reperfusion injury in isolated rat hearts

Peptides 26 (2005) 501–507

Protective effects of intermedin/adrenomedullin2 on ischemia/reperfusion injury in isolated rat hearts Jing-Hui Yanga , Yong-Fen Qia,b,c,∗ , Yue-Xia Jiaa , Chun-Shui Pana , Jing Zhaob , Jun Yangd , Jaw-Kang Changd , Chao-Shu Tanga,c a

b

Institute of Cardiovascular Research, Peking University First Hospital, Beijing 100034, China Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100083, China c Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100083, China d Phoenix Pharmaceutical Inc., 530 Harbor Boulevard Belmont, CA 94002, USA Received 15 September 2004; received in revised form 22 October 2004; accepted 25 October 2004 Available online 10 December 2004

Abstract Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family identified from human and other vertebrate tissues. Preprointermedin can generate a 47-amino acid mature peptide (IMD1–47 ) and a shorter 40-amino acid one (IMD8–47 ) by proteolytic cleavage. The present study was designed to determine the protective effect of IMD on cardiac ischemia/reperfusion (I/R) injury and its possible mechanism. Isolated rat hearts were perfused on a Langendorff apparatus and subjected to 45-min global ischemia and 30-min reperfusion. Cardiac function was measured. The release of myocardial protein and lactate dehydrogenase (LDH) and the formation of malondialdehyde (MDA) were assayed. Myocardial cAMP content was determined by radioimmunoassay (RIA). Cardiac I/R induced a marked inhibition of cardiac function and myocardial injury. Reperfusion with IMD significantly attenuated the I/R injury. Compared with I/R alone, perfusion with 10−8 mol/L IMD1–47 and IMD8–47 induced a 36% and 33% increase in  left ventricular pressure (LVP), 30% and 28% in maximal rate of increase of LV pressure (+LVdP/dt max), and 34% and 31% in maximal rate of decrease of LV pressure (−LVdP/dt max), respectively (all P < 0.01) but an approximately 58% and 51% decrease in LV diastolic pressure, respectively (P < 0.01). In addition, perfusion with IMD markedly attenuated the leakage of LDH, total protein and myoglobin from myocardia compared with I/R alone. The contents of ventricular myocardia cAMP after reperfusion with 10−8 mol/L IMD1–47 and IMD8–47 were 130% and 91% higher, respectively, than that with I/R alone (all P < 0.01). However, formations of myocardial MDA were 52% and 50% lower than that with I/R alone (all P < 0.01), respectively. Interestingly, the above IMD effects were similar to those of adrenomedullin (10−8 mol/L). These results suggest that IMD, like adrenomedullin, exerts cardio-protective effects against myocardial I/R injury. © 2004 Elsevier Inc. All rights reserved. Keywords: Intermedin; Ischemia/reperfusion; cAMP; Heart

1. Introduction Since the discovery of calcitonin in the 1960s, many bioactive peptides such as calcitonin gene-related peptide (CGRP), adrenomedullin (ADM) and amylin were discovered to be structurally related to each other and therefore grouped into the CGRP superfamily [1]. This group of peptide hormones ∗

Corresponding author. Tel.: +86 10 82802183; fax: +86 10 66551036. E-mail address: [email protected] (Y.-F. Qi).

0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.10.025

act on diverse organs and tissues and regulate body homeostasis. ADM and CGRP are important endocrine and neurocrine integrators of homeostasis in the cardiovascular, renal and respiratory systems, whereas amylin is essential for optimal glucose metabolism [9,10,21]. Recently, Roh et al. identified a novel calcitonin/CGRP family peptide, intermedin (IMD), from the genomes of human and other vertebrates [22]. Human intermedin encodes a prepropeptide of 148 amino acids, with a signal peptide for secretion at the N terminus. Preprointermedin can generate a 47-amino acid mature peptide

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(IMD1–47 ) and a shorter 40-amino acid one (IMD8–47 ) by proteolytic cleavage at the N-terminal proximate basic residues followed by an amidated C terminus. Preliminary studies showed that IMD had vasodilatory and hypotensive actions identical to those of ADM and CGRP. Pharmacological analysis showed that IMD exerts its effects through calcitonin receptors such as the calcitonin receptor-like receptor/receptor activity modifying protein (CL/RAMP) system, a common receptor system of the CGRP superfamily [21]. Recently, growing evidence has affirmed that ADM and CGRP are potent endogenous cardio-renal-protective substances. Exogenous administration of ADM and CGRP peptides or their genes delivery is a new preventive and therapeutic strategy for cardiovascular diseases such as hypertension, myocardial ischemia, heart failure and renal failure [2,7,9,20]. The cardiovascular protective effects of ADM and CGRP are considered to be mediated by a CL/RAMP system [12]. We hypothesized that IMD may also play a cardioprotective role through its receptor CL/RAMP system. In this work, we developed an ischemia/reperfusion (I/R) model of isolated rat hearts to explore the cardioprotective effects of IMD1–47 and IMD8–47 and their mechanism.

2. Materials and methods

Langendorff technique. Hearts were perfused at 85 cm H2 O pressure with Krebs-Henseleit (KH) buffer solution in the following composition (mmol/L): NaCl 118, KCl 4.7, CaCl2 2.0, MgSO4 1.2, KH2 PO4 1.2, glucose 11.0, and NaHCO3 25, pH 7.4. The perfusion was bubbled continuously with 95% O2 /5% CO2 , maintaining a pH of 7.2. A saline-filled latex balloon was inserted into the left ventricle (LV) through the left atrium and connected to a pressure transducer to measure LV pressure. The LV diastolic pressure (LVDP) was adjusted to 6 mmHg at the beginning of the experiments. The maximal rate of increase and decrease of LV pressure (+/−LVdP/dt max) was monitored on a Powerlab (4S, Australia). Coronary perfusion flow (CPF) was determined by timed collections. Control hearts underwent continued perfusion with KH buffer, whereas other hearts were perfused for 30 min before being subjected to 45-min no-flow ischemia and 30-min reperfusion. After 45 min of global ischemia, the experimental hearts were randomly divided into the following groups: ischemia/reperfusion (I/R) group, I/R + IMD1–47 group (reperfusion with KH buffer containing 10−10 and 10−8 mol/L IMD1–47 ), I/R + IMD8–47 group (reperfusion with 10−10 and 10−8 mol/L IMD8–47 ), and I/R + ADM group (reperfusion with 10−8 mol/L ADM), with eight animals in each group. At the end of reperfusion, the hearts were freeze-clamped and stored at −70 ◦ C for other myocardial tissue assays.

2.1. Materials 2.3. Radioimmunoassay for cAMP content All animal care and experimental protocols complied with the Animal Management Rule of the Ministry of Health, People’s Republic of China (documentation 55, 2001) and the Animal Care Committee of the First Hospital, Peking University. Sprague-Dawley (SD) rats (weight 212 ± 2 g) were obtained from the Animal Center, Health Science Center, Peking University (Beijing, China). The rats were housed in plastic cages in a room with a controlled humidity of 40% and a temperature of 22 ◦ C. A 12-h light/12-h dark environmental light cycle was maintained. Synthetic rat IMD1–47 , IMD8–47 , adrenomedullin (ADM), 125 I-IMD and rat anti-IMD antibody were kindly provided by Phoenix Pharmaceuticals (Belmont, CA, USA). The cyclic adenosine monophosphate (cAMP) [125 I]-radioimmunoassay kit was purchased from NEN Life Sci. Products Inc. (Boston, MA). Rat anti-preproIMD primary antibody and secondary antibody were from Phoenix Pharmaceuticals (Belmont, CA, USA), nitrocellulose membrane was from Hybond-C (Amersham Life Science, England), and ECL was from Beijing Applygen Technologies Corp. (Beijing, China). Other chemicals and reagents were of analytical grade. 2.2. Ischemia/reperfusion protocol in isolated rat hearts Rat hearts were isolated and perfused as described previously [23]. Briefly, rats were anesthetized with urethane (1 g/kg, given intraperitoneally) and hearts were quickly removed and arranged for aortic retrograde perfusion by the

After the end of reperfusion, an adequate amount of LV tissues was immediately immersed in 0.4 mL of 0.1 mol/L HCl to stop the reaction and then collected in glass tubes and boiled for 5 min. Tissue homogenate was prepared on Polytron and centrifuged at 2500 × g for 15 min at 4 ◦ C. The supernatant was decanted, and 0.05 mL of 50 mmol/L sodium acetate was added. The samples were kept at −70 ◦ C until being assayed for cAMP content. The tissue sample was dissolved in 0.4 mL of 1% sodium dodecyl sulfate (SDS) for determining protein content. Myocardial cAMP content was measured with a commercial radioimmunoassay kit according to the manufacturer’s high-sensitive protocol. The lower limit of detection was 4 fmol per tube. The value was normalized to protein content. 2.4. [125 I]-IMD binding to myocardial sarcolemmal membrane Myocardial sarcolemmal membrane was prepared as previously described [26] with minor modifications, and protein content was determined by the Bradford method. The activity of membrane marker enzymes and protein yield were measured as described previously by our laboratory [8]. And,marker enzyme activity and protein yield of membrane fractions isolated from control hearts and hearts subjected to I/R showed that the sarcolemmal membrane fractions were minimally contaminated with other organelles.

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The IMD receptor binding assay was carried out with use of [125 I]-IMD as a radioligand. The standard assay mixture contained 10 mmol/L MgCl2 , 50 mmol/L Tris–HCl (pH 7.4), [125 I]-IMD (1–40 nmol/L) in the presence or absence of unlabeled IMD (20 ␮mol/L) in a final volume of 0.1 mL. The assay mixture was preincubated at 37 ◦ C for 2 min, and the binding assay was initiated by the addition of sarcolemmal membrane (50 ␮g protein) and allowed to proceed for 20 min at 37 ◦ C. At the end of the incubation, the reaction mixture was diluted with 4 mL of ice-cold washing buffer (10 mmol/L MgCl2 , 50 mmol/L Tris–HCl; pH 7.4) and filtered immediately on Millipore through a 0.45 ␮m glass fiber filter paper (Baxter Healthcare, Ireland) under suction. The filter paper was washed three times with 5 mL of washing buffer and dried and the radioactivity determined with use of a ␥Counter (Perkin Elmer Wizard 1470, Norwalk). The specific binding was defined as the bound radioactivity displaceable by 20 ␮mol/L of IMD. The maximal binding capacity (Bmax ) and the affinity (the reciprocal of the dissociation constant (Kd )) for 125 I-IMD were calculated from Scatchard plot analysis results. 2.5. Western blot analysis Protein extracts from control and I/R myocardia were resuspended in sample buffer containing 2% SDS, 2% ␤mercaptoethanol, 50 mmol/L Tris–HCl (pH 6.8), 10% glycerol and 0.05% bromophenol blue. The protein mixture was then placed in boiling water for 10 min and briefly centrifuged at low speed to collect the denatured proteins. Protein samples were resolved on a 15% Tris/glycine SDS-polyacrylamide gel in running buffer containing 25 mmol/L Tris, 192 mmol/L glycine, and 0.1% SDS. The proteins were then transferred to a nitrocellulose membrane for 2 h at 4 ◦ C at 100 mA with use of a transfer buffer containing 20 mmol/L Tris–HCl (pH 8.0), 150 mmol/L glycine, and 20% methanol. Non-specific proteins were blocked by incubating the membrane with 5% non-fat dry milk in TBS-T (20 mmol/L Tris–HCl [pH 7.6], 150 mmol/L NaCl, and 0.02% Tween 20) for 1 h at room temperature with agitation. Rat anti-preproIMD primary antibody was added to the membrane at a 1:200 dilution in TBS-T and incubated at room temperature for overnight with agitation. The secondary antibody, fluorescein-linked rabbit anti-IMD antibody at a 1:2000 dilution in TBS-T was added to the membrane and incubated at room temperature for 1 h with agitation. Between each of the three proceeding steps (primary, secondary, and tertiary antibody) the membrane was washed three times with TBS-T at room temperature, once for 10 min. The membrane was immediately visualized on a phosphor imager after the addition of the ECL substrate. 2.6. Other measurements Measurement of leakage of myocardial lactate dehydrogenase (LDH) and myoglobin in collected perfusate was per-

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formed as described [11,25]. Myocardial malondialdehyde (MDA) content was quantified as described previously [3,14]. The protein content of myocardia was determined by the method of Bradford [5]. 2.7. Statistical analyses Data are expressed as means ± S.D. One-factor analysis of variance was performed when more than two groups were compared, and when significant (P < 0.05), the Student–Newman–Keuls test was applied to test for differences between individual groups. A P value less than 0.05 was considered statistically significant.

3. Results 3.1. Reperfusion with IMD improved the I/R-induced inhibited cardiac function Before ischemia (preperfusion 15 min), the parameters of cardiac function and CPF were not statistically different among the groups (all P > 0.05). During the experimental period, the cardiac function in the control group was steady. However, in the I/R group, cardiac function was severely inhibited during reperfusion; LV systolic pressure, +/−LVdP/dt max, heart rate and CPF were lower; and LVDP was higher than that in the control group (P < 0.01). Reperfusion with IMD1–47 or IMD8–47 significantly ameliorated the inhibited cardiac function and bradycardia induced by I/R. Compared with the I/R-alone group, the IMD reperfusion groups showed augmented CPF, LVSP and +/–LVdP/dt max and decreased LVDP (P < 0.05 or P < 0.01). The improving effects of equimolar amounts of IMD1–47 , IMD8–47 and ADM on cardiac function were close (all P > 0.05). Data are shown in Table 1. 3.2. Reperfusion with IMD ameliorated I/R-induced myocardial injury I/R resulted in severe myocardial injury: LDH activity and total protein and myoglobin content in collected reperfusate were higher by 2.6-, 5.4- and 5.3-fold, respectively, than that in controls (P < 0.01). Myocardial MDA content was higher by 6.9-fold than that of controls (P < 0.01). Reperfusion with IMD1–47 or IMD8–47 (10−10 and 10−8 mol/L) significantly attenuated myocardial injury induced by I/R. Compared with I/R alone, reperfusion with IMD significantly decreased LDH activity and total protein and myoglobin leakage in reperfusate and myocardial MDA content (all P < 0.01), the effects with 10−8 mol/L IMD being superior to that with 10−10 mol/L (P < 0.01 or P < 0.05). The improving effects of equimolar amounts of IMD1–47 , IMD8–47 and ADM on the myocardial injury were similar (all P > 0.05). Data are shown in Table 2.

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Table 1 Effects of IMD and ADM on cardiac function in isolated, perfused rat hearts Control LVDP LVP LV + dP/dt LV – dP/dt Heart rate Cardiac function

3.4 74.84 1541 1134 268 8.8

± ± ± ± ± ±

I/R 2.1** 9.5** 266** 172** 36* 2.3**

IMD1–47 (10−8 mol/L)

20.1 47.89 1076 796 219 5.0

± ± ± ± ± ±

3.0 6.1 200 112 30 0.9

8.5 65.5 1403 1071 269 7.52

± ± ± ± ± ±

3.0** 5.5** 231* 131** 33* 1.2*

IMD1–47 (10−10 mol/L) 13.1 59.2 1340 1014 253 6.96

± ± ± ± ± ±

2.7**§§ 5.0* 205* 105** 28 1.4*

IMD8-47 (10−8 mol/L) 9.8 63.69 1375 1046 264 7.14

± ± ± ± ± ±

1.7** 8.8** 189* 155** 35* 1.6*

IMD8–47 (10−10 mol/L) 13.9 58.44 1351 1024 250 6.66

± ± ± ± ± ±

1.7**## 7.3 199* 139** 26 1.5*

ADM (10−8 mol/L) 9.9 61.2 1394 1039 267 7.11

± ± ± ± ± ±

2.0** 10.1** 187* 144** 29* 1.8*

Data are means ± S.D. (n = 8). *P < 0.05, **P < 0.01 vs. I/R; § P < 0.01, §§ P < 0.01 vs. IMD1–47 (10−8 mol/L); # P < 0.05, ## P < 0.01 vs. IMD8–47 (10−8 mol/L); LV = left ventriculum; DP = diastolic pressure; LVP = Left ventricular pressure. Table 2 Effects of IMD and ADM on myocardial injury following I/R in isolated, perfused rat hearts Experimental groups

MDA (nmol/gww)

Control I/R IMD1–47 (10−8 mol/L) IMD1–47 (10−10 mol/L) IMD8–47 (10−8 mol/L) IMD8–47 (10−10 mol/L) ADM (10−8 mol/L)

24.0 189.8 90.9 117.0 95.1 128.7 95.3

± ± ± ± ± ± ±

2.7* 30.8 23.1* 10.5*§ 18.4* 20.7*## 6.3*

Total protein (␮g/mL) 14.0 89.9 31.1 48.0 37.5 54.4 38.2

± ± ± ± ± ± ±

2.0* 10.5 5.1* 4.7*§§ 5.0* 6.1*## 4.5*

Mb (␮g/mL) 0.6 3.8 1.34 1.68 1.42 1.82 1.30

± ± ± ± ± ± ±

0.09* 0.50 0.06* 0.11*§§ 0.11* 0.19*## 0.13*

LDH (IU/L) 43.2 156.8 77.8 100.0 83.0 117.8 91.7

± ± ± ± ± ± ±

7.1* 16.3 10.5* 14.8*§§ 11.7* 10.8*## 13.9*

Data are means ± S.D. (n = 8). *P < 0.01 vs. I/R; § P < 0.05, §§ P < 0.01 vs. IMD1–47 (10−8 mol/L); # P < 0.05, ## P < 0.01 vs. IMD8–47 (10−8 mol/L); LDH = lactate dehydrogenase.

3.3. Reperfusion with IMD increased myocardial cAMP content

tively, P < 0.01) and negatively with MDA content (r = −0.79, P < 0.05) and LDH release (r = −0.86, P < 0.05).

Compared with controls, the I/R-alone group had a lower cAMP content (P < 0.01), but after reperfusion with IMD1–47 , IMD8–47 and ADM, myocardial cAMP content was significantly increased (P < 0.01). Administration of equimolar amounts of IMD1–47 , IMD8–47 and ADM resulted in nearly identical increases in cAMP content (P > 0.05). Data are shown in Fig. 1. Myocardial cAMP content correlated positively with +/–LVdP/dt max (r = 0.643 and 0.628, respec-

3.4. I/R potentiated the binding of [125 I]–IMD to myocardial sarcolemmal membranes Fig. 2 shows the effects of I/R on the dynamics of IMD receptors in cardiac sarcolemmal membrane fractions, based on [125 I]-IMD binding studies. As shown in Fig. 2A, [125 I]-IMD binding to cardiac sarcolemmal membranes exhibited a saturable process with a single-component binding characteristic of all experimental groups. In the sarcolemmal membrane, the maximum binding capacity calculated from results of a Scatchard plot (Fig. 2B) was increased by 118% during I/R compared with that in controls (Bmax : 83.05 ± 5.75 fmol/mg protein versus 38.10 ± 1.85 fmol/mg protein. P < 0.01). The affinity (the reciprocal of the dissociation constant [Kd ]) for [125 I]-IMD binding in sarcolemmal membranes (Fig. 2B) was increased with I/R. 3.5. Western blot analysis Western blot analysis showed that the myocardial preproIMD protein level in I/R group was lower than that of controls (Fig. 3). 4. Discussion

Fig. 1. Myocardial cyclic AMP content in isolated, perfused rat hearts. Cyclic AMP was measured by radioimmunoassay of myocardial extracts as described in Section 2. ** P < 0.01 vs. I/R; $$ P < 0.01 vs. IMD1–47 (10−8 mol/L); ## P < 0.01 vs. IMD8–47 (10−8 mol/L).

Roh et al., using a phylogenetic profiling approach to analyze the GenBank TM/EBI Data Bank, identified a novel calcitonin/CGRP family peptide, intermedin, from the genomes of human and other vertebrates [22]. RT-PCR, Northern blot

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Fig. 2. Representative saturation isotherms (A) and Scatchard plots (B) of [125 I]-IMD binding to membranes from rat myocardium. Experiments were performed by incubating a fixed amount of membrane protein (50 ␮g per tube) with increasing concentrations of radiolabeled [125 I]-IMD without (total binding) or with 20 ␮mol/L of unlabeled IMD to determine non-specific binding. Specific binding values were obtained by subtracting non-specific binding from total binding. The saturation curves of specific binding were analyzed by Scatchard analysis to calculate the maximum binding capacities and the Kd values.

and immunoanalysis showed that IMD is primarily expressed in the pituitary and gastrointestinal tract. Genomic analysis showed that the IMD gene is located on the distal arm of human chromosome 22q3 and encodes a prepropeptide of 148 amino acids, which generates a 47-amino acid mature peptide IMD1–47 and a shorter 40-amino acid peptide IMD8–47 . Sequence alignment of the IMD precursor from mammals and teleosts indicated that sequence conservation in orthologous IMD is restricted to the mature region. The mature IMDs of humans and fish share >60% similarity, whereas human and rodent IMDs are 87% identical. This conserved amino acid sequence suggests that IMD plays an important regulatory role in homeostasis [22]. It is well known that calcitonin/CGRP family members are low-molecular-form peptides. In addition to their specific receptors, they can bind to the CL/RAMP system, a common receptor system of the calcitonin/CGRP family [13]. CL is a member of the orphan G-protein coupled receptor (oGPCR) family; it exerts its biological effects by activating G␣s protein and elevating adenylate cyclase [17]. Roh et al. [22] found that in HEK 293T cells, treatment with various concentrations of IMD, ADM and CGRP alone did not change cellular cAMP content; however, on co-transfection with different RAMPs, treatment with IMD resulted in dose-dependent increases in cAMP production. Thus, IMD represents a non-

Fig. 3. Western blot analysis of preproIMD. Protein levels in ventricular tissues. Protein extracts were prepared from control and I/R myocardium (lanes 1and 2). Aliquots (15 mg) were fractionated by SDS-PAGE, transferred to nitrocellulose. membranes, and incubated with a 1:200 dilution of purified antibodies.

selective agonist for the three CL/RAMPs complexes and exerts its biological effects by activating the CL/RAMPs system and elevating the level of intracellular cAMP [22]. Takei et al. [24] identified a novel member of the CGRP family, ADM2, in mammals (mice, rats and humans). However, the amino acid sequences are identical between human ADM2 and preproIMD, so ADM2 and IMD might be the same peptide. ADM2 has been reported to exhibit potent hypotensive action, which is accordance with IMD [22,24]. Our previous work found that intravenous administration of IMD in rats decreased blood pressure in a dose-dependent manner, and IMD had a dilatory effect in the isolated perfused rat aortic ring, which was in accordance with the biological effects of ADM and CGRP (unpublished data). It is well known that ADM and CGRP levels in plasma and cardiovascular tissues are compensatively elevated in cardiovascular diseases such as myocardial ischemia, I/R, hypertension, and heart failure [19]. Administration of the ADM and CGRP peptides or ADM and CGRP gene delivery has significant therapeutic effects on these diseases [7,14,18]. These results suggest that endogenous ADM and CGRP, as potent cardio-protective factors, are important factors in regulating cardiovascular homeostasis and have potential clinical prospects. So, we hypothesized that IMD, as a novel member of CGRP family, could have similar cardio-protective effects. In this work, we observed the cardio-protective role of IMD on the in vitro model of myocardial injury induced by I/R. After 45 min of ischemia in isolated rat hearts, reperfusion resulted in obvious inhibition of cardiac function, the values of LVSP, +/–LVdP/dt max, heart rate and CPF were significantly lowered, but the LVDP was elevated. The heart subjected to I/R showed severe myocardial injury, with a high leakage of myocardial intracellular LDH and proteins and increased formation of myocardial lipid oxide product (MDA) [18]. Treatment with IMD1–47 or IMD8–47 significantly ameliorated cardiac function inhibition and bradycardia induced by I/R. The values of CPF, LVP, and +/–LVdP/dt max

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were augmented, and those of LVDP decreased. Similarly, IMD1–47 or IMD8–47 attenuated the myocardial injury induced by I/R. LDH activity and total protein and myoglobin content in reperfusate and myocardial MDA formation were significantly decreased with IMD treatment compared with I/R alone. Administration of equimolar amounts of IMD1–47 , IMD8–47 and ADM resulted in almost identical cardioprotective effects. Receptor-binding assay demonstrated that IMD binding to sarcolemmal membranes exhibited singlecomponent binding characteristics, which suggests that IMD interacts with a pattern of receptor. Interestingly, the maximum binding capacity of IMD to sarcolemmal membranes was significantly increased after I/R. Regression analysis however showed that the affinity of IMD for its receptor was decreased, which indicted that IMD exerts important effects during I/R. The mechanism by which IMD protects the heart from I/R injury is unclear. I/R injury is a complex process; calcium overload and excessive production of oxygen-free radicals are the main mechanisms involved in I/R injury [15]. ADM can antagonize myocardial injury induced by I/R via inhibiting oxidative stress and reducing calcium overload [6,16]. In this work, we found that treatment with IMD1–47 and IMD 8–47 , just like ADM, significantly decreased myocardial lipid peroxide production of MDA content and indicated that IMD could have an inhibiting effect on oxidative stress. cAMP is a second messenger of ADM and CGRP, which exert their biological effects by binding with their receptor system, CL/RAMPs. The cytoprotective effects of ADM and CGRP were mediated, at least in part, by the cAMP pathway [4]. Roh and associates have reported that IMD can activate the cAMP-dependent signal transduction pathway by activating the CL/RAMP system [22]. We measured the cAMP content in myocardia after reperfusion and showed that IMD1–47 , IMD8–47 and ADM all increased myocardial cAMP content significantly, which suggests that IMD might antagonize the myocardial injury through the cAMP signal pathway. Intermedin is a newly isolated calcitonin/CGRP family peptide. The other two calcitonin/CGRP family peptides, ADM and CGRP, have potent cardiovascular protective roles and a potential prospect on clinical treatment. Intermedin might be a new endogenous substance antagonizing cardiovascular diseases and a new target for the prevention and treatment of cardiovascular diseases. In our study, IMD had strong cardio-protective effects on I/R-induced myocardial injury. The cardio-protective mechanisms of IDM and its significance in terms of pathophysiology are worth further study.

Acknowledgements This work was supported by the State Major Basic Research Development Program of the People’s Republic of China (G2000056905) and the National Natural Science Foundation of China (30470693 and 30170347).

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