Skip Navigation

Cardiovascular Research 2005 68(1):75-86; doi:10.1016/j.cardiores.2005.05.015
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Barac, Y. D.
Right arrow Articles by Binah, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barac, Y. D.
Right arrow Articles by Binah, O.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2005, European Society of Cardiology

The 1,4,5-inositol trisphosphate pathway is a key component in Fas-mediated hypertrophy in neonatal rat ventricular myocytes

Yaron D. Barac, Naama Zeevi-Levin, Gal Yaniv, Irina Reiter, Felix Milman, Mark Shilkrut, Raymond Coleman, Zaid Abassi and Ofer Binah*

Rappaport Institute, Rappaport Faculty of Medicine, Technion, Haifa, Israel

* Corresponding author. Rappaport Institute, P.O.B 9697, Haifa 31096, Israel. Tel.: +972 4 8295262. Email address: binah{at}tx.technion.ac.il

Received 9 February 2005; revised 17 May 2005; accepted 18 May 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Cardiac hypertrophy is a compensatory response to increased mechanical load. Since Fas receptor activation is an important component in hypertrophy induced by pressure- and volume-overload, deciphering the underlying signaling pathways is of prime importance. Based on our previous work showing that in mice and rats ventricular myocytes the electrophysiological disturbances and diastolic [Ca2+]i-rise caused by 3 h of Fas activation are dependent on the Fas->phospholipase C (PLC)->1,4,5-inositol trisphosphate (1,4,5-IP3)->sarcoplasmic reticulum (SR) [Ca2+]i release pathway, we tested the hypothesis that this pathway is also critical for Fas-mediated hypertrophy.

Methods: The effects of 24 h Fas activation in cultured neonatal rat ventricular myocytes (NRVM) were analyzed by means of RT-PCR, Western blot, immunofluorescence and fura-2 fluorescence.

Results: Fas activation increased nuclei surface area, atrial natriuretic peptide and connexin43 (Cx43) mRNA, the protein levels of total Cx43 and non-phosphorylated Cx43, and sarcomeric actin, all indicating hypertrophy. Concomitantly, Fas activation decreased mRNA of SERCA2a, the ryanodine receptor (RyR) and nuclear IP3R3. Further, Fas activation caused NFAT nuclear translocation. The hypertrophy was abolished by U73122 [GenBank] , xestospongin C (blockers of the 1,4,5-IP3 pathway), genistein and by the PI3K blocker LY294002.

Conclusions: Fas-mediated hypertrophy is dependent on the 1,4,5-IP3 pathway, which is functionally inter-connected to the PI3K/AKT/GSK3β pathway. Both pathways act in concert to cause NFAT nuclear translocation and subsequent hypertrophy.

KEYWORDS Calcium (cellular); Cell culture; Hypertrophy; Myocytes; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Recent studies have shown that activation of the cardiac Fas receptor (Fas) causes both apoptotic and non-apoptotic deleterious effects [1–4]. In contrast to many Fas-baring cell lines and tissues, Fas activation in ventricular myocytes does not cause apoptosis, indicating that Fas expression per se does not necessarily commit cells to apoptosis. Specifically, we and others have demonstrated that whereas normoxic myocytes are resistant to apoptosis induced by Fas activation, hypoxia predisposes myocytes to Fas-mediated apoptosis [1,3,4]. In a series of studies we found that in adult murine and neonatal rat ventricular myocytes (NRVM), 3 h of Fas activation caused a wide range of functional disturbances, including action potential changes, early- and delayed after depolarizations, arrhythmias, intracellular Ca2+([Ca2+]i) rise, decreased Ito and increased ICa,L [5,6]. Our key finding was that these effects were exclusively dependent on the Fas->phospholipase C (PLC)->1,4,5-IP3->SR [Ca2+]i release pathway and on tyrosine kinases (TK) phosphorylation [5,6]. In brief, we showed that: (1) the above mentioned effects were blocked by the PLC inhibitor U73122 [GenBank] and by xestospongin C, a specific blocker of the SR 1,4,5-IP3-operated Ca2+ channels, and were reproduced by intracellular application of 1,4,5-IP3; (2) Fas activation increased 1,4,5-IP3 levels.

Recently, Dimmeler's group has shown that 4 h of Fas activation in NRVM caused hypertrophy, and that lpr mice lacking functional Fas demonstrated rapid-onset of left ventricular dilatation and failure, absence of compensatory hypertrophy and increased mortality [1], indicating that the hypertrophy was Fas-dependent. Because of the key role of hypertrophy in major heart diseases, and based on our previous studies, we tested the hypothesis that the 1,4,5-IP3 pathway is critical for Fas-mediated hypertrophy. This work shows for the first time the involvement of the ubiquitous 1,4,5-IP3 pathway in ventricular hypertrophy, suggesting that modulators of this pathway specifically targeted to cardiac muscle may have an important therapeutic value.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Cultures preparation, Fas activation, apoptosis determination and [Ca2+]i transients measurement
NRVM cultures were prepared from ventricles of 1–2-day-old Sprague–Dawley rats [4], and experiments performed on cultures 4–6 days after plating. 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). To inhibit non-cardiac muscle growth, BrdU was included in the culture medium. Fas was activated by recombinant Fas ligand (rFasL) (10 ng/mL) and an enhancing antibody (1 µg/mL) (Alexis, San Diego, CA, USA). This concentration was chosen based on a study [1] showing that in the concentration range of 1–100 ng/ml, maximal hypertrophy (indicated by [3H] leucine incorporation) was attained at 10 ng/mL. For each protocol apoptosis was measured by the DAPI assay [4], in two wells (500 myocytes measured in each well), prepared from 3 different litters, altogether counting 3000 myocytes. [Ca2+]i transients were measured by means of fura-2 fluorescence from cultures maintained at 37 °C and stimulated at 0.5 Hz [4].

2.2 Immunoblotting
NRVM were harvested in lysis buffer, and 40 µg of total protein were separated by 12% SDS-PAGE gels, transferred to nitrocellulose membranes, and immunoblotted [4]. The primary antibodies were anti-Cx43 (Chemicon International, Temecula, CA, USA) and anti-non-phosphorylated (NP) Cx43 antibody (Zymed Laboratories, San Francisco, CA, USA). Immune complexes were detected using the Enhanced Chemiluminescence detection system (Perkin Elmer Life Sciences, Boston, MA, USA) [7].

2.3 Immunofluorescence staining
Actin measurements: NRVM were stained with Alexa Phalloidin (Alexis, Cornerstone Court, San Diego, CA, USA). Actin was quantified by measuring the fluorescence intensity using custom-made "Open View" software, from 12 different areas in each slide, from 4 experiments. Nuclei were visualized by DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). NFAT translocation: NRVM were stained using indirect immunofluorescence staining with polyclonal anti-NFAT (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA), monoclonal anti-{alpha}-actinin (Sigma, St. Louis, MO, USA) antibodies, and anti mouse-FITC+anti-rabbit rhodamin (Jackson ImmunoResearch Laboratories, Inc., Baltimore, PA, USA). Nuclei were visualized by Topro stain (Jackson ImmunoResearch Laboratories). The slides were visualized using a Provis AX70 fluorescence microscope at x 40 magnification (Olympus, Tokyo, Japan).

2.4 RT-PCR assay
Total RNA was isolated using EZ-RNATM kit (Biological Industries, Beit-Haemek, Israel) and Reverse Transcriptase (RT) reaction was conducted [8]. The nucleotides used and the cycling conditions are presented in Table 1. All PCR products were given a final elongation stage at 72 °C for 10 min. Products were resolved on 1.5% agarose gel.


View this table:
[in this window]
[in a new window]

  		Table 1 PCR primers and protocols

 
2.5 Nuclear measurements
For nuclear staining, slides were stained with hematoxylin and eosin. Morphometric determination of myocytes nuclear dimensions was performed using image analysis software (analySIS docu®3.0, Soft Imaging System, Munster, Germany). The system consists of an Olympus CH-40 bright field microscope (x 100 objective) fitted with a CCD camera. The peripheries of selected nuclei were traced with the mouse cursor and the values (in µm2) were determined. Special care was taken to ensure that the areas selected for analysis on the slides were comparable between the treated and control samples. Altogether, 60 nuclei were measured from two different litters for each group.

2.6 Chemicals
Unless otherwise indicated, chemicals were purchased from Sigma (Sigma Chemical Co, St. Louis, MO, USA).

2.7 Statistical analysis
Results were expressed as mean ± SEM. Means of two populations were compared using Student's t-test for unpaired observations. A value of P<0.05 was considered significantly different.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Fas-induced hypertrophy
Firstly, we confirmed that Fas activation in NRVM does not cause apoptosis, although the apoptotic machinery is responsive, as evidenced by the ability of H2O2 to cause apoptosis (Fig. 1A). Next, we characterized the time course (3, 6 and 24 h) of the hypertrophy induced by Fas activation. As shown in Fig. 1C, rFasL increased ANP mRNA in a time-dependent fashion, to the extent comparable to that caused by endothelin-1 (ET-1). Cx43 mRNA levels (known to be affected by hypertrophic stimuli [9–11]) were also increased by rFasL in a time-dependent fashion, reaching a 2-fold change at 24 h (Fig. 1D). Since both ANP and Cx43 mRNA levels peaked at 24 h, subsequent experiments were performed at this time point. In agreement with the Cx43 mRNA data (Fig. 1D), total and non-phosphorylated (NP) Cx43 protein expression were increased at 24 h Fas activation (Fig. 2B–D). Two additional markers of hypertrophy, nuclear area [12,13] and sarcomeric actin were also augmented by Fas activation (Fig. 1B and E). It is noteworthy that due to the pronounced overlap between myocytes in culture, accurate measurement of cell area in confluent NRVM cultures is practically impossible.


Figure 1
View larger version (35K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Fas activation in NRVM causes hypertrophy but not apoptosis. (A) NRVM were treated with rFasL or with 50 µM H2O2, and apoptosis assessed by the DAPI assay. (B) Fas activation for 24 h increased actin immunofluorescence. Next to the graph, corresponding actin immunofluorescence pictures are depicted. Actin fluorescence is expressed in arbitrary units (n = 4). (C) Time dependent changes in ANP mRNA. The upper panel depicts representative gels from control, 3, 6 and 24 h of Fas activation, and from a culture treated for 24 h with endothelin-1 (50 nM) (n = 6). (D) Time dependent changes in Cx43 mRNA. The upper panel depicts representative gels from control, 3, 6 and 24 h of Fas activation (n = 6). Each value was divided by its corresponding β-actin value. Values were normalized to control cultures, which were set as 1.0. (E) Effect of Fas activation on nuclear area. Altogether, 60 nuclei were measured from two different litters for each group. *P<0.05.

 

Figure 2
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 The PLC blocker U73122 and genistein block the increase in actin immunofluorescence and Cx43 protein expression caused by 24 h Fas activation. U73122 (2 µM) or genistein (50 µM) were added to the culture 30 min before, and throughout exposure to rFasL. (A) U73122 blocks the increase in actin immunofluorescence caused by Fas activation. Above the graph, corresponding actin immunofluorescence pictures are depicted (n = 4). (B) Representative Western blots of total Cx43, NP-Cx43 and β-actin under different experimental conditions. The increase in total and NP-Cx43 protein expression induced by Fas activation was blocked by U73122 or genistein. U – U73122; Gen – Genistein. (C) and (D): Summary of the effects of U73122 and genistein on Fas-mediated increase in total and in NP-Cx43, respectively (n = 4). The equivalency of loading was verified with an antibody against β-actin. Each value was divided by its corresponding β-actin value. Values were normalized to control cultures, which were set as 1.0. *P<0.05.

 
3.2 The effect of Fas activation on [Ca2+]i handling proteins
Following a previous study showing that hypertrophy caused by aortic stenosis [14] is associated with alterations in key components of the intracellular Ca2+ handling machinery, we tested whether similar changes occur in Fas-mediated hypertrophy. Firstly, we tested the mRNA levels of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), responsible for pumping Ca2+ back into the SR. Of the three cardiac SERCA isoforms, SERCA2a, SERCA2b, and SERCA3, the first is the major isoform in cardiomyocytes. As depicted in Fig. 3A, Fas activation decreased SERCA2a mRNA level by 24%. Next, we tested the effect of Fas activation on the cardiac ryanodine receptor (RyR) SR isoform-RyR2, which is central to the Ca2+-induced release of Ca2+(CIRC) machinery. As shown in Fig. 3B, RyR2 was decreased by Fas activation to a similar extent as SERCA2a (~20%). Finally, we determined the effect of Fas activation on the intracellular IP3 receptors (IP3R). Of the three known isoforms, we focused on the two that exist in neonatal and adult rat ventricular myocytes [15]. Whereas mRNA level of the type 2 cytosolic-localized IP3R [15] was unchanged by Fas activation (Fig. 3C), that of the nuclear IP3R type 3 was decreased by 25% (Fig. 3D).


Figure 3
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 The effect of 24 h Fas activation on SERCA2a, RyR2, IP3R2, and IP3R3 mRNA content in NRVM. In each panel, the upper section depicts representative gels, and the lower section depicts quantitative densitometric analysis from 4 experiments. (A) SERCA2a; (B) RyR2; (C) IP3R2; (D) IP3R3. *P<0.05. Each value was divided by its corresponding β-actin value. Values were normalized to control cultures, which were set as 1.0. *P<0.05.

 
3.3 The involvement of the 1,4,5-IP3 pathway in Fas-mediated hypertrophy
The first step in testing the 1,4,5-IP3 hypothesis was to determine whether the specific PLC blocker U73122 [GenBank] [4] interferes with Fas-mediated hypertrophy. In support of the hypothesis, U73122 [GenBank] blocked rFasL-induced increase in actin fluorescence (Fig. 2A), as well as total and NP Cx43 protein (Fig. 2B–D), and ANP and Cx43 mRNA (Fig. 4A–C). Furthermore, blocking the SR 1,4,5-IP3-operated Ca2+ release channel with xestospongin C [4] also prevented rFasL-mediated increase in ANP and Cx43 mRNA levels (Fig. 4A–C). In support of our notion that 1,4,5-IP3-mediated Ca2+ release is the main source triggering the hypertrophic program, we found that the RyR blocker ryanodine (10–5 mmol/l) did not prevent rFasL-induced increase in ANP mRNA level (Fig. 4D).


Figure 4
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Inhibitors of the 1,4,5-IP3 pathway and genistein block the increase in ANP and Cx43 mRNA caused by 24 h rFasL-induced. U73122 (2 µM), xestospongin C (10 µM) or genistein (50 µM) were added to the culture medium 30 min before, and throughout exposure to rFasL. (A) Representative gels of ANP and Cx43 mRNA under the different experimental conditions. A summary of the effect of U73122, xestospongin C and genistein on Fas-mediated increase in ANP mRNA (B, n = 7 experiments) and Cx43 mRNA (C, n = 4 experiments). (D) The increase in ANP mRNA induced by Fas activation was not blocked by ryanodine (10 µM). n = 4 experiments. Each value was divided by its corresponding β-actin value. Values were normalized to control cultures, which were set as 1.0. *P<0.05.

 
Since the only known function of the 1,4,5-IP3-operated SR channel is to release Ca2+, it is conceivable that this increase in [Ca2+]i occurring at a certain point in time during Fas activation is instrumental to the hypertrophic response. To decipher this key issue we measured [Ca2+]i transients, and determined diastolic [Ca2+]i at 0, 3 and 24 h of Fas activation (Fig. 5). The effects of Fas activation were arrhythmias (as we previously reported [4]) occurring throughout the test period, and time-dependent changes in diastolic [Ca2+]i, firstly increasing and then decreasing towards control levels (Fig. 5). In agreement with our previous report (focusing on 3 h of Fas activation) [4], the arrhythmias occurring at 24 h of Fas activation were also blocked by U73122 [GenBank] and genistein (Fig. 5D and E). The persistence of the arrhythmias and the increased ANP and Cx43 mRNA levels (Fig. 5F) in the presence of a transient increase in diastolic [Ca2+]i will be addressed in the Discussion.


Figure 5
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 The effect of Fas activation on [Ca2+]i transients; (A) Control. (B, C) Representative [Ca2+]i transients from 2 different experiments in which Fas was activated for 3 h and 24 h. (D, E) Representative [Ca2+]i transients illustrating that addition of U73122 or genistein prevented the development of arrhythmias. (F) A summary of diastolic [Ca2+]i levels, ANP mRNA and Cx43 mRNA at 0, 3, 6 and 24 h of Fas activation. In panels (A)–(E), the paced beats are marked by the asterisks. In panels (B) and (C) the horizontal bar marks arrhythmogenic activity. In panel (B) the dotted line marks the diastolic [Ca2+]i level in the control experiment.

 
3.4 Fas-mediated hypertrophy is blocked by tyrosine kinases inhibition
Because of the clinical significance of Fas-mediated hypertrophy, identifying drug-responsive elements in this pathway which can serve as potential targets for attenuating Fas-based myocardial dysfunction is of prime importance. Based on our recent study showing that the omnipotent tyrosine kinases (TK) inhibitor genistein prevented Fas-mediated acute effects [6], we tested whether inhibiting TK will also attenuate Fas-mediated hypertrophy. Indeed, genistein blocked the hypertrophy, as evidenced by lack of change in ANP and Cx43 mRNA (Fig. 4A–C), and total Cx43 protein (Fig. 2B–C). Concomitantly, genistein increased the NP-Cx43 level, which is probably due to its inhibitory effect of TK phosphorylation site(s) located on the Cx43 molecule [16].

3.5 The effect of Fas activation on the nuclear factor of activated T cell (NFAT)
The NFAT family of transcription factors is dephosphorylated upon activation of the Ca2+/CaM-dependent phosphatase calcineurin, resulting in unmasking of their nuclear localization signals thereby permitting nuclear import [17]. The requirement for NFAT translocation in calcineurin-mediated hypertrophy was confirmed [18], as well as the fact that cardiac hypertrophy is suppressed by activated glycogen synthase-3β (GSK3β) which is upstream to NFAT and causes its nuclear export [19]. Based on previous studies showing that hypertrophic signals such as endothelin-1 [18] cause NFAT nuclear translocation (and hypertrophy), we tested whether NFAT is involved in Fas-mediated hypertrophy. As seen in Fig. 6 (panel B, left side), while in non-stimulated myocytes, NFATc1 (an isoform translocated by endothelin-1) displayed a predominant cytosolic localization (the stained nuclei are shown in the lower panel), Fas activation for 3 h caused intense NFATc1 nuclear translocation (panel B, right side), as evidenced by the Topro-stained nuclei (panel C, right side).


Figure 6
View larger version (89K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Fas activation causes NFAT nuclear translocation. Control (left side) or Fas activated (3 h) cultured NRVM (right side) were immunostained for subcellular localization of NFATc1. (A) Positive staining for the cardiomyocyte-specific sarcomeric {alpha}-actinin. (B) NFATc1 staining. In the control culture, NFATc1 is localized in the cytoplasm (left side), whereas in the presence of rFasL, NFATc1 translocated to the nucleus (right side, indicated by the white arrows). (C) Nuclei were stained with Topro in order to verify NFAT translocation. The arrows indicate NFATc1-labeled nuclei. Magnification x 40.

 
3.6 The interaction between the 1,4,5-IP3 and PI3K/AKT pathways
Earlier studies have shown that activation of the hypertrophic program by Fas activation, and β-adrenergic and Gq-coupled receptors leads to GSK3β inactivation, predominantly through the PI3K/Akt pathway [1,19–22]. To test whether the 1,4,5-IP3 and the PI3K/Akt pathways operate independently or in concert to cause Fas-mediated hypertrophy, we determined whether Fas activation can induce hypertrophy in the presence of LY294002, a PI3K/Akt inhibitor. In agreement with Schlüter et al. [23], LY294002 blocked Fas-mediated hypertrophy, indicated by ANP mRNA level (Fig. 7A). Importantly, LY294002 also prevented the development of Fas-mediated arrhythmias (Fig. 7B), and the diastolic [Ca2+]i rise commonly seen at 3 h Fas activation (4 different experiments, total of 20 myocytes measured). As will be discussed below, these findings suggest that the 1,4,5-IP3 and the PI3K/Akt pathways are functionally inter-connected.


Figure 7
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 The PI3K/Akt pathway blocker LY294002 inhibits Fas-mediated increase in ANP mRNA and in diastolic [Ca2+]i rise. (A) The upper panel depicts representative gels, and the lower panel depicts quantitative densitometric analysis from a control culture and from a culture treated for 24 h with rFasL+ LY294002 (n = 4). (B) Representative [Ca2+]i transients from a culture treated with rFasL+ LY294002 for 3 h. Please refer to Fig. 5 for comparison. Twenty µM of LY294002 were added to the culture medium 60 min before, and throughout exposure to rFasL.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
In support of our working hypothesis that the Fas->PLC->1,4,5-IP3->SR [Ca2+]i release pathway is responsible for Fas-mediated hypertrophy, we found that the hypertrophy was blocked by U73122 [GenBank] – a PLC inhibitor, and xestospongin C – a specific inhibitor of the 1,4,5-IP3-operated SR Ca2+ release channels. Importantly, we found that the 1,4,5-IP3 and the PI3K pathways are inter-connected, causing NFAT nuclear translocation.

4.1 The hypertrophy markers
Fas-mediated hypertrophy was associated with increased ANP mRNA, sarcomeric actin and nuclear area. At 24 h of Fas activation the first two markers increased by ~2–2.5-fold, resembling the effects of the hypertrophy inducers norepinephrine, and the renin-angiotensin system activation [24,25]. Nuclear area was increased by 50%, similar to the effect seen in cardiac hypertrophy caused in rats by aortocaval fistula or pulmonary artery constriction [12,13]. A novel effect of Fas activation is a marked increase in Cx43, the main gap junctional protein in ventricular myocytes [26]. At 24 h of Fas activation, Cx43 mRNA, total Cx43 and NP Cx43 increased by 80% (Fig. 1D), 70% (Fig. 2C) and 130% (Fig. 2D), respectively. These results resemble those reported by Saffitz's group who showed that in NRVM, angiotensin II and the membrane-permeant cAMP analogue db-cAMP (hypertrophy inducers) caused a 2–4 fold increase in Cx43 protein expression, which resulted from increased synthesis [26]. Since in our study both Cx43 mRNA and protein were augmented, it is likely that Fas activation also increased Cx43 synthesis. These changes in Cx43 concomitant with the hypertrophy can contribute under pathophysiological conditions to development of anatomical and functional substrates of arrhythmias, frequently seen in the hypertrophic myocardium [28]. In addition to the changes in the hypertrophy markers, Fas activation decreased the expression of three key proteins associated with [Ca2+]i handling: SERCA (24%, Fig. 3A), RyR2 (Fig. 3B, 18%) and IP3R type 3 (Fig. 3D, 25%). In agreement with our findings, in cultures treated for 24 h with angiotensin II, SERCA and RyR were decreased by 50% and 45% respectively [29]. Similarly, in a volume-overload model in rats, the decrease in SERCA mRNA was associated with a decrease in RyR mRNA and with a progressive left ventricular dysfunction [30]. Our IP3R findings differ from those of Go et al. who showed that in failing human hearts RyR mRNA decreased whereas IP3R mRNA increased [31]. While we did not determine how Fas activation affected RyR, SERCA and the IP3R, we propose that the combined decrease in these proteins may contribute to Fas-mediated cardiac dysfunction.

4.2 The involvement of the 1,4,5-IP3 pathway in Fas-induced hypertrophy
Our working hypothesis that 1,4,5-IP3 is a key participant in Fas-mediated hypertrophy is supported by the findings that both U73122 [GenBank] and xestospongin C prevented Fas-mediated hypertrophy. This is the first demonstration that the ubiquitous second messenger 1,4,5-IP3 is involved in ventricular hypertrophy. These findings are in close agreement with our previous work showing that 1,4,5-IP3 mediates the acute (3 h) effects of Fas activation, which included electrophysiological disturbances and diastolic [Ca2+]i rise [4–6]. Specifically, U73122 [GenBank] and xestospongin C prevented the prominent arrhythmias and the diastolic [Ca2+]i rise in NRVM cultures treated for 3 h with rFasL. Thus, increased 1,4,5-IP3 and the consequent SR Ca2+ release are responsible for both the acute and chronic effects of Fas activation. To further elucidate the signaling events (downstream of the 1,4,5-IP3-mediated SR Ca2+ release) leading to hypertrophy, we determined whether the well established calcineurin-mediated, NFAT nuclear translocation is involved in Fas-mediated hypertrophy. Indeed, as shown in Fig. 6, Fas activation caused nuclear translocation of NFATc1. The NFAT proteins contain an N-terminal regulatory domain that controls their nuclear import in response to calcineurin activation [32]. While in non-stimulated cells this regulatory domain is phosphorylated and masks the nuclear localization sequence, causing NFAT proteins to be sequestered in the cytoplasm, calcineurin activation causes NFAT nuclear localization which is sufficient to induce cardiac hypertrophy [33]. Our findings are in close agreement with previous reports, such as that by Van Rooij et al., showing that endothelin-1 caused NFATc1 nuclear translocation and hypertrophy [18].

In the present work we identified two serially-connected signaling elements involved in Fas-mediated hypertrophy: the 1,4,5-IP3 and the NFAT; the issue which should be addressed is the link between the two pathways. According to our proposed model (Fig. 8), the link is 1,4,5-IP3-mediated Ca2+ release, which activates calcineurin, subsequently triggering NFAT nuclear translocation and the consequent hypertrophy [32]. In this regard, it is noteworthy that diastolic [Ca2+]i peaked at 3 h of Fas activation, and then returned back to control levels at 24 h. At the same time, the extent of hypertrophy steadily increased during the 24 h period (Fig. 5). This lack of complete association between diastolic [Ca2+]i changes and the increase in the hypertrophic response does not necessarily negate the ‘calcium connection’ concept. Hence, we propose that the rise in diastolic [Ca2+]i (at 3 h) activated various intracellular targets such as calcineurin (which triggered hypertrophy), and, although later on, diastolic [Ca2+]i declined towards control levels, the hypertrophic response persisted and even gradually increased (compared to 3 h). This concept is supported by the occurrence of arrhythmias (which were blocked by U73122 [GenBank] and genistein, Fig. 5) at 24 h of Fas activation despite the decline of diastolic [Ca2+]i to control levels. Therefore, we propose that 1,4,5-IP3-mediated Ca2+ release is the trigger that initiates the hypertrophic program.


Figure 8
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 A proposed model of the signaling pathways underlying Fas-mediated hypertrophy: the functional inter-connection of the 1,4,5-IP3 and PI3K/Akt pathways (see Discussion for detailed description of the model). Solid arrows: common elements; dotted arrows: the PI3K/Akt–GSK3β pathway; interrupted arrows: the 1,4,5-IP3 pathway.

 
4.3 The interaction between the 1,4,5-IP3 and the PI3K/AKT pathway
An important finding was that both the 1,4,5-IP3 pathway (U73122 [GenBank] and xestospongin C) and the PI3K/Akt pathway (LY294002) inhibitors completely blocked Fas-mediated hypertrophy, indicating that these pathways share a common link (Fig. 8). These findings are in accord with a report showing that LY294002 blocked the increase in diastolic [Ca2+]i and the accompanying arrhythmias in neonatal cardiac myocytes as well as [Ca2+]i rise in human hepatoma-derived cell line HepG2 [34,35]. According to our model (Fig. 8), the first step in Fas-based hypertrophy is Fas engagement, which via TK phosphorylation leads to PI3K activation. This in turn results in PLC activation by PIP2 – a PI3K product, which anchors PLC to the cell membrane [34], leading to increased 1,4,5-IP3 production. Binding of 1,4,5-IP3 to its SR receptors causes Ca2+ release, a phenomenon demonstrated by us [4] and others [15,36]. Subsequently, the [Ca2+]i rise activates calcineurin which causes translocation to the nucleus of NFAT by its dephosphorylation, which is an essential step in the hypertrophic response [37,38]. Concurrently, Fas-mediated PI3K activation leads to inactivation of GSK3β (by its phosphorylation) [1], which is an established target of the PI3K/Akt signaling pathway [20]. Thus, inhibiting GSK3β – a negative regulator of cardiac hypertrophy, prevents the nuclear export of NFAT [19,37], which promotes hypertrophy. This scheme is consistent with a report by Antos et al. who showed that localization of NFATc1 to the nucleus in response to activated calcineurin was diminished in the presence of a GSK3β inhibitor [39]. Further, it was recently shown that an increase in PI3K activity can induce cardiac hypertrophy, whereas transgenic overexpression of a dominant–negative PI3K decreases cardiac mass [40]. Thus, by so doing, Fas-activated PI3K signaling can potentiate the Ca2+-activated calcineurin hypertrophic pathway by removing the inhibitory influence of GSK3β.

4.4 The involvement of TK phosphorylation in Fas-induced hypertrophy
In agreement with our previous work showing that TK inhibitors block the acute effects of Fas activation, genistein also prevented Fas-mediated hypertrophy. Since a discussion of the potential sites possibly affected by genistein has been presented recently [6], this issue is addressed here only in brief. In principle, genistein can affect the hypertrophic pathway at 3 different sites: (1) The Fas receptor. Although the TK associated with Fas activation have not been fully characterized, they can be inhibited by genistein [41,42] (2) PLC. A number of studies proposed a possible interaction between the TK Fyn and Lck, and PLC. Some studies claim to have identified specific phosphorylation sites on PLC{gamma} [43], while others suggest that PLC activation is related to TK phosphorylation, by forming a stable complex between these two [44,45]. (3) The 1,4,5-IP3 receptor and the PI3K pathway. Gulbins et al. reported that PI3K, shown to be stimulated by Fas activation, [1] is dependent upon TK phosphorylation [46]. Furthermore, it was recently reported that PI3K might act as a regulator of [Ca2+]i in cardiac cells [34]. PIP3 (a PI3K product synthesized from PIP2) anchors Tec – a tyrosine kinase and PLC{gamma} to the membrane, thus enabling 1,4,5-IP3 production from PIP2 [34]. Accordingly, Jayaraman et al. showed that: (1) during T lymphocyte stimulation the IP3R is physically associated with Fyn, and (2) in Fyn knockout mice, IP3R phosphorylation was reduced [47]. Collectively, these and other studies support the notion that TK phosphorylation participates, probably at multiple sites, in the Fas signaling pathway leading to hypertrophy.

In summary, we have identified the PLC->1,4,5 IP3->SR [Ca2+]i release pathway as a novel signaling cascade underlying Fas-induced hypertrophy in ventricular myocytes. Based on these findings, we suggest that modulators of the 1,4,5-IP3 pathway specifically targeted to cardiac muscle may have an important therapeutic value.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by Servier through the Center of Molecular Therapeutics at Columbia University–New York, the Israel Academy of Sciences, Minerva Foundation through the Bernard Katz Center for Cell Biophysics and the Rappaport Institute. We wish to acknowledge the help of Ofer Shenkar and Edith Suss from the Department of Interdisciplinary Equipment, and Dr. Noam Ziv from the Department of Anatomy and Cell Biology.


   Notes
 
Time for primary review 23 days


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

  1. Badorff C., Ruetten H., Mueller S., Stahmer M., Gehring D., Jung F., et al. Fas receptor signaling inhibits glycogen synthase kinase 3β and induces cardiac hypertrophy following pressure overload. J Clin Invest (2002) 109:373–381.[CrossRef][ISI][Medline]
  2. Binah O. Cytotoxic lymphocytes and cardiac electrophysiology. J Mol Cell Cardiol (2002) 34:1147–1161.[CrossRef][ISI][Medline]
  3. Wollert K.C., Heineke J., Westermann J., Ludde M., Fiedler B., Zierhut W., et al. The cardiac Fas (APO-1/CD95) receptor/Fas ligand system: relation to diastolic wall stress in volume-overload hypertrophy in vivo and activation of the transcription factor AP-1 in cardiac myocytes. Circulation (2000) 101:1172–1178.[Abstract/Free Full Text]
  4. Yaniv G., Shilkrut M., Lotan R., Berke G., Larisch S., Binah O. Hypoxia predisposes neonatal rat ventricular myocytes to apoptosis induced by activation of the Fas (CD95/Apo-1) receptor: Fas activation and apoptosis in hypoxic myocytes. Cardiovasc Res (2002) 54:611–623.[Abstract/Free Full Text]
  5. Felzen B., Shilkrut M., Less H., Sarapov I., Maor G., Coleman R., et al. Fas (CD95/Apo-1) mediated damage to ventricular myocytes induced by cytotoxic T lymphocytes from perforin-deficient mice: a major role for inositol 1,4,5-trisphosphate. Circ Res (1998) 82:438–450.[Abstract/Free Full Text]
  6. Shilkrut M., Yaniv G., Asleh R., Levy A.P., Larisch S., Binah O. Tyrosine kinases inhibitors block Fas-mediated deleterious effects in normoxic and hypoxic ventricular myocytes. J Mol Cell Cardiol (2003) 35:1229–1240.[CrossRef][ISI][Medline]
  7. Zeevi-Levin N., Barac Y.D., Reisner Y., Reiter I., Yaniv G., Meiry G., et al. Gap junctional remodeling by hypoxia in cultured neonatal rat ventricular myocytes. Cardiovasc Res (2005) 66:64–73.[Abstract/Free Full Text]
  8. Golomb E., Keiser H.R., Trachewsky D., Abassi Z.A., Friedman E. Rare renal transcription of the atrial natriuretic factor gene in rats. Demonstration by polymerase chain reaction. Am J Hypertens (1993) 6:867–872.[ISI][Medline]
  9. Dodge S.M., Beardslee M.A., Darrow B.J., Green K.G., Beyer E.C., Saffitz J.E. Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes. J Am Coll Cardiol (1998) 32:800–807.[Abstract/Free Full Text]
  10. Hossain M.Z., Jagdale A.B., Ao P., Kazlauskas A., Boynton A.L. Disruption of gap junctional communication by the platelet-derived growth factor is mediated via multiple signaling pathways. J Biol Chem (1999) 274:10489–10496.[Abstract/Free Full Text]
  11. Zhuang J., Yamada K.A., Saffitz J.E., Kleber A.G. Pulsatile stretch remodels cell-to-cell communication in cultured myocytes. Circ Res (2000) 87:316–322.[Abstract/Free Full Text]
  12. Rivello H.G., Meckert P.C., Vigliano C., Favaloro R., Laguens R.P. Cardiac myocyte nuclear size and ploidy status decrease after mechanical support. Cardiovasc Pathol (2001) 10:53–57.[CrossRef][ISI][Medline]
  13. Yan S.M., Finato N., Di Loreto C., Beltrami C.A. Nuclear size of myocardial cells in end-stage cardiomyopathies. Anal Quant Cytol Histol (1999) 21:174–180.[ISI][Medline]
  14. Anger M., Lompre A.M., Vallot O., Marotte F., Rappaport L., Samuel J.S. Cellular distribution of Ca2+ pumps and Ca2+ release channels in rat cardiac hypertrophy induced by aortic stenosis. Circulation (1998) 98:2477–2486.[Abstract/Free Full Text]
  15. Ibarra C., Estrada M., Carrasco L., Chiong M., Liberona J.L., Cardenas C., et al. Insulin-like growth factor-1 induces an inositol 1,4,5-trisphosphate-dependent increase in nuclear and cytosolic calcium in cultured rat cardiac myocytes. J Biol Chem (2004) 279:7554–7565.[Abstract/Free Full Text]
  16. Swenson K.I., Piwnica-Worms H., McNamee H., Paul D.L. Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication. Cell Regul (1990) 1:989–1002.[ISI][Medline]
  17. Aramburu J., Yaffe M.B., Pez R., Cantley L.C., Hogan P.G., Rao A. Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science (1999) 285:2129–2133.[Abstract/Free Full Text]
  18. Van Rooij E., Doevendans P.A., de Theije C.C., Babiker F.A., Molkentin J.D., De Windt L.J. Requirement of nuclear factor of activated T-cells in calcineurin-mediated cardiomyocyte hypertrophy. J Biol Chem (2002) 277:48617–48626.[Abstract/Free Full Text]
  19. Antos C.L., McKinsey T.A., Frey N., Kutschke W., McAnally J., Shelton J.M., et al. Activated glycogen synthase-3β suppresses cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A (2002) 99:907–912.[Abstract/Free Full Text]
  20. Morisco C., Zebrowski D., Condorelli G., Tsichlis P., Vatner S.F., Sadoshima J. The Akt-glycogen synthase kinase 3β pathway regulates transcription of atrial natriuretic factor induced by β-adrenergic receptor stimulation in cardiac myocytes. J Biol Chem (2000) 275:14466–14475.[Abstract/Free Full Text]
  21. Morisco C., Seta K., Hardt S.E., Lee Y., Vatner S.F., Sadoshima J. Glycogen synthase kinase 3β regulates GATA4 in cardiac myocytes. J Biol Chem (2001) 276:28586–28597.[Abstract/Free Full Text]
  22. Haq S., Choukroun G., Kang Z.B., Ranu H., Matsui T., Rosenzweig A., et al. Glycogen synthase kinase-3β is a negative regulator of cardiomyocyte hypertrophy. J Cell Biol (2000) 151:117–130.[Abstract/Free Full Text]
  23. Schlüter K.D., Goldberg Y., Taimor G., Schafer M., Piper H. Role of phosphatidylinositol 3-kinase activation in the hypertrophic growth of adult ventricular cardiomyocytes. Cardiovasc Res (1998) 40:174–181.[Abstract/Free Full Text]
  24. Tsoporis J.N., Marks A., Kahn H.J., Butany J.W., Liu P.P., O'Hanlon D., et al. Inhibition of norepinephrine-induced cardiac hypertrophy in S100β transgenic mice. J Clin Invest (1998) 102:1609–1616.[ISI][Medline]
  25. Ohta K., Kim S., Wanibuchi H., Ganten D., Iwao H. Contribution of local renin-angiotensin system to cardiac hypertrophy, phenotypic modulation, and remodeling in TGR(mRen2)27 transgenic rats. Circulation (1996) 94:785–791.[Abstract/Free Full Text]
  26. Lerner D.L., Beardslee M.A., Saffitz J.E. The role of altered intercellular coupling in arrhythmias induced by acute myocardial ischemia. Cardiovasc Res (2001) 50:263–269.[Free Full Text]
  27. McLenachan J.M., Henderson E., Morris K.I., Dargie H.J. Ventricular arrhythmias in patients with hypertensive left ventricular hypertrophy. N Engl J Med (1987) 317:782–792.
  28. Ju H., Scammell-La Fleur T., Dixon I.M.C. Altered mRNA abundance of calcium transport genes in cardiac myocytes induced by angiotensin II. J Mol Cell Cardiol (1996) 28:1119–1128.[CrossRef][ISI][Medline]
  29. Hashida H., Hamada M., Hiwada K. Serial changes in sarcoplasmic reticulum gene expression in volume-overloaded cardiac hypertrophy in the rat: effect of an angiotensin II receptor antagonist. Clin Sci (Lond) (1996) 96:387–395.
  30. Go L.O., Moschella M.C., Watras J., Handa K.K., Fyfe B.S., Marks A.R. Differential regulation of two types of intracellular calcium release channels during end-stage heart failure. J Clin Invest (1995) 95:888–894.[ISI][Medline]
  31. Bueno O.F., van Rooij E., Molkentin J.D., Doevendans P.A., De Windt L.J. Calcineurin and hypertrophic heart disease: novel insights and remaining questions. Cardiovasc Res (2002) 53:806–821.[Abstract/Free Full Text]
  32. Molkentin J.D., Lu J.R., Antos C.L., Markham B., Richardson J., Robbins J., et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell (1998) 93:215–228.[CrossRef][ISI][Medline]
  33. Bony C., Roche S., Shuichi U., Sasaki T., Crackower M.A., Penninger J., et al. A specific role of phosphatidylinositol 3-Kinase {gamma}: a regulation of autonomic Ca2+ oscillations in cardiac cells. J Cell Biol (2001) 152:717–728.[Abstract/Free Full Text]
  34. Rameh L.E., Rhee S.G., Spokes K., Kazlauskas A., Cantley L.C., Cantley L.G. Phosphoinositide 3-kinase regulates phospholipase C{gamma}-mediated calcium signaling. J Biol Chem (1998) 273:23750–23757.[Abstract/Free Full Text]
  35. Peppiatt C.M., Collins T.J., Mackenzie L., Conway S.J., Holmes A.B., Bootman M.D., et al. 2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels. Cell Calcium (2003) 34:97–108.[ISI][Medline]
  36. Crabtree G.R. Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell (1999) 96:611–614.[CrossRef][ISI][Medline]
  37. Ritter O., Hack S., Schuh K., Rothlein N., Perrot A., Osterziel K.J., et al. Calcineurin in human heart hypertrophy. Circulation (2002) 105:2265–2269.[Abstract/Free Full Text]
  38. Antos C.L., McKinsey T.A., Frey N., Kutschke W., McAnally J., Shelton J.M., et al. Activated glycogen synthase-3β suppresses cardiac hypertrophy in vivo. PNAS (2002) 99:907–912.[Abstract/Free Full Text]
  39. Shioi T., Kang P.M., Douglas P.S., Hampe J., Yballe C.M., Lawitts J., et al. The conserved phosphoinositide 3-kinase pathway determines heart size in mice. EMBO J (2000) 19:2537–2548.[CrossRef][ISI][Medline]
  40. Atkinson E.A., Ostergaard H., Kane K., Pinkoski M.J., Caputo A., Olszowy M.W., et al. A physical interaction between the cell death protein Fas and the tyrosine kinase p59fynT. J Biol Chem (1996) 271:5968–5971.[Abstract/Free Full Text]
  41. Gradl G., Grandison P., Lindridge E. The CD95 (Fas/APO-1) receptor in phosphorylated in vitro and in vivo and constitutively associates with several cellular proteins. Apoptosis (1996) 1:131–138.[CrossRef]
  42. Ozdener F., Dangelmaier C., Ashby B., Kunapuli S.P., Daniel J.L. Activation of phospholipase C{gamma}2 by tyrosine phosphorylation. Mol Pharmacol (2002) 62:672–679.[Abstract/Free Full Text]
  43. Ting A.T., Dick C.J., Schoon R.A., Karnitz L.M., Abraham R.T., Leibson P.J. Interaction between lck and syk family tyrosine kinases in Fc {gamma} receptor-initiated activation of natural killer cells. J Biol Chem (1995) 270:16415–16421.[Abstract/Free Full Text]
  44. Kinsey W.H., Shen S.S. Role of the fyn kinase in calcium release during fertilization of the sea urchin egg. Dev Biol (2000) 225:253–264.[CrossRef][ISI][Medline]
  45. Gulbins E., Hermisson M., Brenner B., Grassm H.U.C., Linderkamp O., Dichgans J., et al. Cellular stimulation via CD95 involves activation of phospho-inositide-3-kinase. Arch Eur J Physiol (1998) 435:546–554.[CrossRef]
  46. Jayaraman T., Ondrias K., Ondriasova E., Marks A.R. Regulation of the inositol 1,4,5-triphophate receptor by tyrosine phosphorylation. Science (1996) 272:1492–1494.[Abstract]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
L. Formisano, M. Saggese, A. Secondo, R. Sirabella, P. Vito, V. Valsecchi, P. Molinaro, G. Di Renzo, and L. Annunziato
The Two Isoforms of the Na+/Ca2+ Exchanger, NCX1 and NCX3, Constitute Novel Additional Targets for the Prosurvival Action of Akt/Protein Kinase B Pathway
Mol. Pharmacol., March 1, 2008; 73(3): 727 - 737.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. Padmasekar, R. Nandigama, M. Wartenberg, K.-D. Schluter, and H. Sauer
The acute phase protein {alpha}2-macroglobulin induces rat ventricular cardiomyocyte hypertrophy via ERK1,2 and PI3-kinase/Akt pathways*
Cardiovasc Res, July 1, 2007; 75(1): 118 - 128.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Barac, Y. D.
Right arrow Articles by Binah, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barac, Y. D.
Right arrow Articles by Binah, O.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?