INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

UROCORTIN is a 41-amino acid peptide member of the corticotrophin-releasing factor (CRF) family. Urocortin binds with high affinity to the CRF receptor 2 subtype (CRF-R2), which is expressed in both human (5) and rat (23) hearts. Urocortin exerts positive inotropic effects in the heart when administered intravenously including concentration-dependent increases in cardiac contractility, coronary blood flow, cardiac output, and heart rate (16). Recently, urocortin has been shown to protect an immature phenotype of cardiomyocytes against simulated ischemia (SI) via a mitogen-activated protein kinase (MAPK)-dependent pathway (2, 15), thereby reducing both the number of trypan blue-stained cells and lactate dehydrogenase (LDH) release (15). We proposed to study the potential of urocortin to protect adult cardiomyocytes from SI.

Fifteen years ago, the phenomenon of ischemic preconditioning was described whereby transient, sublethal episodes of ischemia result in a greater tolerance of the heart to a normally lethal ischemic insult (13). Two phases of ischemic preconditioning are documented. Immediate preconditioning is evident within minutes of the first episode of ischemia and typically lasts 1-2 h (8). A second phase, delayed preconditioning, is observed 1-3 days after the first ischemic period (12). Cardioprotection resulting from ischemic preconditioning is reflected in reduced infarct size (18), improved recovery of contractile function (3), reduced incidence of arrhythmias (28), and reductions in creatine kinase (CK) and LDH levels (21). Adenosine, via A₁ receptor activation, has been shown to be an endogenous trigger of ischemic preconditioning through activation of both protein kinase C (PKC) and ATP-sensitive K⁺ (K_ATP) channels (18, 22, 29).

Current cellular models of SI have a number of limitations. These include the use of immature (neonatal) cells (24) and the long-term (7 days) incubation of cells in serum-containing medium before study (14), which potentially leads to dedifferentiation (7). Other ischemic conditions may cause irreversible rather than reversible cellular injury, such as that caused by O₂ deprivation (10), elevated K⁺, and sodium hydrosulfite (14). A previous model that simulated ischemic preconditioning in isolated rabbit cardiomyocytes utilized a milieu without glucose but did not include a reduction in pH of the buffer or an increase in lactate as occurs with ischemia in vivo (1). We therefore developed a reversible model of simulated ischemia-reperfusion (I/R) injury in adult rat cardiomyocytes to determine whether urocortin exerts direct cardioprotective actions against SI at the level of the cardiomyocyte. We also investigated whether the immediate and delayed phases of ischemic preconditioning were evident in this model and whether urocortin was able to induce protection equivalent to ischemic preconditioning in cardiomyocytes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation of cardiomyocytes. Adult male Sprague-Dawley rats (220-280 g body wt) were anesthetized via administration of ketamine hydrochloride (100 mg/kg ip) plus xylazine (12 mg/kg ip) before hearts were excised. Cardiomyocytes were isolated by enzymatic digestion as previously described (19). In brief, the aorta was cannulated and the heart retrogradely perfused with an enzyme buffer that contained 178 U/ml type II collagenase and 0.1 mg/ml hyaluronidase in a calcium-free Krebs-Henseleit buffer. After 20 min of perfusion, the heart was finely minced and tritriated in an incubation buffer that contained 0.015 mg/ml trypsin, 178 U/ml type II collagenase, and 0.1 mg/ml hyaluronidase. Cells were resuspended in bovine serum albumin and allowed to settle for 12 min. Supernatant was discarded, and the cardiomyocytes were resuspended in fresh albumin. This step was repeated four more times, and each time calcium was gradually added back to yield a final calcium concentration of 100 µM. This sequential process removes nonviable cardiomyocytes. Cardiomyocytes were finally resuspended in defined serum-free medium 199 supplemented with 2% albumin, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 25 µg/ml gentamycin. This technique routinely yields >93% cardiomyocyte content (19). Cardiomyocytes were incubated in suspension with ~3-4 × 10⁵ rod-shaped cells/35-mm well in six-well tissue-culture plates (Becton Dickinson; Franklin Lakes, NJ) and were allowed to equilibrate at 37°C with 95% air-5% CO₂ for 48 h before study. To allow for direct comparison between treatment groups (for example, the different time points of urocortin treatment), cells from the same preparation (i.e., from the same heart) were exposed to each of the different treatments. This allowed for paired comparisons when the statistical analysis was performed. Each cardiomyocyte preparation allowed for comparison of six treatment conditions.

Simulated I/R. Pilot studies were performed to determine the optimal conditions for simulating I/R. We compared two SI buffers at two different incubation times. Both ischemia buffers were based on a HEPES buffer that contained (in mM) 137 NaCl, 3.5 KCl, 0.88 CaCl₂ · 2H₂O, 0.51 MgS0₄ · 7H₂O, 5.55 D-glucose, and 4 HEPES and 2% FCS. SI buffer A contained HEPES buffer supplemented with (in mM) 10 2-deoxy-D-glucose, 20 DL-lactic acid, 0.75 sodium hydrosulfite, and 12 potassium chloride. For SI buffer B, HEPES buffer was supplemented with 10 mM 2-deoxy-D-glucose and 20 mM DL-lactic acid. Both were adjusted to pH 6.5. Cardiomyocytes were exposed to either SI buffer A for 2 or 4 h or SI buffer B for 4 h at 37°C with subsequent recovery for 2.5 h in defined serum-free medium. Paired control cardiomyocytes were exposed to normal HEPES buffer (pH 7.4) in place of SI buffer. Exposure of cardiomyocytes to SI buffer A for 2 and 4 h followed by 2.5 h of recovery increased LDH enzyme levels by 2.7 ± 0.1 and 2.8 ± 0.1 IU/10⁴ cells, respectively (both n = 3). Exposure of cells to SI buffer B for 4 h followed by 2.5 h of recovery induced an increase in LDH levels of 5.0 ± 0.1 IU/10⁴ cells (n = 3). These pilot studies indicated that the most marked increases in LDH levels were observed with 4 h of SI buffer that contained only 2-deoxy-D-glucose and DL-lactic acid with 2.5 h of recovery; i.e., SI buffer B. These conditions were used for all subsequent experiments.

Cardioprotection with urocortin. The effects of urocortin (0.1 µM) during the period of SI were studied by supplementing the SI buffer with urocortin. For comparison, the effects of adenosine (10 µM) during the period of SI were also studied by supplementing the SI buffer with adenosine (Fig. 1). At the conclusion of each experiment, LDH and CK levels were determined in the cardiomyocyte suspensions. Twelve different myocyte preparations were studied.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Experimental protocol for simulated ischemia-reperfusion studies. Exposure to simulated ischemia (SI) buffer B for 4 h and then serum-free medium for 2.5 h at 37°C was used to simulate ischemia. Recovery in defined serum-free medium simulated reperfusion. Delayed and immediate preconditioning (with either simulated ischemia or urocortin) are represented by the small shaded and open bars, respectively.

Preconditioning cardiomyocytes against cellular injury. To determine whether this model exhibits the immediate phase of ischemic preconditioning, cardiomyocytes were exposed to SI buffer for 5 min and recovery in normal medium for 10 min immediately before the 4-h ischemic insult (Fig. 1). The delayed phase of ischemic preconditioning was studied by exposing cardiomyocytes to SI buffer for 20 min with 20 h of recovery in normal serum-free medium before the sustained ischemic insult was invoked (Fig. 1). Up to seven different myocyte preparations were studied. The same time course was used to determine whether cardiomyocytes could be pharmacologically preconditioned with urocortin. For these experiments, cardiomyocytes were exposed to normal HEPES buffer supplemented with urocortin for either 5 min followed by 10 min of recovery in normal serum-free medium or for 20 min with 20 h of recovery in normal serum-free medium before the 4-h ischemic insult (Fig. 1). Again, at the conclusion of the experiments, cardiomyocyte suspensions were collected for assay of LDH and CK levels. Up to five different myocyte preparations were studied.

Effects of PKC and K_ATP channels. To study the contribution of PKC and K_ATP channels to the cardioprotection observed with urocortin, the effects of the PKC inhibitor chelerythrine (10 µM) during the period of SI were studied by supplementing the SI buffer with urocortin and chelerythrine. The effects of the K_ATP channel blocker 5-hydroxydecanoate (500 µM) during the period of SI were also studied by supplementing the SI buffer with urocortin and 5-hydroxydecanoate (Fig. 1). At the conclusion of each experiment, LDH and CK levels were determined in cardiomyocyte suspensions. Up to twelve different myocyte preparations were studied.

Enzyme-level assays. LDH and CK levels were determined by using spectrophotometric detection with commercially available assay kits (Sigma). Separate assays of samples taken from the same wells of each treatment group were performed to determine LDH and CK enzyme levels. LDH assay kit reagents were made up in distilled water to twice the manufacturer's recommended concentration. This reagent (500 µl) was added to 250 µl of sample and 250 µl of distilled water. Samples were incubated in quartz cuvettes (10 × 10 × 45 mm) for 1 min, and absorbance of the samples was measured immediately after incubation (1 min after being placed in the cuvette) and again 1 min after incubation (2 min after being placed in the cuvette). LDH level was measured as the absorbance at 340 nm, which indicates the oxidation of lactate to pyruvate.

Data analysis. For both LDH and CK determinations, the baseline enzyme levels (in IU/l) in control samples were subtracted from each sample (i.e., IU/l), and the levels were expressed relative to the number of cells studied. For assessment of the protective effects of various treatments, the enzyme level for the treatment groups was expressed as a percentage of that induced by SI alone (%SI, where cellular injury caused by SI was defined as 100%; Ref. 14).

Drugs. FCS, penicillin, streptomycin, gentamycin (CSL Biosciences; Parkville, Australia), medium 199 (JRH Biosciences; Lenexa, KS), type II collagenase (Worthington; Lakewood, NJ), hyaluronidase, trypsin (Sigma-Aldrich, St. Louis, MO), bovine fraction V albumin, L-carnitine, creatine, and taurine (Sigma-Aldrich) were of tissue culture grade. Adenosine, 5-hydroxydecanoate, chelerythrine, urocortin, and enzyme level kits (for CK and LDH) were of analytic grade and were obtained from Sigma-Aldrich. Urocortin was a gift from J. Rivier at the Salk Institute.

Statistical analysis. Results are expressed as means ± SE. Statistical evaluation of the data was performed comparing treatment effects with SI using paired t-tests with Bonferroni correction for  where appropriate. P < 0.05 was considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Urocortin protects cardiomyocytes from SI. Exposure of cardiomyocytes for 4 h to SI buffer B followed by 2.5 h of recovery in normal serum-free medium markedly increased both LDH and CK levels. LDH levels were increased by 3.6 ± 0.7 IU/10⁴ cells and CK levels were increased to 5.0 ± 8.1 IU/10⁴ cells above control levels (both n = 39; P < 0.001 vs. control). When present during SI, urocortin (0.1 µM) caused a marked decrease in postrecovery cellular injury; LDH levels were reduced by 85 ± 7% and CK levels by 71 ± 18% of that induced by SI alone (Fig. 2A; both n = 12; P < 0.05 vs. SI). This protective action of urocortin during SI was comparable with that observed with adenosine (10 µM), which decreased cellular injury by 98 ± 20 and 83 ± 13% of that induced by SI alone for LDH and CK levels, respectively (Fig. 2B; both n = 12; P < 0.05 vs. SI).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A: relative lactate dehydrogenase (LDH) and creatine kinase (CK) production by adult rat cardiomyocytes exposed to HEPES buffer (control, n = 12 each) or to SI buffer B either alone (n = 12 each) or in the presence of 0.1 µM urocortin (SI + Uro, n = 12 each). B: relative LDH and CK production by adult rat cardiomyocytes exposed to HEPES buffer (control, n = 12 each) or to SI buffer B either alone (n = 12 each) or in the presence of 10 µM adenosine (SI + A, n = 12 each). * P < 0.05 vs. SI alone.

Preconditioning protects cardiomyocytes from cellular injury. Exposure of the cardiomyocytes for 5 min to urocortin (in normal HEPES buffer) followed by 10 min of recovery in normal serum-free medium before simulated I/R caused a marked decrease in cellular injury as evidenced by both LDH and CK levels, which were reduced by 66 ± 12 and 100 ± 19%, respectively (Fig. 3A; both n = 5; P < 0.05 vs. SI). Similarly, exposure of the cardiomyocytes for 20 min to normal HEPES buffer supplemented with urocortin for 20 h before 4 h of SI also markedly reduced LDH levels by 78 ± 12% and CK levels by 71 ± 15%, respectively (Fig. 3A; both n = 5; P < 0.05 vs. SI).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   A: relative LDH and CK production by adult rat cardiomyocytes exposed to HEPES buffer (control, n = 5 each), or to SI buffer B either alone (n = 5 each) or with immediate pharmacological preconditioning (immediate UPC + SI) consisting of 5 min with 0.1 µM urocortin and 10 min of recovery (n = 5 each), or to delayed pharmacological preconditioning (delayed UPC + SI) consisting of 20 min with 0.1 µM urocortin and 20 h of recovery (n = 5 each). B: relative LDH and CK production by adult rat cardiomyocytes exposed to HEPES buffer (control, n = 7 each), or to SI buffer B either alone (n = 7 each) or with immediate ischemic preconditioning (immediate IPC + SI) consisting of 5 min of SI and 10 min of recovery (n = 7 each), or to delayed ischemic preconditioning (delayed IPC + SI) consisting of 20 min of SI and 20 h of recovery (n = 6 each). * P < 0.05 vs. SI alone.

Cardioprotective effect of urocortin during recovery. Exposure of the cardiomyocytes to urocortin in the 2.5-h recovery period after drug-free SI significantly reduced cellular injury. LDH level was reduced by 44 ± 18% (Fig. 4; n = 15; P < 0.05 vs. SI). A similar but nonsignificant trend was observed with the CK level, which was reduced by 79 ± 23% [Fig. 4; n = 11; P = not significant (NS) vs. SI].

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Relative LDH (A) and CK (B) production by adult rat cardiomyocytes exposed to HEPES buffer (control, n = 15 each) or to SI buffer B either alone (n = 15 each) or with urocortin present during the recovery phase (SI + Uro in recovery: LDH, n = 15; CK, n = 11). * P < 0.05 vs. SI alone.

Mechanism of cardioprotective effect. When present during SI, the PKC inhibitor chelerythrine (10 µM) abrogated the decrease in cellular injury compared with urocortin alone; LDH levels were increased to 105 ± 19% (Fig. 5; n = 9; P = NS vs. SI alone; P < 0.05 vs. urocortin alone during SI) and CK levels by 91 ± 14% above control levels (Fig. 5; n = 12; P = NS vs. SI alone; P < 0.05 vs. urocortin alone during SI). Exposure of cardiomyocytes to the K_ATP channel blocker 5-hydroxydecanoate (500 µM) during SI abolished the decrease in cellular injury that was induced by urocortin alone; CK levels increased by 97 ± 14% and LDH levels increased by 77 ± 9% above control (Fig. 5; both n = 12; P = NS vs. SI alone; P < 0.05 vs. urocortin alone during SI). The cardioprotective actions of urocortin also tended to be reduced by 100 µM 5-hydroxydecanoate; LDH levels were increased by 92 ± 10% above control (n = 10; P = NS vs. SI alone; P < 0.05 vs. urocortin alone during SI), but this was not significantly different on CK analysis vs. urocortin alone during SI (n = 10, results not shown). Exposure of cardiomyocytes to either chelerythrine or 5-hydroxydecanoate had no effect on either CK or LDH levels in the absence of urocortin in control HEPES buffer.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Relative LDH (A) and CK (B) production by adult rat cardiomyocytes exposed to HEPES buffer (control, n = 12 each), or to SI buffer B either alone (n = 12 each) or with 0.1 µM urocortin (SI + Uro) present during SI (n = 12 each), or to 10 µM chelerythrine present with urocortin during SI (SI + Uro + Chel, n = 12 each), or to 500 µM 5-hydroxydecanoate present with urocortin during SI (SI + Uro + 5-HD, n = 10 each). * P < 0.05 vs. SI alone; # P < 0.05 vs. SI + Uro.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This is the first study to demonstrate a cardioprotective effect of the CRF-related peptide urocortin against simulated I/R injury in adult rat cardiomyocytes. Urocortin appears to exert its cardioprotective effects via activation of PKC and K_ATP channels. The cardioprotective effect of urocortin was observed when the cardiomyocytes were exposed to SI buffer supplemented with urocortin for 4 h, which was followed by 2.5 h of recovery in normal, defined serum-free medium. Urocortin that was present during SI exerted protection comparable to adenosine, which is a well-established initiator of preconditioning. Beneficial effects were also observed with urocortin treatment both before and after SI. Cardioprotection was observed when cardiomyocytes were pretreated with urocortin either immediately before or 20 h before SI; this compares favorably with the beneficial effect of both phases of ischemic preconditioning. This is the first study to directly compare the beneficial effect of urocortin in adult rat cardiomyocytes with ischemic preconditioning and to elucidate the mediators that underlie the cardioprotective effect of urocortin.

In a previous study, the effects of pretreating neonatal cardiomyocytes with urocortin for 30 min, 1 h, 2 h, and 24 h before a more severe insult (lethal hypoxic injury) were determined (2). In contrast with our findings, no cardioprotection was observed with a brief exposure (10 min) to urocortin immediately before lethal hypoxia. However, when urocortin was present for 30 min or longer, cell death was reduced (2). In our hands, the addition of urocortin during the 2.5-h recovery period that followed SI was found to be mildly cardioprotective, and this was less marked than the protection previously demonstrated in neonatal cardiomyocytes with urocortin added at the point of reoxygenation (2). In neonatal rat cardiomyocytes, activation of CRF-R2 receptors with urocortin during SI also elicited a cardioprotective effect as measured by a decrease in LDH enzyme levels (15). Of note, our work on adult cardiomyocytes is the only study of the three to demonstrate that urocortin protection during ischemia is comparable with adenosine protection.

The CRF-R2 receptor is the only CRF receptor that has been detected in heart tissue (5), which suggests that the cardioprotective effect of urocortin depends on an action at CRF-R2 receptors. Known cellular actions of urocortin include increases in cAMP production, stimulation of secretion of atrial natriuretic peptide (9), and activation of p42/p44 MAPK in neonatal cardiomyocytes (9, 15); any of these effects might contribute to the cardioprotective effects of urocortin in the adult heart. In the current study, we specifically examined the contributions of PKC and K_ATP channels to the cardioprotective effects of urocortin. Either the PKC inhibitor chelerythrine or the K_ATP channel blocker 5-hydroxydecanoate that was present during the SI period abolished the cardioprotection of urocortin.

Although a number of in vitro models for the study of I/R injury have previously been described (10, 14, 24), most of these result in irreversible myocyte injury. Our model appears to be the first to demonstrate both immediate and delayed phases of ischemic preconditioning in isolated cardiomyocytes (6, 26, 27). Because the cells are used within 48 h after isolation, potential problems of dedifferentiation (which can occur with long-term incubation) are minimized. Our pilot study suggested that SI consisting of exposure of cardiomyocytes to a metabolic inhibition milieu (SI buffer B, a HEPES buffer supplemented with 10 mM 2-deoxy-D-glucose and 20 mM DL-lactic acid) for 4 h followed by recovery in defined serum-free medium caused an optimal cellular injury compared with shorter incubation times or SI buffer A. The protection afforded with adenosine confirmed that this model of SI was reversible, which is in accordance with studies on isolated hearts (4, 30, 31). Exposure of the cells to SI buffer immediately before the 4-h ischemic insult markedly reduced cellular injury, which is indicative of the immediate phase of ischemic preconditioning and in line with the expected cardioprotection that has been reported to last for 1-2 h (8). Likewise, exposure of the cardiomyocytes to SI buffer for 20 h before the ischemic insult also markedly reduced cellular injury. This is in accordance with the reported cardioprotection afforded by delayed preconditioning, which typically lasts for 3 days after sublethal ischemia (29). We have studied I/R injury in an adult model using a simplified nonlethal SI buffer that mimics many of the well-documented conditions that are present during ischemia in vivo including decreased pH (6.5), decreased utilization of substrates (2-deoxy-D-glucose, an analog of glucose that cardiomyocytes cannot utilize for energy), and elevated lactic acid.

Using an adult cardiomyocyte phenotype to study I/R injury as described here is more physiologically relevant than using neonatal cardiomyocytes: ischemia is predominantly an adult disease, and there are marked differences between adult and neonatal myocardia. These include 1) the ability to grow and regenerate, as neonatal cardiomyocytes can more readily maintain and assemble myofibrils than adult cardiomyocytes (20); 2) calcium handling, as immature myocardium has a reduced ability to sequester calcium and produces smaller L-type calcium currents (11); 3) lower phosphodiesterase activity in neonatal cardiomyocytes (17), which implies differences in cyclic nucleotide signaling; and 4) higher resting protein kinase G levels in neonatal myocardium (25).

In conclusion, we have clearly demonstrated a cardioprotective action of the CRF-related peptide urocortin in I/R injury in adult cardiomyocytes. These cardioprotective actions compared favorably with well-established protective agents such as adenosine in both the immediate and delayed phases of ischemic preconditioning. The cardioprotective actions of urocortin were mediated via the activation of PKC and K_ATP channels. Urocortin may thus have potential as a cardioprotective agent during planned episodes of myocardial ischemia such as percutaneous transluminal coronary angioplasty, coronary artery bypass graft surgery, or cardiac transplantation.