor wortmannin (0.5 µM). Increased brightness represents increased immunostaining. (C) Representative images of ob/ob cells under control conditions and after insulin exposure. Scale bars in A also refer to B and C. (D) and (E) High magnification images of sections marked with arrows in B and C, respectively.

 

   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. The OAG-mediated non-selective cation current was smaller in ob/ob cardiomyocytes
In the presence of the membrane permeable non-metabolizable DAG analogue OAG (30 µM), an outwardly rectifying current developed within minutes in both WT and ob/ob cardiomyocytes (Fig. 1A). The maximum amplitude of the OAG-induced current (IOAG) was significantly smaller in ob/ob compared to WT cardiomyocytes (Fig. 1B). The reversal potential of IOAG was not different between the two groups: –9.1±2.6 mV for WT (n=10) vs. –8.2±3.1 mV for ob/ob (n=9) cells. We next tested a battery of pharmacological tools frequently used to inhibit TRP channel activity: 2-APB (30 µM), SK&F96365 (10 µM), and Gd3+ (1 µM) [29]. All these compounds significantly reduced IOAG in WT and ob/ob cardiomyocytes (Fig. 1B).

One possible mechanism by which insulin can increase DAG is by activation of phospholipase C [30,31], which also leads to the formation of another bioactive molecule, inositol trisphosphate (IP3). To assess possible effects of IP3, we used a membrane-permeable IP3 analogue, 2,4,6-tri-O-butyryl-I[1,3,5]P3 (1 µM), which has been shown to prolong electrically evoked cytoplasmic Ca2+ transients in cardiomyocytes [25]. No NSCC was induced when WT cells were exposed to this IP3 analogue and it had no significant effect on the NSCC in the presence of OAG (1.9±0.5 pA/pF at +50 mV; n=4) as compared to application of OAG on its own (2.1±0.2 pA/pF; n=10).

3.2. Insulin activated a non-selective cation current with similar amplitude in WT and ob/ob cardiomyocytes
Recently, insulin was shown to activate a dose- and time-dependent NSCC (Iinsulin) in adult guinea pig cardiomyocytes [32]. Insulin activated an NSCC (Iinsulin) in a dose- and time-dependent manner also in mouse cardiomyocytes and this current was similar in WT and ob/ob cardiomyocytes (Fig. 2A and B). The reversal potential (Erev) of Iinsulin was not different between WT and ob/ob cells (Erev=7.9±2.0 mV for WT (n=10) vs. 8.6±2.2 mV for ob/ob (n=9)), but it was significantly more positive than Erev for IOAG (~–9 mV; see above). In both WT and ob/ob cells, Iinsulin developed gradually and the maximal outward current occurred after ~15 min of insulin exposure (see Fig. 3A). SK&F96365 (10 µM) and Gd3+ (1 µM) significantly decreased Iinsulin in both WT and ob/ob cells (Fig. 2C). Application of 2-APB (30 µM), on the other hand, did not affect Iinsulin in either WT or ob/ob cells. One central and early event in intracellular insulin signalling is the activation of PI3K [3,4]. To determine the involvement of the PI3K pathway, we pretreated cells with the PI3K inhibitor wortmannin (0.5 µM) [28] and this blocked Iinsulin in both cell types. A similar inhibition of Iinsulin was obtained with 0.2 µM wortmannin (n=5; data not shown).


Figure 2
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  		Fig. 2 Insulin triggered an NSCC in both WT and ob/ob cardiomyocytes. A, representative I–V curves obtained after application of insulin (600 nM) in WT (black line) and ob/ob (grey line) cells. B, mean data (± SEM) of the current density at +50 mV at different insulin concentrations (n=5–10 cells in each group). Note that there were no significant differences in the current density between WT and ob/ob cardiomyocytes at this holding potential and this was the situation also at other holding potentials (data not shown). C, mean data (n=7 cells in each conditions) of the effect of pharmacological agents on the current density of the insulin-mediated NSCC (600 nM). *indicates significant difference from insulin alone (P<0.05).

 

Figure 3
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  		Fig. 3 The OAG-induced NSCC was larger after insulin pretreatment in WT but not in ob/ob cardiomyocytes. Mean data (±SEM; n>7 cells in each group) of the time-course of changes in current density induced by addition of insulin (60 nM) followed by OAG (30 µM) in WT cells (Aa) and ob/ob cells (Ba). Measurements were performed at +50 and –80 mV holding potential. Mean data of the current density at +50 mV obtained in WT (Ab) and ob/ob (Bb) cells under steady-state conditions in OAG, in OAG+insulin, or in OAG+insulin+TeNT (10 nM); measurements performed after subtracting the current induced by insulin alone. *indicates significant difference from OAG alone (P<0.05).

 
3.3. Insulin potentiated the OAG-mediated non-selective cation current only in WT cardiomyocytes
The interaction between insulin and OAG was tested by first exposing cardiomyocytes to insulin (60 nM) for 20 min, which gave a stable Iinsulin, and then adding OAG (30 µM). This resulted in a markedly increased current density that reached its maximal amplitude within 15 min (Fig. 3Aa and Ba). In WT cardiomyocytes, IOAG was significantly larger in the presence than in the absence of insulin (Fig. 3Ab). In contrast, insulin did not induce an increase in IOAG in ob/ob cardiomyocytes (Fig. 3Bb). When WT cardiomyocytes were exposed to insulin in the presence of wortmannin, IOAG (2.3±0.3 pA/pF at +50 mV; n= 6) was not different from that recorded in cells not exposed to insulin (2.1±0.2 pA/pF). In summary, pre-treatment with insulin resulted in a wortmannin-dependent increase in IOAG in WT but not in ob/ob cardiomyocytes.

Tetanus toxin (TeNT) cleaves VAMP2 [33,34] and hence prevents the interaction between v-and t-SNARE proteins [22]. We used TeNT in the patch pipette (10 nM) to investigate whether SNARE proteins are involved in the insulin-mediated potentiation of IOAG in WT cardiomyocytes. In the presence of TeNT, insulin pre-treatment failed to increase IOAG in WT cardiomyocytes (Fig. 3Ab), whereas it had no effect in ob/ob cells (Fig. 3Bb).

3.4. The OAG-induced non-selective cation current was inhibited by anti-TRPC3 antibody
It was recently shown that the anti-TRPC3 antibody used in the present study can block cation currents in arterial smooth muscle cells when applied on the inside of the plasma membrane [35]. We therefore measured Iinsulin and IOAG with anti-TRPC3 antibody (+/–an excess of TRPC3 blocking peptide) present in the patch pipette. Iinsulin was not inhibited by addition of anti-TRPC3 antibodies to the patch pipette solution; at +50 mV the insulin-stimulated (60 nM) current density was 0. 79±0.20 pA/pF with antibody (n=6) and 0.57±0.13 pA/pF with antibody plus blocking peptide (n=6). On the other hand, the OAG-induced (30 µM) current was significantly decreased both in the absence and presence of insulin (Fig. 4). Control experiments with an excess of TRPC3 blocking peptide in the patch pipette showed results similar to those obtained in experiments without application antibodies. Thus, these results indicate a direct role of TRPC3 in the OAG-induced but not the insulin-induced NSCC.


Figure 4
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  		Fig. 4 The OAG-induced NSCC was inhibited by an anti-TRPC3 antibody. A. Representative I-V curves obtained after application of OAG (30 µM) with anti-TRPC3 antibody+/–TRPC3 blocking peptide in the patch pipette (a) and mean (±SEM) current density values at +50 mV (b). B. Same as in A but with an initial exposure to insulin (60 nM) followed by application of OAG. The current induced by insulin alone was subtracted from the representative currents and mean data. Data obtained from 6 cells in each condition. *indicates significant difference between the presence and absence of TRPC3 blocking peptide (P<0.05).

 
3.5. TRPC3 was expressed in cardiomyocytes
We used Western blots to determine the expression of TRPC3 in cardiac ventricles of WT and ob/ob hearts (Fig. 5A). The gels showed a single immunoreactive band at the expected molecular weight (http://www.signaling-gateway.org/molecule) and measurements of the intensity of this band revealed a modest, but significant (P<0.05), decrease in the expression of TRPC3 protein in ob/ob (57±6%) compared to WT (100±5%) ventricles (data expressed relative to the mean expression in WT cells, which was set to 100%; n=6 hearts in each group).


Figure 5
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  		Fig. 5 TRPC3 protein is expressed in cardiac ventricles and co-immunoprecipitates with GLUT4. A. Representative TRPC3 Western blot of WT and ob/ob ventricles as indicated above each gel. 30 µg of total protein was loaded into each well. B. Upper part shows a Western blot of TRPC3 performed on GLUT4 immunoprecipitates from ventricles that had been incubated in the presence (Ins) or absence (Con) of insulin. Lower part shows the same membrane after stripping and blotting for GLUT4.

 
3.6. TRPC3 was detected in GLUT4 immunoprecipitates and insulin induced a translocation of TRPC3 in WT cardiomyocytes
Both GLUT4 and TRPC3 can be translocated from intracellular vesicles to the plasma membrane in a VAMP2-dependent manner [2,11,22]. Therefore, we studied the possible co-localization of GLUT4 and TRPC3 in WT cardiomyocytes by immunoprecipitating with anti-GLUT4 antibodies and detecting TRPC3 with Western blotting. Fig. 5B shows that TRPC3 was detected in GLUT4 immunoprecipitates both in the absence and presence of insulin (60 nM). We found no difference in the ratio of TRPC3 to GLUT4 staining between control (n=4) and insulin-treated (n=5) hearts (data not shown).

Immunofluorescence staining was used to study the intracellular localization of TRPC3 in ventricular cardiomyocytes of WT and ob/ob mice. Control experiments to assess the specificity of the anti-TRPC3 antibody showed no staining when cells were exposed to an excess of TRPC3 blocking peptide (Fig. 6A), whereas exposure to the TRPC6 blocking peptide had no effect on the TRPC3 staining (data not shown). Fig. 6B shows representative images of the TRPC3 staining in WT cardiomyocytes. In the absence of insulin, TRPC3 staining was observed directly on as well as immediately beneath the cell surface membrane (Fig. 6D) and measurements of fluorescence intensity gave similar values at 0% and 5% (~1.3 µm) from the edge of the cells (Fig. 7A). Little staining was seen in the centre of the cell and the t-tubular system could not be discerned. Treatment of WT cardiomyocytes for 20 min with insulin (60 nM) concentrated TRPC3 staining to the cell surface membrane and decreased the subsarcolemmal staining; the relative staining at 0% from the cell edge was 0.98±0.02 with insulin and 0.77±0.05 without (P<0.05) and at 5% from the cell edge it was 0.32±0.03 with insulin and 0.75±0.04 without (P<0.05). Moreover, punctuated TRPC3 staining at the subsarcolemmal level was observed under control conditions, whereas the staining was more uniformly distributed along the plasma membrane after insulin treatment (Fig. 6D). This suggests that TRPC3 was stored in vesicles in the vicinity of the sarcolemma before insulin application. We investigated the involvement of PI3K in TRPC3 translocation to the sarcolemma by application of the PI3K inhibitor wortmannin (0.5 µM). This completely abrogated of the insulin-mediated TRPC3 translocation (Figs. 6B and 7AGo); the relative fluorescence at 0% and 5% from the cell edge was 0.70±0.05 and 0.65±0.04, respectively, i.e. significantly (P<0.05) different from the values obtained with insulin alone (see above).


Figure 7
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  		Fig. 7 The cellular distribution of TRPC3 in the presence and absence of insulin. Mean data (±SEM) of the normalized fluorescence staining along the short axis of WT (A) and ob/ob (B) cells. The fluorescence was measured at fixed relative distances from the edge of each cell. Open triangles, control; filled squares, insulin; open circles, wortmannin+insulin. N=18–24 cells from 3–5 mice in each group.

 
Exposure of WT cardiomyocytes to OAG (30 µM) did not cause any detectable translocation of TRPC3; the relative fluorescence at 0% and 5% from the cell edge was 0.68±0.09 and 0.72±0.07 (n=9), respectively.

In the absence of insulin, ob/ob ventricular cardiomyocytes showed a TRPC3 staining pattern similar to that seen in WT cells not exposed to insulin (Fig. 6C and E). However, insulin treatment failed to cause any redistribution of TRPC3 to the plasma membrane in ob/ob cardiomyocytes (Fig. 7B).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The major novel results of the present study are: 1) OAG activates an NSCC (IOAG) that is significantly smaller in insulin-resistant ob/ob as compared to WT control cardiomyocytes; 2) insulin pre-treatment increases IOAG in a wortmannin- and TeNT-dependent manner in control but not in insulin-resistant cells; 3) IOAG is inhibited by introducing an anti-TRPC3 antibody into the cells via the patch pipette; 4) TRPC3 is expressed in both normal and insulin-resistant cardiomyocytes and the expression is somewhat lower in insulin-resistant cardiomyocytes; 5) TRPC3 is detected in GLUT4 immunoprecipitates; 6) insulin induces a translocation of TRPC3 in control but not in insulin-resistant cells.

The TRPC3/6/7 channel subgroup can be activated by the membrane permeable DAG analogue OAG in a protein kinase C-independent manner [16–20]. In the present study, application of OAG activated an NSCC in WT and ob/ob cardiomyocytes, which had a reversal potential at ~–10 mV and exhibited outward rectification, properties that are consistently encountered with members of the TRPC3/6/7 subgroup [17,36]. Moreover, the present results show that application of an IP3 analogue did not induce any NSCC and had no effect on IOAG. Thus, we observed no cross-talk between DAG-and IP3-signalling, which is consistent with most studies on receptor-mediated TRPC3/6/7 activation [17,37].

We focused on the role of TRPC3 in the present study. Our results show a significantly smaller IOAG in ob/ob as compared to WT cardiomyocytes (Fig. 1), which is in accordance with a lower expression of TRPC3 in ob/ob ventricles (Fig. 5A). It should be noted that the decreased IOAG in ob/ob cells might also reflect differences in single channel properties and the present experiments do not allow us to distinguish between alterations in protein expression and channel properties. In various expression systems, TRPC3 has been shown to be sensitive to diverse pharmacological agents, such as 2-APB, SK&F96365 and Gd3+ [38–40]. Each of these compounds significantly reduced IOAG in both WT and ob/ob cardiomyocytes. Thus, these results can be explained by a model where DAG activates the TRPC3 channels. Further support for this model comes from the finding that IOAG was inhibited when the anti-TRPC3 antibody was added to the patch pipette solution (Fig. 4).

Application of insulin activated an NSCC in a dose-dependent manner (Fig. 2), as previously described by Zhang and Hancox [32]. Iinsulin was inhibited by SK&F96365, which is in accordance with the previous results [32], and in addition we show inhibition by a low concentration Gd3+ (1 µM) and wortmannin. However, Iinsulin was neither inhibited by 2-APB, which in many studies has been found to inhibit ligand-induced activation of TRPC3 channels [40], nor by inclusion of the anti-TRPC3 antibody in the patch pipette. Moreover, the amplitude of Iinsulin did not differ between WT and ob/ob ventricular cardiomyocytes despite the fact that TRPC3 protein expression was decreased in ob/ob cells. Taken together these results indicate that Iinsulin was not mediated by TRPC3 channels.

Iinsulin was markedly increased at insulin concentrations >100 nM both in the present study and in the previous study of Zhang and Hancox [32]. Insulin effects mediated via the insulin receptors are generally maximal at an insulin concentration of ~10 nM and at higher concentrations, insulin will also bind to IGF receptors [41]. Thus, the induction of Iinsulin might be a consequence of insulin activation of IGF receptors rather than insulin receptors. Interestingly, Iinsulin shows major similarities to a PI3K-dependent NSCC that has been observed following insulin-like growth factor-1 (IGF-1) exposure [42]. This IGF-1-induced current was mediated via the growth factor related channel (GRC), which is a murine homolog of TRPV2 (VRL-1), a member of the TRPV subfamily [42]. GRC is expressed in adult mouse cardiomyocytes [43]. Thus, Iinsulin might be mediated by IGF receptors and GRC/TRPV2 and this signalling pathway would then not be affected in insulin-resistant cells.

Our results show an insulin-induced potentiation of IOAG in wildtype cardiomyocytes (Fig. 3). This potentiation was inhibited by wortmannin and TeNT, which indicates that it involves PI3K-and VAMP2-dependent vesicle translocation. Activation of ion channels and transporters frequently involves a translocation from intracellular stores to the cell surface [6]. Insulin stimulates the translocation of GLUT4 from storage vesicles to the plasma membrane via a PI3K-dependent mechanism [2,7]. Several proteins have been identified in association with the GLUT4 vesicles and appear to move with GLUT4 to the plasma membrane in an insulin-dependent manner [2,11], including the v-SNARE protein VAMP2. Interestingly, Singh et al. recently showed that TRPC3 binds to SNARE proteins including VAMP2 [22]. These authors also showed that agonist stimulation causes a VAMP2-dependent insertion of TRPC3 into the plasma membrane and inhibition of this process with TeNT results in a decreased OAG-mediated Ca2+ influx. In line with this, we found that insulin promoted a PI3K-dependent translocation of TRPC3 to the cell surface membrane from a subsarcolemmal localization in WT cardiomyocytes (Figs. 6 and 7Go). Moreover, in WT cells TRPC3 was detected in GLUT4 immunoprecipitates both in the absence and presence of insulin (Fig. 5B), which suggests that TRPC3 and GLUT4 are co-localized in vesicles that are translocated to the plasma membrane by insulin. In ob/ob cells, on the other hand, insulin did not modify the TRPC3 intracellular distribution, which is consistent with an impaired intracellular insulin signalling in ob/ob muscle cells [44,45]. Noteworthy, defective insulin-regulated mobilization of GLUT4 to the plasma membrane is generally a key feature in insulin-resistant conditions [8–10]. Taken together, these results indicate that in WT cardiomyocytes insulin activates both GLUT4 and TRPC3 via a VAMP2-dependent translocation to the sarcolemma. Cardiomyocytes of ob/ob mice show severe defects in early steps of insulin signalling, e.g. decreased insulin-mediated phosphorylation of the insulin receptor tyrosine site and protein kinase B [45]. Accordingly, our results show defective insulin-mediated potentiation of IOAG and TRPC3 translocation in ob/ob cardiomyocytes.

Peripheral insulin resistance and hyperinsulinemia are hallmarks of obesity and type 2 diabetes, which are leading causes of coronary heart disease and heart failure [1,2,46,47]. In cardiomyocytes, these changes in insulin signalling would have important effects on ion fluxes, since insulin can activate NSCC:s both directly and indirectly via increased DAG [30,31,48]. We show decreased ion fluxes through TRPC3 channels in cardiomyocytes from insulin resistant ob/ob mice, which may affect intracellular Ca2+ homeostasis and hence cellular function, for instance, via the calcineurin and nuclear factor of activated T cells (NFAT) pathway [49,50]. TRPC3 expression was found to be increased in several rodent models of pathological cardiac hypertrophy and TRPC3 overexpression stimulates the calcineurin-NFAT pathway [51]. Furthermore, cardiac-specific TRPC3 overexpressing mice displayed an increased cardiac hypertrophy in response to prolonged infusion of phenylephrine and angiotensin II or pressure overload induced by transverse aortic constriction [52]. Based on these results it might be speculated that decreased ion fluxes through TRPC3 channels in insulin-resistant conditions are beneficial and limit or delay the development of pathological changes.

In conclusion, OAG induced an NSCC (IOAG) that was smaller in insulin-resistant than in normal cardiomyocytes. IOAG was potentiated by pretreatment with insulin in normal but not in insulin-resistant cardiomyocytes. The differences in IOAG can be explained by decreased TRPC3 expression and defective insulin-mediated trafficking of this protein in insulin-resistant cardiomyocytes.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
The present study was supported by the Swedish Research Council (proj 10842 and 14453), the Swedish Heart and Lung Foundation, Biovitrum Partner Fund, the Swedish Diabetes Foundation, and Funds at the Karolinska Institutet. A.S. was supported by a fellowship from EUROGENDIS.


   Notes
 
Time for primary review 35 days


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

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