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Am J Physiol Heart Circ Physiol 289: H277-H284, 2005. First published March 18, 2005; doi:10.1152/ajpheart.01206.2004
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Activation of regenerating gene Reg in rat and human hearts in response to acute stress

Tatsuya Kiji,1 Yoshiko Dohi,2 Shin Takasawa,3 Hiroshi Okamoto,4 Akitaka Nonomura,5 and Shigeki Taniguchi1

Departments of 1Thoracic and Cardiovascular Surgery and 2Public Health, Nara Medical University School of Medicine, Kashihara, Nara; Departments of 3Biochemistry and 4Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai, Miyagi; and 5Department of Diagnostic Pathology, Nara Medical University School of Medicine, Nara, Japan

Submitted 1 December 2004 ; accepted in final form 10 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recently, the regenerating gene (Reg) has been documented to play an important role in various regenerating tissues, but it is unknown whether the Reg gene could be activated in the heart. The aim of this study was to reveal the transcriptional activation of Reg in the heart in response to heart stress. We first found REG-1 protein expression in human hearts obtained from autopsied patients who died of myocardial infarction. REG protein was immunohistochemically stained in a fine granular pattern in the cytoplasm of cardiomyocytes. To demonstrate the activation profiles of Reg gene expression in the heart, we quantified the levels of Reg-1 mRNA in rat hearts after coronary artery ligation using real-time RT-PCR. Transient Reg-1 mRNA activation, peaking at 12 h after coronary ligation, was observed mainly in the atria, which was sevenfold higher compared with hearts with pressure overload due to aortic constriction. In contrast, Reg receptor mRNA was expressed intensely in damaged ventricles. Furthermore, Western blot analysis showed the corresponding pattern of Reg protein secretion into the serum after loading, and circulating levels of the protein after myocardial infarction were higher than those after aortic constriction. In conclusion, our results demonstrate for the first time the presence of the Reg/Reg receptor system in damaged hearts. In view of emerging evidence of Reg for tissue regeneration in a variety of tissues/organs, it is proposed that the damaged heart may be a target for Reg action and that Reg may protect against acute heart stress.

receptor; heart failure; myocardial infarction; pressure overload

IN 1984, it was discovered that administration of nicotinamide, an inhibitor of poly(ADP-ribose) synthetase (PARS), to 90% depancreatized rats induced regeneration of pancreatic islets (29). In screening the regenerating islet-derived cDNA library, a novel gene was found and named "regenerating gene" (Reg) (25). The regeneration of pancreatic {beta}-cells seems to be primarily regulated by the expression of the Reg gene (1, 22, 26). Recently, Reg has been shown to play an important role in various types of tissue regeneration, for instance, gastric mucosal growth (4) and regeneration of motor neurons (17, 20). Interestingly, cancer cells in digestive organs tend to express Reg protein, and Reg gene expression seems to be positively correlated with the severity of the cancer (6, 8, 28). However, the action of the Reg gene in the cardiovascular system is largely uncertain.

We therefore examined the possibility of Reg gene expression in cryopreserved rat arterial allografts after transplantation (14). Reg expression was not detected in the normal artery but began to be detected on posttransplantation day 3. This change was augmented by daily administration of nicotinamide or immunosuppressant FK506, accompanied by the restoration of the cellular integrity of the cryopreserved allograft. We have proposed the usefulness of Reg-inductive therapy for enhancing the viability of vascular allografts after transplantation. However, the possibility of Reg expression in the heart still remains unknown.

The aim of this study was to explore the possibility of the transcriptional activation of the Reg gene in the heart. First, we tested immunohistochemical staining of human heart tissue obtained from autopsied patients who died from myocardial infarction with anti-human REG-1 protein MAb. Hereby, REG-1 protein expression in the cytoplasm of cardiomyocytes was revealed. We therefore examined cardiac Reg expression at both the mRNA and protein levels in the hearts of experimental rodents to demonstrate the activation profiles of gene expression in response to acute heart stress.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Human tissue specimens. Human heart tissue was obtained from five autopsied patients who died of heart failure in 2003 after myocardial infarction at Nara Medical University Hospital. Four patients had anteroseptal infarction, whereas the other patient had inferior infarction. The mean time to autopsy from the onset of infarction was 19 days (range, 2–45 days). The tissue specimens were fixed in 10% formalin-PBS and embedded in paraffin. We also obtained a heart specimen from one autopsied patient who died of a noncardiac cause (terminal lung cancer) to use in the control experiment.

Immunohistochemical staining for anti-human REG-1 protein MAb. Sections were prepared on glass slides with a 7 µm thickness, processed by deparaffinization/rehydration, and thereafter fixed in cold acetone at 4°C for 10 min. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 15 min, followed by incubation with diluted nonfat milk (BlockAce, Dainippon Pharmaceutical; Osaka, Japan) at room temperature for 30 min. The tissue slices were incubated with an anti-human REG-1 protein MAb (27, 28) (1:250) overnight at 4°C in a humidified environment. After slices were washed twice with PBS-Tween 20, bound antibody was detected by the universal immunoenzyme polymer method [Histofine Simple Stain MAX-PO (M), Nichirei; Tokyo, Japan], followed by hematoxylin staining.

Animals and experimental models. Male Wistar rats weighing 200–250 g were obtained from Charles River Japan (Yokohama, Japan). The animals were housed in a pathogen-free environment and allowed food and water ad libitum. The animals received humane care in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Revised 1996). The planning and performance of animal experiments were supervised under the control of the committee in accordance with the guidelines on animal experiments in Nara Medical University School of Medicine.

A rat acute myocardial infarction (AMI) model was generated by the ligation of the proximal left anterior descending coronary artery (LAD), as previously described (23). The broad anterolateral infarction of the left ventricle (LV) was created by LAD ligation. Sham-operated rats were treated in the same way except for the LAD ligation.

A pressure overload (PO) model was generated by aortic constriction (AoC). Briefly, the suprarenal abdominal aorta was exposed through a median laparotomy under light ether anesthesia. A 21-gauge needle was placed adjacent to the aorta, and the two were tied together with a 5-0 silk; the needle was pulled out, leaving the aorta constricted to the outer diameter of the needle (0.8 mm). The abdominal wall was then closed. Sham-operated rats were treated in the same way except for the AoC.

Removal of the heart. The animals were killed at 3, 6, 12, and 24 h and 3 and 7 days after LAD ligation or AoC (n = 5–7 animals per time point per group).

Rats were anesthetized with pentbarbital sodium (60 mg/kg ip). Body weight was measured, and 0.75 ml of blood were collected from the tail vein. After an additional anesthetic injection (60 mg/kg ip), the heart was exposed via a left thoracotomy and rapidly excised. In chilled (4°C) PBS, the four chambers of the heart were dissected. In the case of the AMI model, the LV was further divided into the infarct area, noninfarcted area, and border zone between them. The specimens were then directly immersed in liquid nitrogen except for a portion of the tissue block that was retained for histological study.

Isolation of total RNA and reverse transcription. The retrieved heart tissue was pulverized using a FastPrep FP-120 instrument (Qbiogene; Carlsbad, CA). The total RNA of each sample was isolated using an ISOGEN RNA extraction kit (Nippon Gene). The extracted total RNA (1–2 µg) was converted into cDNA with avian myeloblastosis virus reverse transcriptase XL (RNA PCR Kit, Takara Bio; Otsu, Japan) and regarded as a template for cDNA in the sample.

Synthesis of cDNAs. A 693-bp fragment (127–819 bp) of rat Reg receptor cDNA was synthesized using primers corresponding to regions 127–144 (5'-CTTCTTCCCCCTCATTGC-3') and 802–819 (5'-TTGTGTCCGTCTGTCCTC-3') from the rat aorta cDNA library. In a similar way, a 351-bp fragment (85–435 bp) of rat brain natriuretic peptide (BNP) cDNA was synthesized using primers corresponding to regions 85–102 (5'-ATGATTCTGCTCCTGCTT-3') and 413–435 (5'-CAGGAGGTCTTCCTAAAACAACC-3'). Rat Reg-1 (429 bp), IL-6 (101 bp), and GAPDH (306 bp) cDNAs were prepared as previously described (14).

Quantification of mRNAs by a real-time quantitative PCR. The theoretical basis of real-time quantitative PCR using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems; Norwalk, CT) has been described elsewhere (11). The sequences of each specific forward and reverse primer and a specific fluorogenic probe (TaqMan) were designed as shown in Table 1. To measure mRNA levels, amplification for the real-time PCR was performed using each respective primer and fluorogenic probe. The known amounts of the cDNA fragment of rat Reg-1 (429 bp), Reg receptor (693 bp), BNP (351 bp), IL-6 (101 bp), and GAPDH (306 bp) mentioned above were used as standards. The thermal cycler conditions were the same as those of a previous report (14). The fluorescence data collected were processed and analyzed with the ABI PRISM Sequence Detection Software (Applied Biosystems). Each mRNA value was normalized to that of the housekeeping gene GAPDH, which was used to account for differences in the efficiency of reverse transcription between samples.


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  		Table 1. Primers and Probes for RT-PCR

 
Northern blot analysis. The denatured total RNA (10 µg) extracted from rat heart tissue was size fractionated by electrophoresis on 1.2% agarose gel containing 16% formaldehyde solution and transferred onto a N+-Nylon membrane (Hybond N+, Amersham Bioscience; Tokyo, Japan) by a capillary method with aquatic resins (Nippon Shokubai; Tokyo, Japan). The resulting blot was prehybridized at 68°C in 5 ml of QuickHyb buffer (Stratagene; La Jolla, CA) for 30 min and hybridized with a 32P-labeled Reg-1 cDNA probe (429 bp) for 90 min under the same conditions described for prehybridization. The blot was washed twice in 2x SSC-0.1% SDS at room temperature for 15 min and twice in 0.1x SSC-0.1% SDS at 60°C for 15 min. Autoradiography was performed using a BAS1000 image analyzer (MacBAS version 2.0, Fuji Photofilm; Tokyo, Japan) on an imaging plate.

Western blot analysis. The 1:10 diluted rat serum and recombinant rat Reg-1 protein (16, 28) as a standard were treated with SDS sample buffer at 85°C for 5 min under reducing conditions, separated by SDS-PAGE using a gradient gel of 15–25%, and transferred to a nitrocellulose filter. Immunoblotting was carried out with a 1:30,000 diluted anti-rat Reg-1 protein MAb (0.23 µg IgG/ml) (1, 24) and then with 1:200 diluted horseradish peroxidase-conjugated goat anti-mouse IgG (IBL; Takasaki, Japan). The membranes were immersed with a chemiluminescence reagent (Renaissence, NEN Life Science; Boston, MA) at room temperature for 1 min and exposed to Hyperfilm ECL (Amersham Pharmacia Biotech) for 30–60 s. The Reg-1 protein concentration was evaluated semiquantitatively by densitometry scanning of signal intensity on the films using NIH Image software (version 4.63). A known amount of recombinant rat Reg-1 protein, purified from the culture medium of Pichia (1, 16), was used as a standard.

Immunohistochemical staining for anti-rat Reg-1 protein MAb. Tissue samples of rat hearts were fixed in 4% paraformaldehyde overnight and immersed in PBS containing 30% sucrose at 4°C for 2–3 h. They were then embedded in OCT compound (Tissue-Tek 4583, Sakura Finetek; Tokyo, Japan) and quickly frozen in liquid nitrogen. The cryostat sections were prepared on glass slides with a 7 µm thickness and fixed in cold acetone at 4°C for 10 min. The following process was similar to the procedure of the human study as previously described except for the use of rat-specific antibody. A monoclonal anti-rat Reg-1 protein antibody (24) was diluted 1:500.

Statistics. All values are presented as means ± SE. All statistical analyses were performed with the statistical program suite Statview (version 5.0, SAS Institute; Cary, NC). Differences between two groups were analyzed with the Mann-Whitney U-test. A P value of <0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Immunohistostaining of human infarcted hearts for REG-1 protein. REG-1 protein-positive cells could be identified in the ventricles (Fig. 1, A and B) and atria (Fig. 1C) of the patients who died of myocardial infarction. Tissue components other than cardiomyocytes (including the diseased coronary arteries) appeared not to be involved in the secretion of REG protein (Fig. 1D). High magnification showed that immunoreactive REG-1 protein was stained in a fine granular pattern in the cytoplasm of human cardiomyocytes (Fig. 1, E and F). These findings were not observed in corresponding negative control slides without the antibody against human REG-1 protein (Fig. 1G). The normal ventricle (Fig. 1H) and atrium (Fig. 1I), obtained from the patient who died of a noncardiac cause were not stained with anti-REG-1 protein MAb at all.



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  		Fig. 1. Immunohistochemical staining of infarcted human hearts for REG-1 protein. REG protein-positive cells were identified in cardiomyocytes of the ventricle (A and B) and atrium (C). REG protein was not detected in the diseased coronary artery (D). E and F: high magnification of the REG protein-positive cells shows the fine granular staining of REG protein in cytoplasm. G: negative control slides without the anti-human REG-1 protein MAb, corresponding to A. The normal ventricle (H) and atrium (I) were not stained with anti-REG protein antibody at all. Scale bars equal 200 µm except for E and F, which equal 50 µm.

 
Reg gene expression in infarcted rat hearts. On the basis of the above results, we explored cardiac Reg expression using acute stress models in the rat (Fig. 2). We found Reg-1 mRNA expression in the infarcted rat ventricles 24 h after the onset of AMI using Northern blot analysis (Fig. 2A). The ventricles remarkably expressed Reg-1 mRNA (lanes 3–5) 24 h after the operation, whereas the mRNA was not detected in the corresponding site of either the nontreated (lanes 1 and 2) or sham rats (lanes 6 and 7).



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  		Fig. 2. Expression of Reg-1 mRNA in rat hearts and its protein secretion in response to acute stress of the heart. A: Northern blot analysis of mRNA from the ventricle 24 h after coronary ligation (lanes 3–5) and corresponding sham groups (lanes 6 and 7) compared with the normal ventricle (lanes 1 and 2). After being washed, the same membrane was rehybridized with the probe for rat GAPDH mRNA to verify the equivalent loading of each sample. The position of 18S rRNA is presented on the right. B and C: time courses of Reg-1 mRNA expression in rat hearts after coronary ligation and aortic constriction measured by real-time quantitative RT-PCR (n = 5–7 for each time point). *P < 0.05 and **P < 0.01 vs. corresponding sham groups. D: temporal changes of immunoreactive Reg-1 protein secretion in rat serum after aortic constriction, detected by Western blot analysis. Two lanes (lanes 7 and 8) of standard were applied: 2.5 and 5 ng of recombinant rat Reg-1 protein purified from the culture medium of Pichia (1, 16), respectively. E: comparison of rat Reg-1 protein secretion in serum between acute myocardial infarction (lanes 1–3) and pressure-overloaded (lanes 4 and 5) models 3 h after operation by Western blot analysis.

 
The time course of Reg-1 mRNA expression in rat hearts after LAD ligation was quantitatively examined using the real-time quantitative RT-PCR method (Fig. 2B). In normal rats, no Reg-1 mRNA expression was observed in the heart. In contrast, Reg-1 mRNA expression obviously began 3 h after LAD ligation, peaking at 12 h. In terms of Reg-1 expression in the ventricles, the strongest site of Reg expression was the right ventricle (RV), but the expression scarcely occurred in the LV including the infarcted area, noninfarcted area, or border zone. Surprisingly, we found that the maximum Reg-1 mRNA levels in right atrium (RA) and left atrium (LA) were ~10 and 3 times higher than that of the highest region in the ventricles, respectively (e.g., 317 ± 54 fg/pg GAPDH in the RA and 89 ± 25 fg/pg GAPDH in the LA vs. 31 ± 10 fg/pg GAPDH in the RV at 12 h after the operation). Reg-1 expression in the heart declined to basal transcriptional levels by the third postoperative day.

To examine the correlation between the level of intracardiac Reg gene expression and the severity of the stress, we studied hearts with PO by AoC. Figure 2C shows the time course of the expression of Reg-1 mRNA in PO hearts. Cardiac Reg-1 mRNA expression after AoC increased more slowly and the peak was delayed by 12 h compared with the infarcted hearts. The expression levels of Reg-1 mRNA after AoC were consistently lower in all sites compared with that found after LAD ligation. The maximum level after AoC was 45 ± 7 fg/pg GAPDH in the RA at 24 h after operation compared with 317 ± 54 fg/pg GAPDH in the RA at 12 h after LAD ligation. In general, the cardiac Reg-1 gene expression after AoC was one-fourth to one-seventh of the levels observed after LAD ligation.

To investigate whether Reg protein is secreted into the systemic circulation with a load on the heart, Western blot analysis of rat serum collected after LAD ligation and AoC was performed. As shown in Fig. 2D, Reg protein was not detected in serum collected from normal rats (lane 1) but started to be recognized 3 h after AoC. Reg protein distinctly appeared in serum 12 h after AoC, and secretion was observed over a period of 24 h (lanes 4 and 5). Regression of Reg protein in the serum was observed until 7 days after AoC (lane 6). The concentration of Reg-1 protein in blood, corrected for known amount of the authentic protein, was 0 (control), 0.5 (3 h), 0.8 (6 h), 5.7 (12 h), 4.4 (24 h), and 0 (7 days) µg/ml after AoC. Figure 2E shows the comparison of Reg-1 protein secretion in blood between AMI (lanes 1–3) and PO (lanes 4 and 5) models 3 h after the operation. Circulating levels of the protein after LAD ligation were higher than those after AoC.

Reg receptor mRNA expression in rat hearts during acute heart failure. Figure 3 shows the time course of Reg receptor mRNA expression in rat burdened hearts. A significant increase in Reg receptor mRNA occurred 24 h after LAD ligation (Fig. 3A), and this was detected in all of the parts in the heart. In particular, Reg receptor mRNA expressions in the infarcted area and the border zone were significantly higher compared with the other regions (P < 0.05 vs. the noninfarcted LV, RV, LA, and RA), in contrast with only traces of Reg-1 mRNA transcription. The high levels of Reg receptor mRNA both in the infarcted area and border zone were still observed 7 days after LAD ligation.



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  		Fig. 3. Time courses of Reg receptor mRNA expression in rat hearts after coronary ligation (A) or aortic constriction (B). The manner of illustration is the same as in Fig. 2, B and C. Note the difference of vertical scales between A and B. *P < 0.05 and **P < 0.01 vs. corresponding sham group; {dagger}P < 0.05 vs. the left ventricle of the corresponding group.

 
The expression pattern of Reg receptor mRNA after AoC was different from that observed after LAD ligation (Fig. 3B). The levels of the gene in the heart after AoC were less than one-third of those found after LAD ligation. In this instance, the increase in Reg receptor mRNA in the RV was more apparent than in the other chambers within 7 days after AoC. However, Reg receptor mRNA expression in both the LV and RV were significantly greater than in the atria after 1 mo (P < 0.05, data not shown).

IL-6 mRNA expression in the heart after LAD ligation. We examined the possible involvement of IL-6, known as a typical in vivo inducer of Reg (1), in the mechanism of Reg activation in the heart. Figure 4 shows the time-course expression of IL-6 mRNA in the rat heart after LAD ligation analyzed by real-time quantitative PCR. The strong and transient IL-6 induction in the heart occurred after coronary ligation, especially in the infarcted area and border zone. However, IL-6 was not significantly induced in any of the cardiac chambers after AoC as well as in the corresponding sham-operated group (data not shown).



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  		Fig. 4. Time course of myocardial IL-6 mRNA expression after coronary ligation. The manner of illustration is the same as in Fig. 2B. *P < 0.05 and **P < 0.01 vs. corresponding sham groups.

 
Immunohistostaining of rat infarcted hearts for Reg-1 protein. Figure 5 shows the representative results of immunohistochemical staining for Reg-1 protein in rat hearts after LAD ligation. Reg protein was not detected in a normal heart at all (data not shown). However, Reg protein-positive cells were recognized 6 h after LAD ligation (Fig. 5, A–E) but not in the atrium or ventricle of the corresponding sham rats (Fig. 5, F and G, respectively). Figure 5, A and B, shows Reg-positive cells in the RA and LA 6 h after LAD ligation, respectively. The dye affinities for atrial tissue seemed to be higher than for the RV (Fig. 5D) and LV (Fig. 5E), which was in good agreement with the quantified Reg-1 mRNA (Fig. 2B). High magnification revealed that immunoreactive Reg protein was stained in a fine granular pattern in the cytoplasm of cardiomyocytes (Fig. 5C) as well as in the human infarcted heart. The vascular endothelia of arterioles, fibroblasts, or other components of the heart appeared not to be involved in Reg protein expression.



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  		Fig. 5. Immunohistochemical staining of infarcted rat hearts for Reg-1 protein. Reg-1 protein was detected 6 h after coronary ligation (A–E) but not in the atrium (F) or ventricle (G) of the corresponding sham rats. The dye affinities for the right (A) and left (B) atria are higher than those for the right (D) and left (E) ventricles. C: high magnification of the left atrium retrieved 6 h after the operation revealed that immunoreactive Reg-1 protein was stained in a fine granular pattern in the cytoplasm. Scale bars equal 100 µm except for C, which equals 25 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We first found REG-1 protein expression in human heart tissue obtained from autopsied patients who died from myocardial infarction. This result raised the possibility that the Reg gene could be induced in response to heart stress. We therefore tried to determine the expression profiles of the gene in the heart after the onset of cardiac load using animal experimental models.

Two animal models of acute heart stress, namely, AMI (10) and PO (5, 12, 18), were used to estimate the relationship between the level of Reg expression and the intensity of the stress. The transcriptional activation level of the myocardial BNP gene, measured by real-time quantitative RT-PCR, was used as a guide for the intensity of acute stress (7, 10, 19). The expression levels of BNP mRNA in the atria and ventricle increased 14.0 and 27.3 times at the time point of 3 h after LAD ligation than those of baseline, respectively. On the other hand, the maximum levels of BNP mRNA during the examination period after AoC were 7.8 and 11.8 times in the atria and ventricles, respectively, compared with baselines, which were significantly lower compared with those of AMI model (P = 0.025 and 0.043, respectively). From these findings, the stress of AMI seems to be more intense compared with that of PO. Actually, the mortality rate of animals that received LAD ligation (26.8% during the experimental period) was much higher compared with that after AoC (2.2%).

According to a previous report (10), LVEDP 24 h after LAD ligation was 1.8 times higher than sham group (P < 0.05). On the other hand, LVEDP 24 h after ascending aortic banding was reported as 1.35 times higher than the sham group (P < 0.05) (3). Moreover, Knecht et al. (15) tested three different heart failure models of rats, namely, aortocaval shunt, aortic banding, and myocardial infarction. Thirty days after the operation, plasma atrial natriuretic peptide (ANP) levels were 314 ± 66 (control), 998 ± 207 (shunt), 921 ± 96 (banding), and 1,675 ± 239 pmol/l (infarction). On the basis of this evidence, mechanical load caused by AMI is deduced to be stronger compared with that by PO.

Reg-1 mRNA levels in the PO model were approximately one-fourth to one-seventh compared with those in the AMI model, although the relative expression of Reg-1 at different sites was similar between the two models. These results indicated that Reg-1 expression levels in the burdened heart could reflect the severity of load in the acute phase. In addition, the time of peak Reg-1 mRNA levels in the AoC model was delayed by ~12 h compared with the AMI model. This might reflect a difference in the progression of the decompensation between these two stress models.

As shown in Fig. 2B, the site of greatest expression of Reg-1 mRNA was the atrium after LAD ligation, and immunohistochemical staining also strongly suggested that Reg-1 protein might be secreted more from cardiomyocytes in the atria than the ventricles (Fig. 5). On the other hand, Reg receptor mRNA expressions in the infarcted area and border zone were higher than those in the other four sites. From these results, we hypothesize that there is a paracrine signaling pathway for Reg between the atrium and the damaged ventricle. In case of PO, similar patterns of Reg and its receptor gene expression was observed. In accordance with our hypothesis, Reg protein levels in serum seemed to correspond to the levels of intracardiac Reg-1 mRNA expression under the condition of acute stress (Fig. 2D). As Fig. 2E shows, we also found that circulating levels of Reg-1 protein in AMI rats were higher compared with those in PO rats, which was consistent with the results of mRNA measurement (Figs. 2, B and C). Reg protein has a hydrophobic region similar to the signal sequence of many secretory proteins (25). In fact, Reg protein is considered to act via autocrine/paracrine secretion in the process of pancreatic regeneration or gastric mucosal healing (2, 16).

We cannot absolutely rule out the possibility of Reg-1 protein production in a source other than the heart in response to acute heart stress because Reg-1 protein detection in plasma seemed to occur earlier than Reg-1 mRNA overexpression in the heart (Fig. 2, C and D). However, we speculated that Reg-1 protein could not be taken up into cardiomyocytes from blood in the early phase, because Reg receptor mRNA was not expressed 6 ~ 12 h after loading, as shown in Fig. 3. In addition, the actual time course of Reg-1 protein secretion in blood remains to be proved not only by Western blot analysis but also by ELISA system.

Although IL-6 is a typical cytokine involved in inflammation or the immune response (13), it has been reported that typical inflammation mediators such as IL-6 and glucocorticoid can induce the Reg gene (1, 22). The 5'-regions of the Reg gene family have the consensus sequences of IL-6 responsive elements (21, 22). However, this illustration seems difficult to apply to the results of the present study. As Fig. 4 shows, activation of the IL-6 gene appeared exclusively in the infarcted area and border zone after coronary artery ligation, whereas Reg-1 expression in these regions was relatively less compared with that observed in the atria (Fig. 2B). Moreover, the IL-6 mRNA expression after AoC could be regarded as almost nil in contrast to the AMI model (data not shown); however, Reg-1 mRNA expression was also observed in the AoC model. In other words, there was no correlation between IL-6 and Reg-1 gene expressions in the failed heart. One of the dominant factors regulating the synthesis and release of some cardiac hormones, such as the natriuretic peptides, is mechanical stretch of the myocardium (9). We analogize that one of the factors responsible for Reg-1 gene expression is the severity of mechanical stretch. Because the wall thickness of the atria is obviously thinner than that of the ventricles, the atria have a possibility for showing great sensitivity to small change in wall stretch. As Fig. 2B shows, the expression of Reg-1 mRNA was not found in the noninfarct area of the LV after LAD ligation despite not having caused infarction like atria, which may be a result of the difference in the wall thickness between these two chambers.

As for the Reg receptor gene, there seems to be no clear-cut correlation between mRNA expression and regional wall thickness (Fig. 3). The damaged ventricle, namely, the infarct area and border zone, expressed the receptor gene obviously higher than others in a series of our animal experiments. Moreover, the expression of the Reg receptor gene in the RV is apparent 24 h and 7 days after AoC compared with that of the LV (P < 0.05; Fig. 3B), although PO to the LV must be very strong after AoC. These results suggest that the mechanism of Reg receptor gene activation differs vastly from that of the Reg gene. The elucidation of the trigger(s) for Reg and its receptor gene expression is one of the most important agenda for revealing Reg function in the heart.

The Reg gene may act in the heart through a paracrine interaction between the atrium and the damaged ventricle as indicated above. In addition, transcriptional activation of the Reg gene in the heart seems to depend on the severity of heart stress. From these results, one possible explanation is that Reg protein might play a role in the restoration of injured myocardium. Actually, Reg is involved in various types of tissue restoration in addition to the originally identified function in pancreatic regeneration (22, 25). The enhancement of Reg and its product in rat enterochromaffin-like cells was seen in water immersion-induced gastric lesions and played important roles in the healing process (2). We also have found that Reg-1 can restore the cellular integrity of transplanted cryopreserved vascular allografts in rats (14). Daily administration of 0.5 g/kg nicotinamide after transplantation induced intragraft Reg mRNA expression in cryopreserved vascular allografts accompanied by augmentation of the intragraft cell population. Although Reg might be viewed as a key to elucidate the ability of the heart to restore damaged myocardium, Reg modulation by administration of Reg inducers needs to be evaluated in future studies.

In conclusion, we report for the first time the transcriptional activation of Reg and its receptor gene in the heart against acute stress caused by myocardial infarction or PO. Furthermore, we advocate an alternative action of Reg, namely, paracrine interaction between the atrium and ventricle. Our preliminary findings may help to elucidate the molecular basis of myocardial injury and suggest the possibility of a new approach for treatment of acute heart injury.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This study was supported in part by Japan Society for the Promotion of Science Grant 16591405 (to S. Taniguchi).


   ACKNOWLEDGMENTS
 
We thank Drs. Kazuhiko Nishizaki, Kunio Yonemasu, and Atsushi Kubo for helpful advices and supplying rat IL-6 cDNA and the specific TaqMan probe. We are grateful to Mamiko Yoshimura for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Kiji, Dept. of Thoracic and Cardiovascular Surgery, Nara Medical Univ. School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan (E-mail: t-kiji{at}naramed-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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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 GRANTS
 REFERENCES
 

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