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Am J Physiol Heart Circ Physiol 290: H2146-H2154, 2006. First published December 30, 2005; doi:10.1152/ajpheart.01142.2005
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Subdepressor dose of benidipine ameliorates diabetic cardiac remodeling accompanied by normalization of upregulated endothelin system in rats

Subrina Jesmin,1 Yuichi Hattori,2 Seiji Maeda,1 Sohel Zaedi,1 Ichiro Sakuma,3 and Takashi Miyauchi1

1Department of Cardiovascular Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan; 2Department of Pharmacology, School of Medicine, University of Toyama, Toyama, Japan; and 3Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Submitted 28 October 2005 ; accepted in final form 16 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated whether benidipine, a long-acting calcium channel blocker (CCB), can normalize cardiac expression profiles of the endothelin (ET)-1 system in insulin-resistant diabetes. Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of human Type 2 diabetes, were treated for 12 wk with vehicle or benidipine (3 mg·kg–1·day–1). OLETF rats exhibited a significant increase in ET-1 in plasma and left ventricular (LV) tissues compared with nondiabetic controls. Expression of prepro-ET-1, ET-converting enzyme, and ETA and ETB receptors in LV tissues was also significantly higher in OLETF rats. The two MAPKs, JNK and p38MAPK, both of which are activated by ET-1, were more abundantly expressed in OLETF rat LV tissues. All these alterations were reversed to nondiabetic levels when OLETF rats were treated with the subdepressor dose of benidipine. Furthermore, benidipine therapy resulted in hindering cardiomyocyte hypertrophy and cardiac perivascular fibrosis in OLETF rats. The beneficial actions of benidipine at the subdepressor dose on cardiac remodeling in insulin-resistant diabetes may involve normalization of the upregulated ET-1 system.

insulin resistance; Otsuka Long-Evans Tokushima Fatty rat heart; calcium channel blocker

CALCIUM CHANNEL BLOCKERS (CCBs) are widely used clinically for the management of hypertension, angina pectoris, and cardiac arrhythmias (45). Their pharmacological and therapeutic properties are attributable to the block of the influx of Ca2+ through L-type Ca2+ channels in vascular smooth muscle and/or cardiac muscle (7). CCBs have been shown to inhibit collagen deposition in the extracellular matrix formed by cultured human fibroblasts (46), to prevent renal fibrosis (12, 29), and to reduce cardiac fibrosis in spontaneously hypertensive rats (6, 52). Furthermore, long-acting CCBs, amlodipine and lacidipine, can reduce cardiac hypertrophy and cardiac hypertrophy-related gene programming in stroke-prone spontaneously hypertensive rats (30, 32), suggesting the preventive effect of CCBs on hypertensive cardiac remodeling.

Cardiac remodeling is characterized by increased cardiomyocyte volume and collagen deposition (54, 57). Endothelin (ET)-1 promotes growth of cardiomyocytes in vitro (22, 38, 58), induces collagen synthesis by cardiac fibroblasts (17, 38), and stimulates the development of left ventricular (LV) hypertrophy (27, 51). Thus ET-1 may be of significant importance in the development of cardiac remodeling. Indeed, the involvement of ET-1 in hypertensive cardiac remodeling has been suggested by the findings that ET-1 blockade therapy can prevent cardiac fibrosis and fibrosis-associated molecular events in hypertensive animal models such as deoxycorticosterone acetate-salt hypertension (1, 2).

Circulating plasma ET-1 levels have been shown to be higher in diabetic subjects than in nondiabetic controls (11, 53), and the plasma concentration of Big ET-1, a precursor of ET-1, has been found to be elevated in patients with diabetes mellitus (55). ET-1 has been implicated in the pathogenesis of insulin resistance-induced cardiovascular complications in human diabetes (15, 42). Notably, cardiac remodeling, including cardiac fibrosis, is promoted from the early stage of developing diabetes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (37). This strain displays stable clinical and pathological features that resemble human Type 2 diabetes, and one of its characteristics is an early manifestation of existence of insulin resistance. Our group (25) previously showed that benidipine, a long-acting dihydropyridine CCB, improved cardiac remodeling in OLETF rats at the insulin-resistance stage. In the present study, we examined the effects of long-term treatment with benidipine at dosage insufficient on blood pressure on cardiac expression levels of prepro-ET-1, ET-1, ETA and ETB receptors, and ET-1 signaling molecules, including p38MAPK and JNK, in this insulin-resistant diabetic model to assess whether the beneficial actions of benidipine involve suppression of the ET-1 signaling pathways, which may be exaggerated in insulin-resistant diabetes independently of the reduction in blood pressure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and drug treatment. The experimental design was approved by the Hokkaido University School of Medicine Animal Care and Use Committee. Animals were obtained from the Tokushima Research Institute, Otsuka Pharmaceutical (Tokushima, Japan) and maintained under constant temperature and lighting conditions with free access to food and water. At 8 wk of age, male OLETF rats were randomly divided into two groups. Only one group of 12 animals was given daily benidipine (3 mg/kg po) in 0.3% carboxymethylcellulose solution. This treatment period was 12 wk, and the animals were used at 20 wk of age, a time when they were in the early stage of Type 2 diabetes with modest hyperglycemia and prominent insulin resistance (see RESULTS). Twelve male Long-Evans Tokushima Otsuka (LETO) rats, which developed from the same colony by selective mating but did not develop diabetes, served as controls. On the day of the experiments, rats were euthanized under gaseous ether anesthesia. The hearts were rapidly excised, and the whole heart and LV weights were measured. Some portion of the LV was dipped into optimum cutting temperature (OCT) compound (Sakura Finetechnical, Tokyo, Japan) and immediately frozen in liquid nitrogen. The remaining portion was preserved at –80°C without OCT compound.

Histopathology examination. For histopathological analysis, the tissues were fixed in 4% buffered formalin solution, dehydrated, embedded in paraffin, and then sliced into 5-µm-thick sections. After being deparaffinized, in addition to the routine hematoxylin and eosin staining, the sections were stained for collagen using van Geison's trichrome method.

Immunofluorescence staining. For immunohistochemical determination of target molecules, the following commercially available antibodies were used: guinea pig anti-ET-1 (Peninsula, San Carlos, CA) and rat anti-ETA and -ETB receptors (Alomone, Jerusalem, Israel). Frozen cryostat sections (8 µm thick) were fixed in acetone and air dried. Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 15 min. To prevent nonspecific staining by the secondary antibody, the sections were blocked by normal goat serum. After an overnight incubation at 4°C with primary antibodies, the sections were rinsed in phosphate-buffered solution and then exposed to the fluorescence secondary antibody Cy3-conjugated AffiniPure anti-rabbit IgG or fluorescein-conjugated AffiniPure goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) for 2 h according to the manufacturer's instructions. The samples processed without primary antibodies served as negative controls. Immunofluorescent images were observed under the Laser Scanning Confocal Imaging System (MRC-1024; Bio-Rad).

Western blot analysis. Immunoblotting was performed as demonstrated in our previous report (35). Samples of tissue homogenate were subjected to electrophoresis on polyacrylamide gels, and proteins were transferred to polyvinylidine difluoride filter membrane. After being blocked with 5% nonfat milk in phosphate-buffered saline, the membranes were incubated with specific antibody recognizing ETA receptor, ETB receptor, p38MAPK, JNK, or phospho-JNK. For the recognition of p38MAPK, JNK, and phospho-JNK, we used the following commercially available antibodies: rabbit anti-p38MAPK polyclonal antibody (Stressgen, Victoria, Canada), rabbit anti-JNK polyclonal IgG (Upstate Biotechnology, Lake Placid, NY), and rabbit anti-phospho-JNK polyclonal antibody (Cell Signaling Technology, Beverly, MA). After being extensively washed with phosphate-buffered saline, containing 0.1% Tween 20 to remove any nonspecifically bound primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit antibody. The blots were visualized with the enhanced chemiluminescence detection system (Amersham Biosciences UK, Little Chalfont, Buckinghamshire, UK), exposed to X-ray film, and analyzed with free software National Institutes of Health image.

Enzyme immunoassay for ET-1. Concentrations of ET-1 in sera and tissue extracts were determined using an ET-1 Enzyme Immuno Assay Kit (Immuno-Biological, Fujioka, Japan). The reported cross-reactivity of the antibody for the former was <0.1% for all Big ETs, <0.1% for ET-3, and 3.3% for ET-2. The assay procedure was carried out according to the manufacture's instructions.

RNA preparation and real-time quantitative PCR. Total RNA was extracted from tissues by the guanidinium thiocynate-phenol-chloroform single-step extraction method with Isogen (Nippon Gene, Toyama, Japan), used routinely in our laboratory (34, 36). After being isolated, treated with DNase I, and quantified, RNA was reverse-transcribed to cDNA with the use of a ReverTra Ace (TOYOBO, Osaka, Japan).

The single-stranded cDNA was then used in real-time quantitative PCR for evaluation of relative expression levels of the six genes of interest. Selected genes and primers are shown in Table 1. DNA amplification was performed in the Applied Biosystem (ABI 7900HT) real-time PCR machine with the GeneAmp 7900HT sequence detection system software (Perkin-Elmer, Foster City, CA), and the detection was made by measuring the binding of the fluorescence dye SYBR Green I to double-stranded DNA. The PCR reactions were set up in microtubes in a volume of 20 µl. The reaction components were 2 µl cDNA synthesized as above, 10 µl 2x SYBR Green master mix (Perkin-Elmer), and 0.4 µl of each pair of oligonucleotide primers (Table 1). The program was as follows: an initial step at 95°C for 10 min and then 40 cycles of 95°C for 15 s and 60°C for 60 s. Regression curves were drawn for each sample, and the relative amount was calculated from the threshold cycles with the instrument's software (SDS 2.0) according to the manufacturer's instructions. The PCR products were analyzed by gel electrophoresis to confirm the specificity of generated products. Relative expression levels of the target genes were normalized to the genometric mean of the two internal control genes, beta-actin and GAPDH.


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  		Table 1. Primers and conditions for real-time quantitative PCR analyses

 
Statistical analysis. Values are means ± SE. Statistical assessment of the data was made by one-way analysis of variance with multiple comparisons by Fisher’s protected least significant difference t-test. Nonparametric data were analyzed by the Mann-Whitney's U-test or Wilcoxon's signed rank test. A P value <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characteristics of experimental animals. The general features of OLETF rats at 20 wk of age have been well described in our group's previous report (25), along with those of age-matched LETO rats. In brief, after 24 h of fasting, OLETF rats were modestly hyperglycemic (197 ± 10 vs. 164 ± 4 mg/dl, n = 12 animals for each, P < 0.01), and their plasma insulin level was much higher than in LETO rats (4.31 ± 0.47 vs. 2.33 ± 0.19 ng/ml, P < 0.01). Treatment of OLETF rats with benidipine for 8 wk altered neither hyperglycemia (201 ± 6 mg/dl, n = 12 animals) nor hyperinsulinemia (4.34 ± 0.41 ng/ml). Heart and LV weights were 1.4-fold higher in OLETF than in LETO rats. Benidipine treatment had no effect on the increases in heart and LV weights. Systolic blood pressure of OLETF rats was slightly but significantly higher than that of LETO rats (140 ± 3 vs. 129 ± 3 mmmHg, P < 0.001). Benidipine therapy lowered the blood pressure (132 ± 4 mmHg) in OLETF rats, but this effect was statistically insignificant.

Manifestation of cardiac remodeling. In Fig. 1A, a general histological architecture of LETO, OLETF, and benidipine-treated OLETF rat hearts is shown. OLETF rats showed significant cardiomyocyte hypertrophy, which was restored by benidipine treatment. Thus the values for the myocyte scan area were 521 ± 43, 626 ± 34, and 503 ± 35 µm2 in LETO, OLETF, and benidipine-treated OLETF rats, respectively (P < 0.01). The van Gieson's method of staining for collagen was used to assess the degree of perivascular fibrosis (Fig. 1B). The area of perivascular fibrosis in coronary arterioles, which was corrected for total vessel area, was much larger in OLETF than in LETO rats (74 ± 13% vs. 26 ± 3%, P < 0.01). The development of fibrosis in the perivascular area in OLETF rats was prevented by benidipine treatment (28 ± 4%, P < 0.01). The wall-to-lumen ratio of coronary arterioles in OLETF rats (2.8 ± 0.6, P < 0.01) was approximately threefold greater than that in LETO rats (1.0 ± 0.2). Benidipine treatment significantly reduced this ratio (1.3 ± 0.2, P < 0.01).


Figure 1
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  		Fig. 1. Photomicrographs of hematoxylin- and eosin-stained (A) and van Gieson's stained (B) left ventricular (LV) sections from Long-Evans Tokushima Otsuka (LETO), Otsuka Long-Evans Tokushima Fatty (OLETF), and benidipine (Ben)-treated OLETF rats. Original magnification is x400.

 
Cardiac expression of ET-1 system. As shown in Fig. 2A, the plasma ET-1 level, as determined by ELISA, was significantly elevated in OLETF compared with LETO rats. Furthermore, the immunoreactive ET-1 level in LV tissues was significantly higher in OLETF than in LETO rats (Fig. 2B). In LV sections from both LETO and OLETF rats, ET-1 was immunocytochemically demonstrable in coronary vessels and cardiomyocytes, but increased immunoreactivity was noted in the LV sections from OLETF rats (Fig. 3A). \. Benidipine treatment of OLETF rats reduced immunoreactive ET-1 in the plasma and tissues to the levels obtained in LETO rats (Figs. 2, A and B, and 3A). When gene expression of prepro-ET-1, a precursor of ET-1, and endothelin-converting enzyme (ECE), which specifically cleaves Big ET-1, was assessed by using real-time PCR, the levels of prepro-ET-1 mRNA and ECE mRNA in LV tissues were significantly greater in OLETF than in LETO rats (Fig. 2, C and D). The increases in their gene expression levels were completely blocked by benidipine treatment.


Figure 2
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  		Fig. 2. Endothelin-1 (ET-1) levels in plasma (A) and LV tissues (B) and gene expression levels of prepro-ET-1 (C) and endothelin-converting enzyme (ECE; D) in LV tissues of LETO, OLETF, and Ben-treated OLETF rats. Plasma and tissue ET-1 levels were determined by ELISA. Expression of prepro-ET-1 mRNA and ECE mRNA was quantitatively evaluated by real-time PCR. Data are means ± SE; n = 12 animals. **P < 0.01 and ***P < 0.001, compared with corresponding values obtained in LETO rats; ##P < 0.01, compared with corresponding values in OLETF rats without any treatment.

 

Figure 3
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  		Fig. 3. Confocal images showing immunofluorescence labeling for ET-1 (A), ETA receptor (B), and ETB receptor (C) in LV sections from LETO, OLETF, and Ben-treated OLETF rats. Both vascular and extravascular regions are more strongly positive in LV sections from OLETF than in those from LETO and Ben-treated rats. Original magnification is x400.

 
On Western blots, the ETA and ETB receptor levels were determined by measuring the density of a single band migrating at 48 and 49.5 kDa, respectively. As revealed by the cumulative data for quantitative immunoblotting (Fig. 4, A and B), both the ETA and ETB receptor protein levels in LV tissues were significantly higher in OLETF than in LETO rats. Immunofluorescence studies showed that positive staining for the ETA receptor was detected not only in coronary vessels but also in cardiomyocytes, whereas the ETB receptor was mainly localized to coronary vessels in nondiabetic LETO rats (Fig. 3, B and C). In LV sections from OLETF rats, however, diffuse and strong immunoreactivity of both the ETA and ETB receptors was observed. In OLETF rats treated with benidipine, the expression levels and patterns of the two ET receptors were returned to those levels seen in LETO rats (Figs. 3, B and C, and 4, A an B). The increases in the ETA and ETB receptor proteins in LV tissues of OLETF rats correlated with increases in their mRNAs, which were obtained from real-time PCR (Fig. 4, C and D). Benidipine treatment of OLETF rats reduced their gene expression levels to those of nondiabetic controls.


Figure 4
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  		Fig. 4. Protein and gene expression levels of ETA (A and C) and ETB (B and D) receptors in LV tissues of LETO, OLETF, and Ben-treated OLETF rats. Traces (A and B, top) show typical Western blots indicating increases in expression of 48-kDa band (ETA) and 49.5-kDa band (ETB) in diabetic LV tissues, respectively. C and D: mRNA expression of ETA and ETB receptors was quantitatively evaluated by real-time PCR. Data are means ± SE; n = 12 animals. **P < 0.01 and ***P < 0.001, compared with corresponding values obtained in LETO rats; ##P < 0.01, compared with corresponding values in OLETF rats without any treatment.

 
The expression level of JNK, a subfamily of MAPK that ET-1 can activate in smooth muscle cells (13) and in cardiomyocytes (9), was significantly enhanced in LV tissues of OLETF rats compared with those of LETO rats (Fig. 5A). The results of Western blot analysis also revealed that LV expression of JNK dually phosphorylated at threonine-183 and tyrosine-185 was increased in OLETF rats (Fig. 5B). The increases in LV expression of JNK and phospho-JNK seen in OLETF rats were reversed by benidipine treatment. As shown in Fig. 5C, Western blot analysis showed a significant increase in the expression of p38MAPK, which is also involved in the signal transduction pathway associated with ET-1 stimulation in cardiomyocytes (10), in LV tissues of OLETF rats. The increase in p38MAPK transcripts in OLETF rat LV tissues was quantitatively confirmed by analysis using the real-time PCR method (Fig. 5D). The increases in protein and mRNA expression levels of p38MAPK were blocked by benidipine treatment.


Figure 5
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  		Fig. 5. Expression levels of JNK (A), phospho-JNK (B), p38MAPK (C), and p38MAPK mRNA (D) in LV tissues of LETO, OLETF, and Ben-treated OLETF rats. Traces (A–C, top) show typical Western blots indicating increases in expression of major band for each target molecule (54 kDa, JNK; 54 kDa, phospho-JNK; and 38 kDa, p38MAPK) in diabetic LV tissues. D: mRNA expression of p38MAPK was quantitatively evaluated by real-time PCR. Data are means ± SE; n = 12 animals. **P < 0.01, compared with corresponding values obtained in LETO rats; ##P < 0.01, compared with corresponding values in OLETF rats without any treatment.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we used the OLETF strain of rats, which has been established as an animal model of congenital diabetes by selective mating (28). This strain has an early manifestation of glucose metabolic disorders. Our group's previous work revealed that OLETF rats exhibited significant insulin resistance and impaired glucose tolerance with hyperglycemia at 20 wk of age. At this age, OLETF rats were found to show modest hyperglycemia and prominent hyperinsulinemia after 24 h of fasting. Thus our subjects were in the stage of insulin resistance of Type 2 diabetes seen early in this strain.

Plasma ET-1 level was significantly higher in OLETF than in age-matched nondiabetic LETO rats. In addition, a significant increase in ET-1 levels was found in LV tissues of OLETF rats. This was further confirmed by our immunofluorescent study showing strongly increased ET-1 labeling of coronary vessels and cardiomyocytes in LV sections from OLETF rats. Possible reasons for increased ET-1 levels may include hyperinsulinemia seen in our model, because several studies in insulin-resistant humans have reported increased ET-1 serum concentrations that directly correlated with the levels of hyperinsulinemia (15, 42). It also should be noted that insulin stimulates ET-1 production in endothelial cells in vitro (40). ET-1 is produced primarily in endothelial cells by gene activation of prepro-ET-1 and subsequent processing by proteolytic enzymes into the biologically active peptide (18). The increase in ET-1 peptide level in LV tissues of rats with heart failure has been shown to originate from upregulation of prepro-ET-1 mRNA (31). We found that gene expression of prepro-ET-1 and ECE was upregulated in LV tissues of OLETF rats. The increase in prepro-ET-1 expression may be associated with an increase in expression of ETA and ETB receptor mRNA. This has been suggested by the previous report (43) showing coordinated increases in prepro-ET-1 and ETA and ETB receptor mRNA levels in LV tissues of rats with heart failure induced by coronary artery ligation. In this study, expression of ETA and ETB receptors was evidently upregulated in LV tissues of OLETF rats at both gene and protein levels. These results suggest that cardiac ET-1 system is greatly accelerated in this animal model at the early insulin-resistant stage of diabetes.

We also found that the two MAPKs, JNK and p38MAPK, were more abundantly expressed in LV tissues of OLETF than in those of LETO rats. JNK is activated by its dual phosphorylation motif, which results in phosphorylating serine residues in the NH2-terminal region of c-jun, thereby increasing in transcriptional activating activity of c-jun (44). Our finding that LV expression of phospho-JNK was significantly higher in OLETF than in LETO rats indicates an increase in activated cardiac JNK in insulin-resistant diabetes. ET-1 can activate the JNK pathway in smooth muscle cells and cardiomyocytes (9, 13). The existence of the signaling pathway linking the ETA receptor to JNK has been documented (36). ET-1 has been also shown to activate p38MAPK via ETA receptors in rat myocardial cells (56). However, studies of COS cells transfected with ETB receptors have indicated that this receptor subtype can stimulate activation of JNK and p38MAPK (3). Thus it remains probable that both MAPKs may be necessary for the signal transduction pathways associated with activation of each ET receptor subtype, but upregulation of the two MAPK signaling pathways could lead to further enhancement of ET-1-induced biological responses of the cell. Interestingly, both JNK and p38MAPK have been implicated in hypertrophy of cardiomyocytes, although the exact roles of each of these pathways have yet to be determined (47, 59).

ET-1 is powerfully hypertrophic in cardiac myocytes (23, 49). ET-1 also activates the procollagen I promoter (8) and collagen synthesis in fibroblasts (19); it can modulate collagenase activity, although it is unclear which ET receptor subtype mediates these effects. Moreover, ET-1 potently stimulates, via ETB receptors, aldosterone release (4) and therefore might increase collagen synthesis via enhanced aldosterone secretion. In this study, our histopathology showed evident cardiomyocyte hypertrophy and prominent perivascular fibrosis in the heart of OLETF rats. Furthermore, our group (25) previously demonstrated a significant increase in cardiac collagen deposition in this animal model. We thus suggest that the profound histopathological changes in the heart (referred to as cardiac remodeling), found in OLETF rats, partly resulted from overproduction of ET-1 and overexpression of ETA and ETB receptors and their signal transduction pathways. Our group's (25, 26) previous studies showed that transforming growth factor-beta1 (TGF-beta1) was highly expressed in the OLETF rat heart at both protein and mRNA levels. TGF-beta1 is known to be a powerful stimulant of cardiomyocyte hypertrophy (48) and collagen synthesis in the heart (5). This implies that superinduction of TGF-beta1 transcription could be causally involved in the development of cardiac remodeling at the early insulin-resistant stage of diabetes seen in this study. ET-1 has been reported to stimulate production of TGF-beta1 in rat liver stellate cells (17). Furthermore, ETA receptor antagonism can prevent the elevation of cardiac TGF-beta1 mRNA and protein levels in deoxycorticosterone acetate-salt hypertensive rats (2). We thus assume that TGF-beta1 may play an adjuvant role in the ET-1-related cardiac remodeling process in OLETF rats.

The key finding in this study is the demonstration that treatment of OLETF rats with benidipine, a long-acting CCB, at the subdepressor dose greatly inhibited the increased plasma and tissue levels of ET-1 and suppressed overexpression of prepro-ET-1, ECE, ETA and ETB receptors, and two ET-1 signal transduction pathways (JNK and p38MAPK) without affecting the plasma glucose and insulin concentrations. The ability of diltiazem and verapamil to decrease ET release from cultured coronary smooth muscle and endothelial cells has been previously demonstrated (20). Furthermore, accumulating evidence suggests that CCB therapy can result in a marked inhibition of overexpression of ET-1 genes in different rat models of hypertension, and this effect plays an important role in the prevention of cardiac remodeling in hypertensive rats (14, 31, 33). Benidipine treatment strongly prevented cardiomyocyte hypertrophy and perivascular fibrosis in the OLETF rat heart. This amelioration may be, at least in part, attributed to reductions in the expression levels of ET-1 and its related molecules in the heart. Potential mechanisms for the regulation of ET-1 gene by CCBs may be considered on the basis of the existence of two putative binding sites identified in the promoter sequence of this gene (41). Moreover, the calcium influx through L-type calcium channels can trigger c-fos activation, and, therefore, calcium channel blockers may modulate indirectly the interaction of the product of this immediate early gene with the activator protein-1 (AP-1) binding site present in the promoter sequence of ET-1 gene (14, 39).

The blood pressure in OLETF rats was modestly reduced, although this reduction was statistically insignificant. The increases in ET-1 and ETA receptor levels in the heart are suggested to be secondary to hypertension (50). Thus the effect of benidipine therapy to normalize the upregulated ET-1 system in OLETF rat hearts may be partly due to the modestly lowering action on blood pressure. Furthermore, we have previously found that angiotensin II is upregulated in coronary vessels of the OLETF rat heart, and the blockade of angiotensin II type 1 receptors with candesartan can greatly reverse diabetes-induced cardiac remodeling (24). In this regard, angiotensin II can induce ET-1 gene expression in rat aortic smooth muscle cells (21), and angiotensin-converting enzyme inhibition can decrease circulatory ET-1 levels in lean noninsulin-dependent diabetic patients (16). Thus the possible mutual cross talk between angiotensin II and ET-1 may exist in our diabetic rat model. Although not addressed in this work, whether angiotensin II type 1 receptor blockers and ET-1 receptor antagonists can prevent the upregulation of the ET-1 system and of angiotensin II in OLETF rat hearts, respectively, would well deserve further study. Finally, the question remains as to whether the beneficial effects of benidipine observed in OLETF rats resulted from its nonspecific actions in addition to its specific action on L-type calcium channels. Further study to examine whether other long-lasting CCBs can also exhibit the same effects on cardiac remodeling and upregulation of cardiac ET-1 system in OLETF rats, because benidipine may clarify this issue.

In conclusion, OLETF rats showed marked increases in local generation of ET-1 and expression of the ET-1 cellular signaling system (ETA and ETB receptors, JNK, and p38MAPK) in the heart at the insulin-resistant stage of Type 2 diabetes. This change in the ET-1 system was completely reversed by treatment with benidipine at the subdepressor dose. Furthermore, benidipine therapy ameliorated cardiac remodeling in OLETF rats. This amelioration may be partly due to the suppressive effect on increased expression of the ET-1 system in the OLETF rat heart. We thus represent the first study that provides a new understanding of the roles of the ET-1 system in the mechanisms for the blood pressure-independent cardioprotective effects of CCBs in insulin-resistant diabetes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and by Health Science Research Grants for Comprehensive Research on Aging and Health from the Ministry of Health, Welfare, and Labor of Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Miyauchi, Cardiovascular Div., Dept. of Internal Medicine, Institute of Clinical Medicine, Univ. of Tsukuba, Tsukuba, Ibaraki 305-8575 Japan (e-mail: t-miyauc{at}md.tsukuba.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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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