Article2 November 2006free access TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy Naoya Onohara Naoya Onohara Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka Search for more papers by this author Motohiro Nishida Motohiro Nishida Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka Search for more papers by this author Ryuji Inoue Ryuji Inoue Department of Physiology, School of Medicine, Fukuoka University, Jonan-ku, Fukuoka, Japan Search for more papers by this author Hiroyuki Kobayashi Hiroyuki Kobayashi Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka Search for more papers by this author Hideki Sumimoto Hideki Sumimoto Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan Search for more papers by this author Yoji Sato Yoji Sato National Institute of Health Sciences, Setagaya, Tokyo, Japan Search for more papers by this author Yasuo Mori Yasuo Mori Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan Search for more papers by this author Taku Nagao Taku Nagao National Institute of Health Sciences, Setagaya, Tokyo, Japan Search for more papers by this author Hitoshi Kurose Corresponding Author Hitoshi Kurose Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka Search for more papers by this author Naoya Onohara Naoya Onohara Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka Search for more papers by this author Motohiro Nishida Motohiro Nishida Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka Search for more papers by this author Ryuji Inoue Ryuji Inoue Department of Physiology, School of Medicine, Fukuoka University, Jonan-ku, Fukuoka, Japan Search for more papers by this author Hiroyuki Kobayashi Hiroyuki Kobayashi Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka Search for more papers by this author Hideki Sumimoto Hideki Sumimoto Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan Search for more papers by this author Yoji Sato Yoji Sato National Institute of Health Sciences, Setagaya, Tokyo, Japan Search for more papers by this author Yasuo Mori Yasuo Mori Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan Search for more papers by this author Taku Nagao Taku Nagao National Institute of Health Sciences, Setagaya, Tokyo, Japan Search for more papers by this author Hitoshi Kurose Corresponding Author Hitoshi Kurose Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka Search for more papers by this author Author Information Naoya Onohara1, Motohiro Nishida1, Ryuji Inoue2, Hiroyuki Kobayashi1, Hideki Sumimoto3, Yoji Sato4, Yasuo Mori5, Taku Nagao4 and Hitoshi Kurose 1 1Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka 2Department of Physiology, School of Medicine, Fukuoka University, Jonan-ku, Fukuoka, Japan 3Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan 4National Institute of Health Sciences, Setagaya, Tokyo, Japan 5Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan *Corresponding author. Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel./Fax: +81 92 642 6884; E-mail: [email protected] The EMBO Journal (2006)25:5305-5316https://doi.org/10.1038/sj.emboj.7601417 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca2+-responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP3) is responsible for Ca2+ increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP3 is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca2+ oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca2+ channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca2+ signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy. Introduction Regulators of cardiac function such as vasoactive neurotransmitters and hormones activate phospholipase C (PLC) and thereby generate inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These agonists elevate the concentration of cytoplasmic free Ca2+ ([Ca2+]i) in cardiomyocytes, which induces positive inotropic effects on the heart and activates several transcriptional pathways that lead to cardiac hypertrophy (Wilkins and Molkentin, 2004; Woodcock and Matkovich, 2005). NFAT is one of the transcriptional factors regulated by [Ca2+]i (Crabtree and Olson, 2002). The relevance of the NFAT signaling pathway to cardiac hypertrophy is underscored by the observation that cardiac-targeted transgenic animals expressing constitutively activated forms of either calcineurin or NFAT produced ventricular hypertrophy (Molkentin et al, 1998; Taigen et al, 2000). The Ca2+-sensitive serine/threonine phosphatase (calcineurin) primarily regulates NFAT activity by rapid dephosphorylation of NFAT proteins and their translocation to the nucleus. A drop in nuclear Ca2+ deactivates calcineurin and allows one of several NFAT kinases to rephosphorylate NFAT, causing it to leave the nucleus and thereby inactivating transcription (Timmerman et al, 1996; Dolmetsch et al, 1997). Therefore, a sustained elevation of [Ca2+]i is required for NFAT-dependent transcription. The importance of agonists that activate PLC for cardiac hypertrophy is well established (Molkentin and Dorn, 2001). Many lines of evidence have shown that stimulation of PLC-linked G protein-coupled receptors, such as α1-adrenergic receptor (Maruyama et al, 2002), Ang II receptor (Nishida et al, 2005) and endothelin receptor (Arai et al, 2003), induce hypertrophic growth of rat cardiac myocytes. More clinically relevant, hypertrophied hearts induced by volume overload are commonly characterized by high levels of IP3-generating agonists such as Ang II (Dostal et al, 1992; Sadoshima et al, 1993). Numerous studies have demonstrated the need for sustained or periodic increases in [Ca2+]i to cause the nuclear localization of NFAT (Dolmetsch et al, 1997; Tomida et al, 2003). In nonexcitable cells, IP3 is generally accepted to function as a mediator of sustained Ca2+ responses (Timmerman et al, 1996; Dolmetsch et al, 1997). The sustained Ca2+ signaling requires the store-operated Ca2+ channel (SOC), which opens in response to depletion of intracellular stores through IP3 receptor (IP3R). Therefore, it is currently believed that Ca2+ entry through SOC regulates NFAT translocation. In the heart, however, the expression level of IP3R is much lower than that of ryanodine receptor (Moschella and Marks, 1993). Voltage-dependent L-type Ca2+ channel and ryanodine receptor function as the major source of Ca2+ for normal Ca2+-induced Ca2+ release of excitation–contraction (E–C) coupling, but many reports do not support the idea that the increase in [Ca2+]i through E–C coupling between L-type Ca2+ channel and ryanodine receptor is coupled to NFAT activation (Wilkins and Molkentin, 2004). A possible source of Ca2+ for activation of calcineurin is Ca2+ influx through transient receptor potential (TRP) proteins that are involved in store-operated Ca2+ entry (Clapham, 2003). Upregulation of canonical transient receptor potential (TRPC) proteins is recently reported to contribute to the development of cardiac hypertrophy (Seth et al, 2004). Other groups reported that TRPM7 regulates Mg2+ homeostasis, and TRPM6 and TRPM7 are differentially regulated by Ang II in vascular smooth muscle cells (He et al, 2005; Touyz et al, 2006). However, it is still unknown whether TRP channels contribute to receptor-stimulated activation of calcineurin-NFAT pathway in the heart. In this study, we investigated the mechanism of how Ang II stimulation induces the sustained Ca2+ signaling leading to NFAT activation and hypertrophic growth of rat neonatal cardiomyocytes. Results Essential role of DAG in Ang II-induced NFAT activation and cardiac hypertrophy We first examined whether IP3 or DAG is involved in Ang II-induced NFAT activation in rat neonatal cardiomyocytes. As it has been reported that pressure overload- and Ang II-induced cardiac hypertrophy are attenuated in NFAT4 (NFATc3)-null mice (Wilkins et al, 2002), the translocation of NFAT4 was determined in this study. Stimulation of cardiac myocytes with Ang II for 30 min increased the maximal nuclear predominant fluorescence of GFP-fused amino-terminal region of NFAT4 protein (GFP-NFAT4) (Figure 1A–C). The Ang II-induced NFAT translocation was completely suppressed by the expression of DAG kinase β (DGKβ), an enzyme that decreases the cellular DAG level by converting DAG to phosphatidic acid. Treatment with RHC80267, a DAG lipase inhibitor, significantly increased the Ang II-induced nuclear translocation of GFP-NFAT4. However, treatment with xestospongin C, an IP3R blocker, did not affect the Ang II-induced translocation of GFP-NFAT4 to the nucleus. To directly inhibit IP3-mediated signaling, we expressed the ligand-binding region of type 1 IP3R (IP3-sponge) (Uchiyama et al, 2002). The Ang II-induced transient increase in [Ca2+]i (or Ca2+ release) was completely suppressed by the treatment with xestospongin C and by the expression of IP3-sponge but not DGKβ (Supplementary Figure S1), suggesting the efficient inhibition of IP3-mediated Ca2+ signaling. The Ang II-induced increase in NFAT-dependent luciferase reporter activity was suppressed by DGKβ, but not by xestospongin C and IP3-sponge (Figure 1D and E). Treatment with RHC80267 promoted the Ang II-induced NFAT activation (Figure 1E). These results suggest the involvement of DAG in Ang II-induced NFAT activation. We also examined the involvement of DAG in Ang II-induced hypertrophic responses. Expression of DGKβ, but not IP3-sponge, completely suppressed Ang II-induced hypertrophic responses, such as actin reorganization (Figure 1F), protein synthesis (Figure 1G), and expression of brain natriuretic peptide (BNP) (Figure 1H). These results suggest that DAG, but not IP3, is essential for Ang II-induced NFAT activation and hypertrophic responses of neonatal cardiomyocytes. Figure 1.Essential role of DAG in Ang II-induced cardiomyocyte hypertrophy. (A) Nuclear translocation of GFP-NFAT4 by Ang II stimulation. A portion of cells was treated with RHC80267 (30 μM) or xestospongin C (XestC, 20 μM) for 30 min before the addition of Ang II (100 nM), and a portion of cells was infected with DGKβ for 48 h before Ang II stimulation. (B, C) Quantification of nuclear predominant fluorescence of GFP-NFAT4 after Ang II stimulation. (D, E) Effects of DGKβ, RHC80267, and XestC on the increase in NFAT-dependent luciferase activity by Ang II stimulation for 6 h. The fold activation was calculated by the values of untreated cells set as 1. (F–H) Effects of DGKβ and GFP-IP3-sponge on Ang II-induced actin reorganization (F), protein synthesis (G), and BNP expression (H). Scale bar=20 μm. *P<0.05, **P<0.01 versus control or LacZ-expressing cells. Download figure Download PowerPoint Involvement of Ang II type 1 receptor, Gαq, and PLC in Ang II-induced NFAT activation In contrast to the absence of extracellular Ca2+ (Supplementary Figure S1A), myocytes showed spontaneous increases in [Ca2+]i in the presence of extracellular Ca2+. Treatment with Ang II induced the transient increase in [Ca2+]i followed by sustained oscillatory increase in [Ca2+]i (Figure 2A; the former can more clearly be seen in Supplementary Figure S1A). The Ca2+ oscillation represents a spontaneous activity of myocytes, and Ang II stimulation increased its frequency (Supplementary Figure S1C). The Ang II-induced Ca2+ response and NFAT activation were greatly suppressed by U73122, a PLC inhibitor, but not by U73343, an inactive analog of U73122 (Figure 2A–C). Thus, PLC primarily regulates Ang II-induced Ca2+ signal generation. The Ang II-induced translocation of GFP-NFAT4 was suppressed by CV11974, an Ang II type 1 receptor (AT1R) blocker, but not by PD123319, an AT2R blocker (Figure 2D). These results indicate that AT1R-mediated PLC activation is involved in Ang II-induced NFAT4 activation. We next examined which G proteins are involved in Ang II-induced NFAT activation. It has been generally believed that Gαq plays an important role in agonist-induced cardiac hypertrophy (Molkentin and Dorn, 2001). To examine the involvement of Gαq, we expressed regulator of G protein signaling (RGS) domain that is ∼200 amino acids, specifically binds GTP-bound form of Gα and accelerates GTPase activity. When RGS domain is expressed in cells, it competes with activated form of Gα for endogenous effectors and accelerates turn-off reaction of Gα. Therefore, RGS domain can work as a specific inhibitor of Gα. As expected, the expression of a Gαq-specific RGS domain of G protein-coupled receptor kinase 2 (GRK2-RGS) completely suppressed the Ang II-induced translocation of GFP-NFAT4 (Figure 2E). However, the expression of a Gα12/13-specific RGS domain of p115RhoGEF (p115-RGS) did not affect the Ang II-induced translocation of GFP-NFAT4. Pertussis toxin (PTX) and carboxyl terminal region of GRK2 (GRK2-ct), a βγ subunit of G protein (Gβγ)-sequestering polypeptide, did not inhibit the Ang II-induced translocation of GFP-NFAT4 (Figure 2E). Thus, these results support the evidence that agonist-induced Ca2+-dependent NFAT activation is predominantly regulated by Gαq, but not by Gα12/13, Gi or Gβγ in cardiomyocytes. Figure 2.Involvement of AT1R, Gαq, and PLC in Ang II-induced NFAT activation. (A–C) Effects of U73122 and U73343 on Ang II-induced Ca2+ responses (A), translocation of GFP-NFAT4 (B), and NFAT activation (C). (A) Effects of U73122 and U73343 on the increases in the frequency of Ca2+ oscillation during 5 min Ang II stimulation. The digital images were obtained every 1 s. (D) Effects of CV11974 and PD123319 on Ang II-induced NFAT translocation. Cells were treated with U73122 (5 μM), U73343 (5 μM), CV11974 (CV, 5 μM), or PD123319 (PD, 5 μM) for 30 min before the addition of Ang II (100 nM). (E) Effects of PTX, GRK2-RGS, p115-RGS, and GRK2-ct on Ang II-induced NFAT translocation. Cells were infected with adenovirus encoding LacZ (100 MOI), p115-RGS or GRK2-ct (100 MOI), or GRK2-RGS (300 MOI) for 48 h. A portion of cells was treated with PTX (100 ng/ml) for 24 h before Ang II stimulation. *P<0.05, **P<0.01 versus Ang II stimulation of control or LacZ-expressing cells. Download figure Download PowerPoint Requirement of Ca2+ influx through L-type Ca2+ channels and nonselective cation channels in Ang II-induced NFAT activation It has been reported that DAG induces Ca2+ influx through activation of cation channels (Hofmann et al, 1999; Clapham, 2003). As the Ang II-induced periodic increase in [Ca2+]i likely results from enhanced spontaneous activity of myocytes (which are dependent on extracellular Ca2+; see above), and these were suppressed by DGKβ (Supplementary Figure S1), we next examined whether Ca2+ influx is involved in DAG-mediated responses. Treatment of cardiac myocytes with Ang II or with a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), increased the nuclear translocation of GFP-NFAT4 and NFAT activity, both of which were almost completely suppressed by the voltage-dependent Ca2+ channel blocker nitrendipine and a receptor-activated cation channel (RACC) inhibitor SK&F96365 (Figure 3A–C). As OAG-induced NFAT activation was also completely suppressed by cyclosporine A, a calcineurin inhibitor (Figure 3C), DAG increases NFAT activity through calcineurin activation. These results suggest that RACC and Ca2+ influx through L-type Ca2+ channel mediate Ang II- or DAG-induced NFAT activation. Figure 3.Requirement of RACC and L-type Ca2+ channel in DAG-mediated NFAT translocation. (A) Effects of SK&F96365 (SKF), nitrendipine (Nit) and valinomycin (Val) on Ang II- or OAG-induced NFAT translocation. (B) Quantification of the nuclear predominant fluorescence of GFP-NFAT4 without (None) or with Ang II or OAG stimulation. (C) Effects of SKF, Nit, and cyclosporine A (CysA) on Ang II- or OAG-induced increase in NFAT-luciferase activity. Cells were treated with SKF (10 μM), Nit (1 μM), Val (1 μM), or CysA (1 μM) for 30 min before the addition of Ang II (100 nM) or OAG (25 μM). **P<0.01 versus Ang II stimulation of control cells. ##; P<0.01 versus OAG stimulation of control cells. Download figure Download PowerPoint Ang II activates DAG-sensitive cation channels in cardiac myocytes To directly demonstrate that Ang II activates DAG-sensitive RACC, whole-cell patch-clamp experiments were performed. In quasi-physiological ionic conditions, administration of Ang II into the bath activated inward currents at −80 mV, which were further enhanced by RHC80267 (Figure 4A and B). These currents were completely abolished by N-methyl-D-glucamine substitution for all external cations (data not shown), and showed an outward-rectifying property with the reversal potential of ca. 0 mV (1.0±1.0 mV, n=6), when Cs+ was intracellularly dialyzed via patch pipette and TTX (3 μM) and nitrendipine (1 μM) were added into K+-free external solution to block voltage-dependent K+, Na+, and L-type Ca2+ channels, respectively (see inset in Figure 4C). Administration of OAG (25 μM) also activated inward currents showing indistinguishable properties from those activated by Ang II, whereas application of myo-IP3 (10 μM) in the internal solution was unable to activate any discernible currents by itself (data not shown). These results collectively suggest that Ang II activates DAG-sensitive nonselective cation currents in cardiomyocytes via an IP3-independent pathway, which bears considerable resemblance to heterologously expressed TRPC channels. Figure 4.Activation of DAG-sensitive currents from single cardiomyocytes by Ang II stimulation. (A) Representative traces of ionic currents recorded from Ang II-treated cardiomyocytes at a holding potential of −80 mV under conventional whole-cell patch-clamp with K+-internal solution. The nonselective cation currents are activated by Ang II (1 μM), and potentiated by RHC80267 (30 μM). The dotted line represents the zero current level. (B) Current density of inward current (at −80 mV) averaged for the period of 60–65 s after Ang II stimulation or RHC80267 treatment (n>8). **P<0.01. (C) I–V relationship of ionic currents from unstimulated (Control) and Ang II-stimulated myocytes with Cs+-internal solution containing myo-IP3 (10 μM). TTX (3 μM) and nitrendipine (1 μM) are included in the K+-free external solution. (Inset) I–V relationship of TRPC-like currents induced by Ang II (differences between Ang II and Control). (D) Representative traces of time-dependent changes in the membrane potential and the frequency of action potential by Ang II stimulation in the current-clamp mode. Download figure Download PowerPoint In the next step, we examined Ang II-induced changes in membrane potential by using the current-clamp technique, since the treatment with valinomycin, a K+ ionophore, which causes inactivation of voltage-dependent channels via stabilization of membrane potential (Linares-Hernandez et al, 1998), completely suppressed the Ang II-induced translocation of GFP-NFAT4 (Figure 3A and B), and in general, the activation of RACC causes membrane depolarization (Large, 2002). As expected, membrane potential recording from single myocytes with current-clamp mode clearly demonstrated that Ang II increased the frequency of action potentials, which eventually led to continuous burstic firing superimposed on concomitant sustained depolarization (22.2±5.6 mV, n=5) (Figure 4D). It is noteworthy that the time course of these effects is very similar to that observed for the enhanced frequency of Ca2+ oscillations induced by Ang II (see above). Properties of DAG induced membrane depolarization in rat cardiac myocytes Current-clamp recordings were technically little feasible to monitor the membrane potential for a long period of time, because of rhythmical contractions of myocytes evoked by Ang II. To circumvent this problem, we adopted a voltage-sensitive fluorescent probe DiBAC4(3). After DiBAC4(3) enters the cells, it binds to cellular proteins and membrane lipids. Then, DiBAC4(3) enhances fluorescence. Because of its slow dissociating nature, DiBAC4(3) can only detect slow cumulative changes in resting potential rather than rapid changes in membrane potential generated by action potential. Ang II stimulation gradually increased the fluorescence intensity of DiBAC4(3) (Figure 5A and B), indicating the shift of membrane potential to positive (BACzkó et al, 2004). The averaged changes in membrane potential induced by Ang II were estimated to be ∼15 mV. Treatment with RHC80267 enhanced the Ang II-induced increases in the fluorescence intensity of DiBAC4(3) (Figure 5C). These results indicate that DAG generated by Ang II stimulation shifts the membrane potential of cardiac myocytes more positively. DAG also activates other signaling molecules including protein kinase C (PKC). PKC is known to potentiate the extent of L-type Ca2+ channel activation, and both OAG and phorbor 12-myristrate 13-acetate (PMA) have been reported to increase the channel open probability in rat cardiomyocytes (Guinamard et al, 2004). However, treatment with PMA did not increase the fluorescence intensity of DiBAC4(3) (Figure 5A and B) and OAG-induced translocation and activation of NFAT were not affected by bisindolylmaleimide, a selective PKC inhibitor (Supplementary Figure S2). It is possible that the metabolites of DAG work as mediators for NFAT translocation. However, treatment with arachidonic acid (AA) or phospholipase A2 (PLA2) inhibitors did not affect Ang II-induced NFAT translocation (Supplementary Figure S2). These results suggest that PKCs and DAG metabolites do not participate in Ang II-induced depolarization and NFAT translocation. The Ang II-induced increases in the fluorescence intensity of DiBAC4(3) were completely suppressed by SK&F96365, but not by nitrendipine and xestospongin C (Figure 5D). Figure 5.Changes in membrane potential through RACC activation by DAG. (A) Representative time courses of changes in Ang II-, OAG-, or PMA-induced F/F0 of DiBAC4(3) fluorescence from time course experiments. Cells were stimulated with Ang II (1 μM), OAG (25 μM), PMA (1 μM), or KCl (10 mM). F0 means the initial value of fluorescence. (B) Maximal changes in resting membrane potential calculated from the changes in DiBAC4(3) fluorescence intensity during 15 min drug treatment. For the in vivo calibration of the membrane potentials, the KCl-induced maximal changes in fluorescence were fitted to the theoretical potentials obtained from Nernst equation, and then the changes in membrane potential by Ang II stimulation was calculated based on the fitting fomula. (C) Effects of RHC80267 on the concentration-dependent changes in resting membrane potentials induced by Ang II stimulation. (D) Involvement of RACC in Ang II-induced increases in the resting membrane potential. Cells were treated with SK&F96365 (SKF, 10 μM), nitredipine (Nit, 1 μM), or xestospongin C (XestC, 20 μM) for 30 min before the addition of Ang II. **P<0.01 versus Ang II stimulation of control cells. (E) Effects of SK&F96365 (SKF), nitrendipine (Nit), and xestospongin C (XestC) on Ang II-induced Ca2+ responses. The digital images were obtained every 1 s during 0–3 min under basal conditions and during 25–28 min after Ang II stimulation. (F) Number of Ca2+ spikes was normalized to per minute. **P<0.01 versus Ang II stimulation of control cells. Download figure Download PowerPoint We next examined whether periodic increase in [Ca2+]i is regulated by RACC. The myocytes showed spontaneous Ca2+ oscillations in the presence of extracellular Ca2+ (top panel in Figure 5E). The frequency of Ca2+ oscillations was increased by Ang II stimulation and this was suppressed by SK&F96365 and nitrendipine, but not by xestspongin C (middle and bottom panels in Figure 5E and F). These results support the idea that DAG generated by Ang II-induced PLC activation causes membrane depolarization through RACC activation and thereby secondarily activates L-type Ca2+ channel, leading to increased frequency of Ca2+ oscillations. Requirement of TRPC3 and TRPC6 in Ang II-induced membrane depolarization TRPC proteins are thought to be molecular candidates for RACC (Clapham, 2003). We found the expression of at least five TRP canonical (TRPC) mRNAs (TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7) in rat neonatal cardiomyocytes by RT–PCR analysis (data not shown). Recent reports have demonstrated that three TRPC channels (TRPC3, TRPC6, and TRPC7) are activated directly by DAG (Hofmann et al, 1999; Clapham, 2003). Thus, we next examined which DAG-sensitive TRPC protein is involved in Ang II-induced NFAT activation. We overexpressed TRPC3, TRPC6, or TRPC7, and examined the Ang II-induced changes in membrane potential with DiBAC4(3) (Figure 6A and B). Among three TRPC proteins, Ang II-induced increases in the fluorescence intensity of DiBAC4(3) were significantly enhanced by the expression of TRPC3 and TRPC6 but not by TRPC7 (Figure 6B), although the latter enhanced OAG-induced [Ca2+]i increases to the same extent as the former two did (Supplementary Figure S3). These results indicate that TRPC3 and TRPC6, but not TRPC7, likely regulate the Ang II-induced membrane depolarization. This conclusion was further corroborated by siRNA-mediated knockdown of TRPC3 (siRNA 1397, 1992, and 2043) and TRPC6 (siRNA 1609 and 1786) in the cardiomyocytes; this procedure decreased the expression level of endogenous TRPC3 and TRPC6 proteins without affecting other TRPC proteins (Figure 6C–F), and simultaneously caused significant suppression of Ang II-induced increases in the fluorescence intensity of DiBAC4(3) (Figure 6G). Taken together, the above results strongly suggest that DAG-mediated activation of TRPC3 and TRPC6 channels contributes to the enhanced Ca2+ oscillation by Ang II via their membrane depolarizing actions. Figure 6.Requirement of TRPC3 and TRPC6 in Ang II-induced increases in membrane potential. (A) Western blots of the respective TRPC proteins. To identify the sizes of TRPC3 (C3), TRPC6 (C6), and TRPC7 (C7), each TRPC was overexpressed with recombinant adenoviruses. (B) Potentiating effects of TRPC3 and TRPC6 on changes in membrane potential by Ang II stimulation in LacZ-, TRPC3-, TRPC6-, and TRPC7-expressing cells. **P<0.01 versus Ang II stimulation of LacZ-expressing cells. NS means no significance from LacZ-expressing cells. (C–F) Effects of TRPC3 siRNAs (C, D) and TRPC6 siRNAs (E, F) on the expression of the respective TRPC proteins. (C, E) Representative Western blots with anti-TRPC3 (C) and anti-TRPC6 (E). (D, F) Effects of siRNAs of TRPC3 and TRPC6 on the average expression of native TRPC3, TRPC6, and TRPC7 proteins. (G) Effects of siRNAs of TRPC3 and TRPC6 on the maximal changes in DiBAC4(3) fluorescence intensity by Ang II (100 nM). Data are shown as the changes in membrane potentials (mV) calculated by in vivo calibration. **P<0.01 versus Ang II stimulation of control siRNA-treated cells (Control). Download figure Download PowerPoint In addition, siRNA silencing of TRPC3 and TRPC6 also significantly suppressed Ca2+ entry-mediated [Ca2+]i elevation induced by the addition of Ca2+ into the bath after Ang II stimulation (Supplementary Figure S3). Thus, some role of direct Ca2+ entry via TRPC3/TRPC6-associated pathway cannot completely be excluded in the Ang II-enhanced Ca2+ oscillation. Requirement of TRPC3 and TRPC6 in Ang II-induced NFAT translocation and hypertrophic responses We next examined whether TRPC3 and TRPC6 are involved in Ang II-induced hypertrophic responses. Treatment with siRNAs of TRPC3 and TRPC6 significantly suppressed Ang II-induced NFAT translocation (Figure 7A
