AJP - Heart Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H1105-H1112, 2007. First published October 27, 2006; doi:10.1152/ajpheart.00927.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/2/H1105    most recent
00927.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Niizeki, T.
Right arrow Articles by Kubota, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Niizeki, T.
Right arrow Articles by Kubota, I.

Cardiac-specific overexpression of diacylglycerol kinase {zeta} attenuates left ventricular remodeling and improves survival after myocardial infarction

Takeshi Niizeki,1 Yasuchika Takeishi,1 Takanori Arimoto,1 Hiroki Takahashi,1 Tetsuro Shishido,1 Yo Koyama,1 Kaoru Goto,2 Richard A. Walsh,3 and Isao Kubota1

Departments of 1Cardiology, Pulmonology, and Nephrology, and 2Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata, Japan; and 3Department of Medicine, Case Western Reserve University, Cleveland, Ohio

Submitted 25 August 2006 ; accepted in final form 17 October 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Left ventricular (LV) remodeling, including cardiomyocyte necrosis, scar formation, LV geometric changes, and cardiomyocyte hypertrophy, contributes to cardiac dysfunction and mortality after myocardial infarction (MI). Although precise cellular signaling mechanisms for LV remodeling are not fully elucidated, Gq protein-coupled receptor signaling pathway, including diacylglycerol (DAG) and PKC, are involved in this process. DAG kinase (DGK) phosphorylates DAG and controls cellular DAG levels, thus acting as a negative regulator of PKC and subsequent cellular signaling. We previously reported that DGK inhibited angiotensin II and phenylephrine-induced activation of the DAG-PKC signaling and subsequent cardiac hypertrophy. The purpose of this study was to examine whether DGK modifies LV remodeling after MI. Left anterior descending coronary artery was ligated in transgenic mice with cardiac-specific overexpression of DGK{zeta} (DGK{zeta}-TG) and wild-type (WT) mice. LV chamber dilatation (4.12 ± 0.10 vs. 4.53 ± 0.32 mm, P < 0.01), reduction of LV systolic function (34.8 ± 8.3% vs. 28.3 ± 4.8%, P < 0.01), and increases in LV weight (95 ± 3.6 vs. 111 ± 4.1 mg, P < 0.05) and lung weight (160 ± 15 vs. 221 ± 25 mg, P < 0.05) at 4 wk after MI were attenuated in DGK{zeta}-TG mice compared with WT mice. In the noninfarct area, fibrosis fraction (0.51 ± 0.04, P < 0.01) and upregulation of profibrotic genes, such as transforming growth factor-beta1 (P < 0.01), collagen type I (P < 0.05), and collagen type III (P < 0.01), were blocked in DGK{zeta}-TG mice. The survival rate at 4 wk after MI was higher in DGK{zeta}-TG mice than in WT mice (61% vs. 37%, P < 0.01). In conclusion, these results demonstrate the first evidence that DGK{zeta} suppresses LV structural remodeling and fibrosis and improves survival after MI. DGK{zeta} may be a potential novel therapeutic target to prevent LV remodeling after MI.

protein kinase C; cardiac hypertrophy; signal transduction

MYOCARDIAL INFARCTION (MI) induces left ventricular (LV) remodeling, including myocyte necrosis, thinning of the infarcted myocardium, dilatation of the ventricular cavity, eccentric myocardial hypertrophy, interstitial fibrosis, etc. (22). Early changes in the LV architecture after MI are compensatory phenomena of the heart to adapt the consequences of loss of functional myocardium and initially preserve cardiac performance. However, when these processes develop once after large MI, the infarcted heart progressively dilates and cardiac fibrosis is markedly formed in the noninfarcted area. These progressive changes in LV geometry, including infarct expansion, ventricular dilation, and fibrosis of viable myocardium, are all maladaptive processes that contribute to LV dysfunction, progression to congestive heart failure, and increased mortality after MI (15, 22, 23). Indeed, patients who escape death during the acute stage of MI are at high risk of developing heart failure during the chronic stage (14). Thus LV remodeling not only compensates for the deficiency in function of the infarcted region but also may play a pivotal role in the prognosis after MI. Accordingly, it is important to elucidate the mechanisms and to develop therapeutic strategies that will effectively suppress this adverse process.

Although the cellular signaling and molecular mechanisms of LV remodeling are not fully elucidated, Gq protein-coupled receptor signaling pathway, including PKC, plays a critical role in this process. The binding of diacylglycerol (DAG) to the C1 domain of PKC induces an active conformation, and activated PKC phosphorylates several myocardial proteins, leading to myocardial hypertrophy, cell growth, differentiation, and heart failure. We and others have previously demonstrated that PKC plays an important role in the development of cardiac hypertrophy and progression to heart failure (2, 31, 33, 36). On the other hand, DAG kinase (DGK) is an enzyme that is responsible for controlling the cellular level of DAG by converting it to phosphatidic acid (9, 10, 24, 30, 34). We recently demonstrated in cultured rat neonatal cardiomyocytes that adenoviral-mediated overexpression of DGK{zeta} blocked endothelin-1-induced increases in protein synthesis concomitant with increases in cell size and reactivation of fetal genes via the inhibition of PKC-{epsilon}-ERK-activator protein-1 signaling pathway (29). Furthermore, we have generated transgenic mice with cardiac-specific overexpression of DGK{zeta} (DGK{zeta}-TG) using an {alpha}-myosin heavy chain (MHC) promoter and demonstrated that DGK{zeta} prevents angiotensin II and phenylephrine-induced activation of the DAG-PKC downstream signaling cascades and subsequent cardiac hypertrophy (1). These results suggest that DGK may play a pivotal role in the process of ventricular remodeling after MI.

In the present study, we tested the hypothesis that DGK{zeta} attenuates LV remodeling after MI. We created MI by ligation of the left anterior descending coronary artery and compared cardiac function, translocation of PKC, gene expression, fibrosis, and survival between DGK{zeta}-TG and wild-type (WT) mice.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. DGK{zeta}-TG mice were created in our institution as previously reported (1), and DGK{zeta}-TG (line 70) mice and WT littermates 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 specifically 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.

Surgery of left anterior descending coronary artery ligation. Induction of MI was performed as described previously in 8- to 10-wk-old mice with minor modifications (6, 12, 25). Briefly, mice (~20 to 25 g body wt) were anesthetized by intraperitoneal injection with a mixture of ketamine (80 mg/kg) and xylazine (8 mg/kg). Animals were intubated with a 20-gauge polyethylene catheter and were ventilated with a rodent ventilator (Harvard Apparatus, Holliston, MA). An incision was performed along the left sternal border, and the fourth rib was cut proximal to the sternum. The left anterior descending coronary artery was identified, and an 8-0 proline suture was passed around the artery and subsequently tied off (6, 12, 25). Successful ligation of the coronary artery was verified visually by the discoloration of the LV myocardium. In sham-operated animals, the same procedure was performed except the coronary artery ligation. Finally, the heart was repositioned in the chest, and the chest wall was closed. The animals remained in a supervised setting until fully conscious.

After surgery, mice were inspected at least three times daily to determine the cause of death in each animal found dead. The presence of a large amount of blood clot around the heart and within the chest cavity in the postmortem examination, as well as a perforation of the infarcted myocardial wall, indicated death by cardiac rupture (6, 7). Death was considered due to congestive heart failure in mice with the following signs: presence of pulmonary congestion (increased lung weight) and massive chest fluid accumulation as described previously (6, 7).

Hemodynamic measurements and echocardiography. Heart rate (in beats/min) and blood pressure (in mmHg) were determined in the conscious state at baseline, 1 wk, and 4 wk after surgery using a computerized tail-cuff manometer (MK-1030, Muromachi Kikai, Tokyo, Japan) as reported previously (33).

Transthoracic echocardiography was recorded as described previously with an FFsonic 8900 (Fukuda Denshi, Tokyo, Japan) equipped with a 13-MHz phased-array transducer at baseline, 1 wk, and 4 wk after surgery (12, 21, 25). LV internal dimensions at end systole and end diastole (LVESD and LVEDD) and LV posterior wall thickness were measured digitally on the M-mode tracings and averaged from at least three cardiac cycles (12, 21, 25). LV fractional shortening (LVFS) was calculated as [(LVEDD – LVESD)/LVEDD] x 100.

Morphological examination and infarct size measurement. At 4 wk after surgery, mice were euthanized, coronary arteries were retrogradely flushed with saline, and the heart and lungs 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 (21, 25). Three sections were stained with hematoxylin-eosin or van Gieson stain. The infarct size was calculated and expressed as a percentage of the total LV wall circumference from each of the three LV sections as previously described (6, 25). Animals with <30% of the infarct size were excluded from the analysis because they did not show typical LV remodeling (12, 25). Transverse sections were captured digitally, and cardiomyocyte cross-sectional area was measured by using a Scion imaging system (Scion, Frederick, MD) (25, 38). We traced the outline of at least 200 cardiomyocytes in each section, and the data were averaged.

To assess the degree of fibrosis in the noninfarct area, the sections stained with van Gieson stain were scanned with computer-assisted videodensitometry, and the images from at least 10 fields for each heart were analyzed as described previously (1, 25). The fibrosis fraction was obtained by calculating the ratio of van Gieson-stained connective tissue area (stained red) to total myocardial area with an image analysis software as described previously (25, 38).

Separation of membranous and cytosolic fractions and Western blot analysis. Membranous and cytosolic fractions of detergent-extracted PKC were prepared from the noninfarction area of the LV myocardium as described previously (28, 31, 32). Protein concentration of myocardial samples was carefully determined by the protein assay, and equal amounts of protein extracts from membranous and cytosolic fractions were loaded on each gel lane. Equal amounts of membranous and cytosolic protein were subjected to 10% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membranes. To ensure equivalent protein loading and quantitative transfer efficiency of proteins, membranes were stained with Ponceau S before incubating with primary antibodies. Subcellular localization of PKC isoforms was examined by quantitative immunoblotting using isoform-specific antibodies (mouse monoclonal anti-PKC-{alpha}, -beta, -{delta}, and -{epsilon}, Santa Cruz Biotechnology, Santa Cruz, CA) as reported previously (28, 31, 32). Immunoreactive bands were detected by an 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 (28, 31, 32).

Extraction of RNA and real-time RT-PCR. Total RNAs were extracted from the noninfarction area of 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 (6, 25, 29). To examine mRNA expression levels of atrial natriuretic factor (ANF), beta-MHC, brain natriuretic peptide (BNP), transforming growth factor-beta1 (TGF-beta1), collagen type I, and collagen type III, real-time PCR amplification was performed as reported previously (25, 29). Reagents were purchased from Roche Diagnostics Japan (Tokyo, Japan). Amplification was performed by using LightCycler DNA Master SYBR Green I in a 20-µl volume reaction and analyzed by using LightCycler software v. 3.5 (Roche Diagnostics Japan). Standard curves of these genes were generated by full sequence plasmid of known concentrations. Gene expressions were normalized to GAPDH. Primers were designed based on GenBank sequences (ANF, K-02781; beta-MHC, AY-056464; BNP, NM-008726; TGF-beta1, NM-011577; collagen type I, NM-007742; collagen type III, NM-009930; and GAPDH, NM-001001303).

Statistical analysis. All values are reported as means ± SE. Effects of MI on histological data, PKC translocation, body weight, LV weight, lung weight, LVEDD, LVESD, LVFS, and RT-PCR data between WT and 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 after MI 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
 
LV remodeling after MI. There were no differences in body weight, systolic blood pressure, and heart rate at 4 wk after MI between DGK{zeta}-TG and WT mice (Table 1). Heart weight and LV weight corrected for body weight were not different between sham-operated WT and sham-operated DGK{zeta}-TG mice. At 4 wk after MI, the ratio of heart weight to body weight and the ratio of LV weight to body weight were significantly lower in DGK{zeta}-TG mice than in WT mice (P < 0.01 and P < 0.01, respectively) as shown in Table 1. Lung weight and right ventricular weight at 4 wk after MI were also significantly lower in DGK{zeta}-TG mice than in WT mice (P < 0.05 and P < 0.01, respectively). Infarct size at 4 wk after coronary ligation was similar between WT and DGK{zeta}-TG mice as shown in Fig. 1 (52.7 ± 3.0% and 48.0 ± 2.7%, P = 0.3793).


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

  		Table 1. Gravimetric data in DGK{zeta}-TG and WT mice at 4 wk after surgery

 

Figure 1
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 1. Histological analyses of the left ventricular (LV) myocardium in transgenic mice with cardiac-specific overexpression of diacylglycerol kinase (DGK{zeta}-TG) and wild-type (WT) mice at 4 wk after myocardial infarction (MI). LV transverse sections were stained by van Gieson. The infarct size, expressed as a percentage of the total LV wall circumference, at 4 wk after MI was similar between WT and DGK{zeta}-TG mice (52.5 ± 9.5% vs. 48.1 ± 7.3%, P = 0.3217). Data were obtained from 10 to 12 mice for each group.

 
Echocardiographic measurements. Echocardiography was performed at baseline, 1 wk, and 4 wk after surgery in WT and DGK{zeta}-TG mice. While mice were under anesthesia, the heart rate was found to be similar between WT and DGK{zeta}-TG mice at each point (data not shown). The representative M-mode echocardiograms of WT and DGK{zeta}-TG mice at baseline, 1 wk, and 4 wk after surgery are shown in Fig. 2A. As shown in Fig. 2 and Table 2, before surgery, LVEDD, LVFS, and other echocardiographic parameters were similar between WT and DGK{zeta}-TG mice. LVEDD was significantly larger in WT MI mice than in sham-operated WT mice at 1 wk (P ≤ 0.01), and LVEDD was further dilated at 4 wk after MI as shown in Fig. 2. However, ventricular dilatation after MI was prevented in DGK{zeta}-TG mice compared with WT mice (3.54 ± 0.13 vs. 4.01 ± 0.15 mm, P ≤ 0.05 at 1 wk; and 4.12 ± 0.03 vs. 4.53 ± 0.11 mm, P ≤ 0.01 at 4 wk). Moreover, the reduction of LVFS after MI was attenuated in DGK{zeta}-TG mice compared with WT mice at 4 wk after MI (LVFS: 34.8 ± 2.4% vs. 28.3 ± 1.3%, P ≤ 0.01). As shown in Table 2, LV dysfunction and remodeling after MI were significantly attenuated in DGK{zeta}-TG mice compared with WT mice.


Figure 2
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 2. A: representative M-mode echocardiograms of WT and DGK{zeta}-TG mice at baseline, 1 wk (1 W), and 4 wk (4 W) after MI. AW, anterior wall; PW, posterior wall; and LVD or LVEDD, LV end-diastolic dimension. B: quantitative data for echocardiographic measurements. All data were obtained from 12–15 animals in each group. Although LVEDD was increased in both WT and DGK{zeta}-TG mice after MI, LVEDD in DGK{zeta}-TG mice was significantly smaller than that in WT mice. Although LV fractional shortening (LVFS) was reduced in both WT and DGK{zeta}-TG mice after MI, LVFS in DGK{zeta}-TG mice was significantly higher than that in WT mice at 4 wk after MI. **P < 0.01 vs. sham-operated mice with same strain. TG, DGK{zeta}-TG mice.

 

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

  		Table 2. Echocardiographic data in DGK{zeta}-TG and WT mice at 4 wk after surgery

 
Translocation of PKC isoforms, expressions of fetal genes, and cardiomyocyte hypertrophy. We examined translocation of PKC isoforms in the noninfarct area of DGK{zeta}-TG and WT mice. As shown in Fig. 3, we detected translocation of PKC-{alpha}, -beta, -{delta}, and -{epsilon} isoforms at 4 wk after MI in the noninfarct area of WT mouse hearts. However, in DGK{zeta}-TG mice, translocation of PKC-{alpha} and -{epsilon}, but not -beta and -{delta}, were significantly attenuated as shown in Fig. 3 (P < 0.01 and P < 0.01 vs. WT MI, respectively).


Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 3. Translocation of PKC-{alpha} (A), PKC-beta (B), PKC-{delta} (C), and PKC-{epsilon} (D) in the noninfarct area of DGK{zeta}-TG and WT mice at 4 wk after MI. Membrane-to-cytosol (M/C) ratio of immunoreactivity was used as an index of PKC activation. Translocation of PKC-{alpha}, -beta, -{delta}, and -{epsilon} isoforms was detected in WT mouse hearts after MI. However, in DGK{zeta}-TG mice, translocation of PKC-{alpha} and -{epsilon} isoforms, but not -beta and -{delta}, was attenuated significantly compared with that in WT mice. Data are reported as means ± SE obtained from 8 mice for each group. **P < 0.01 vs. sham-operated mice with same strain.

 
We next examined mRNA expressions of fetal type genes, such as ANF, beta-MHC, and BNP, in the noninfarct area at 4 wk after MI in WT and DGK{zeta}-TG mice. Expressions of ANF, beta-MHC, and BNP were significantly upregulated in WT MI mice compared with WT sham-operated mice as demonstrated in Fig. 4. Conversely, in DGK{zeta}-TG mouse hearts, gene induction of ANF, beta-MHC, and BNP in response to MI was significantly attenuated compared with that in WT mice as shown in Fig. 4.


Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 4. Quantitative analyses of atrial natriuretic factor (ANF; A), beta-myosin heavy chain (beta-MHC; B), and brain natriuretic peptide (BNP; C) gene expressions by real-time PCR in DGK{zeta}-TG and WT mice at 4 wk after MI. Data of ANF, beta-MHC, and BNP were normalized by GAPDH. Expressions of ANF, beta-MHC, and BNP were increased in the noninfarct area of WT mice after MI. However, increases in gene expressions were significantly attenuated in DGK{zeta}-TG mice compared with WT mice. **P ≤ 0.01 vs. sham-operated WT mice. Data are reported as means ± SE obtained from 8 mice for each group.

 
Microscopic observations revealed that no significant difference in cardiomyocyte cross-sectional area was seen between sham-operated WT (216 ± 12 µm2) and DGK{zeta}-TG (224 ± 13 µm2) mice (Fig. 5). Cardiomyocyte cross-sectional area in the noninfarct area was profoundly increased in WT mice at 4 wk after MI. However, increases in cardiomyocyte cross-sectional area after MI were significantly attenuated in DGK{zeta}-TG mice compared with WT mice (311 ± 19 vs. 459 ± 42 µm2, P < 0.01).


Figure 5
View larger version (62K):
[in this window]
[in a new window]

  		Fig. 5. Histological analyses in DGK{zeta}-TG and WT mice after MI. Left: quantitative analysis of cardiomyocyte cross-sectional area isolated from the noninfarction area of the left ventricle. Data are reported as means ± SE obtained from 10–12 mice for each group. **P < 0.01 vs. sham-operated WT mice. Right: hematoxylin-eosin micrographs of cardiomyocyte cross sections (magnification, x400; bar = 20 µm).

 
Myocardial fibrosis in the noninfarct area and expressions of profibrotic genes. Since reactive interstitial and perivascular fibrosis in the noninfarcted area adversely alters myocardial stiffness and ultimately leads to LV dysfunction (12, 28), we examined myocardial fibrosis in the noninfarct area at 4 wk after MI in WT and DGK{zeta}-TG mice using sections stained with van Gieson stain. In the noninfarct area, prominent perivascular and interstitial fibrosis was observed in WT mice at 4 wk after MI as reported in previous studies (12, 25). However, the degree of myocardial fibrosis at the noninfarct area was much less in DGK{zeta}-TG mice than in WT mice at 4 wk after MI as shown in Fig. 6, A and B (fibrosis fraction, 0.51 ± 0.04 vs. 1.00 ± 0.09, P < 0.01). These results indicated that, although coronary ligation caused fibrous formation in the infarct area and the size of MI was similar between WT and DGK{zeta}-TG mouse hearts, reactive fibrosis in the noninfarct area was significantly attenuated by DGK{zeta}.


Figure 6
View larger version (52K):
[in this window]
[in a new window]

  		Fig. 6. Histological analyses in DGK{zeta}-TG and WT mice at 4 wk after MI. A: representative sections stained with van Gieson stain (magnification, x400; bar = 20 µm) in the noninfarct area of WT and DGK{zeta}-TG mice after MI. B: comparison of the fibrosis fraction between DGK{zeta}-TG and WT mice. The fibrosis fraction was calculated as the ratio of van Gieson-stained connective tissue area (stained red) to total myocardial area. In DGK{zeta}-TG mice, myocardial fibrosis at 4 wk after MI was significantly attenuated compared with that in WT mice. Data are means ± SE obtained from 10–12 mice for each group. C: quantitative analyses of transforming growth factor-beta1 (TGF-beta1), collagen type I, and collagen type III gene expressions in the noninfarct area of WT and DGK{zeta}-TG mice at 4 wk after MI. Expressions of these genes were normalized by GAPDH. Expressions of TGF-beta1, collagen type I, and collagen type III mRNA were increased in WT mice after MI. However, upregulation of these gene expressions was significantly attenuated in DGK{zeta}-TG mice compared with WT mice. **P < 0.01 vs. sham-operated WT mice. Data were obtained from 8 mice for each group.

 
We next examined expressions of profibrotic genes, such as TGF-beta1, collagen type I, and collagen type III, to investigate whether these morphological observations were accompanied by alterations in gene expressions relevant to fibrotic changes. Real-time PCR revealed that TGF-beta1, collagen type I, and collagen type III mRNA in the noninfarct area at 4 wk after MI were markedly upregulated in WT mice. However, these responses were significantly attenuated in DGK{zeta}-TG mice compared with WT mice as shown in Fig. 6C.

Survival rates after MI in DGK{zeta}-TG mice. MI was created by coronary artery ligation in 93 WT mice and 82 DGK{zeta}-TG mice. The rate of acute surgical death was not different between WT mice [14%, 13 deaths out of 93 mice (13/93)] and DGK{zeta}-TG mice (15%, 12/82). The survival rates until 4 wk after MI were 45% (36/80) in WT mice and 66% (46/70) in DGK{zeta}-TG mice (P < 0.01). The survival rates until 4 wk after MI in WT mice were almost similar to previous reports (8, 20, 27). After euthanasia at 4 wk after MI, 10 WT mice (13%) and 8 DGK{zeta}-TG mice (11%) were excluded from the analysis because the size of infarction was <30%. Kaplan-Meier survival curves were then created from 70 WT mice and 62 DGK{zeta}-TG mice as shown in Fig. 7. The survival rate of DGK{zeta}-TG mice was significantly higher than that of WT mice (61%, 38/62 vs. 37%, 26/70, P < 0.01). The major cause of death in the first 10 days after MI was cardiac rupture, and the incidence of cardiac rupture was not different between WT and DGK{zeta}-TG mice. These observed differences in survival were due to less lethal congestive heart failure in DGK{zeta}-TG mice compared with WT mice.


Figure 7
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 7. Survival curves after MI in WT and DGK{zeta}-TG mice. Survival curves were created by a Kaplan-Meier method and compared by a log-rank test. Percentages of surviving WT and DGK{zeta}-TG mice were plotted. At 4 wk after MI, survival rates were significantly higher in DGK{zeta}-TG mice than in WT mice (61% vs. 37%, P < 0.01).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we demonstrated that LV chamber dilatation and impaired systolic function after MI were attenuated in DGK{zeta}-TG mice compared with WT littermates. We also showed that fibrosis, induction of profibrotic genes, translocation of PKC isoforms, and cardiomyocyte hypertrophy in the noninfarct area were blocked in DGK{zeta}-TG mice. Attenuation of postinfarction LV remodeling was associated with substantial improvement in survival in DGK{zeta}-TG mice.

MI causes a complex sequence of structural changes of the left ventricle, determined as postinfarction remodeling (15, 22, 23). These include progressive chamber dilatation, eccentric cardiac hypertrophy, and fibrosis. Recent studies have reported the importance of fibrosis in the pathogenesis of postinfarction cardiac dysfunction (16, 18, 19, 37). Although fibrosis is an integral component of the reparative process to maintain structural integrity of the infarct area, the accumulation of this collagenous material remote from infarction is maladaptive and is associated with reduced myocardial elasticity and contractility. Recently, Boyle et al. (3) have reported that pharmacological inhibition of PKC attenuated matrix accumulation and fibrosis in the noninfarct area and prevented cardiac dysfunction. In our present study, histological examinations demonstrated that fibrosis in the noninfarct area was suppressed in DGK{zeta}-TG mice (Fig. 6, A and B). Furthermore, gene expressions of TGF-beta1, collagen type I, and collagen type III, which were responsible for fibrosis formation (16, 18, 38), were attenuated in DGK{zeta}-TG mice compared with WT mice after MI (Fig. 6C). Because excessive fibrosis in the noninfarct area causes the lethal heart failure (38), substantial attenuation of cardiac dysfunction contributed to improved survival in DGK{zeta}-TG mice observed in the present study (Fig. 7). Inhibition of fibrosis in the postinfarction period may potentially impair the healing process in the infarct area, resulting in a weaker scar and a possible increase in mortality and heart failure. In the present study, overexpression of DGK{zeta} reduced the ongoing remodeling in the noninfarct myocardium but was not associated with an increase in infarct scar size, indicating that there was no interference with the reparative scar formation at the infarct area. Indeed, survival rate after MI was higher in DGK{zeta}-TG mice than in WT mice. It has been reported that elevation of the endogenous DGK{zeta} signal was observed in the acute phase of rat MI hearts, and this expression became less 21 days later (30). Thus this molecule may be regulated during cardiac remodeling after MI and seems to be a new therapeutic target for MI.

We did not measure myocardial DAG levels in the present study. However, in a previous study, we observed marked increases in myocardial DAG levels after continuous administration of phenylephrine in WT mice, but these increases were completely suppressed in DGK{zeta}-TG mice (1). Thus we speculate that increases in myocardial DAG levels after MI are similarly inhibited in DGK{zeta}-TG mice compared with WT mice.

It has been reported that PKC-{alpha} reduces cardiac contractility and induces a propensity toward heart failure (4) and negatively regulates ventricular systolic and diastolic function in pathological hypertrophy (11). Furthermore, transgenic overexpression of PKC-{alpha} causes lethal heart failure with marked interstitial fibrosis (11, 13). Therefore, the inhibition of PKC-{alpha} translocation by DGK{zeta} might be responsible for attenuation of fibrosis in the noninfarct area observed in the present study. Our present data, the inhibition of PKC-{alpha} translocation by DGK{zeta}, are supported by an in vitro study demonstrating spatial association of DGK{zeta} with PKC-{alpha} in HEK293 cells (17). On the other hand, cardiac specific overexpression of PKC-beta causes LV hypertrophy, fibrosis, and decreased LV performance (31, 35). In the present study, DGK{zeta} blocked translocation of PKC-{alpha}, but not -beta, in the noninfarct myocardium. Therefore, it appears that the inhibition of PKC-{alpha} plays an important role for protective effects of DGK{zeta} to prevent ventricular remodeling and fibrosis after MI in transgenic mouse hearts. Although both PKC-{alpha} and -beta isoforms are activated by intracellular increases in DAG levels, our data may suggest that activation of PKC isoforms in response to MI is differentially regulated in in vivo mouse hearts.

In the present study, DGK{zeta} also blocked activation of PKC-{epsilon} in the noninfarct area. It has been reported that the PKC-{epsilon} isoform has been implicated in cardiac hypertrophy (33). An in vitro study using neonatal cardiomyocytes has shown that PKC is critical for angiotensin II-induced activation of ERK, which promotes cardiac hypertrophy by activating several transcriptional factors (26). Among PKC isoforms, PKC-{epsilon}, but not PKC-{alpha}, is a mediator for ERK activation induced by endothelin-1 and phenylephrine. Therefore, these data suggest that the inhibition of PKC-{epsilon} by DGK{zeta} might contribute to the suppression of gene induction of ANF, BNP, and beta-MHC and the attenuation of increases in cross-sectional cardiomyocyte surface area observed in the present study. However, we could not exclude the possibility of other (PKC independent) effects of DGK. We will have to examine this effect in the future experiments.

In this study, although not statistically significant, the infarct size of WT mice tended to be larger than that of DGK{zeta}-TG mice. It has been recently reported that compensatory development of cardiac hypertrophy and normalization of wall stress may not be necessary to preserve cardiac function as previously hypothesized (5). Therefore, it is possible that the decreased tendency of infarct size and the inhibition of excessive cardiac hypertrophy and cardiac remodeling in DGK{zeta}-TG mice may cause improved cardiac function observed in the present study.

In mice that died in the first 10 days after MI, the infarct size was larger and cardiac rupture was the major cause of death as reported previously (8, 20, 27). However, there was no significant difference in the incidence of cardiac rupture between WT and DGK{zeta}-TG mice. Importantly, during a follow-up period up to 4 wk after MI, the rate of death by congestive heart failure in DGK{zeta}-TG mice was lower compared with WT mice. Therefore, we think that the reduced rate of lethal heart failure due to the inhibition cardiac remodeling accounts for the improved survival in DGK{zeta}-TG mice.

In conclusion, we demonstrated that cardiac-specific overexpression of DGK{zeta} blocked fibrosis in the noninfarct area, attenuated postinfarction LV remodeling, and improved survival after MI. These results will provide us a novel approach to investigate the pathogenesis of LV remodeling after MI and a potential novel therapeutic target to prevent cardiac structural remodeling.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
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 program of the Japan Society for the Promotion of Science; and grants from the Takeda Science Foundation and the Fukuda Foundation for Medical Technology.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Takeishi, Dept. of Cardiology, Pulmonology, and Nephrology, Yamagata Univ. School of Medicine, 2-2-2 Iida-Nishi, Yamagata, Japan 990-9585 (e-mail: takeishi{at}med.id.yamagata-u.ac.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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Arimoto T, Takeishi Y, Takahashi H, Shishido T, Niizeki T, Koyama Y, Shiga R, Nozaki N, Nakajima O, Nishimaru K, Abe J, Endoh M, Walsh RA, Goto K, Kubota I. Cardiac-specific overexpression of diacylglycerol kinase zeta prevents Gq protein-coupled receptor agonist-induced cardiac hypertrophy in transgenic mice. Circulation 113: 60–66, 2006.
  2. 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 99: 384–391, 1999.
  3. Boyle AJ, Kelly DJ, Zhang Y, Cox AJ, Gow RM, Way K, Itescu S, Krum H, Gilbert RE. Inhibition of protein kinase C reduces left ventricular fibrosis and dysfunction following myocardial infarction. J Mol Cell Cardiol 39: 213–221, 2005.[CrossRef][ISI][Medline]
  4. Braz JC, Gregory K, Pathak A, Zhao W, Sahin B, Klevitsky R, Kimball TF, Lorenz JN, Nairn AC, Liggett SB, Bodi I, Wang S, Schwartz A, Lakatta EG, DePaoli-Roach AA, Robbins J, Hewett TE, Bibb JA, Westfall MV, Kranias EG, Molkentin JD. PKC-alpha regulates cardiac contractility and propensity toward heart failure. Nat Med 10: 248–254, 2004.[CrossRef][ISI][Medline]
  5. Esposito G, Rapacciuolo A, Naga Prasad SV, Takaoka H, Thomas SA, Koch WJ, Rockman HA. Genetic alterations that inhibit in vivo pressure-overload hypertrophy prevent cardiac dysfunction despite increased wall stress. Circulation 105: 85–92, 2002.
  6. Gao XM, Dart AM, Dewar E, Jennings G, Du XJ. Serial echocardiographic assessment of left ventricular dimensions and function after myocardial infarction in mice. Cardiovasc Res 45: 330–338, 2000.[Abstract/Free Full Text]
  7. Gao XM, Dilley RJ, Samuel CS, Percy E, Fullerton MJ, Dart AM, Du XJ. Lower risk of postinfarct rupture in mouse heart overexpressing beta 2-adrenergic receptors: importance of collagen content. J Cardiovasc Pharmacol 40: 632–640, 2002.[CrossRef][ISI][Medline]
  8. Gao XM, Xu Q, Kiriazis H, Dart AM, Du XJ. Mouse model of post-infarct ventricular rupture: time course, strain- and gender-dependency, tensile strength, and histopathology. Cardiovasc Res 65: 469–477, 2005.[Abstract/Free Full Text]
  9. Goto K, Kondo H. A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus. Proc Natl Acad Sci USA 93: 11196–11201, 1996.[Abstract/Free Full Text]
  10. Goto K, Kondo H. Diacylglycerol kinase in the central nervous system—molecular heterogeneity and gene expression. Chem Phys Lipids 98: 109–117, 1999.[CrossRef][ISI][Medline]
  11. Hahn HS, Marreez Y, Odley A, Sterbling A, Yussman MG, Hilty KC, Bodi I, Liggett SB, Schwartz A, Dorn GW 2nd. Protein kinase C alpha negatively regulates systolic and diastolic function in pathological hypertrophy. Circ Res 93: 1111–1119, 2003.[Abstract/Free Full Text]
  12. Harada K, Sugaya T, Murakami K, Yazaki Y, Komuro I. Angiotensin II type 1A receptor knockout mice display less left ventricular remodeling and improved survival after myocardial infarction. Circulation 100: 2093–2099, 1999.
  13. He Z, Way KJ, Arikawa E, Chou E, Opland DM, Clermont A, Isshiki K, Ma RC, Scott JA, Schoen FJ, Feener EP, King GL. Differential regulation of angiotensin II-induced expression of connective tissue growth factor by protein kinase C isoforms in the myocardium. J Biol Chem 280: 15719–15726, 2005.[Abstract/Free Full Text]
  14. Hosenpud JD, Bennett LE, Keck BM, Boucek MM, Novick RJ. The Registry of the International Society for Heart and Lung Transplantation: seventeenth official report–2000. J Heart Lung Transplant 19: 909–931, 2000.[CrossRef][ISI][Medline]
  15. ISIS-4 Collaborative Group. ISIS4: a randomised factorial trial assessing early oral captopril, oral mononitrate, and intravenous magnesium sulphate in 58,050 patients with suspected acute myocardial infarction. ISIS-4 (Fourth International Study of Infarct Survival) Collaborative Group. Lancet 345: 669–685, 1995.[CrossRef][ISI][Medline]
  16. Lijnen PJ, Petrov VV, Fagard RH. Induction of cardiac fibrosis by transforming growth factor-beta(1). Mol Genet Metab 71: 418–435, 2000.[CrossRef][ISI][Medline]
  17. Luo B, Prescott SM, Topham MK. Association of diacylglycerol kinase zeta with protein kinase C alpha: spatial regulation of diacylglycerol signaling. J Cell Biol 160: 929–937, 2003.[Abstract/Free Full Text]
  18. Manabe I, Shindo T, Nagai R. Gene expression in fibroblasts and fibrosis: involvement in cardiac hypertrophy. Circ Res 91: 1103–1113, 2002.[Abstract/Free Full Text]
  19. Matsumoto R, Yoshiyama M, Omura T, Kim S, Nakamura Y, Izumi Y, Akioka K, Iwao H, Takeuchi K, Yoshikawa J. Effects of aldosterone receptor antagonist and angiotensin II type I receptor blocker on cardiac transcriptional factors and mRNA expression in rats with myocardial infarction. Circ J 68: 376–382, 2004.[CrossRef][ISI][Medline]
  20. Matsumura S, Iwanaga S, Mochizuki S, Okamoto H, Ogawa S, Okada Y. Targeted deletion or pharmacological inhibition of MMP-2 prevents cardiac rupture after myocardial infarction in mice. J Clin Invest 115: 599–609, 2005.[CrossRef][ISI][Medline]
  21. Nozaki N, Shishido T, Takeishi Y, Kubota I. Modulation of doxorubicin-induced cardiac dysfunction in toll-like receptor-2-knockout mice. Circulation 110: 2869–2874, 2004.
  22. Pfeffer JM, Fischer TA, Pfeffer MA. Angiotensin-converting enzyme inhibition and ventricular remodeling after myocardial infarction. Annu Rev Physiol 57: 805–826, 1995.[CrossRef][ISI][Medline]
  23. Pfeffer MA, Braunwald E, Moye LA, Basta L, Brown EJ Jr, Cuddy TE, Davis BR, Geltman EM, Goldman S, Flaker GC, Klein M, Lamas GA, Packer M, Rouleau J, Rouleau JL, Rutherford J, Wertheimer JH, Hawkins CM. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. Results of the survival and ventricular enlargement trial. The SAVE Investigators. N Engl J Med 327: 669–677, 1992.[Abstract]
  24. Sakane F, Yamada K, Kanoh H, Yokoyama C, Tanabe T. Porcine diacylglycerol kinase sequence has zinc finger and E-F hand motifs. Nature 344: 345–348, 1990.[CrossRef][Medline]
  25. 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 108: 2905–2910, 2003.
  26. Sugden PH, Clerk A. Cellular mechanisms of cardiac hypertrophy. J Mol Med 76: 725–746, 1998.[CrossRef][ISI][Medline]
  27. Sun M, Dawood F, Wen WH, Chen M, Dixon I, Kirshenbaum LA, Liu PP. Excessive tumor necrosis factor activation after infarction contributes to susceptibility of myocardial rupture and left ventricular dysfunction. Circulation 110: 3221–3328, 2004.
  28. 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 34: 85–93, 2004.[CrossRef][ISI][Medline]
  29. Takahashi H, Takeishi Y, Seidler T, Arimoto T, Akiyama H, Hozumi Y, Koyama Y, Shishido T, Tsunoda Y, Niizeki T, Nozaki N, Abe J, Hasenfuss G, Goto K, Kubota I. Adenovirus-mediated overexpression of diacylglycerol kinase-zeta inhibits endothelin-1-induced cardiomyocyte hypertrophy. Circulation 111: 1510–1516, 2005.
  30. 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 89: 265–272, 2001.[Abstract/Free Full Text]
  31. Takeishi Y, Chu G, Kirkpatrick DM, Li Z, Wakasaki H, Kranias EG, King GL, Walsh RA. In vivo phosphorylation of cardiac troponin I by protein kinase Cbeta2 decreases cardiomyocyte calcium responsiveness and contractility in transgenic mouse hearts. J Clin Invest 102: 72–78, 1998.[ISI][Medline]
  32. Takeishi Y, Jalili T, Ball NA, Walsh RA. Responses of cardiac protein kinase C isoforms to distinct pathological stimuli are differentially regulated. Circ Res 85: 264–271, 1999.[Abstract/Free Full Text]
  33. Takeishi Y, Ping P, Bolli R, Kirkpatrick DL, Hoit BD, Walsh RA. Transgenic overexpression of constitutively active protein kinase C epsilon causes concentric cardiac hypertrophy. Circ Res 86: 1218–1223, 2000.[Abstract/Free Full Text]
  34. Topham MK, Bunting M, Zimmerman GA, McIntyre TM, Blackshear PJ, Prescott SM. Protein kinase C regulates the nuclear localization of diacylglycerol kinase-zeta. Nature 394: 697–700, 1998.[CrossRef][Medline]
  35. Wakasaki H, Koya D, Schoen FJ, Jirousek MR, Ways DK, Hoit BD, Walsh RA, King GL. Targeted overexpression of protein kinase C beta2 isoform in myocardium causes cardiomyopathy. Proc Natl Acad Sci USA 94: 9320–9325, 1997.[Abstract/Free Full Text]
  36. Wang J, Liu X, Sentex E, Takeda N, Dhalla NS. Increased expression of protein kinase C isoforms in heart failure due to myocardial infarction. Am J Physiol Heart Circ Physiol 284: H2277–H2287, 2003.[Abstract/Free Full Text]
  37. Weber KT. Extracellular matrix remodeling in heart failure: a role for de novo angiotensin II generation. Circulation 96: 4065–4082, 1997.
  38. Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium. Fibrosis and renin-angiotensin-aldosterone system. Circulation 83: 1849–1865, 1991.



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
T. Kitahara, Y. Takeishi, M. Harada, T. Niizeki, S. Suzuki, T. Sasaki, M. Ishino, O. Bilim, O. Nakajima, and I. Kubota
High-mobility group box 1 restores cardiac function after myocardial infarction in transgenic mice
Cardiovasc Res, October 1, 2008; 80(1): 40 - 46.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. Sonin, S.-Y. Zhou, C. Cronin, T. Sonina, J. Wu, K. A. Jacobson, A. Pappano, and B. T. Liang
Role of P2X purinergic receptors in the rescue of ischemic heart failure
Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1191 - H1197.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. Niizeki, Y. Takeishi, T. Kitahara, T. Arimoto, M. Ishino, O. Bilim, S. Suzuki, T. Sasaki, O. Nakajima, R. A. Walsh, et al.
Diacylglycerol kinase-{varepsilon} restores cardiac dysfunction under chronic pressure overload: a new specific regulator of G{alpha}q signaling cascade
Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H245 - H255.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/2/H1105    most recent
00927.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Niizeki, T.
Right arrow Articles by Kubota, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Niizeki, T.
Right arrow Articles by Kubota, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.