Calmodulin Kinases II and IV and Calcineurin Are Involved in Leukemia Inhibitory Factor–Induced Cardiac Hypertrophy in Rats
Abstract—We recently reported that leukemia inhibitory factor (LIF) enhances Ca2+]i through an increase in L-type Ca2+ current (ICa,L) in adult cardiomyocytes. The aim of this study was to investigate whether LIF activates Ca2+-dependent signaling molecules, such as calcineurin and calmodulin kinases II and IV (CaMKII and CaMKIV), and, if so, whether these Ca2+-mediated signaling events contribute to LIF-mediated cardiac hypertrophy. We first confirmed that LIF increased ICa,L and [Ca2+]i in primary cultured rat neonatal cardiomyocytes. Calcineurin, CaMKII, and CaMKIV activities increased at 2 minutes and peaked by 1.6-, 2.2-, and 2.2-fold, respectively, at 15 minutes. Nicardipine or verapamil fully inhibited these activities. Autophosphorylation of CaMKII was also observed to parallel the timing of CaMKII activity, and this phosphorylation was blocked by nicardipine, verapamil, or EGTA. LIF treatment led to a 3-fold increase in nuclear factor of activated T cell–luciferase activity. To confirm that inositol triphosphate (IP3)-induced Ca2+ release from sarcoplasmic reticulum was not involved in this process, IP3 content and phosphorylation of phospholipase Cγ were investigated. LIF did not increase IP3 content or phosphorylate phospholipase Cγ. KN62 (an inhibitor of CaMKII and CaMKIV) attenuated c-fos, brain natriuretic peptide, α-skeletal actin, and atrial natriuretic peptide expression. KN62 suppressed the LIF-induced increase in [3H]phenylalanine uptake and cell size. Cyclosporin A and FK506 slightly attenuated brain natriuretic peptide but did not affect c-fos or atrial natriuretic peptide expression. Cyclosporin A significantly reduced the LIF-induced increase in [3H]phenylalanine uptake. These findings indicated that LIF activated CaMKII, CaMKIV, and calcineurin through an increase in ICa,L and [Ca2+]i and that CaMKII, CaMKIV, and calcineurin are critically involved in LIF-induced cardiac hypertrophy.
Leukemia inhibitory factor (LIF), a member of the interleukin-6 family of cytokines, has a potent hypertrophic effect on cardiomyocytes. However, the underlying molecular mechanisms that couple hypertrophic signals initiated at the cell membrane receptor to the reprogramming of cardiomyocyte gene expression remain poorly understood. We and others have demonstrated that the Janus kinase and signal transducers and activators of transcription pathway, mitogen-activated protein kinase (MAPK) pathway, and phosphatidyl inositol 3 (PI3) kinase pathway were present downstream of gp130 in cardiomyocytes.1 2 3 4 We recently reported that LIF enhanced L-type Ca2+ current by 42% and [Ca2+]i transient by 63% in cardiomyocytes.5 Interestingly, LIF-induced increases in L-type Ca2+ current and [Ca2+]i transient took a unique time course, which gradually increased from 2 minutes and peaked at 15 minutes. Moreover, although the precise mechanism remains unknown, these increases were not mediated by protein kinase A (PKA) or protein kinase C (PKC) pathways.
There is a growing body of evidence to suggest that Ca2+ signaling plays an important role in the pathogenesis of cardiac hypertrophy and heart failure. Recently, the Ca2+/calmodulin–dependent protein phosphatase calcineurin has attracted attention as a new signal transducer of hypertrophic stimuli in vitro and in vivo. Calcineurin dephosphorylates the nuclear factor of activated T cells (NFAT)-3 transcription factor, which is then translocated into the nucleus. Recently, Molkentin et al6 reported that NFAT-3, GATA-4, and calcineurin synergistically activate a marker gene of cardiac hypertrophy. In cultured cardiomyocytes, cyclosporin A (CsA), an inhibitor of calcineurin activity, inhibited angiotensin II–induced and phenylephrine-induced cardiac hypertrophy. Cardiac overexpression of the constitutively active form of calcineurin and NFAT-3 caused marked hypertrophy and heart failure in transgenic mice, which was blocked by CsA. Sussman et al7 reported that CsA and FK506 prevent cardiac hypertrophy attributable to genetic perturbations in contractile proteins in hypertrophic cardiomyopathy model mice. However, they also reported that these blockers did not block hypertrophy attributable to overexpression of the constitutively active form of retinoic acid receptor.7 Moreover, the effects of CsA and FK506 remain controversial in pressure-overloaded cardiac hypertrophy models.8 9 10 11 12 Thus, it remains to be determined whether the calcineurin pathway is universally implicated in cardiac hypertrophy in response to all kinds of hypertrophic stimuli.
Calmodulin-dependent kinase II (CaMKII) is an intracellular enzyme that was discovered several years ago in the brain. CaMKII is known to have several isoforms (α to δ), and the gene products are additionally divided into splicing variants.13 All α and β isoforms are found only in the brain, with the exception of the γ and δ isoforms, which have been detected in rat heart.14 15 CaMKII has also been implicated in the transduction of hypertrophic signals in cultured cardiomyocytes.16 17 18 Expression of the δB isoform of CaMKII specifically activates atrial natriuretic peptide (ANP) expression without inducing cellular hypertrophy.19
Recently, Lu et al20 reported that hypertrophic growth of cardiomyocytes in response to phenylephrine and serum is accompanied by muscle enhancer factor 2 (MEF2) activation through a posttranslational mechanism mediated by CaMKI, CaMKIV, and p38 MAPK, and these CaMKs stimulate MEF2 activity by dissociating class II histone deacetylase from the DNA-binding domain. They concluded that these CaMKs and p38 MAPK–dependent activation of MEF2 were endpoints for the hypertrophic stimuli in cardiomyocytes.
These findings suggested that calcium-regulated signaling molecules play a central regulatory role in coordinating the activities of multiple hypertrophic signaling pathways. On the basis of these findings, we investigated whether LIF activates the calcineurin, CaMKII, and CaMKIV pathways via an increase in ICa,L and [Ca2+]i transient in rat cardiomyocytes, and if so, whether this calcium-regulated signaling is involved in the hypertrophic response induced by LIF.
Materials and Methods
Primary cultures of cardiomyocytes were prepared from the ventricles of 1-day-old neonatal Wistar rats as described.1 Cells were stimulated with LIF 1000 U/mL.
Perforated Patch-Clamp Recording
Perforated patch-clamp recording was performed using gramicidin, as described in the online data supplement.
Measurement of the [Ca2+]i Transient
The [Ca2+]i transient was monitored by use of the fluorescent calcium indicator Fluo-4, as described in the online data supplement.
Measurement of CaMKII and CaMKIV Activity
Cells were collected in an assay dilution buffer and lysed by sonication. Anti-CaMKII (Santa Cruz) or CaMKIV (Transduction Laboratory) antibody and protein A Sepharose beads were added to the lysates and incubated overnight at 4°C. CaMKII activity was assayed with CaMKII assay kits (Upstate Biotechnology Inc) using a peptide substrate (KKALRRQETVDAL) according to the manufacturer’s instructions. CaMKIV activity was assayed using a peptide substrate (KSDGGVKKRKSSSS).
Measurement of Calcineurin Activity
Calcineurin activity was determined by the protocol of Shibasaki and McKeon,21 with minor modifications. Cells were lysed in 400 μL of lysis buffer and freeze-thawed. After removing of cell debris, lysate was incubated in the assay buffer and [32P]-labeled RII peptide for 30 minutes at 30°C. The released 32P-phosphate in 500 μL of supernatant was determined by Cherenkov’s method. Because the phosphatase activity using this assay buffer represents the mixed activity of PP2B and PP2C, we obtained the PP2B (calcineurin) activity by subtracting PP2C activity from the mixture activity. The PP2C activity was measured by a similar method using the same buffer by chelating Ca2+ with 5 mmol/L EGTA.
Immunoprecipitation (IP)-Western Blot Analysis
Antibody to phospholipase C (PLC)-γ1 was purchased from Santa Cruz Biolabs. Monoclonal antibody to phosphotyrosine (4G10) was purchased from Chemicon International Inc. IP Western blot was performed as described.1
Transfection and Luciferase Assay
Transient transfection was performed using Effectene transfection reagent (Qiagen). Within 24 hours after plating, cells were incubated with a transfection mixture with 0.24 μg of NFAT-luciferase reporter plasmids, 0.08 μg of plasmid encoding the constitutively active form of MEKK1 (Stratagene), and 0.08 μg of pRL-SV40 (Promega) as an internal control plasmid. Total cell lysates were collected 6 hour after LIF stimulation, and luciferase activity was measured by Dual Luciferase reporter assay system (Promega).
Radioimmunoassay of Inositol Triphosphate
Radioimmunoassay kit for inositol triphosphate (IP3) was purchased from Amersham Pharmacia. IP3 content was measured according to the manufacturer’s instructions.
Incorporation of [3H]Phenylalanine
The effects of LIF on [3H]phenylalanine uptake were determined as described.1 After 24 hours of serum depletion, cells were pretreated with or without KN62 or CsA, and then the cells were stimulated with LIF. Each data point was the mean of 5 separate experiments.
Immunofluorescence Microscopy and Cell Sizing Protocol
Cells grown on glass coverslips were permeabilized in cold methanol (1:1, −20°C) for 10 minutes and air dried. Immunofluorescence staining and measurement of the size (cell area, perimeter) were performed as described previously.2
RNA Extraction and Northern Blot Analysis
Northern blots were performed as described.2 Rat ANP and brain natriuretic peptide (BNP) cDNA was obtained by reverse transcriptase–polymerase chain reaction from the heart RNA and cloned into the pCR II plasmid. Rat α-skeletal actin cDNA was provided by Hiroshi Ito (Tokyo Medical and Dental University, Tokyo, Japan). Rat GAPDH cDNA was used as an internal control.
Values are presented as mean±SD. Statistical significance among mean values was evaluated with an ANOVA. Student’s t test was used when 2 values were compared. Differences were considered to be significant when P<0.05.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
LIF Increased ICa,L and [Ca2+]i in Neonatal Rat Cardiomyocytes
Because our previous study used cultured adult rat cardiomyocytes, we first confirmed whether LIF also increases ICa,L in neonatal rat cardiomyocytes using perforated patch-clamp techniques. Figure 1A⇓ (inset) shows the representative tracings of ICa,L of the control cell and the cells stimulated with LIF for 15 minutes, and Figure 1A⇓ shows the time course of the effect of LIF on ICa,L in the neonatal rat ventricular myocyte. ICa,L started to increase as early as 2 minutes after application of LIF and reached a maximal level at 10 to 15 minutes. The maximal increase in ICa,L induced by LIF was 33.3±5.6% (n=4).
We next measured the effect of LIF on [Ca2+]i in neonatal rat ventricular cardiomyocytes. Figure 1B⇑ shows a representative tracing of the Fluo-4 fluorescence at the control levels and 15 minutes after LIF stimulation, which confirms that LIF increased the [Ca2+]i in cardiomyocytes. Figure 1C⇑ shows the time course of the percent changes in amplitude of the [Ca2+]i after LIF stimulation. The amplitude of the [Ca2+]i increased significantly with exposure to LIF. These findings indicated that LIF also increases ICa,L and [Ca2+]i in neonatal rat cardiomyocytes.
LIF Activates the CaMKII and CaMKIV Pathways in Cardiomyocytes
To determine whether the LIF-induced increase in [Ca2+] transient can augment CaMKII and CaMKIV activities in cardiomyocytes, we initially measured the CaMKII activity in LIF-stimulated cardiomyocytes (Figure 2A⇓). CaMKII activity increased from 2 minutes, peaked by 2.2-fold at 15 minutes, and decreased thereafter.
Once CaMKII is activated by the Ca2+/calmodulin complex, CaMKII is known to autophosphorylate and does not need calmodulin for its activity. To confirm that LIF really activates the CaMKII, we performed IP Western blot analysis to detect the phosphorylation of CaMKII with anti-phosphoCaMKII antibody (Figure 2B⇑). LIF augmented phosphorylation of CaMKII from 2 minutes, peaked at 15 minutes, and then decreased thereafter.
Next, we measured the CaMKIV activity in LIF-stimulated cells (Figure 3⇓). LIF increased CaMKIV from 2 minutes and peaked by 2.2-fold at 15 minutes. These finding indicated that LIF activated CaMKII and CaMKIV in cardiomyocytes.
LIF Activates Calcineurin and NFAT-3 Luciferase Activity in Cardiomyocytes
We measured the calcineurin activity in LIF-stimulated cardiomyocytes (Figure 4⇓). The calcineurin activity increased at 2 minutes, peaked by 1.6-fold at 15 minutes, and decreased thereafter. The time course of the activation of calcineurin corresponded to that of CaMKII and CaMKIV. It is well known that cytosolic NFAT is dephosphorylated by calcineurin and is translocated to the nucleus. NFAT proteins form cooperative DNA-binding complexes with dimers of the activator protein-1 (AP-1) (Fos/Jun) family at composite NFAT:AP-1 DNA elements that have been identified in multiple NFAT-regulated genes, including interleukin-2. To additionally confirm whether LIF activates calcineurin in cardiomyocytes, we examined NFAT transcriptional activity using an NFAT:AP-1 luciferase plasmid derived from interleukin-2 gene promoter. To investigate the activation of the Ca2+/calcineurin–NFAT pathway elicited by LIF independent of AP-1 activation, we cotransfected an active form of MEKK1, which actives c-Jun N-terminal kinase cascades, to constitutively activate AP-1 activity. In unstimulated cells, FK506-inhibitable NFAT-luciferase activity was observed. LIF increased this NFAT-luciferase activity by 3-fold, and this increase was completely inhibited by CsA (Figure 4B⇓). These results suggested that LIF can activate calcineurin, which is in turn sufficient for dephosphorylation and activation of NFAT in cardiomyocytes.
L-Type Ca2+ Channel Mediates the LIF-Induced Activation of CaMKII, CaMKIV, and Calcineurin
To confirm that the LIF-induced increase in CaMKII, CaMKIV, and calcineurin activities were caused by Ca2+-induced Ca2+ release via an L-type Ca2+ current, we preincubated the cells with 10–6 mol/L nicardipine or 10–6 mol/L verapamil, stimulated with LIF, and measured CaMKII CaMKIV and calcineurin activities (Figures 5A⇓ through 5C). KN62 (CaMKII and CaMKIV inhibitor) and CsA were used as a control. KN62 and CsA inhibited the LIF-induced increase in CaMKII and CaMKIV and CN activities, respectively. LIF-induced increase in CaMKII, CaMKIV, and calcineurin activity was almost completely inhibited by nicardipine or verapamil. These results were reproducible in 3 separate experiments. We also observed the effect of nicardipine, verapamil, or EGTA (1 mmol/L) on LIF-induced autophosphorylation of CaMKII. Nicardipine, verapamil, and EGTA fully inhibited this phosphorylation (Figure 5C⇓). We additionally examined whether preincubation of nicardipine, verapamil, and EGTA can inhibit LIF-induced autophosphorylation of CaMKII (Figure 5D⇓). These reagents strongly inhibited the autophosphorylation of CaMKII. These findings indicated that the L-type Ca2+ currents mediate LIF-induced activation of CaMKII, CaMKIV, and calcineurin.
Next, we examined whether sarcoplasmic reticulum Ca2+ store was involved in the LIF-induced activation of CaMKIV in cardiomyocytes. We preincubated the cells with thapsigargin (1 μmol/L) for 8 hours, stimulated with LIF, and measured CaMKIV activity. Thapsigargin completely inhibited LIF-induced increase in CaMKIV activity, and its level was lower than that of the control (data not shown), indicating that the sarcoplasmic reticulum Ca2+ store was involved in LIF-induced activation of CaMKIV. Taken together, these findings indicated that Ca2+-induced Ca2+ release might be involved in activation of these kinases and phosphatase.
Nicardipine and Verapamil Inhibited LIF-Induced Increase in [3H]Phenylalanine Uptake
To confirm that a LIF-induced increase in L-type Ca2+ current was involved in LIF-induced cardiac hypertrophy, we performed [3H]phenylalanine uptake experiments to test whether nicardipine or verapamil can inhibit LIF-induced hypertrophy (Figure 5E⇑). The LIF-induced increase in [3H]phenylalanine uptake was significantly attenuated by preincubation of nicardipine and verapamil.
LIF-Induced Activation of CaMKII, CaMKIV, and Calcineurin Was Independent of PLC/IP3 Pathway
To date, there is no evidence to suggest that the gp130 receptor mediates its signal through the PLC/IP3 pathway. To confirm that LIF-induced activation of Ca2+-dependent pathway was not mediated by the PLC/IP3 pathway, we performed IP Western blot analysis to detect the tyrosine phosphorylation of PLC-γ1. Platelet-derived growth factor (PDGF) was used as a positive control. PDGF strongly phosphorylated PLC-γ1, but LIF did not phosphorylate PLC-γ1 (Figure 6A⇓). We also confirmed that LIF did not increase IP3 content in cardiomyocytes (Figure 6B⇓). Together, these results indicated that LIF-induced activation of CaMKII, CaMKIV, and calcineurin was mediated not by the PLC/IP3 pathway but by L-type Ca2+ current-induced Ca2+ release.
Role of CaMKII and CaMKIV in LIF-Induced Cardiac Hypertrophy
To determine whether LIF-induced activation of CaMKII and CaMKIV plays an important role in mediating cardiac hypertrophy, we investigated the effect of KN62 on the LIF-induced increase in [3H]phenylalanine uptake and cell size and induction of hypertrophic marker gene expression. LIF induced c-fos (30 minutes), BNP (1 hour), skeletal α-actin (24 hours), and ANP (24 hours). KN62 slightly decreased LIF-induced expression of c-fos, BNP, α-skeletal actin, and ANP (Figure 7A⇓). LIF caused a 43% increase in [3H]phenylalanine uptake compared with control. KN-62 dose-dependently inhibited the LIF-induced [3H]phenylalanine uptake by 78.5%, whereas KN62 at this concentration had no effect on the baseline [3H]phenylalanine uptake (Figure 7B⇓). LIF caused a 48% and 27% increase in cell area and perimeter compared with the control cells, respectively. KN-62 (1 μmol/L) significantly decreased the LIF-induced increase in cell area and perimeter by 46% and 57%, respectively, whereas KN-62 alone did not significantly attenuate the cell size (Figures 7C⇓ and 7D⇓). The results were fully reproducible and indicated that the LIF-induced increase in cardiac hypertrophy was partially mediated by CaMKII or CaMKIV.
Role of Calcineurin in LIF-Induced Cardiac Hypertrophy
To investigate the role of calcineurin in LIF-induced cardiac hypertrophy, we also performed the same assay using FK506 or CsA. Northern blotting revealed that both FK506 and CsA did not affect the LIF-induced induction of c-fos, BNP (1 hour), or ANP (Figure 8A⇓) expression. Because a previous report found that the promoter sequences of BNP contained NFAT-binding sequences and that calcineurin plays an important role in BNP expression, we additionally analyzed the effect of CsA on LIF-induced induction of the BNP genes. CsA did not affect LIF-induced induction of the BNP gene at 30 minutes; however, it attenuated BNP expression from 2 to 24 hours (Figure 8B⇓). CsA inhibited the LIF-induced increase in [3H]phenylalanine uptake by 37% at a concentration of 50 ng/mL (Figure 8C⇓). CsA also decreased the LIF-induced increase in cell area and perimeter of the cells by 39% and 46%, respectively, at a concentration of 50 ng/mL (Figures 8D⇓ and 8E⇓). These findings indicated that calcineurin might be involved in LIF-induced cardiac hypertrophy.
In cardiomyocytes, extracellular Ca2+ entering through L-type Ca2+ channels during depolarization triggers additional elevation of intracellular Ca2+ by stimulating Ca2+ release from the sarcoplasmic reticulum (Ca2+-induced Ca2+ release) and induces muscle contraction. β-Adrenergic stimulation of cardiomyocytes increases L-type Ca2+ current (ICa,L)and augments muscle contraction. In neuronal cells, transient changes in the concentration of intracellular Ca2+ attributable to membrane depolarization and activation of voltage-dependent L-type Ca2+ channels can lead to various physiological responses, including neurotransmitter release, modulation of synaptic transmission, and changes in gene expression.22 However, it is not known whether increased intracellular Ca2+ concentration through L-type Ca2+ channels can cause Ca2+-sensitive signal transduction, leading to gene expression and protein synthesis in cardiomyocytes. Stimulation with a Ca2+-channel agonist induces cardiac hypertrophy, and, conversely, blockade of ICa,L prevents cardiac hypertrophy, suggested that Ca2+ entry through L-type Ca2+ channels plays a critical role in cardiac hypertrophy.23
Several lines of evidence suggest that the activity of voltage-dependent L-type Ca2+ channels is regulated by various second messengers, including PKA, PKC, CaMKII,24 and tyrosine kinases. In a previous study, we reported that LIF augmented [Ca2+]i via an increase in L-type Ca2+ in adult rat cardiomyocytes. It is worth noting that an LIF-induced increase in [Ca2+]i and L-type Ca2+ was observed from 2 minutes and peaked at 15 minutes and was therefore a slower activation process than that mediated by PKA or PKC. We confirmed that the PKA, PKC, and CaMKII pathways were not involved in this activation, which suggested that this slow increase was caused by another mechanism. In the present study, we demonstrated that LIF augmented ICa,L and [Ca2+]i in neonatal rat cardiomyocytes. The time course of the activation of CaMKII, CaMKIV, and calcineurin was slow and in accordance with that of the increase in [Ca2+]i and L-type Ca2+. We also confirmed that the LIF-induced activation of CaMKII, CaMKIV, and calcineurin was completely abrogated by blocking of L-type Ca2+ with nicardipine or verapamil, and that LIF does not increase IP3 in cardiomyocytes. IP3-induced Ca2+ release plays an important role in intracellular Ca2+ signaling in a wide variety of cell types.25 Activation of PLCβ is mediated by the α or βγ subunit of the heterotrimeric G proteins, whereas the γ-type enzymes are activated by phosphorylation with receptor- or nonreceptor-type protein tyrosine kinases, such as the Src family proteins. The present study also demonstrated that LIF did not phosphorylate PLCγ in cardiomyocytes. These findings suggested that IP3-induced Ca2+ release was not involved in augmentation of [Ca2+]i transient.
A previous report implicated calmodulin and CaMKII kinases in transduction of hypertrophic signals in cultured cardiomyocytes. Gruver et al26 reported that targeted developmental overexpression of calmodulin induces proliferative and hypertrophic growth of cardiomyocytes in transgenic mice. Ramirez et al19 showed that the CaMKII inhibitors M7 and KN93 prevent myocardial hypertrophy and upregulation of ANP in response to phenylephrine, which indicates that CaMKII activation is an essential step in phenylephrine-mediated hypertrophy.22 Abraham et al27 reported that angiotensin II, vasopressin, and PDGF increased CaMKII activity by 4.6-, 2-, and 1.7-fold, respectively, in rat vascular smooth muscle cells. In this study, we determined that CaMKII was activated by LIF via an L-type Ca2+ current and that inhibition of CaMKII by KN62 partially prevented LIF-induced protein synthesis, induction of immediate early gene, and upregulation of fetal gene expression. These findings indicated that CaMKII is critically involved in LIF-induced cardiac hypertrophy.
Lu et al20 recently suggested the new idea that MEF2 is an endpoint for the hypertrophic stimuli in cardiomyocytes and that MEF2 mediates synergistic transcriptional response to the CaMKs and p38 MAPK signaling pathways by signal-dependent dissociation of histone deacetylase. They proposed a model for the regulation of MEF2 by CaMKI and CaMKIV. Hypertrophic signals that activate CaMKI, CaMKIV, and p38 MAPK lead to MEF2 activation by a different mechanism. Some stimuli, such as phenylephrine, may activate both pathways. Association of histone deacetylase 4/5 with the DNA-binding domain of MEF2 represses MEF transcriptional activity. CaMKI and CaMKIV activates MEF2 by preventing association of histone deacetylase 4/5 with MEF2. p38 MAPK stimulates MEF2 by direct phosphorylation of the transcription activation domain. Together, the CaMKs and p38 MAPK pathways synergize to activate MEF2. The present study demonstrated that LIF activated CaMKIV in cardiomyocytes and KN62 inhibited LIF-induced cardiac hypertrophy. Because KN62 can inhibit both CaMKII and CaMKIV, the inhibitory effect of KN62 might be an additive effect of the inhibition of the 2 kinases. We have already confirmed that LIF activated p38 MAPK in cardiomyocytes (data not shown). Our findings indicated that the CaMK–p38 MAPK–MEF2 theory might also play an important role in gp130-mediated cardiac hypertrophy, although this theory is still controversial.
Since Molkentin et al6 reported that this calcineurin-NFAT3 pathway was critical to the induction of cardiac hypertrophy, several studies of this pathway have been reported. Sussman et al7 reported that inhibition of this pathway by CsA or FK506 could prevent pressure overload–induced in vivo cardiac hypertrophy produced by aortic banding. Luo et al,9 Ding et al,10 and Zhang et al11 independently reported that these calcineurin inhibitors did not inhibit pressure overload–induced cardiac hypertrophy, and Meguro et al12 reported that CsA attenuates pressure-overload hypertrophy in mice while enhancing susceptibility to decompensation and heart failure. Moreover, Force et al28 reported that human transplanted heart shows long-term cardiac hypertrophy despite the use of these compounds. CsA and FK506 are well known to cause hypertension in vivo and have several side effects. Thus, it is difficult to conclude that inhibition of this pathway in vivo is sufficient to prevent cardiac hypertrophy. In the present study, we have shown that LIF activated calcineurin in cardiomyocytes and that inhibition of this pathway by CsA or FK506 partially blocked protein synthesis and slightly attenuated BNP expression but did not affect c-fos and ANP expression. On the basis of these findings, we suspect that the calcineurin pathway might well be involved in LIF-induced cardiac hypertrophy.
In conclusion, we have demonstrated for the first time, to our knowledge, that LIF activates CaMKII, CaMKIV, and calcineurin by an increase in ICa,L. in cardiomyocytes and that CaMKII, CaMKIV, and calcineurin contributed significantly to LIF-induced cardiac hypertrophy in vitro. The importance of the calcineurin/NFAT3 and CaMKIV-MEF2 pathways in cardiac hypertrophy is still controversial, and additional investigation is needed to clarify their role.
This study was supported in part by research grants from Research for the Future Program from the Japan Society for the Promotion of Science (JSPS-RFTF97I00201) and by research grants from the Ministry of Education, Science and Culture, Japan and Health Science Research Grants for Advanced Medical Technology from the Ministry of Welfare, Japan. The authors greatly appreciate the very useful discussions with Professor Shigeo Koyasu and Satoshi Matsuda. The authors also wish to acknowledge Kio Nakamaru for technical assistance.
↵1 Both authors contributed equally to this study.
- Received May 31, 2000.
- Revision received September 13, 2000.
- Accepted September 13, 2000.
- © 2000 American Heart Association, Inc.
Kodama H, Fukuda K, Pan J, Makino S, Baba A, Hori S, Ogawa S. Leukemia inhibitory factor, a potent cardiac hypertrophic cytokine, activates JAK/STAT pathway in rat cardiomyocytes. Circ Res. 1997;81:656–663.
Kunisada K, Hirota H, Fujio Y, Matsui H, Tani Y, Yamauchi-Takihara K, Kishimoto T. Activation of JAK-STAT and MAP kinases by leukemia inhibitory factor through gp130 in cardiac myocytes. Circulation. 1996;94:2626–2632.
Kodama H, Fukuda K, Pan J, Sano M, Takahashi T, Kato T, Makino S, Manabe T, Murata M, Ogawa S. Significance of ERK cascade compared with JAK/STAT and PI3-K pathway in gp130-mediated cardiac hypertrophy. Am J Physiol Heart Circ Physiol. 2000;279:H1635–H1644.
Oh H, Fujio Y, Kunisada K, Hirota H, Matsui H, Kishimoto T, Yamauchi-Takihara K. Activation of phosphatidylinositol 3-kinase through gp130 induces protein kinase B and p70 S6 kinase phosphorylation in cardiac myocytes. J Biol Chem. 1998;273:9703–9710.
Sussman MA, Lim HW, Gude N, Taigen T, Olson EN, Robbins J, Colbert MC, Gualberto A, Wieczorek DF, Molkentin JD. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science. 1998;281:1690–1693.
Mende U, Kagen A, Cohen A, Aramburu J, Schoen FJ, Neer EJ. Transient cardiac expression of constitutively active Gαq leads to hypertrophy and dilated cardiomyopathy by calcineurin-dependent and independent pathways. Proc Natl Acad Sci U S A. 1998;95:13893–13898.
Ding B, Price RL, Borg TK, Weinberg EO, Halloran PF, Lorell BH. Pressure overload induces severe hypertrophy in mice treated with cyclosporine, an inhibitor of calcineurin. Circ Res. 1999;84:729–734.
Zhang W, Kowal RC, Rusnak F, Sikkink RA, Olson EN, Victor RG. Failure of calcineurin inhibitors to prevent pressure-overload left ventricular hypertrophy in rats. Circ Res. 1999;84:722–728.
Meguro T, Hong C, Asai K, Takagi G, McKinsey TA, Olson EN, Vatner SF. Cyclosporine attenuates pressure-overload hypertrophy in mice while enhancing susceptibility to decompensation and heart failure. Circ Res. 1999;84:735–740.
Edman CF, Schulman H. Identification and characterization of δB-CaM kinase and δC-CaM kinase from rat heart, two new multifunctional Ca2+/calmodulin-dependent protein kinase isoforms. Biochim Biophys Acta. 1994;1221:89–101.
Mayer P, Mohlig M, Schatz H, Pfeiffer A. Additional isoforms of multifunctional calcium/calmodulin-dependent protein kinase II in rat heart tissue. Biochem J. 1994;298:757–758.
Sei CA, Irons CE, Sprenkle AB, McDonough PM, Brown JH, Glembotski CC. The α-adrenergic stimulation of atrial natriuretic factor expression in cardiac myocytes requires calcium influx, protein kinase C, and calmodulin-regulated pathways. J Biol Chem. 1991;266:15910–15916.
McDonough PM, Glembotski CC. Induction of atrial natriuretic factor and myosin light chain-2 gene expression in cultured ventricular myocytes by electrical stimulation of contraction. J Biol Chem. 1992;267:11665–11668.
Samarel AM, Spragia ML, Maloney V, Kamal SA, Engelmann GL Contractile arrest accelerates myosin heavy chain degradation in neonatal rat heart cells. Am J Physiol. 1992;263:C642–652.
Ramirez MT, Zhao XL, Schulman H, Brown JH. The Nuclear B Isoform of Ca2+/calmodulin-dependent protein kinase II regulates atrial natriuretic factor gene expression in ventricular myocytes. J Biol Chem. 1997;272:31203–31208.
Lu J, McKinsey TA, Nicol RL, Olson EN. Signal-dependent activation of the MEF2 transcription factor from histone deacetylase. Proc Natl Acad Sci U S A. 2000;97:4070–4075.
Shibasaki F, McKeon FJ. Calcineurin functions in Ca2+-activated cell death in mammalian cells. Cell Biol. 1995;131:735–743.
Xiao RP, Cheng H, Lederer WJ, Suzuki T, Lakatta EG. Dual regulation of Ca2+/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx. Proc Natl Acad Sci U S A. 1994;91:9659–9663.
Keizer J, Li YX, Stojilkovic S, Rinzel J. IP3-induced Ca2+ excitability of the endoplasmic reticulum. Mol Biol Cell. 1995;6:945–951.
Abraham ST, Benscoter HA, Schworer CM, Singer HA. A role for Ca2+/calmodulin-dependent protein kinase II in the mitogen-activated protein kinase signaling cascade of cultured rat aortic vascular smooth muscle cells. Circ Res. 1997;81:575–584.
Force T, Rosenzweig A, Choukroun G, Hajjar R. Calcineurin inhibitors and cardiac hypertrophy. Lancet. 1993;353:1290–1292.