INTRODUCTION

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MYOCARDIAL ISCHEMIA followed by reperfusion results in cardiac contractile dysfunction (5, 28, 29). The sequential events that produce this cardiac dysfunction are a decreased release of nitric oxide (NO), upregulation of adhesion molecules on the endothelial cell surface (i.e., P-selectin), leading to polymorphonuclear leukocyte (PMN) rolling and adherence to the endothelium, and infiltration of PMNs into the myocardium, resulting in cardiac dysfunction and enhanced necrosis (19, 29). The time course of these events begins 2.5-5 min postreperfusion (i.e., reduced NO release), and the PMNs start to transmigrate from the coronary vasculature and infiltrate into cardiac tissue at 20 min (19, 29, 33). PMNs induce endothelial and myocardial injury by releasing cytotoxic substances such as oxygen-derived free radicals, inflammatory cytokines, and proteolytic enzymes (2, 30, 32). Previous studies have shown that endogenous NO or administration of NO donors can attenuate endothelial dysfunction (8, 22, 26, 34) and protect against cardiac contractile dysfunction (1, 4, 21). Thus maintenance of basal NO release from the coronary endothelium attenuates PMN adherence to the coronary endothelium, leading to preservation of cardiac contractile function in ischemia-reperfusion (I/R) injury (20, 21). These findings also suggest that compounds that enhance NO production from the vascular endothelium may exert cardioprotective actions against PMN-induced I/R injury (19, 24).

C-peptide is a 31-amino acid peptide cleaved from proinsulin as it is converted to insulin. Proinsulin consists of an A chain, a connecting peptide (C-peptide), and a B chain. After proinsulin is cleaved, C-peptide remains in the secretory granule of beta cells in the pancreas and is cosecreted with insulin in response to glucose stimulation (27, 31). Originally C-peptide was only thought to promote alignment of the A and B chains of the insulin molecule (27, 31). Recent studies have shown that systemic administration of C-peptide improves vascular, neural, and renal dysfunction in diabetic rats (11, 25), decreases glomerular filtration, and increases renal plasma flow in type 1 diabetic patients (6, 13). In addition, it has been shown that C-peptide promotes arteriolar dilation in skeletal muscle (12) and inhibits leukocyte-endothelium interaction in the mesenteric microcirculation (24) by a NO-mediated mechanism (12, 24).

Therefore, the purpose of the present study was to examine the effect of C-peptide on cardiac contractile function in the isolated perfused rat heart following PMN-induced I/R injury. Our findings show that systemic administration of C-peptide 4 or 24 h before cardiac perfusion is able to significantly attenuate cardiac contractile dysfunction in PMN-induced I/R injury. This cardioprotective action of C-peptide was associated with inhibition of PMN infiltration into the I/R myocardium. Finally, PMN adherence to the vascular endothelium was significantly attenuated in C-peptide-injected rats compared with 0.9% NaCl-injected rats. Our data indicate that C-peptide is able to attenuate cardiac contractile dysfunction by inhibition of neutrophil infiltration into the I/R myocardium.

	METHODS

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Isolated rat heart preparation. Male Sprague-Dawley rats (275-325 g) were anesthetized with 60 mg/kg pentobarbital sodium intraperitoneally. Heparin sodium (1,000 U) was also administered intraperitoneally. Hearts were rapidly excised, the ascending aorta was cannulated, and retrograde perfusion of the heart was initiated with a modified Krebs buffer maintained at 37°C and at a constant pressure of 80 mmHg. The Krebs buffer had the following composition (in mmol/l): 17 dextrose, 120 NaCl, 25 NaHCO₃, 2.5 CaCl₂, 0.5 EDTA, 5.9 KCl, and 1.2 MgCl₂. The perfusate was aerated with 95% O₂-5% CO₂ and equilibrated at a pH of 7.3-7.4. Two side arms in the perfusion line proximal to the heart inflow cannula allowed for infusion of PMNs and plasma directly into the coronary inflow line. Coronary flow was monitored by a flowmeter (model T106, Transonic Systems). Left ventricular developed pressure (LVDP) and the maximal rate of development of LVDP (+dP/dt_max) were monitored using a pressure transducer (model SPR-524, 2.5-F, Millar Instruments) that was positioned in the left ventricular cavity. Coronary flow, LVDP, and +dP/dt_max were recorded by using a MacLab data acquisition system (ADI Diagnostics) in conjunction with a Power Macintosh 7600 computer (Apple Computers). LVDP, +dP/dt_max, and coronary flow were measured every 5 min for 15 min to equilibrate the hearts and obtain a baseline measurement. LVDP was defined as left ventricular end-systolic minus left ventricular end-diastolic pressure. After 15 min, the flow of Krebs buffer was reduced to zero to induce global ischemia for 20 min. After ischemia, hearts were infused for 5 min with 200 × 10⁶ PMNs resuspended in 5 ml of Krebs buffer along with 5 ml of plasma at a rate of 1 ml/min. Sham I/R hearts were not perfused with PMNs and received only plasma. Previous studies showed that sham I/R hearts given PMNs exhibited no changes from initial control values (18). Data were recorded every 5 min for the first 30 min after reperfusion and at the 45-min time point. Rats were anesthetized with ethyl ether and then given 70 nmol/kg C-peptide or an equal volume of 0.9% NaCl injected into the sublingual vein of rats 4 or 24 h before experiments. After each experiment, hearts were placed in 4% paraformaldehyde and stored at 4°C for later histological analysis. C-peptide produced by solid-state synthesis and having a purity of 98% (Eli Lilly, Indianapolis, IN) was freshly weighed and diluted in 0.9% NaCl just before intravenous injection. In additional perfused hearts, N^G-nitro-L-arginine methyl ester (L-NAME, 50 µmol/l) was added to the perfusate for the entire 45-min reperfusion period.

Isolation of plasma. Blood was collected in citrate phosphate buffer before isolation of the rat heart. The blood was centrifuged at 10,000 g for 10 min. Thereafter, the plasma was decanted and used for infusion to I/R hearts. Plasma (5 ml) taken from a single rat was used for each perfused heart.

Isolation of PMNs. Sprague-Dawley rats (350-400 g) were anesthetized with ethyl ether and given 14 ml of 0.5% glycogen (Sigma Chemical) intraperitoneally dissolved in PBS. Sixteen to eighteen hours later, the rats were anesthetized with ethyl ether, and the neutrophils were harvested from these donor rats by peritoneal lavage in 30 ml of 0.9% NaCl as previously described (18). The peritoneal lavage fluid was centrifuged at 250 g for 20 min at 4°C. The PMNs were then washed several times in 15 ml of PBS. Thereafter, the PMNs were resuspended in 2.5 ml of PBS and 10 samples were pooled before use in cardiac perfusion experiments. The neutrophil preparations were >90% pure and >95% viable according to microscopic analysis and exclusion of 0.3% trypan blue, respectively.

Determination of PMN infiltration of cardiac tissue. Three rat hearts from each of the six experimental groups were used for histological analysis. Ten areas of each rat heart were counted for PMN infiltration. Hearts were dehydrated in graded ice-cold acetone washes (50-100%). The heart tissue was then embedded in plastic, sectioned into 4-µm serial sections, and placed onto glass slides. Sections were then placed in 100% ethanol for 5 min to remove plastic and were rehydrated in tap water for 1 min. Thereafter, hematoxylin was applied to the sections for 7 min, and the sections were rinsed in running tap water for 30 s. Eosin stain was then applied to the sections for 2 min, followed by a running tap water rinse for 30 s. The number of infiltrated PMNs was counted by light microscopy, and the results are expressed as infiltrated PMNs/mm² cardiac tissue area.

PMN adherence to superior mesenteric artery endothelium. Rat PMNs were isolated as reported in Isolation of PMNs. Segments of the superior mesenteric artery (SMA) were removed from control rats and C-peptide-treated rats, sectioned into 2- to 3-mm rings, opened, and placed into wells containing 2 ml of Krebs-Henseleit (KH) buffer. The SMA tissue was challenged with 2 U/ml thrombin to activate the endothelium by inducing P-selectin surface expression on the endothelium. The thrombin-activated endothelium was then coincubated with fluorescent dye-labeled PMNs (2 × 10⁶ cells) as previously described (20). The number of adherent PMNs was counted by epifluorescence microscopy. Five different fields of each endothelial surface were counted, and the results are expressed as adherent PMNs/mm² endothelium. An organic NO donor, 4-hydroxymethyl-furazan-3-carboxylic acid-2-oxide (CAS-1609, Casella, Frankfurt, Germany) was used as a positive control (9).

Measurement of NO release from rat aortic segments. Rats were given 70 nmol/kg C-peptide or an equal volume of 0.9% NaCl intravenously as described in Isolated rat heart preparation. Twenty-four hours later, the aortas were isolated after pentobarbital sodium anesthesia, and the excised aortas were immersed in warm, oxygenated KH solution. Aortas were cleaned of adherent fat and connective tissue, and rings 6-7 mm in length were carefully prepared. Aortic rings were cut, opened, and fixed by small pins with the endothelial surface facing up in 24-well culture dishes containing 1 ml of KH solution. After equilibration at 37°C, NO released into the KH solution was measured. NO was measured according to the method of Guo et al. (10) using a NO meter (Iso-NO; World Precision Instruments, Sarasota, FL) connected to a polarographic internally shielded NO electrode. NO released into the medium is reported as nanomoles per gram of tissue.

Statistical Analysis. All data in the text and figures are presented as means ± SE. Data on coronary flow, LVDP, and +dP/dt_max were analyzed by ANOVA using post hoc analysis with the Bonferroni/Dunn test. Student's t-test was used to compare final coronary flow, LVDP, and +dP/dt_max values between two groups. Probability values of 0.05 were considered to be statistically significant.

	RESULTS

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To determine whether systemic administration of 70 nmol/kg C-peptide exerted direct effects on cardiac contractile function, we compared nonischemic control rat hearts isolated from rats that received C-peptide 4 h before perfusion with nonischemic control rat hearts not receiving C-peptide. Perfusion of C-peptide-treated hearts did not result in any change in coronary flow, LVDP, or +dP/dt_max at the end of the observation period compared with rat hearts not receiving C-peptide, indicating that C-peptide did not exert any direct effect on cardiac function in nonischemic perfused rat hearts. Moreover, perfusion of untreated I/R hearts without PMNs did not result in any significant long-term alteration in any of the cardiac function variables measured, indicating that global ischemia did not provoke prolonged cardiac dysfunction in this model of I/R. Ischemia for 20 min produces a transient cardiac dysfunction, such that LVDP is only depressed by one-third (i.e., 67 ± 6% of control) 15 min after reperfusion. This transient cardiac dysfunction recovers to 92 ± 5% of control 45 min after reperfusion. However, I/R rat hearts perfused with PMNs experienced a marked and sustained reduction in cardiac contractile function and coronary flow compared with hearts in the other five groups, the LVDP recovering to only 53 ± 8% of control 45 min after reperfusion. In contrast, I/R rat hearts obtained from rats that received 70 nmol/kg C-peptide at either 4 or 24 h and perfused with PMNs exhibited significant attenuation of cardiac contractile dysfunction (i.e., markedly higher coronary flow, LVDP, and +dP/dt_max; Figs. 1-3). The 4-h C-peptide-treated group exhibited a significant improvement in final coronary flow compared with the 0.9% NaCl-treated group (P < 0.05, Fig. 1). The 24-h C-peptide-treated group also exhibited an improvement in final coronary flow compared with the 0.9% NaCl group. Moreover, the 4- and 24-h C-peptide groups exhibited an improvement in final LVDP and +dP/dt_max compared with the 0.9% NaCl group when perfused with PMNs (P < 0.01, Figs. 2 and 3). All of these cardioprotective effects of C-peptide were blocked in I/R hearts perfused with L-NAME (in the presence of PMNs and plasma) (Figs. 1-3), although L-NAME only reduced LVDP in sham I/R hearts by 10% (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Initial and final coronary flow expressed in ml/min in isolated perfused rat hearts before ischemia and after reperfusion. Ischemic hearts were perfused in the presence or absence of polymorphonuclear leukocytes (PMNs). All values are expressed as means ± SE. Numbers of hearts are shown at the bottom of the bars. I/R, ischemia-reperfusion; L-NAME, NG-nitro-L-arginine methyl ester; NS, not significant. *P < 0.05 vs. I/R + PMN + vehicle group.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Initial and final left ventricular developed pressure (LVDP) expressed in mmHg in isolated perfused rat hearts before ischemia and after reperfusion. Hearts were perfused in the presence or absence of PMNs. PMNs induced a significant contractile dysfunction, which was attenuated by C-peptide. All values are expressed as means ± SE. Numbers of hearts are shown at the bottom of the bars. **P < 0.01, ***P < 0.001 vs. I/R + PMN + vehicle group.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Initial and final maximal rate of development of LVDP (+dP/dtmax) expressed in mmHg/s in isolated perfused rat hearts before ischemia and after reperfusion. Hearts were perfused in the presence or absence of PMNs. PMNs induced a significant contractile dysfunction, which was attenuated by C-peptide. All values are expressed as means ± SE. Numbers of hearts are shown at the bottom of the bars. **P < 0.01 vs. I/R + PMN + vehicle group.

I/R hearts perfused with PMNs and plasma in the 0.9% NaCl group exhibited a significant reduction of 42 ± 15% in coronary flow from initial values (P < 0.01) (Fig. 1), LVDP decreased 47 ± 8% from initial values (P < 0.001) (Fig. 2), and +dP/dt_max decreased 61 ± 10% from initial values (P < 0.001) (Fig. 3). The reductions in cardiac performance in both C-peptide groups were significantly attenuated compared with the reductions in cardiac performance in the 0.9% NaCl-treated group. Thus coronary flow decreased only 18 ± 6% and 8 ± 13% in the 4-h and 24-h C-peptide-treated rats. Similarly, LVDP decreased only 17 ± 4% and 20 ± 5%, and +dP/dt_max decreased only 22 ± 7% and 27 ± 6%, in the 4- and 24-h C-peptide-treated rats, respectively. These values are significantly different from the corresponding values for the 0.9% NaCl-treated rats for coronary flow, LVDP, and +dP/dt_max in the 4-h C-peptide-treated group and for LVDP and +dP/dt_max in the 24-h C-peptide-treated group.

The significant deficit in coronary flow and cardiac performance can be associated with the presence of PMNs at the time of reperfusion where a marked attenuation of PMN infiltration into postreperfused cardiac tissue was observed in the two C-peptide-treated groups compared with 0.9% NaCl-treated rats (P < 0.001) (Fig. 4).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Histological assessment of extravascular infiltrated PMNs in isolated perfused rat heart samples taken from 3 rats per group and 10 areas per heart. All values are mean numbers of PMNs/mm2 heart area ± SE. The number of PMNs infiltrated into postreperfusion cardiac tissue was significantly attenuated by C-peptide. ***P < 0.001 vs. I/R + PMN + vehicle group.

A significant reduction in PMN adherence (30% ± 6%) was also observed in vascular segments from 4-h C-peptide-treated rats compared with those isolated from untreated rats (P < 0.001) (Fig. 5). In addition, the NO donor CAS-1609, which served as a positive control, also significantly reduced PMN adherence in vascular segments compared with those isolated from untreated rats by 58 ± 8% (P < 0.001) (Fig. 5).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Adherence of rat PMNs to rat superior mesenteric artery (SMA) endothelium is expressed as PMNs/mm2. Rat SMAs were isolated from rats given C-peptide or vehicle (0.9% NaCl) and were either nonstimulated or stimulated with thrombin (2 U/ml). PMNs that adhered to endothelium were counted and analyzed. All values are expressed as means ± SE. Numbers shown at the bottom of the bars indicate numbers of SMA segments analyzed in each group. KH, Krebs-Henseleit buffer. ***P < 0.001 vs. thrombin group.

Figure 6 summarizes the effects of C-peptide on the release of NO in isolated rat aortic segments. As shown, C-peptide more than doubled the release of NO from these vascular segments (P < 0.01 from control). Thus C-peptide enhances basal NO release even 24 h after intravenous administration to rats.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effect of C-peptide on basal release of nitric oxide (NO) from isolated rat aortic segments. C-peptide (70 nmol/kg) was injected intravenously in rats 24 h before isolation of the aorta. All values are means ± SE of 12 segments isolated from 4 rats/group.

	DISCUSSION

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The present study demonstrates that C-peptide exerts significant cardioprotective effects against PMN-mediated reperfusion injury in the isolated rat heart. The cardioprotective effect of C-peptide was characterized by a significant restoration of postreperfusion coronary flow, LVDP and +dP/dt_max compared with PMN-perfused I/R hearts obtained from rats receiving 0.9% NaCl. These C-peptide effects are most likely due to significantly reducing PMN adherence to the vascular endothelium, thereby leading to a significant reduction in PMN infiltration into postreperfused cardiac tissue (24). C-peptide may exert its cardioprotective effects by stimulating NO production from the vascular endothelium (12, 24). NO attenuates PMN adherence to the vascular endothelium (16), thus reducing PMN infiltration into cardiac tissue. In this regard, NO has been shown to act as a physiological inhibitor of leukocyte-endothelial cell interaction by suppressing upregulation of endothelial cell adhesion molecules (i.e., P-selectin) (3, 8, 19) and intercellular adhesion molecule-1 in the mesenteric circulation as well (8, 16).

Recently Jensen and Messina (12) showed that C-peptide was able to increase the diameter of rat skeletal muscle arterioles in a concentration-dependent manner in the presence of insulin, and this could be blocked by L-NAME, thus implicating NO as the vasodilator. Our study, in contrast, did not require coinfusion of insulin with C-peptide to produce cardioprotective effects of C-peptide. However, the rats in our study were not fasted so that insulin secretion presumably still occurred after the rats consumed food, and perhaps endogenous insulin could have synergized with C-peptide to achieve cardioprotective effects. Scalia et al. (24) recently showed that a 70 nmol/kg bolus intravenous C-peptide dose to rats increased endothelial NO synthase expression and produced a threefold increase in the basal release of NO from the aortic endothelium at 3-4 h. In the present study, a significant increase in basal NO release was shown even at 24 h. Moreover, L-NAME blocked the C-peptide-induced improvement in coronary flow, LVDP, and +dP/dt_max that occurred after I/R, further implicating NO in these processes. It is unlikely that C-peptide exerted its cardioprotective effects by directly influencing coronary vasodilation or increasing cardiac contractility because there were no increases in coronary flow, LVDP, or +dP/dt_max in nonischemic perfused rat hearts given C-peptide compared with control nonischemic rat hearts not given C-peptide.

C-peptide is normally released from pancreatic -cells in equimolar concentrations to insulin (31). C-peptide was initially thought to fulfill an important function in the assembly of the A and B chains within the mature insulin protein. However, it was not considered to be biologically active (27). More recently, animal and clinical studies have shown that systemic administration of C-peptide to streptozotocin-induced diabetic animals (11, 25) and type 1 diabetic patients (6, 7, 13-15, 17) may have some additional beneficial effects. Injection of 130 nmol/kg C-peptide to diabetic rats prevented the vascular and neuronal dysfunction associated with this type 1 animal model of diabetes (11). In type 1 diabetic patients, systemic administration of C-peptide decreased glomerular hyperfiltration (15), improved skeletal muscle glucose utilization (13), improved red blood cell deformability (17), improved microvascular skin blood flow (5, 6, 7), and improved resting forearm blood flow (14). More recently, C-peptide was shown to bind to specific G protein-coupled receptors on human cell membranes (23) as confirmed by inhibition of C-peptide binding by pertussis toxin (23). The existence of a putative C-peptide receptor may provide a biochemical basis for the biological effects of C-peptide.

In summary, our results are the first to show a cardioprotective effect of C-peptide in myocardial I/R injury. C-peptide was able to significantly attenuate cardiac contractile dysfunction in the isolated perfused rat heart when infused with PMNs compared with 0.9% NaCl-treated hearts. These cardioprotective effects appear to be related to inhibition of PMN adherence to the vascular endothelium, resulting in fewer PMNs infiltrating the cardiac tissue. C-peptide also promotes NO release by the endothelium, which significantly contributes to these effects via downregulation of leukocyte-endothelium interaction.