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.)
Department of Medicine, Case Western Reserve University, Cleveland, Ohio (R.A.W.).
Abstract
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
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
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.
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, , , 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 previously26,27 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
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.
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 II2,3 and phenylephrine31,32 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.
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 PKC 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).
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 , , 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.
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.
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.
Discussion
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.
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Glucose-6 Phosphate Dehydrogenase Deficiency Decreases the Vascular Response to Angiotensin II
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Pharmacogenetic Association of the Angiotensin-Converting Enzyme Insertion/Deletion Polymorphism on Blood Pressure and Cardiovascular Risk in Relation to Antihypertensive Treatment
Persistent Cardiac Aldosterone Synthesis in Angiotensin II Type 1A Receptor–Knockout Mice After Myocardial Infarction