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Am J Physiol Heart Circ Physiol 295: H245-H255, 2008. First published May 16, 2008; doi:10.1152/ajpheart.00066.2008
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Diacylglycerol kinase-{varepsilon} restores cardiac dysfunction under chronic pressure overload: a new specific regulator of G{alpha}q signaling cascade

Takeshi Niizeki,1 Yasuchika Takeishi,4 Tatsuro Kitahara,1 Takanori Arimoto,1 Mitsunori Ishino,1 Olga Bilim,1 Satoshi Suzuki,1 Toshiki Sasaki,1 Osamu Nakajima,2 Richard A. Walsh,5 Kaoru Goto,3 and Isao Kubota1

1Department of Cardiology, Pulmonology, and Nephrology, 2Research Laboratory for Molecular Genetics, and 3Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata, and 4First Department of Internal Medicine, Fukushima Medical University, Fukushima, Japan; and 5Department of Medicine, Case Western Reserve University, Cleveland, Ohio

Submitted 22 January 2008 ; accepted in final form 13 May 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
G{alpha}q protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in cardiac hypertrophy. DAG kinase (DGK) catalyzes DAG phosphorylation and controls cellular DAG levels, thus acting as a regulator of GPCR signaling. It has been reported that DGK{varepsilon} acts specifically on DAG produced by inositol cycling. In this study, we examined whether DGK{varepsilon} prevents cardiac hypertrophy and progression to heart failure under chronic pressure overload. We generated transgenic mice with cardiac-specific overexpression of DGK{varepsilon} (DGK{varepsilon}-TG) using an {alpha}-myosin heavy chain promoter. There were no differences in cardiac morphology and function between wild-type (WT) and DGK{varepsilon}-TG mice at the basal condition. Either continuous phenylephrine infusion or thoracic transverse aortic constriction (TAC) was performed in WT and DGK{varepsilon}-TG mice. Increases in heart weight after phenylephrine infusion and TAC were abolished in DGK{varepsilon}-TG mice compared with WT mice. Cardiac dysfunction after TAC was prevented in DGK{varepsilon}-TG mice, and the survival rate after TAC was higher in DGK{varepsilon}-TG mice than in WT mice. Phenylephrine- and TAC-induced DAG accumulation, the translocation of PKC isoforms, and the induction of fetal genes were blocked in DGK{varepsilon}-TG mouse hearts. The upregulation of transient receptor potential channel (TRPC)-6 expression after TAC was attenuated in DGK{varepsilon}-TG mice. In conclusion, these results demonstrate the first evidence that DGK{varepsilon} restores cardiac dysfunction and improves survival under chronic pressure overload by controlling cellular DAG levels and TRPC-6 expression. DGK{varepsilon} may be a novel therapeutic target to prevent cardiac hypertrophy and progression to heart failure.

hypertrophy; heart failure; diacylglycerol; protein kinase C

CARDIOVASCULAR DISEASE REPRESENTS an important and growing public health problem and is the cause of substantial morbidity and mortality. Cardiac hypertrophy is initially a compensatory response to a variety of stimuli such as chronic hypertension, stenotic valves, and loss of contractile mass from previous myocardial infarction. Conversely, sustained cardiac hypertrophy can eventually lead to decompensation characterized by heart failure, arrhythmias, and cardiac death (16). Therefore, the identification of novel molecular mechanisms underlying the development of cardiac hypertrophy and its transition to heart failure is an important challenge of cardiovascular biology and medicine. Multiple lines of experimental and clinical evidence have suggested the importance of the G{alpha}q protein-coupled receptor (GPCR) signaling in the development of pathological cardiac hypertrophy and heart failure (11, 31). GPCR agonists such as angiotensin II (25), endothelin-1 (26), and phenylephrine (20) activate phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces inositol 1,4, 5-trisphosphate and diacylglycerol (DAG). DAG functions as a potent activator of protein kinase C (PKC). We and others have previously demonstrated that PKC plays an important role in the development of cardiac hypertrophy and progression to heart failure (3, 30, 32).

One major route for terminating DAG signaling is thought to be its phosphorylation and inactivation by DAG kinase (DGK), producing phosphatidic acid (PA) (7, 14, 33). Three DGK isoforms, {alpha}, {varepsilon}, and {zeta}, are expressed in the heart, and we have recently demonstrated in cultured rat neonatal cardiomyocytes that adenoviral-mediated expression of DGK{zeta} blocks endothelin-1-induced increases in cell size and the reactivation of fetal genes via the inhibition of PKC{varepsilon} extracellular signal-regulated kinase (ERK)-activator protein-1 (AP-1) signaling pathway (28). Furthermore, we have generated transgenic mice with cardiac-specific overexpression of DGK{zeta} using an {alpha}-myosin heavy chain (MHC) promoter and demonstrated that DGK{zeta} negatively regulates the hypertrophic signaling cascade and resultant cardiac hypertrophy after GPCR agonist infusion without any detectable adverse effects in in vivo hearts (1). On the other hand, it has been reported that DGK{varepsilon} acts specifically on DAG produced by inositol cycling compared with DGK{zeta}, suggesting differences in two DGK isoforms about substrate specificity and the functional roles in signal transduction pathway (22). In addition, there are differences in subcellular localization between two isoforms, DGK{zeta} in the nucleus and DGK{varepsilon} in cytoplasm (14). In this study, to characterize the functional role of DGK{varepsilon} in the heart, we generated transgenic mice with cardiac-specific overexpression of DGK{varepsilon} (DGK{varepsilon}-TG) using an {alpha}-MHC promoter and examined whether DGK{varepsilon} prevents cardiac hypertrophy and progression to heart failure under chronic pressure overload. We demonstrated that DGK{varepsilon} restores cardiac dysfunction and improves survival under chronic pressure overload by controlling cellular DAG levels and transient receptor potential channel (TRPC)-6 expression.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of DGK{varepsilon}-TG mice. DGK{varepsilon}-TG mice were created in Yamagata University by standard techniques as described previously (1). Briefly, a 5.5-kb fragment of murine {alpha}-MHC gene promoter (a kind gift from Dr J. Robbins, Children's Hospital Research Foundation, Cincinnati, OH) and rat DGK{varepsilon} cDNA (7) were subcloned into pBsIISK (+) plasmids. The plasmid was digested with SpeI to generate a DNA fragment composed of the {alpha}-MHC gene promoter, DGK{varepsilon} cDNA, and a poly-A tail of the human growth hormone, as illustrated in Fig. 1A. We microinjected the DNA construct into the pronuclei of single-cell fertilized mouse embryos to generate transgenic mice as previously described (1). To detect the exogenous DGK{varepsilon} gene, genomic DNA was extracted from the tail tissues of 4-wk-old pups, and polymerase chain reaction (PCR) was performed with one primer specific for the {alpha}-MHC gene promoter and another primer specific for the DGK{varepsilon}, as shown in Fig. 1A.


Figure 1
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  		Fig. 1. A: transgene construct used for the generation of diacylglycerol (DAG) kinase-transgenic (DGK{varepsilon}-TG) mice. The transgene contains the {alpha}-myosin heavy chain (MHC) gene promoter, the full-length rat DGK{varepsilon} cDNA clone, and a human growth hormone (Hgh) poly-A sequence. Solid line, region amplified by PCR for genotyping. B: cardiac-specific expression of transgene was confirmed by RT-PCR. C: quantitative analysis of DGK{varepsilon} mRNA expression of wild-type (WT) and DGK{varepsilon}-TG mice by real-time RT-PCR. Data are reported as means ± SE obtained from 6 mice for each group. * P < 0.05 vs. WT mice. D: representative Western blots of DGK{varepsilon} protein from WT and DGK{varepsilon}-TG mouse hearts.

 
Wild-type (WT) littermate mice and DGK{varepsilon}-TG mice aged 8–10-wk old with a BDF1 background were used in the present study. Mice were housed in a facility with a 12-h:12-h light-dark cycle and were given free access to water and standard rodent chow. The room was kept specific pathogen-free. All experimental procedures were performed according to the animal welfare regulations of Yamagata University School of Medicine, and the study protocol was approved by the Animal Subjects Committee of Yamagata University School of Medicine. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Western blot analysis. Membranous and cytosolic fractions of detergent-extracted PKC were prepared from the left ventricular myocardium as described previously (1, 30, 31). The protein concentration of myocardial samples was carefully determined by the protein assay, and equal amounts of protein were subjected to 10% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membranes. To ensure an equivalent protein loading and quantitative transfer efficiency of proteins, the membranes were stained with Ponceau S before incubating with primary antibodies. The subcellular localization of PKC isoforms was examined by quantitative immunoblotting using isoform-specific antibodies (mouse monoclonal anti-PKC{alpha}, β, {delta}, and {varepsilon}, Santa Cruz Biotechnology, Santa Cruz, CA) as reported previously (1, 30, 31). Immunoreactive bands were detected by an enhanced chemiluminescence (ECL) kit (Amersham Biosciences, Piscataway, NJ), and membrane-to-cytosol ratios of immunoreactivity were used as indexes for the extent of translocation of PKC isoforms.

We prepared total protein from the left ventricular myocardium using a cell lysis buffer (Cell Signaling Technology, Danvers, MA) to examine protein expressions of TRPC isoforms. TRPC isoforms (TRPC 1, 3, 4, and 6) expressions were examined by quantitative immunoblotting using isoform-specific antibodies (Sigma, St. Louis, MO). Immunoreactive bands were detected by an ECL kit, and TRPC isoform expressions were normalized to actin.

We prepared nuclear and cytosolic protein from left ventricular myocardium using a nuclear and cytoplasmic extraction reagents (Pierce, Tokyo, Japan) to examine localization of DGK isoforms.

Real-time RT-PCR. Total RNAs were extracted from the left ventricle using TRIzol (Invitrogen, Tokyo, Japan), and first-strand cDNA was synthesized from 1 µg of RNA sample with oligo (dT) primers and superscript II RT as previously described (1, 28). To examine mRNA expression levels of DGK{varepsilon}, atrial natriuretic factor (ANF), β-MHC, brain natriuretic peptide (BNP), {alpha}-skeletal actin, collagen type I, collagen type III, and TRPC isoforms (TRPC 1, 3, 4, and 6), real-time RT-PCR amplification was performed as reported previously (1, 28). Standard curves of these genes were generated by full-sequence plasmid of known concentrations. Gene expressions were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers were designed based on GenBank sequences (ANF, K-02781; β-MHC, AY-056464; BNP, NM-008726; {alpha}-skeletal actin, NM-009606; collagen type I, NM-007742; collagen type III, NM-009930; TRPC 1, NM-011643; TRPC 3, NM-019510; TRPC 4, NM-016984; TRPC 6, NM-013838; and GAPDH, NM-001001303). Sense and anti-sense primers for DGK{varepsilon} were 5'-TTCCTTCCTAGCATTGTGTG-3' and 5'-AGGTCCAGGAAGATGAAACA-3'. The reaction conditions for the RT are a denaturing step at 95°C for 10 min followed by a three-step PCR amplification to quantify expression. The steps are 95°C for 10 s, 55–62°C for 10 s, and 72°C for 5–9 s (depending on the gene) for 45 cycles.

Lipid extraction and measurements of myocardial DAG levels. Myocardial lipid extract was prepared from the left ventricle, and DAG levels were measured as previously reported (1). Briefly, with the use of the DAG within myocardial lipid extract as substrate and with the use of [{gamma}-32P]ATP, the myocardial DAG level was quantified by production of [32P]PA. After 30 min of incubation, the reaction was terminated and the radiolabeled product was separated by thin-layer chromatography on silica plates. The [32P]PA was identified by autoradiography. Silica corresponding to PA was scraped and counted by liquid scintillation counting (1).

Caspase-3 activity. Caspase-3 activity in myocardial tissues was measured with a APOPCYTO/caspase-3 colorimetric assay kit (Medical & Biological Laboratories, Nagoya, Japan) that recognizes the sequence DEVD. The assay was from the labeled substrate DEVD-p-nitroanilide.

Subcutaneous implantation of osmotic minipump and thoracic transverse aortic constriction. A subpressor dose of phenylephrine (20 mg·kg–1·day–1) dissolved in saline or saline alone (control) was continuously infused into mice subcutaneously via an osmotic minipump (ALZET Osmotic Pump, DURECT) for 3 days. This dose did not increase systolic blood pressure as previous studies (1, 20). Systolic blood pressure was determined in the conscious state with the use of a computerized tail-cuff monometer, MK-1030 (Muromachi Kikai), as reported previously (1).

Thoracic transverse aortic constriction (TAC) was performed to produce chronic pressure overload as described previously (9). Briefly, mice (20–25 g body wt) were anesthetized and intubated with a 20-gauge polyethylene catheter and ventilated with a rodent ventilator (Harvard Apparatus, Holliston, MA). The chest cavity was opened at the second intercostals space at the left upper sternal border. The transverse section of the aorta was freed, an 8-0 prolene suture was passed around the aorta between the right innominate and left common carotid arteries, a tight ligature was tied against a 27-gauge needle, and the needle was then promptly removed. In sham-operated animals, the same procedure was performed except for the ligation. Finally, the lungs were reexpanded, and the chest wall was closed. The animals remained in a supervised setting until fully conscious.

Echocardiography and cardiac catheterization. Transthoracic echocardiography was recorded as described previously with an FFsonic 8900 (Fukuda Denshi, Tokyo, Japan) equipped with a 13-MHz phased-array transducer (1, 9). Left ventricular internal dimensions at end systole and end diastole (LVESD and LVEDD, respectively), interventricular septum (IVS), and left ventricular posterior wall thickness (PW) were measured digitally on the M-mode tracings and averaged from at least three cardiac cycles. Left ventricular fractional shortening (LVFS) was calculated as [(LVEDD – LVESD)/LVEDD] x 100.

A closed-chest approach by cardiac catheter was performed as described previously (40). The right carotid artery was cannulated under anesthesia by the micropressure transducer with an outer diameter of 0.42 mm (Samba 3200, Samba Sensors, Göteborg, Sweden), which was then advanced into the left ventricle. Heart rate, left ventricular end-diastolic pressure (LVEDP) and end-systolic pressure, the maximal and minimum rates of left ventricular pressure development (±dP/dt), and the time constant of left ventricular isovolumic relaxation ({tau}) were measured using an Acknowledge version 3.8.1 system with a sampling rate of 2,000 Hz (37).

Morphological and histopathological examinations. After mice were euthanized, the coronary arteries were retrogradely flushed with saline and the heart, lungs, and liver were excised and weighed. The heart was fixed with a 10% solution of formalin in PBS at 4°C for 24 h, embedded in paraffin, and then cut serially from the apex to the base. The sections were stained with hematoxylin-eosin or Masson's trichrome stain for histopathological analysis. Transverse sections were captured digitally, and cardiomyocyte cross-sectional area was measured using a Scion imaging system (Scion, Frederick, MD) (9). We traced the outline of at least 200 cardiomyocytes in each section, and the data were averaged.

To assess the degree of fibrosis, the sections stained with Masson's trichrome stain were scanned with computer-assisted videodensitometry, and the images from at least 10 fields for each heart were analyzed. The fibrosis fraction was obtained by calculating the ratio of Masson's trichrome-stained connective tissue area (stained blue) to total myocardial area (stained red) with image analysis software as described previously (9).

In immunohistochemical analysis, myocardial sections from DGK{varepsilon}-TG and DGK{zeta}-TG (1) mice were stained with anti-DGK{varepsilon} and anti-DGK{zeta} antibodies, respectively, to examine the differences in subcellular localization of two DGK isoforms. The staining was visualized by treatment for 15–20 s in the solution of 3,3'-diaminobenzidine (Dako Cytomation Liquid DAB Substrate Chromogen System, Dako Japan, Tokyo, Japan). Control reactions included the omission of the primary antibody, which was substituted by nonimmune rabbit serum.

Statistical analysis. All values are reported as means ± SE. The effects of phenylephrine or TAC on gravimetric data, histological data, PKC translocation, TRPC isoform expressions, echocardiographic data, cardiac catheter data, and RT-PCR data between WT and DGK{varepsilon}-TG mice were analyzed by two-way ANOVA or the Friedman test followed by multiple comparisons with the Fisher protected least significant difference test. Survival curves were created by the Kaplan-Meier method and compared by a log-rank test. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac-specific overexpression of DGK{varepsilon} does not affect cardiac function at basal condition. After microinjection and embryo implantation, five transgenic mice lines were successfully established. Cardiac-specific expression of transgene was confirmed by RT-PCR as shown in Fig. 1B. We confirmed the expression levels of DGK{varepsilon} in the left ventricle by real-time RT-PCR (Fig. 1C) and Western blot analysis (Fig. 1D) in five DGK{varepsilon}-TG lines. Among them, line 2 with the moderate expression level and high-breeding capacity was characterized in the following experiments.

Both male and female DGK{varepsilon}-TG mice had normal life spans and no evidence of morphogenic defects in cardiac or skeletal muscle. To characterize mouse phenotypes, all experiments were performed with age- and sex-matched (8–10 wk old) WT littermate mice and DGK{varepsilon}-TG mice. Body weight, systolic blood pressure, and heart rate were similar between WT and DGK{varepsilon}-TG mice. The absolute heart weight, ratio of heart weight to body weight, and ratio of the left ventricular weight to body weight were not different between WT and DGK{varepsilon}-TG mice. Echocardiography demonstrated that cardiac dimensions, wall thickness, and LVFS were normal in DGK{varepsilon}-TG mice. There was no evidence of fibrosis on microscopic examinations of multiple histological sections. In immunohistochemical staining and Western blot analysis, we detected differences in subcellular localization between DGK isoforms in cardiomyocytes from hearts of DGK{zeta}-TG and DGK{varepsilon}-TG mice, DGK{zeta} in the nucleus, and DGK{varepsilon} in cytoplasm and nucleus as shown in Fig. 2.


Figure 2
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  		Fig. 2. Immunohistochemical staining and Western blot of left ventricular myocardium of DGK{varepsilon}-TG and DGK{zeta}-TG mice. Bars = 20 µm.

 
Effects of DGK{varepsilon} on hypertrophic programs in response to phenylephrine infusion. WT and DGK{varepsilon}-TG mice were assessed with respect to their susceptibility to hypertrophic response to subpressor doses of subcutaneous phenylephrine administration. No significant changes in body weight, heart rate, and systolic blood pressure were observed between WT and DGK{varepsilon}-TG mice after subcutaneous infusion of phenylephrine as previous studied (1, 20). As shown in Table 1, subcutaneous infusion of phenylephrine caused significant increases in the ratio of heart weight to body weight and the ratio of the left ventricular weight to body weight in WT mice. However, these increases were completely blocked in DGK{varepsilon}-TG mice.


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  		Table 1. Gravimetric parameters and real-time RT-PCR data in WT and DGK{varepsilon}-TG mice ays after PE infusion

 
We next examined cardiac mRNA expressions of fetal-type genes after phenylephrine infusion. As shown in Table 1, the mRNA expressions of ANF and β-MHC were increased in WT mice given phenylephrine compared with WT mice infused with saline. However, phenylephrine failed to cause gene induction of ANF in DGK{varepsilon}-TG mice.

Effects of DGK{varepsilon} on phenylephrine-induced activation of the DAG-PKC signaling. Lipid extracts were then prepared from the left ventricle, and we quantified myocardial DAG levels in WT and DGK{varepsilon}-TG mouse hearts. As shown in Fig. 3A, DAG levels were not different between WT and DGK{varepsilon}-TG mice at the basal condition. In WT mouse hearts, myocardial DAG level increased markedly after continuous administration of phenylephrine. On the other hand, this effect of phenylephrine on myocardial DAG levels was completely suppressed in DGK{varepsilon}-TG mouse hearts.


Figure 3
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  		Fig. 3. A, left: representative autoradiogram for DAG levels. A, right: quantitative data of DAG by scintillation counting. B: translocation of PKC{alpha} and -{varepsilon} in WT mice after phenylephrine infusion was abolished in DGK{varepsilon}-TG mice. Data are reported as means ± SE obtained from 8 mice for each group. M/C, membrane-to-cytosol ratio.

 
As shown in Fig. 3B, the membrane-associated immunoreactivities of PKC{alpha} and -{varepsilon} were significantly increased in phenylephrine-treated WT mice compared with saline-infused WT mice. However, phenylephrine-induced translocation of PKC{alpha} and -{varepsilon} was significantly attenuated in DGK{varepsilon}-TG mice. The translocation of PKCβ and -{delta} was not detected after continuous administration of phenylephrine (data not shown). These data suggest that DGK{varepsilon} regulates PKC{alpha} and -{varepsilon} activity by controlling cellular DAG levels.

Cardiac hypertrophy after TAC. Because mechanical stimuli such as pressure and volume overload are clinically relevant and important in cardiac hypertrophy and heart failure, we next examined whether DGK{varepsilon} modifies cardiac remodeling in response to pressure overload using a TAC mouse model (9). Figure 4A shows representative transverse sections of WT and DGK{varepsilon}-TG mouse hearts after TAC or sham operation. Extensive cardiac hypertrophy was observed in WT mice at 4 wk after TAC. However, cardiac hypertrophy after TAC was attenuated in DGK{varepsilon}-TG mice. As shown in Table 2, the pressure gradient across the aortic stenosis at 4 wk after TAC surgery was similar between WT and DGK{varepsilon}-TG mice (71 ± 3 vs. 74 ± 4 mmHg). Similarly, the ascending aortic systolic pressure (170 ± 6 vs. 167 ± 7 mmHg) and left ventricular systolic pressure (167 ± 6 vs. 175 ± 9 mmHg) were not different between WT and DGK{varepsilon}-TG mice as in Table 2. These data clearly demonstrated that surgical intervention was equal between WT and DGK{varepsilon}-TG mice. Body weight was not different at 4 wk after TAC, whereas heart weight was markedly increased in WT mice after TAC surgery as reported in Table 2. However, increases in heart weight and left ventricular weight corrected for body weight after TAC were attenuated in DGK{varepsilon}-TG mice compared with WT mice.


Figure 4
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  		Fig. 4. Histological observations at 4 wk after thoracic transverse aortic constriction (TAC) or sham operation. A: representative left ventricular transverse sections stained by hematoxylin-eosin stain. B, left: representative images of hematoxylin-eosin micrographs of cardiomyocyte cross sections (magnification, x400; bar = 20 µm). B, right: quantitative analysis of cardiomyocyte cross-sectional area isolated from the left ventricular myocardium. Data are reported as means ± SE obtained from 10–12 mice for each group.

 

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  		Table 2. Gravimetric data and cardiac function (echocardiographic and cardiac catheter) at 4 wk after TAC or sham operation

 
Microscopic observations revealed that the cardiomyocyte cross-sectional area was profoundly increased in WT mice at 4 wk after TAC compared with sham-operated WT mice (Fig. 4B). However, this increase after TAC was significantly attenuated in DGK{varepsilon}-TG mice compared with WT mice.

DGK{varepsilon} restores cardiac dysfunction under chronic pressure overload. Echocardiography was performed at baseline, 1 wk, and 4 wk after TAC surgery. Under anesthesia, the heart rate was similar between WT and DGK{varepsilon}-TG mice (data not shown). The representative M-mode echocardiograms are shown in Fig. 5, and Table 2 summarizes the echocardiographic data. IVS and PW thickness were significantly increased in WT mice after TAC. However, these increases were significantly attenuated in DGK{varepsilon}-TG mice. Furthermore, the reduction of LVFS and dilatation of LVEDD in WT mice at 4 wk after TAC were prevented in DGK{varepsilon}-TG mice.


Figure 5
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  		Fig. 5. Representative M-mode echocardiograms at baseline, 1 wk (1w) and 4 wk (4w) after TAC surgery. IVS, interventricular septum; EDD, left ventricular end-diastolic dimension; PW, posterior wall.

 
Cardiac catheter was performed at baseline and 4 wk after surgery. As shown in Table 2, WT mice after TAC exhibited severe cardiac dysfunction at 4 wk after TAC as demonstrated by increased LVEDP and reduced ±dP/dt compared with sham-operated WT mice. However, LVEDP after TAC was significantly reduced in DGK{varepsilon}-TG mice. The ±dP/dt after TAC improved in DGK{varepsilon}-TG mice, but these changes were not statistically significant.

Activation of the DAG-PKC signaling and fetal gene induction after TAC. The DAG level was significantly increased in WT mice at 4 wk after TAC compared with sham-operated WT mice (Fig. 6A). On the other hand, the DAG level was completely suppressed in DGK{varepsilon}-TG mice compared with WT mice at 4 wk after TAC.


Figure 6
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  		Fig. 6. A: increase of DAG level in WT mice at 4 wk after TAC was significantly attenuated in DGK{varepsilon}-TG mice. B: translocation of PKC{alpha} (top, left), -β (top, right), -{delta} (bottom, left), and -{varepsilon} (bottom, right) isoforms in WT mice after TAC was significantly attenuated in DGK{varepsilon}-TG mice. Data are reported as means ± SE obtained from 8 mice for each group.

 
As shown in Fig. 6B, we detected the translocation of PKC{alpha}, -β, -{delta}, and -{varepsilon} isoforms in WT mice after TAC compared with sham-operated mice. However, in DGK{varepsilon}-TG mice, the translocation of these PKC isoforms after TAC was completely abolished compared with that in the WT mice.

We next examined mRNA expressions of fetal type genes such as ANF, β-MHC, BNP, and {alpha}-skeletal actin at 4 wk after surgery. The expressions of ANF, β-MHC, BNP, and {alpha}-skeletal actin were significantly upregulated in WT mice after TAC compared with sham-operated WT mice as demonstrated in Table 3. Conversely, in DGK{varepsilon}-TG mice, the gene inductions of ANF, β-MHC, and BNP, but not {alpha}-skeletal actin, were significantly attenuated compared with WT mice.


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  		Table 3. Fetal type genes, profibrotic genes, and TRPC isoform expressions by real-time RT-PCR analysis at 4 wk after TAC or sham operation

 
Myocardial fibrosis and expressions of profibrotic genes after TAC. Since reactive interstitial and perivascular fibrosis adversely alters myocardial stiffness and ultimately leads to left ventricular dysfunction, we examined myocardial fibrosis at 4 wk after surgery using sections stained with Masson's trichrome stain (Fig. 7). Prominent perivascular and interstitial fibrosis was observed in WT mice after TAC as reported in previous studies (9). However, the degree of myocardial fibrosis after TAC was much less in DGK{varepsilon}-TG mice than in WT mice as shown in Fig. 7. Furthermore, although caspase-3 activity associated with fibrosis was significantly increased in WT mice after TAC as previous studied (23), this increase was attenuated in DGK{varepsilon}-TG mice.


Figure 7
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  		Fig. 7. Histological analyses at 4 wk after surgery. Left: representative images of Masson's trichrome stain (Magnification, x400; bar = 20 µm) in the left ventricular myocardium. Right, top: comparison of the fibrosis fraction. Right, bottom: comparison of caspase-3 activity. Data are reported as means ± SE obtained from 8 mice for each group.

 
We next examined the expressions of profibrotic genes such as collagen types I and III to investigate whether these morphological observations were accompanied by alterations in gene expressions relevant to fibrotic changes. Real-time RT-PCR revealed that collagen types I and III mRNA and the ratio of collagen type I to type III were markedly upregulated in WT mice at 4 wk after TAC compared with sham-operated WT mice (Table 3). However, these responses after TAC were significantly attenuated in DGK{varepsilon}-TG mice compared with WT mice.

TRPC isoform expressions after TAC surgery by real-time RT-PCR and Western blot analysis. Recently, it has been reported that a DAG-mediated increase in cytosolic Ca2+ via TRPC participates in a positive regulatory circuit in the calcineurin-nuclear factor of activated T cells (NFAT) pathway leading to pathological cardiac hypertrophy (15). Since DAG produced by PLC activation directly activates TRPC 6 (19), we next examined TRPC isoform expressions in WT and DGK{varepsilon}-TG mice at 4 wk after TAC surgery.

We detected cardiac expressions of TRPC 1, 3, 4 and 6, and only TRPC-6 expression was significantly upregulated in WT mice at 4 wk after TAC by both real-time RT-PCR analysis (Table 3) and Western blot analysis (Fig. 8). However, in DGK{varepsilon}-TG mice, this upregulation of TRPC 6 after TAC was significantly attenuated compared with WT mice.


Figure 8
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  		Fig. 8. Expressions of transient receptor potential channel (TRPC) isoforms at 4 wk after TAC or sham operation. Data of TRPC were normalized by actin. Upregulation of TRPC 6 detected in WT mice after TAC was significantly attenuated in DGK{varepsilon}-TG mice. Data are reported as means ± SE obtained from 8 mice for each group.

 
DGK{varepsilon} improves survival after TAC. As shown in Fig. 9, the Kaplan-Meier survival curves were created from 38 TAC-operated WT mice, 31 TAC-operated DGK{varepsilon}-TG mice, 14 sham-operated WT mice, and 15 sham-operated DGK{varepsilon}-TG mice. Some TAC mice died suddenly, and an autopsy revealed that those mice had enlarged hearts with severe myocardial fibrosis accompanied by pericardial and pleural effusion. The survival rate in TAC-operated WT mice was 42.8% at 4 wk after surgery. However, cardiac DGK{varepsilon} expression improved the survival rate after TAC to 79.2% (P = 0.0334). Furthermore, the presence of a pleural effusion was less in DGK{varepsilon}-TG mice compared with WT mice at 4 wk after TAC [5/31 (16%) vs. 17/38 (45%), P = 0.0345]. Thus these observed differences in survival rate might be due to less lethal congestive heart failure in DGK{varepsilon}-TG mice than in WT mice.


Figure 9
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  		Fig. 9. Survival curves in WT and DGK{varepsilon}-TG mice after TAC or sham operation. Percentages of surviving WT and DGK{varepsilon}-TG mice were plotted. During follow-up period, survival rates after TAC were significantly higher in DGK{varepsilon}-TG mice than in WT mice. n, Number of mice.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first report characterizing a functional role of DGK{varepsilon} in in vivo mouse hearts. We demonstrated that DGK{varepsilon} blocks the hypertrophic signaling cascade and the resultant cardiac hypertrophy in response to phenylephrine infusion and chronic pressure overload. We showed that cardiac fibrosis, an increase of myocardial DAG level, the translocation of PKC isoforms, caspase-3 activity, the induction of fetal and profibrotic genes, and the upregulation of TRPC 6 in WT mice after TAC were significantly blocked by DGK{varepsilon}. Furthermore, DGK{varepsilon} prevented the progression to heart failure after TAC without detectable adverse effects in in vivo hearts. The fact that controlling the GPCR signaling pathway by DGK{varepsilon} prevents developing heart failure after TAC suggests that DGK{varepsilon} may represent a novel therapeutic target for cardiac hypertrophy and the progression to subsequent heart failure.

Numerous investigations have demonstrated the importance of G{alpha}q-mediated signaling in the development of cardiac hypertrophy (6, 11). We have previously shown that DGK{zeta} inhibits GPCR agonist-induced hypertrophic signaling pathway and the resultant cardiac hypertrophy in vitro and in vivo (1, 28). On the other hand, previous reports suggest that DGK isoforms have different functional roles and substrate specificity in signal transduction pathway (22, 29, 38). In infarcted rat hearts, the DGK{zeta} expression was enhanced in the infarcted area and border zone (29). In contrast, the DGK{varepsilon} expression was decreased in viable myocardium and was completely restored by treatment with captopril. No significant difference in the expression of DGK{zeta} was observed in left ventricular myocardium between TAC-operated and sham-operated rats at 3, 7, and 28 days after surgery (38). However, the DGK{varepsilon} expression in left ventricular myocardium was significantly decreased after TAC. Furthermore, the overexpressing DGK{varepsilon} especially reduced the polyunsaturated DAG levels and caused the redistribution of PKC{alpha} and -{varepsilon} isoforms in porcine aortic endothelial cells (22), whereas the overexpression of DGK{zeta} caused very little changes in DAG levels and PKC distribution. In contrast to DGK{zeta} (1, 28), DGK{varepsilon} inhibited the translocation of PKCβ and -{delta} after TAC in the present study. In addition, DGK{varepsilon} completely blocked phenylephrine-induced cardiac hypertrophy (Table 1). Since the localization of the enzymes has a marked impact on signaling cascade (36), these results might be due to substrate specificity toward arachidonoyl-containing DAG in DGK{varepsilon} (22) and differences in subcellular localization (14), DGK{zeta} in the nucleus, and DGK{varepsilon} in cytoplasm and nucleus (Fig. 2). Because the DAG-binding site in DGK has not yet been identified with certainty, this needs to be further examined in the future. Since this study employed only the overexpression approach, future experiments of a loss of DGK function using knockout mice are necessary to elucidate further the role of DGK{varepsilon} in signaling cascade in vivo.

The systolic blood pressure after the infusion of phenylephrine was not different among DGK{varepsilon}-TG and WT mice, indicating that the overexpression of DGK{varepsilon} does not affect hemodynamic regulations in response to phenylephrine. Thus the hypertrophic response in this phenylephrine model occurred independently of the hemodynamic effects of phenylephrine because the systemic blood pressure was not elevated after the infusion. The dose of phenylephrine used in this study (20 mg·kg–1·day–1) was lower compared with the dose (100 mg·kg–1·day–1) of the other study (13).

In this study, the DAG level was significantly increased in WT mice at 4 wk after TAC compared with sham-operated WT mice. However, this increase was significantly attenuated in DGK{varepsilon}-TG mice. Wang et al. (35) reported that PLC activity was substantially increased in WT mice at 6 wk after TAC compared with sham-operated mice. Both PLC activity and reduction in DAG clearance might influence myocardial DAG levels and explain the effectiveness of DGK{varepsilon}.

TRPC is one of the candidate channel subunits responsible for receptor-activated Ca2+ entry and store-operated Ca2+ entry. Cytoplasmic free Ca2+ in cardiomyocytes induces positive inotropic effects on the heart and activates several transcriptional pathways, such as NFAT signaling, that lead to cardiac hypertrophy. Recent reports have demonstrated that TRPC 6 is activated directly by DAG and stretch stimuli and that TRPC 6 is involved in NFAT activation during pathological cardiac remodeling in in vivo hearts (19, 27). Furthermore, it has been reported that TRPC 6 expression was upregulated in mouse hearts expressing constitutively active calcineurin and subjected to pressure overload by TAC (15) and the infusion of phenylephrine and endothelin-1 (5), because the promoter of the TRPC 6 gene contains two conserved NFAT-binding sites that confer responsiveness to calcineurin-NFAT signaling translationally. The overexpression of TRPC 6 in cardiomyocytes activates the NFAT and increases in the expression of β-MHC (15). Furthermore, small-interfering RNA knockdown of TRPC 6 reduces hypertrophic signaling by phenylephrine and endothelin-1, suggesting that TRPC 6 participates in a positive regulatory circuit in the calcineurin-NFAT pathway in response to GPCR signaling (15). We demonstrated for the first time that DAG-mediated expression of TRPC 6 after TAC was blocked by DGK{varepsilon} in this study. The attenuation of DAG signaling by DGK{varepsilon} might inhibit TRPC-6 expression by the regulation of calcineurin-NFAT signaling. Although we were unable to measure TRPC-6 activity, the altered DAG metabolism may impact on the posttranslational regulation of TRPC activity. These data suggest that TRPC might contribute to cardiac hypertrophy and the progression to heart failure under chronic pressure overload.

Previous studies have reported that the PKC{delta} and -{varepsilon} isoforms play an important role in cardiac hypertrophy (4, 32). The PKC{varepsilon} activates ERK-AP-1 signaling pathway, leading to myocardial hypertrophy and increases in protein synthesis (28). It has been reported that PKCβ plays a critical role in the development of heart failure (30) and that PKC{alpha} induces reactive fibrosis, impairing both systolic and diastolic function and leading to early heart failure and premature lethality (8). Furthermore, the GPCR agonist activates c-jun NH2-terminal kinase and p38 mitogen-activated protein kinase through PLC-PKC pathways in the cardiomyocyte, and these pathways act as potent signals for cardiac hypertrophy and the progression to heart failure (24). Thus the inhibition of PKC translocation by DGK{varepsilon} in this study might attenuate cardiac hypertrophy and fibrosis and prevent progression to heart failure after TAC. Membrane and cytosolic Western blot analysis for PKC suggested that the transcriptional activation of PKC protein synthesis upon TAC had occurred to coordinate the increase of protein in the course of hypertrophic development as previously studied (4, 12).

DGK{varepsilon}-TG mice showed a complete inhibition of hypertrophy in response to phenylephrine but only a partial inhibition of the hypertrophic response to TAC. Because the cardiac hypertrophy caused by TAC was induced by various signaling pathways such as gp130-STAT3 (34), Gs protein-coupled receptor-adenylyl cyclase signaling pathway (10), and glycogen synthase kinase-3 (39), DGK{varepsilon} could not inhibit hypertrophy after TAC completely. In this study the survival rate after TAC was lower compared with that in other reports (2, 21). Because other studies used C57BL6/J (2) and FVB mice (21), the difference of background might contribute to the survival rate in this study. The cause of death was considered due to congestive heart failure with the following signs: the presence of pulmonary congestion (increased lung weight) and massive chest fluid accumulation as described previously (18). Future studies are needed to examine survival rate after longer periods of TAC between DGK{varepsilon}-TG and WT mice.

Conclusions. We demonstrated that DGK{varepsilon} is a new specific regulator of GPCR signaling in cardiomyocytes. DGK{varepsilon} inhibits the activation of PKC and the expression of TRPC by controlling cellular DAG levels. These results will allow us a novel approach to investigate the pathogenesis of cardiac hypertrophy and heart failure, and DGK{varepsilon} may be a potential novel therapeutic target to prevent heart failure.


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 ABSTRACT
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This study was supported in part by the Ministry of Education, Science, Sports and Culture, Japan, Grant-in-Aid for Scientific Research No. 19590804.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Niizeki, Dept. of Cardiology, Pulmonology, and Nephrology, Yamagata Univ. School of Medicine, 2-2-2 Iida-Nishi, Yamagata, Japan 990-9585 (e-mail: tniizeki{at}nihonkai.gr.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 METHODS
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 DISCUSSION
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