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(Circulation. 2006;113:60-66.)
© 2006 American Heart Association, Inc.

Heart Failure

Cardiac-Specific Overexpression of Diacylglycerol Kinase Prevents Gq Protein-Coupled Receptor Agonist-Induced Cardiac Hypertrophy in Transgenic Mice

Takanori Arimoto, MD; Yasuchika Takeishi, MD; Hiroki Takahashi, MD; Tetsuro Shishido, MD; Takeshi Niizeki, MD; Yo Koyama, MD; Ryoko Shiga, MD; Naoki Nozaki, MD; Osamu Nakajima, PhD; Kazuhide Nishimaru, PhD; Jun-ichi Abe, MD; Masao Endoh, MD; Richard A. Walsh, MD; Kaoru Goto, MD; Isao Kubota, MD

From the First Department of Internal Medicine (T.A., Y.T., H.T., T.S., T.N., Y.K., R.S., N.N., I.K.), Research Laboratory for Molecular Genetics (O.N.), Department of Cardiovascular Pharmacology (K.N., M.E.), and Department of Anatomy and Cell Biology (K.G.), Yamagata University School of Medicine, Yamagata, Japan; Center for Cardiovascular Research, University of Rochester, Rochester, NY (J.A.); and Department of Medicine, Case Western Reserve University, Cleveland, Ohio (R.A.W.).

Reprint requests to Yasuchika Takeishi, MD, First Department of Internal Medicine, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata, Japan 990-9585. E-mail takeishi{at}med.id.yamagata-u.ac.jp

Received July 20, 2004; de novo received May 10, 2005; revision received October 6, 2005; accepted October 17, 2005.

	Abstract

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Background— Diacylglycerol is a lipid second messenger that accumulates in cardiomyocytes when stimulated by Gq protein-coupled receptor (GPCR) agonists such as angiotensin II, phenylephrine, and others. Diacylglycerol functions as a potent activator of protein kinase C (PKC) and is catalyzed by diacylglycerol kinase (DGK) to form phosphatidic acid and inactivated. However, the functional roles of DGK have not been previously examined in the heart. We hypothesized that DGK might prevent GPCR agonist-induced activation of diacylglycerol downstream signaling cascades and subsequent cardiac hypertrophy.

Methods and Results— To test this hypothesis, we generated transgenic (DGK-TG) mice with cardiac-specific overexpression of DGK. There were no differences in heart size and heart weight between DGK-TG and wild-type littermate mice. The left ventricular function was normal in DGK-TG mice. Continuous administration of subpressor doses of angiotensin II and phenylephrine caused PKC translocation, gene induction of atrial natriuretic factor, and subsequent cardiac hypertrophy in WT mice. However, in DGK-TG mice, neither translocation of PKC nor upregulation of atrial natriuretic factor gene expression was observed after angiotensin II and phenylephrine infusion. Furthermore, in DGK-TG mice, angiotensin II and phenylephrine failed to increase cross-sectional cardiomyocyte areas and heart to body weight ratios. Phenylephrine-induced increases in myocardial diacylglycerol levels were completely blocked in DGK-TG mouse hearts, suggesting that DGK regulated PKC activity by controlling cellular diacylglycerol levels.

Conclusions— These results demonstrated the first evidence that DGK negatively regulated the hypertrophic signaling cascade and resultant cardiac hypertrophy in response to GPCR agonists without detectable adverse effects in in vivo hearts.

Key Words: angiotensin • hypertrophy • enzymes • signal transduction

	Introduction

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Cardiac hypertrophy is an initially adaptive response in several forms of cardiac disease, whereas sustained hypertrophy is a powerful independent risk factor for cardiac morbidity and mortality.¹ Therefore, to identify the critical molecular mechanisms involved in cardiac hypertrophy is an important challenge of cardiovascular biology and medicine. Multiple lines of experimental and clinical evidence have suggested the importance of the Gq-phosphoinositide signaling system in the development of pathological cardiac hypertrophy and heart failure.^2–6 Gq protein-coupled receptor (GPCR) agonists such as angiotensin II,⁷ endothelin-1,⁸ and phenylephrine⁹ activate phospholipase C-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). The binding of DAG to the C1 domain of PKC induces an active conformation, and activated PKC regulates a variety of cellular functions including cell growth and differentiation. We and others have previously demonstrated that PKC plays an important role in the development and progression of cardiac hypertrophy.^10–12

One major route for terminating DAG signaling is thought to be its phosphorylation and inactivation by DAG kinase (DGK), producing phosphatidic acid.^13–16 A previous study has shown that of the , , and isoforms of DGK expressed in the myocardium, DGK is the predominant isoform.¹⁷ We have recently demonstrated using cultured rat neonatal cardiomyocytes that DGK blocks endothelin-1–induced activation of PKC, extracellular signal-regulated kinase (ERK), and activator protein-1.¹⁸ DGK also inhibits gene induction of atrial natriuretic factor (ANF), increases in protein synthesis, and resultant cardiomyocyte hypertrophy in response to endothelin-1 in neonatal cardiomyocytes. However, the in vivo role of DGK has not been previously investigated in the heart.¹⁹

We hypothesized that DGK may act as a negative regulator for GPCR agonist-induced activation of the DAG-PKC signaling cascade and subsequent cardiac hypertrophy in vivo. To test this hypothesis, we generated transgenic mice with cardiac-specific overexpression of DGK using an -myosin heavy chain (MHC) promoter. We examined the functional role of DGK to interfere with hypertrophic responses by GPCR agonists such as angiotensin II and phenylephrine in transgenic mouse hearts.

	Methods

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Generation of DGK Transgenic Mice
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 US National Institutes of Health.

Transgenic mice with cardiac-specific overexpression of DGK (DGK-TG) were created in Yamagata University by standard techniques.^10,12,20 Briefly, a 5.5-kb fragment of murine -MHC gene promoter (a kind gift from Dr J. Robbins, Children’s Hospital Research Foundation, Cincinnati, Ohio) and 3.4-kb rat DGK cDNA¹³ were subcloned into pBsIISK(+) plasmids. The plasmid was digested with SpeI to generate a 9.5-kb DNA fragment composed of the -MHC gene promoter, DGK cDNA, and a poly A tail of the human growth hormone, as illustrated in Figure 1A. We microinjected the construct into the pronuclei of single-cell fertilized mouse embryos to generate transgenic mice as previously described.^10,12,20 To detect the exogenous DGK gene, genomic DNA was extracted from the tail tissues of 3- to 4-week-old pups, and polymerase chain reaction (PCR) was performed with one primer specific for the -MHC gene promoter and another primer specific for the DGK, as shown in Figure 1A.

View larger version (31K): [in this window] [in a new window]   Figure 1. A, Diagram of the transgene construct used for the generation of DGK-TG mice. The construct contains the -MHC gene promoter, the full-length rat DGK cDNA clone, and a human growth hormone (Hgh) polyadenylylation sequence. The solid line indicates the region amplified by PCR for genotyping. B, Quantitative analysis of DGK mRNA expression in the left ventricle of DGK-TG and WT mice. Left ventricular myocardium of 8-week-old mice was examined by real-time PCR analysis and normalized with the use of GAPDH mRNA. Data are reported as fold increase over WT and obtained from 3 mice for each. C, Representative Western blot analysis of DGK protein from the left ventricle of DGK-TG-High, DGK-TG-Low, and WT mice. D, Representative autoradiograms of DGK assay of DGK-TG-High, DGK-TG-Low, and WT mice.

DGK Activity
DGK activity in the left ventricle was measured by octyl glucoside mixed-micelle assay as described previously.^21,22 The 1,2-dioleoyl-sn-glycerol (18:1/18:1 DAG) and 1-stearoyl-2- linoleoyl-sn-glycerol (18:0/18:2 DAG) were used as substrates for kinase assays. Phosphatidic acid separated by thin layer chromatography was scraped and counted by liquid scintillation.^21,22

Western Blotting
Total protein was extracted from the left ventricle with ice-cold lysis buffer as described previously.^23–25 Protein concentration of myocardial samples was carefully determined by the protein assay, and equal amounts of protein extracts were loaded on each gel lane. To ensure equivalent protein loading and quantitative transfer efficiency of proteins, membranes were stained with Ponceau S before incubating with primary antibodies. Western blotting was performed as reported previously, and DGK protein levels in TG mice were expressed as fold increase over wild-type (WT) mice. Membrane and cytosolic fractions were also prepared from left ventricular myocardium as previously reported.^10,12,25 Membrane/cytosol ratios of immunoreactivity with the use of isoform-specific antibodies (mouse monoclonal anti-PKC, ß, , and ; Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) were used as indices for the extent of translocation of PKC isoforms.^10,12,25

Extraction of Total RNA and Real-time Reverse Transcriptase-PCR
Total RNAs were extracted from the left ventricle, and first-strand cDNA was synthesized as previously described.^26,27 To examine mRNA levels of DGK and ANF quantitatively, real-time reverse transcriptase-PCR (RT-PCR) amplification was performed.¹⁸ Amplification was performed with the use of LightCycler and analyzed with the use of LightCycler Software Version 3.5 (Roche Diagnostics Japan). Standard curves of DGK and ANF were generated by full sequence plasmid of known concentrations. Gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as fold increase over WT mice. Primers were designed on the basis of GenBank sequences (DGK, BC049228; ANF, K02781; and GAPDH, NM_001001303).

Isolated Cardiomyocyte Function and Ca²⁺ Transient
Ventricular myocytes were isolated from hearts from WT mice and mouse lines with the high expression of transgene mRNA (DGK-TG-High), and cardiomyocyte mechanical properties were examined with the use of a computerized edge-detection analyzer as previously reported.²⁸ Cells were paced at 0.5 Hz throughout the experiments. Same isolated cells were used for measurements of cytosolic free Ca²⁺ by indo-1 with a previously described method.²⁸ Data from at least 5 to 6 cardiomyocytes were averaged for each mouse heart, and the statistical analysis was performed on the basis of the number of hearts studied (n=6 for each group).

Histological Examinations
Coronary arteries were retrogradely flushed with saline, and the heart was fixed with 4% paraformaldehyde at 4°C for 24 hours and then embedded in paraffin.^26,27 Three sections were stained with hematoxylin-eosin or elastica Goldner stain. Transverse sections were captured digitally, and cardiomyocyte cross-sectional area was measured with the use of a Scion imaging system (Scion Corporation).²⁹ At least 300 cardiomyocytes were examined in each heart, and the data were averaged.

Hemodynamic Measurements and Echocardiography
Heart rate (bpm) and blood pressure (mm Hg) were determined with animals in the conscious state with the use of a computerized tail-cuff manometer, MK-1030 (Muromachi Kikai Co, Ltd), as reported previously.¹² Echocardiography was performed as described previously^26,27 with an FFsonic 8900 (Fukuda Denshi Co) equipped with a 13-MHz phased-array transducer. Left ventricular wall thickness and internal dimensions at end-systole and end-diastole were measured digitally on the M-mode tracings and averaged for 3 cardiac cycles. Left ventricular fractional shortening was calculated as previously reported.^26,27

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.³⁰ Briefly, with the use of the DAG within myocardial lipid extract as substrate and with the use of [-³²P]ATP, myocardial DAG level was quantified by production of [³²P]phosphatidic acid. Lipid extract was solubilized in 50 µL of 0.6% (wt/vol) Triton X/288 µmol/L phosphatidylserine. The reaction mixture contained 0.3% (wt/vol) Triton X, 144 µmol/L phosphatidylserine, 50 mmol/L imidazole/HCl, pH 6.6, 50 mmol/L NaCl, 12.5 mmol/L MgCl₂, 1 mmol/L EGTA, 10 mmol/L dithiothreitol, 0.5 mmol/L ATP (1 µCi of [-³²P]ATP), and 5 m-unit of DGK (Escherichia coli). After 30 minutes of incubation, the reaction was terminated, and the radiolabeled product was separated by TLC on silica plates. The [³²P]phosphatidic acid was identified by autoradiography. Silica corresponding to phosphatidic acid was scraped and counted by liquid scintillation counting.³⁰

Subcutaneous Implantation of Osmotic Minipump
A subpressor dose of angiotensin II (100 ng/kg per minute) or phenylephrine (20 mg/kg per day) dissolved in saline or saline alone (control) was continuously infused into mice subcutaneously via an osmotic minipump (ALZET Osmotic Pumps, DURECT Corporation) for 14 or 3 days, respectively.^2,3,31,32 Heart rate and blood pressure were measured before and after subcutaneous infusion of angiotensin II or phenylephrine.

Statistical Analysis
All values are reported as mean±SD. Comparisons of hemodynamic data and gravimetric and echocardiographic measurements at basal conditions among WT, DGK-TG-High, and DGK-TG-Low mice were made by the Kruskal-Wallis test. Isolated cardiomyocyte mechanical properties and calcium transient data between WT and DGK-TG-High mice were compared by the Mann-Whitney U test. Effects of angiotensin II and phenylephrine on body weight, blood pressure, heart rate, heart weight, left ventricular weight, PKC translocation, DAG level, ANF expression, and cardiomyocyte surface area in animal groups were compared by 2-way ANOVA followed by multiple comparisons with the Bonferroni test. Probability values of <0.05 were considered statistically significant.

	Results

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Generation of DGK Transgenic Mice
After microinjection and embryo implantation, 2 lines of transgenic mice (lines 70 and 45) were successfully established. Figure 1B shows the real-time PCR results of DGK in the left ventricle of TG and WT mice. Heterozygous mouse lines with the high expression of transgene mRNA (DGK-TG-High) and the low expression of transgene mRNA (DGK-TG-Low) in the left ventricle were characterized in detail in the following experiments. RNA was extracted from brain, heart, lungs, liver, kidney, spleen, intestine, and skeletal muscle tissues of DGK-TG mice, and cardiac-specific expression of transgene was confirmed by RT-PCR (data not shown). Protein levels of DGK were augmented 21- and 5.5-fold in DGK-TG-High and DGK-TG-Low hearts compared with control WT littermates, respectively (Figure 1C). Additionally, we confirmed that kinase activities of DGK in the heart were also augmented in both DGK-TG mouse lines (Figure 1D). No neonatal and adult deaths were observed in DGK-TG mice.

Gravimetric Data, Cardiac Function, and Isolated Cardiomyocyte Mechanical Properties of DGK-TG Mice at Basal Condition
To characterize mouse phenotype, all experiments were performed with age- and sex-matched (8- to 10-week-old) DGK-TG and WT littermate mice. Body weight, blood pressure, and heart rate were similar between DGK-TG and WT mice (Table 1). There was no evidence of fibrosis on microscopic examinations of multiple histological sections (data not shown). The absolute heart weight, ratio of heart to body weight, and ratio of the left ventricle to body weight were not different between DGK-TG and WT mice (Table 1). Echocardiography demonstrated that cardiac dimensions, wall thickness, and fractional shortening were normal in DGK-TG mice, as shown in Table 1. Isolated cardiomyocyte mechanical properties and Ca²⁺ transients were examined with the use of 6 WT mice and 6 DGK-TG-High mice. Cell shortening (4.10±0.47% versus 4.71±0.49%), time to peak shortening (52±4 versus 54±6 ms), and time to 80% relaxation (105±17 versus 89±17 ms) were not different between DGK-TG-High and WT mice. The amplitude of the Ca²⁺ signal (0.084±0.011 versus 0.077±0.007) and half-life decay of the Ca²⁺ signal (156±13 versus 162±13 ms) were also same between DGK-TG-High and WT mice.

View this table: [in this window] [in a new window]   TABLE 1. Gravimetric Data and In Vivo Cardiac Function of DGK-TG Mice at Basal Condition

Effects of DGK on GPCR Agonist-Induced Activation of DAG-PKC Signaling
DGK-TG and WT mice were assessed with respect to their susceptibility to hypertrophic response to subpressor doses of subcutaneous angiotensin II^2,3 and phenylephrine^31,32 administration. No significant changes in body weight, heart rate, and blood pressure were observed between WT and DGK-TG mice after subcutaneous infusion of angiotensin II or phenylephrine, as shown in Table 2.

View this table: [in this window] [in a new window]   TABLE 2. Changes in Hemodynamic and Gravimetric Parameters in WT and DGK-TG Mice After Angiotensin II or Phenylephrine Infusion

In the present study there were no significant changes in total protein abundance of PKC isoforms after angiotensin II or phenylephrine infusion (data not shown). We have previously demonstrated angiotensin II-induced translocation of PKC isoforms through pathways involving phospholipase C in the guinea pig ex vivo heart.⁵ As shown in Figure 2, the membrane-associated immunoreactivities of PKC and PKC, but not PKCß and PKC, were significantly increased in angiotensin II-treated WT mice compared with saline-infused WT mice (P<0.05). However, angiotensin II-induced translocation of PKC and PKC was blocked in both DGK-TG-High and DGK-TG-Low mice (P<0.05 versus angiotensin II-infused WT mice).

View larger version (55K): [in this window] [in a new window]   Figure 2. Translocation of PKC (A), PKCß (B), PKC (C), and PKC (D) in DGK-TG-High, DGK-TG-Low, and WT mice in response to angiotensin II (Ang II). A subpressor dose of angiotensin II (100 ng/kg per minute) was continuously infused with an osmotic minipump for 14 days.2,3 Membrane/cytosol (M/C) ratio of immunoreactivity was used as an index of PKC activation. Data were analyzed by 2-way ANOVA followed by multiple comparisons with the Bonferroni test and are reported as mean±SD obtained from 6 to 7 mice for each group.

Next, we examined effects of another GPCR agonist, phenylephrine, in TGK-TG mice. Phenylephrine induced translocation of the PKC isoform (P<0.01), but not , ß, and isoforms, in WT mouse hearts, as shown in Figure 3. However, in both DGK-TG-High and DGK-TG-Low mice, translocation of the PKC by phenylephrine was completely blocked (P<0.01 versus phenylephrine-infused WT mice). These data suggested that DGK had an inhibitory effect on GPCR agonist-induced translocation of PKC isoforms in in vivo mouse hearts.

View larger version (51K): [in this window] [in a new window]   Figure 3. Translocation of PKC (A), PKCß (B), PKC (C), and PKC (D) and in DGK-TG-High, DGK-TG-Low, and WT mice in response to phenylephrine (PE). A dose of phenylephrine (20 mg/kg per day) was infused into mice subcutaneously via an osmotic minipump for 3 days.31,32 Membrane/cytosol (M/C) ratio of immunoreactivity was used as an index of PKC activation. Data were analyzed by 2-way ANOVA followed by multiple comparisons with the Bonferroni test and are reported as mean±SD obtained from 6 to 7 mice for each group.

Lipid extracts were then prepared from the left ventricular myocardium, and we quantified myocardial DAG levels in WT and DGK-TG-High mouse hearts (n=6 for each group). At basal condition, DAG levels were similar between WT and DGK-TG-High mice (51±15 versus 66±18 pmol/mg tissue). In WT mouse hearts, myocardial DAG level increased significantly after continuous administration of phenylephrine for 3 days (from 51±15 to 103±27 pmol/mg tissue; P<0.001). On the other hand, this effect of phenylephrine on myocardial DAG levels was completely suppressed in DGK-TG-High mouse hearts (from 66±18 to 34±7 pmol/mg tissue; P=NS). These data suggest that DGK regulates PKC activity by controlling cellular DAG levels.

Effects of DGK on Hypertrophic Programs in Response to GPCR Agonists
Ventricular hypertrophy induced by continuous infusion of angiotensin II or phenylephrine is accompanied by the induction of several specific genes such as ANF.^2,18 As shown in Figure 4, the mRNA expression of ANF was increased in WT mice given angiotensin II and phenylephrine compared with saline-infused WT mice (P<0.01). However, in DGK-TG-High and DGK-TG-Low mice, angiotensin II failed to cause gene induction of ANF (P<0.01). Phenylephrine-induced ANF gene induction was significantly blocked in DGK-TG-High mice (P<0.01), but this inhibitory effect was not statistically significant in DGK-TG-Low mice (P=0.0704). These data suggested that DGK blocked hypertrophic gene induction by angiotensin II and phenylephrine in in vivo mouse hearts.

View larger version (19K): [in this window] [in a new window]   Figure 4. Cardiac ANF gene expressions in DGK-TG-High, DGK-TG-Low, and WT mice in response to angiotensin II (Ang II) (A) and phenylephrine (PE) (B). Mean value in saline-infused WT mice is presented as 1.0. Data were analyzed by 2-way ANOVA followed by multiple comparisons with the Bonferroni test. Data reported are mean±SD obtained from 6 to 8 mice for each group.

As shown in Table 2, heart weight and left ventricular weight corrected for body weight were not significantly different between saline-infused WT and saline-infused DGK-TG mice. Subcutaneous infusion of angiotensin II and phenylephrine caused significant increases in the ratio of heart to body weight and ratio of the left ventricle to body weight in WT mice (P<0.01). However, in both DGK-TG-High and DGK-TG-Low mice, neither angiotensin II nor phenylephrine produced increases in the ratio of heart to body weight and ratio of the left ventricle to body weight (Table 2).

Microscopic observations revealed that no significant difference in cardiomyocyte cross-sectional area was seen between saline-infused WT and saline-infused DGK-TG mice (Figures 5 and 6). In WT mice, cardiomyocyte cross-sectional area was significantly increased by angiotensin II and phenylephrine infusion (P<0.01 and P<0.05, respectively). However, in both DGK-TG-High and DGK-TG-Low mice, neither angiotensin II nor phenylephrine caused increases in cardiomyocyte cross-sectional area (P<0.01 versus angiotensin II-infused WT and P<0.05 versus phenylephrine-infused WT mice). Obvious fibrosis in the myocardium was not observed in DGK-TG and WT mice given angiotensin II or phenylephrine (data not shown). Taken together, these data clearly demonstrated that DGK might interfere with GPCR agonist-induced cardiac hypertrophy.

View larger version (61K): [in this window] [in a new window]   Figure 5. Histological analysis in DGK-TG-High, DGK-TG-Low, and WT mice after angiotensin II (AngII) infusion. Bottom panels show representative images of hematoxylin-eosin micrographs of cardiomyocyte cross sections. (Magnification x200, bar=20 µm). Top bar graph shows quantitative analysis of cardiomyocyte cross-sectional area from the left ventricle. Data were analyzed by 2-way ANOVA followed by multiple comparisons with the Bonferroni test. Data reported are mean±SD obtained from 7 mice for each group.

View larger version (62K): [in this window] [in a new window]   Figure 6. Histological analysis in DGK-TG-High, DGK-TG-Low, and WT mice after phenylephrine (PE) infusion. Bottom panels show representative images of hematoxylin-eosin micrographs of cardiomyocyte cross sections. (magnification x200, bar=20 µm). Top bar graph shows quantitative analysis of cardiomyocyte cross-sectional area from the left ventricle. Data were analyzed by 2-way ANOVA followed by multiple comparisons with the Bonferroni test. Data are reported as mean±SD obtained from 7 mice for each group.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This is the first report characterizing a functional role of DGK in the in vivo mouse heart. Molecular, gravimetric, and morphological analyses clearly showed that cardiac-specific overexpression of DGK abrogated cardiac hypertrophy in response to GPCR agonists such as angiotensin II and phenylephrine by regulating the DAG-PKC signaling in transgenic mouse hearts.

DGK-TG mice were indistinguishable in appearance from WT mice, and no baseline cardiac effects were observed in physiological or histological analyses. In addition, we found that isolated cardiomyocyte function and in vivo cardiac function evaluated by echocardiography were normal in DGK-TG mice. Blood pressure and heart rate after subcutaneous infusion of angiotensin II and phenylephrine were not different among DGK-TG-High, DGK-TG-Low, and WT mice, indicating that the overexpression of DGK does not affect hemodynamic regulations in response to angiotensin II and phenylephrine. Consistent with previous works,^2,3,31,32 the hypertrophic response in this study occurred independently of the hemodynamic effects of angiotensin II and phenylephrine because systemic blood pressure was not elevated after infusion.

Previous studies in human heart failure and animal models of heart failure have clearly demonstrated that activation of PKC isoforms plays a critical role in the development of cardiac hypertrophy and progression to heart failure.^5,6,10–12 GPCR agonists increase production of DAG, resulting in sustained PKC activation in the cardiomyocyte.^7,33 DGK is an enzyme that is responsible for controlling the cellular levels of DAG by converting it to phosphatidic acid, and thus is thought to be acting as an endogenous regulator of PKC activity. Continuous infusion of phenylephrine for 3 days increased myocardial DAG levels in WT mice, but this effect was completely abolished by overexpression of DGK. These data suggest that DGK regulates PKC activity by controlling cellular DAG levels.

Phosphatidic acid is yielded not only by DGK but also by the action of phospholipase D. Phospholipase D hydrolyzes phosphatidylcholine to form phosphatidic acid, and phosphatidic acid itself also has signaling function, stimulates DNA synthesis, and modulates activity of several enzymes, including phosphatidylinositol 5-kinases, ERK, and others.³⁴ However, because the bulk of the signaling pool of phosphatidic acid is mainly derived from the action of phospholipase D in cardiomyocytes,³⁵ overexpression of DGK may not affect phosphatidic acid pool and its signaling function. We previously demonstrated that activation of downstream ERK and protein synthesis by endothelin-1 were abolished by DGK in isolated neonatal rat cardiomyocytes.¹⁸ These results suggested the importance of inhibiting the DAG-PKC signaling pathway by DGK to prevent cardiomyocyte hypertrophy.

The in vitro studies have reported that DGK isoforms modulate the DAG-PKC signaling in several types of cells.^36,37 In particular, Luo et al³⁸ have recently showed that DGK spatially regulates PKC activity by attenuating local accumulation of DAG in HEK293 cells. It has been reported that the DGK isoform reduces cellular DAG levels in aortic endothelial cells.³⁶ We have recently found that adenovirus-mediated overexpression of the DGK isoform blocked endothelin-1–induced activation of the DAG-PKC signaling and resultant cardiomyocyte hypertrophy in cultured rat neonatal cardiomyocytes.¹⁸ To further elucidate these issues obtained from in vitro studies, isoform-specific regulation of DGK in an in vivo study with the use of a transgenic approach, as we employed in the present study, is necessary. In our present study, angiotensin II- and phenylephrine-induced translocation of PKC was blocked in DGK-TG mice (Figures 2 and 3). These data suggest that DGK has an inhibitory effect on PKC translocation, which depends on kinase activity, in the left ventricular myocardium. Furthermore, angiotensin II- and phenylephrine-induced hypertrophic programs determined by gene induction of ANF, increases in heart weight, and enlargements of cardiomyocyte surface area were abolished in DGK-TG mice (Table 2 and Figures 4 to 6). These data suggest that DGK functions as a negative regulator of the DAG-PKC signaling and prevents subsequent cardiomyocyte hypertrophy. Because this study used only the overexpression approach, future experiments of a loss of DGK function with the use of knockout mice are necessary to elucidate further the role of DGK in signaling cascade in vivo.

In conclusion, our study provides the first in vivo evidence that DGK blocks cardiac hypertrophy in response to GPCR agonists by regulating DAG-PKC signaling. Further studies will be necessary to examine whether and to what extent DGK may prevent pressure overload-induced cardiac hypertrophy.

	Acknowledgments

 
This study was supported in part by a grant-in-aid for scientific research (No. 17590702) from the Ministry of Education, Science, Sports, and Culture, Japan; a grant-in-aid from the 21st Century Center of Excellence (COE) program of the Japan Society for the Promotion of Science; and grants from the Japan Heart Foundation, the Mochida Memorial Foundation, and Takeda Science Foundation. We thank Shuku Takahashi and Sachi Adachi for their excellent technical assistance.

Disclosures

None.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med. 1990; 322: 1561–1566.[Abstract]
   
2. Harada K, Komuro I, Shiojima I, Hayashi D, Kudoh S, Mizuno T, Kijima K, Matsubara H, Sugaya T, Murakami K, Yazaki Y. Pressure overload induces cardiac hypertrophy in angiotensin II type 1A receptor knockout mice. Circulation. 1998; 97: 1952–1959.[Abstract/Free Full Text]
   
3. Schultz Jel J, Witt SA, Glascock BJ, Nieman ML, Reiser PJ, Nix SL, Kimball TR, Doetschman T. TGF-ß1 mediates the hypertrophic cardiomyocyte growth induced by angiotensin II. J Clin Invest. 2002; 109: 787–796.[CrossRef][Medline] [Order article via Infotrieve]
   
4. Izumiya Y, Kim S, Izumi Y, Yoshida K, Yoshiyama M, Matsuzawa A, Ichijo H, Iwao H. Apoptosis signal-regulating kinase 1 plays a pivotal role in angiotensin II-induced cardiac hypertrophy and remodeling. Circ Res. 2003; 93: 874–883.[Abstract/Free Full Text]
   
5. Takeishi Y, Jalili T, Ball NA, Walsh RA. Responses of cardiac protein kinase C isoforms to distinct pathological stimuli are differentially regulated. Circ Res. 1999; 85: 264–271.[Abstract/Free Full Text]
   
6. Hunter JJ, Chien KR. Signaling pathways for cardiac hypertrophy and failure. N Engl J Med. 1999; 341: 1276–1283.[Free Full Text]
   
7. Sadoshima J, Izumo S. Signal transduction pathways of angiotensin II-induced c-fos gene expression in cardiac myocytes in vitro: roles of phospholipid-derived second messengers. Circ Res. 1993; 73: 424–438.[Abstract/Free Full Text]
   
8. Shubeita HE, McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Brown JH, Chien KR. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes: a paracrine mechanism for myocardial cell hypertrophy. J Biol Chem. 1990; 265: 20555–20562.[Abstract/Free Full Text]
   
9. Otani H, Otani H, Das DK. 1-Adrenoceptor-mediated phosphoinositide breakdown and inotropic response in rat left ventricular papillary muscles. Circ Res. 1988; 62: 8–17.[Abstract/Free Full Text]
   
10. Takeishi Y, Chu G, Kirkpatrick DL, Wakasaki H, Li Z, Kranias EG, King GL, Walsh RA. In vivo phosphorylation of cardiac troponin I by PKCß2 decreases cardiomyocyte calcium responsiveness and contractility in transgenic mouse heart. J Clin Invest. 1998; 102: 72–78.[Medline] [Order article via Infotrieve]
    
11. Bowling N, Walsh RA, Song G, Estridge T, Sandusky GE, Fouts RL, Mintze K, Pickard T, Roden R, Bristow MR, Sabbah HN, Mizrahi JL, Gromo G, King GL, Vlahos CJ. Increased protein kinase C activity and expression of Ca²⁺-sensitive isoforms in the failing human heart. Circulation. 1999; 99: 384–391.[Abstract/Free Full Text]
    
12. Takeishi Y, Ping P, Bolli R, Kirkpatrick DL, Hoit BD, Walsh RA. Transgenic overexpression of constitutively active protein kinase C- causes concentric cardiac hypertrophy. Circ Res. 2000; 86: 1218–1223.[Abstract/Free Full Text]
    
13. Goto K, Kondo H. A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus. Proc Natl Acad Sci U S A. 1996; 93: 11196–11201.[Abstract/Free Full Text]
    
14. Topham MK, Bunting M, Zimmerman GA, McIntyre TM, Blackshear PJ, Prescott SM. Protein kinase C regulates the nuclear localization of diacylglycerol kinase-. Nature. 1998; 394: 697–700.[CrossRef][Medline] [Order article via Infotrieve]
    
15. Topham MK, Prescott SM. Mammalian diacylglycerol kinases, a family of lipid kinases with signaling functions. J Biol Chem. 1999; 274: 11447–11450.[Free Full Text]
    
16. Goto K, Kondo H. Diacylglycerol kinase in the central nervous system: molecular heterogeneity and gene expression. Chem Phys Lipids. 1999; 98: 109–117.[CrossRef][Medline] [Order article via Infotrieve]
    
17. Takeda M, Kagaya Y, Takahashi J, Sugie T, Ohta J, Watanabe J, Shirato K, Kondo H, Goto K. Gene expression and in situ localization of diacylglycerol kinase isozymes in normal and infarcted rat hearts: effects of captopril treatment. Circ Res. 2001; 89: 265–272.[Abstract/Free Full Text]
    
18. Takahashi H, Takeishi Y, Seidler T, Arimoto T, Akiyama H, Koyama Y, Shishido T, Tsunoda Y, Niizeki T, Hozumi Y, Abe J, Hasenfuss G, Goto K, Kubota I. Adenovirus-mediated overexpression of diacylglycerol kinase inhibits endothelin-1–induced cardiomyocyte hypertrophy. Circulation. 2005; 111: 1510–1516.[Abstract/Free Full Text]
    
19. Arimoto T, Takahashi H, Shishido T, Niizeki T, Koyama Y, Nakajima O, Goto K, Takeishi Y. Cardiac-specific overexpression of diacylglycerol prevents angiotensin II-induced cardiac hypertrophy in transgenic mice. Circulation. 2004; 110: III-226.(Abstract)
    
20. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A. 1980; 77: 7380–7384.[Abstract/Free Full Text]
    
21. Goto K, Kondo H. Molecular cloning and expression of a 90-kDa diacylglycerol kinase that predominantly localizes in neurons. Proc Natl Acad Sci U S A. 1993; 90: 7598–7602.[Abstract/Free Full Text]
    
22. Goto K, Funayama M, Kondo H. Cloning and expression of a cytoskeleton-associated diacylglycerol kinase that is dominantly expressed in cerebellum. Proc Natl Acad Sci U S A. 1994; 91: 13042–13046.[Abstract/Free Full Text]
    
23. Takeishi Y, Abe J, Lee JD, Kawakatsu H, Walsh RA, Berk BC. Differential regulation of p90 ribosomal S6 kinase and big mitogen-activated protein kinase-1 by ischemia/reperfusion and oxidative stress in perfused guinea pig hearts. Circ Res. 1999; 85: 1164–1172.[Abstract/Free Full Text]
    
24. Takeishi Y, Huang Q, Abe J, Glassman M, Che W, Lee JD, Kawakatsu H, Lawrence EG, Hoit BD, Berk BC, Walsh RA. Src and multiple MAP kinase activation in cardiac hypertrophy and congestive heart failure under chronic pressure-overload: comparison with acute mechanical stretch. J Mol Cell Cardiol. 2001; 33: 1637–1648.[CrossRef][Medline] [Order article via Infotrieve]
    
25. Takahashi H, Takeishi Y, Miyamoto T, Shishido T, Arimoto T, Konta T, Miyashita T, Ito M, Kubota I. Protein kinase C and extracellular signal regulated kinase are involved in cardiac hypertrophy of rats with progressive renal injury. Eur J Clin Invest. 2004; 34: 85–93.[CrossRef][Medline] [Order article via Infotrieve]
    
26. Shishido T, Nozaki N, Yamaguchi S, Shibata Y, Nitobe J, Miyamoto T, Takahashi H, Arimoto T, Maeda K, Yamakawa M, Takeuchi O, Akira S, Takeishi Y, Kubota I. Toll-like receptor-2 modulates ventricular remodeling after myocardial infarction. Circulation. 2003; 108: 2905–2910.[Abstract/Free Full Text]
    
27. Nozaki N, Shishido T, Takeishi Y, Kubota I. Modulation of doxorubicin-induced cardiac dysfunction in toll-like receptor-2 knockout mice. Circulation. 2004; 110: 2869–2874.[Abstract/Free Full Text]
    
28. Kubota I, Tomoike H, Han X, Sakurai K, Endoh M. The Na⁺-Ca²⁺ exchanger contributes to ß-adrenoceptor mediated positive inotropy in mouse heart. Jpn Heart J. 2002; 43: 399–407.[CrossRef][Medline] [Order article via Infotrieve]
    
29. Bendall JK, Cave AC, Heymes C, Gall N, Shah AM. Pivotal role of a gp91(phox)-containing NADPH oxidase in angiotensin II-induced cardiac hypertrophy in mice. Circulation. 2002; 105: 293–296.[Abstract/Free Full Text]
    
30. Paterson A, Plevin R, Wakelam MJO. Accurate measurement of sn-1,2-diradyl-glycerol mass in cell lipid extracts. Biochem J. 1991; 280: 829–836.[Medline] [Order article via Infotrieve]
    
31. Asakura M, Kitakaze M, Takashima S, Liao Y, Ishikura F, Yoshinaka T, Ohmoto H, Node K, Yoshino K, Ishiguro H, Asanuma H, Sanada S, Matsumura Y, Takeda H, Beppu S, Tada M, Hori M, Higashiyama S. Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy. Nat Med. 2002; 8: 35–40.[CrossRef][Medline] [Order article via Infotrieve]
    
32. Roman BB, Geenen DL, Leitges M, Buttrick PM. PKC-beta is not necessary for cardiac hypertrophy. Am J Physiol. 2001; 280: H2264–2270.
    
33. Ohanian J, Ollerenshaw J, Collins P, Heagerty A. Agonist-induced production of 1,2-diacylglycerol and phosphatidic acid in intact resistance arteries: evidence that accumulation of diacylglycerol is not a prerequisite for contraction. J Biol Chem. 1990; 265: 8921–8928.[Abstract/Free Full Text]
    
34. Dhalla NS, Xu YJ, Sheu SS, Tappia PS, Panagia V. Phosphatidic acid: a potential signal transducer for cardiac hypertrophy. J Mol Cell Cardiol. 1997; 29: 2865–2871.[CrossRef][Medline] [Order article via Infotrieve]
    
35. Exton JH. Phospholipase D: enzymology, mechanisms of regulation, and function. Physiol Rev. 1997; 77: 303–320.[Abstract/Free Full Text]
    
36. Pettitt TR, Wakelam MJ. Diacylglycerol kinase , but not , selectively removes polyunsaturated diacylglycerol, inducing altered protein kinase C distribution in vivo. J Biol Chem. 1999; 274: 36181–36186.[Abstract/Free Full Text]
    
37. Verrier E, Wang L, Wadham C, Albanese N, Hahn C, Gamble JR, Chatterjee VK, Vadas MA, Xia P. PPAR agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway: role of diacylglycerol kinase. Circ Res. 2004; 94: 1515–1522.[Abstract/Free Full Text]
    
38. Luo B, Prescott SM, Topham MK. Association of diacylglycerol kinase with protein kinase C : spatial regulation of diacylglycerol signaling. J Cell Biol. 2003; 160: 929–937.[Abstract/Free Full Text]
    

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