INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PROTECTION AGAINST MYOCARDIAL damage due to prolonged ischemia occurs in hearts pretreated with various neurohormonal agents or transient ischemia. Murry et al. (23) were the first to show that short-duration ischemia protected intact dog hearts from damage due to a sustained period of ischemia. They found that tissue necrosis was decreased by 22% in those hearts that had been pretreated with four 5-min episodes of ischemia before 40 min of ischemia. The use of short-duration transient ischemia to protect the heart against damage from a subsequent and more prolonged ischemic event is known as "ischemic preconditioning." Since then, a number of studies have demonstrated the beneficial effects of ischemic preconditioning in other animals, including rat (3), rabbit (5), swine (33), and humans (1, 4, 46).

Cardioprotection can also be brought about through the activation of A₁-adenosine receptors in rabbits (5) and dogs (16) as well as -adrenergic (2) and opioid (12, 32, 34, 35) receptor activation in the rat heart. The cardioprotective nature of protein kinase C (PKC) activators such as phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-sn-glycerol (DOG) suggests that activation of PKC may be an integral part of the cardioprotective mechanism of action (18, 37). In addition to reduced tissue necrosis (23, 41), pretreatment with cardioprotective agents or transient ischemia improves postischemic contractile function (3) and decreases occurrences of postischemic dysrhythmias (36, 42).

A number of theories have been put forth to explain the cellular basis of cardioprotection. These include opening of ATP-sensitive K⁺ channels (14), closure of L-type Ca²⁺ channels, and an increased availability of ATP (24, 29). Although it has been demonstrated that protected hearts have relatively greater high-energy phosphate stores than control hearts during ischemia and reperfusion (10, 28), the mechanism responsible for the increase in high-energy phosphates and the contribution of this energy conservation to cardioprotection are unknown. The first aim of the present study was to investigate a possible source of the excess high-energy phosphate in the form of the metabolic slowing of the actomyosin Mg²⁺-ATPase. The Ca²⁺-dependent actomyosin Mg²⁺-ATPase consumes ATP during cross-bridge cycling with actin. It has been estimated that in the rat heart, cross-bridge cycling consumes ~80% of the ATP produced (6). Thus we wished to determine whether ATP consumption by the actomyosin ATPase of the cardioprotected heart was less than that of control hearts subjected to the same ischemic stress.

Opioid agonists decrease tissue necrosis normally associated with a prolonged ischemic insult (33). The most abundant opioid receptor in rat heart is the -opioid subtype (48). Thus the second aim of our study was to investigate the potentially protective role of the -opioid agonist U-50488H {trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide} in the rat heart and to determine whether its effects, if any, occurred concomitant with a decrease in ATP consumption by the Ca²⁺-dependent actomyosin Mg²⁺-ATPase. For these studies, we also wished to determine whether the single ventricular myocyte velocity of unloaded shortening, a direct measure of cross-bridge cycling rate, would be reduced with U-50488H exposure.

Phosphorylation of myofibrillar proteins has been shown to alter the activity of the actomyosin Mg²⁺-ATPase. Phosphorylation of troponin I (TnI), troponin T (TnT), and/or C protein by PKC slows actin-myosin cross-bridge cycling (40, 25), whereas PKC-dependent phosphorylation of myosin light chain-2 (LC₂) increases actomyosin Mg²⁺-ATPase activity (25). Thus the third aim of this study was to investigate the changes in myofibrillar protein phosphorylation with -opioid and -adrenergic receptor activation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Langendorff-perfused heart preparation. Hearts were removed from female Wistar rats anesthetized by methoxyflurane (Metofane) inhalation. The isolated hearts were cannulated in ice-cold modified Krebs-Henseleit solution and mounted on a Langendorff perfusion apparatus. Modified Krebs-Henseleit solution contained 4.7 mM KCl, 118 mM NaCl, 1.2 mM MgSO₄, 1.3 mM CaCl₂, 25 mM NaHCO₃, 11 mM glucose, 1.2 mM KH₂PO₄, 0.05 mM EDTA, and 2 mM lactic acid, pH 7.4. Hearts were perfused with oxygenated (95% O₂-5% CO₂), 37°C modified Krebs-Henseleit solution and placed in a 100-ml organ bath that contained oxygenated, 37°C modified Krebs-Henseleit solution. A pressure transducer (BLPR; World Precision Instruments, Sarasota, FL) was inserted through the left atrium into the left ventricle, and pacing at 300 beats/min was initiated. Pacing voltage was set at twice the threshold value. A cellophane balloon on the end of the pressure transducer was inflated until left ventricular end-diastolic pressure (EDP) was 5-15 mmHg.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Top: experimental protocol for left ventricular developed pressure (LVDP) and Ca2+-dependent actomyosin Mg2+-ATPase activity determination. Bottom: representative examples of pre- and postischemic LVDP with respect to time. For measurement of postischemic LVDP recovery, hearts underwent 20 min of baseline perfusion, 20 min of global ischemia, and 60 min of reperfusion. During baseline perfusion, some hearts underwent 2 or 4 min of agonist/antagonist perfusion. For Ca2+-dependent actomyosin Mg2+-ATPase activity, hearts underwent 20 min of baseline perfusion and 20 min of global ischemia before myofibril isolation. Before ischemia, hearts were treated with 10 µM phenylephrine (PHE) + 3 µM propranolol (PRO) (), 1 µM U-50488H (U50; ), or 1 µM U-50488H + 1 µM nor-binaltorphimine (nor-BNI) () or were untreated controls ().

Myofibrillar isolation. After 20 min of global ischemia, hearts that were to be used for myofibrillar ATPase measurements were removed from the Langendorff perfusion apparatus. Myofibrils were isolated according to a modified protocol described by Murphy and Solaro (22). The ventricles were removed and placed in iced standard phosphate buffer. Standard phosphate buffer contained 60 mM KCl, 30 mM imidazole (pH 7.0), 2 mM MgCl₂, 4.2 µM aprotoninin, 14.6 µM pepstatin A, and 20.3 µM leupeptin hemisulfate. The ventricles were then cut into 10-12 pieces and homogenized in a Waring blender for 1 min. The phosphatase inhibitor calyculin A (100 nM) was added to the homogenate to inhibit type-1 and -2A phosphatases. The homogenate was pelleted, and the resulting pellets were dissolved in iced solution containing 10 mM EGTA, 8.2 mM MgCl₂, 14.4 mM KCl, 60 mM imidazole (pH 7.0), 5.5 mM ATP, 12 mM creatinine phosphate, 10 U/ml creatinine phosphokinase, 100 nM calyculin A, and 1% Triton X-100. The suspension was placed on ice for 30 min, followed by centrifugation until it was pelleted. The pellet was washed and resuspended in iced standard phosphate buffer with 100 nM calyculin A. Protein concentration was determined with a Biuret assay, and the myofibrils were diluted to give a concentration between 4 and 8 mg/ml.

Myofibrillar ATPase measurement. ATPase buffers with Ca²⁺ concentrations of pCa 4.0 and 9.0 were used. The pCa 4.0 buffer contained 23.48 mM KCl, 5 mM MgCl₂, 3.22 mM ATP, 2 mM EGTA, 20 mM imidazole, and 2.15 mM CaCl₂, pH 7.0. The pCa 9.0 buffer contained 25.96 mM KCl, 5.13 mM MgCl₂, 3.16 mM ATP, 2 mM EGTA, 20 mM imidazole, and 4.86 µM CaCl₂, pH 7.0. Free Ca²⁺ concentration was calculated by use of the program of Fabiato (9). Myofibrils were added to the 32°C buffers. After 2 min of incubation, the reaction was quenched with 2 ml of 20% trichloroacetic acid. Inorganic phosphate (P_i) levels were determined according to the method of Fiske and Subbarow (11). P_i production was found to be linear with respect to time under conditions of 32°C with a final protein concentration of 1.0-2.0 mg/ml (data not shown).

Enzymatic isolation of myocytes. Ventricular myocytes were isolated by use of the protocol described by Lester et al. (17). The yield was calculated as the percentage of rod-shaped myocytes as a fraction of the total population of cells in 1 mM Ca²⁺-enriched Ringer solution (Ca²⁺-Ringer). Yields >40% were used for drug treatment and subsequent velocity analysis.

Measurement of shortening velocity. Maximal shortening velocity was determined by the slack-test method (7, 17). Isolated cardiac myocytes were attached via glass micropipettes to a force transducer (model 403; Cambridge Technology, Watertown, MA) and a piezoelectric translator (model 173; Physik Institute, Waldbronn, Germany) with Great Stuff adhesive (Insta-Foam, Marrietta, GA) (Fig. 2). During maximum activation in a pCa 4.5 solution, the cells were rapidly slackened by various shortening length steps for determination of the velocity of unloaded shortening. The pCa 4.5 solution contained 7 mM EGTA, 20 mM imidazole (pH 7.0), 5.46 mM MgCl₂, 77.63 mM KCl, 6.58 mM CaCl₂, 14.5 mM creatinine phosphate, and 4.65 mM ATP. Duration of unloaded shortening was taken as the difference in time between the introduction of slack and the redevelopment of tension.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Postischemic LVDP (A) and end-diastolic pressure (EDP; B). Values were obtained after 20 min of global ischemia and 60 min of reperfusion. Before ischemia, hearts were treated with 10 µM phenylephrine + 3 µM propranolol (n = 5), 10 µM phenylephrine (n = 5), or 1 µM U-50488H (n = 5) or were untreated controls (n = 8). For investigation of -opioid receptor antagonism, hearts were treated with 1 µM U-50488H (n = 3), 1 µM U-50488H + 1 µM nor-BNI, or 1 µM nor-BNI (n = 3) or were untreated controls (n = 4). Values are expressed as means ± SE. * P < 0.05 and § P < 0.01 compared with postischemic controls.

Myofibrillar protein phosphorylation. Changes in myofibrillar protein phosphorylation were determined by ³²P autoradiography. Ventricular myocytes were isolated as previously described in Enzymatic isolation of myocytes. Myocytes were incubated in 1 mM Ca²⁺-Ringer containing 1 mmol ATP, 20 µCi of [-³²P]orthophosphate (NEN), and 0.05% bovine serum albumin (BSA). After 35 min, 1 µM okadaic acid (inhibitor of type I/IIa phosphatases) was added. After 55 min, agonists/antagonists were added. Reactions were quenched after 5 min with the addition of electrophoresis sample buffer. Samples were heated at 95°C for 4 min and run on 12.5% SDS-PAGE. Gels were stained with Coomassie stain, dried between cellophane paper, and subjected to autoradiography with the use of a 7-day exposure with X-OMAT film (Eastman Kodak, Rochester, NY).

Rationale for agonist/antagonist dosages. Phenylephrine (10 µM) plus propranolol (3 µM) has previously been shown to produce maximal increases in intracellular Ca²⁺ concentration and twitch amplitude (8), whereas the dosage for U-50488H (1 µM) was chosen to selectively activate the -opioid receptors (27). Nor-BNI (1 µM) has been shown to block -opioid receptor activation by U-50488H (47). PMA (1 µM) has previously been shown to activate the PKC isoforms found in the rat heart (40).

Statistical analysis. All values are reported as means ± SE, and P < 0.05 was chosen to indicate statistical significance. All data were analyzed by two-way analysis of variance and Fisher's protected least-significant differences post hoc test except for maximum velocity of unloaded shortening (V_max) data. Velocity data were analyzed by two-way analysis of variance and Student's t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolated heart LVDP. No statistical differences were found in the values for preischemic LVDP and EDP between groups before any intervention, i.e., baseline values (Fig. 2). After 20 min of global ischemia and 60 min of reperfusion, control hearts recovered 34.2 ± 11.7% of preischemic LVDP. U-50488H-treated hearts recovered significantly better than the control group, with a mean postischemic LVDP of 58.6 ± 14.4% of preischemic LVDP. Both the phenylephrine and phenylephrine plus propranolol groups also showed significantly greater postischemic recovery compared with the control group. Phenylephrine-treated hearts had a mean postischemic LVDP of 59.7 ± 14.8% of preischemic LVDP, and the phenylephrine plus propranolol-treated hearts had a mean postischemic LVDP of 62.5 ± 12.9%. There were no significant differences among the postischemic LVDP values for the U-50488H-, phenylephrine-, and phenylephrine plus propranolol-treated hearts. Mean postischemic EDP was found to be significantly elevated compared with preischemic values in all groups, with no statistically significant intergroup differences (Fig. 2).

Actomyosin Mg²⁺-ATPase. Mean Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity from myofibrils isolated from hearts obtained after 20 min of global ischemia was 271.1 ± 15.5 nmol P_i · min¹ · mg protein¹ in the control group (Fig. 3). Compared with control hearts, the postischemic U-50488H-treated group had a significantly lower myofibrillar mean Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity of 184.2 ± 27.2 nmol P_i · min¹ · mg protein¹. Similarly, both the phenylephrine plus propranolol-treated hearts and the phenylephrine group had statistically lower myofibrillar mean Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity compared with control hearts. The Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity was 191.8 ± 43.0 nmol P_i · min¹ · mg protein¹ for myofibrils obtained from ischemic hearts treated with phenylephrine plus propranolol and 199.5 ± 29.4 nmol P_i · min¹ · mg protein¹ for the phenylephrine group. No significant difference was detected between the drug-treated groups.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Myofibrillar Ca2+-dependent actomyosin Mg2+-ATPase activity from agonist-treated hearts. Ca2+-dependent actomyosin Mg2+-ATPase activity was determined with the use of myofibrils isolated after 20 min of global ischemia of the hearts. A: before ischemia, hearts were treated with 10 µM phenylephrine + 3 µM propranolol (n = 3), 10 µM phenylephrine (n = 3), or 1 µM U-50488H (n = 3) or were left as untreated controls (n = 4). B: for investigation of -opioid receptor antagonism, hearts were treated with 1 µM U-50488H (n = 3), 1 µM U-50488H + 1 µM nor-BNI, or 1 µM nor-BNI (n = 3) or were left as untreated controls (n = 3). Pi, inorganic phosphate. Values are expressed as means ± SE. * P < 0.05 and § P < 0.01 compared with controls.

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Ventricular myocyte velocity of unloaded shortening. Velocity of unloaded shortening was determined for nonischemic, enzymatically isolated myocytes treated with U-50488H or PMA and subsequently skinned. Isometric attachments to micropipettes of single myocytes were obtained for all groups. Table 1 presents the characteristics of these myocytes. Control myocytes had a mean V_max of 2.99 ± 0.24 muscle lengths/s (MLs/s) (Fig. 4). U-50488H- and PMA-treated myocytes had significantly lower V_max values compared with control myocytes. The V_max for U-50488H-treated myocytes was 2.50 ± 0.20 MLs/s, and it was 2.29 ± 0.38 MLs/s for PMA-treated myocytes. Maximum force production was not significantly different between groups (Table 1).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Maximal velocity of unloaded shortening (Vmax) in agonist-treated and skinned ventricular myocytes. Hearts were treated for 5 min with 1 µM U-50488H (n = 9) or 1 µM phorbol 12-myristate 13-acetate (PMA; n = 6) or were untreated controls (n = 10); myocytes were then chemically skinned. Values are expressed as means ± SE. * P < 0.05 compared with controls.

Myofibrillar protein phosphorylation. The -opioid receptor agonist U-50488H increased the phosphorylation of C protein and TnI but not LC₂ or tropomyosin (Fig. 5 and Table 2). Isoproterenol treatment of isolated ventricular myocytes resulted in an increase in the phosphorylation of C protein, TnI, and myosin LC₂ but not tropomyosin. Phenylephrine plus propranolol treatment increased the phosphorylation levels of C protein and TnI and had no effect on the phosphorylation of LC₂ or tropomyosin.

View larger version (93K): [in this window] [in a new window]   Fig. 5.   Autoradiograph (A) and corresponding SDS-polyacrylamide gel (B) of ventricular myocytes after isoproterenol (ISO), phenylephrine + propranolol (PP), U-50488H (U50), or no treatment (control; CON) in the presence of [32P]orthophosphate and 1 µM okadaic acid. Tm, tropomyosin; TnI, troponin I; LC2, light chain-2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study we have demonstrated that postischemic LVDP is significantly elevated by the -opioid receptor agonist U-50488H and the -adrenergic receptor agonist phenylephrine, and that this improvement occurs concomitant with a postischemic reduction in myofibrillar Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity. The ability of nor-BNI (a -opioid receptor antagonist) to block the actions of U-50488H suggests that these effects are mediated through -opioid receptor activation. Consistent with these observations was the demonstration that the V_max of nonischemic, isolated ventricular myocytes is significantly reduced when they are treated with U-50488H or PMA. Both U-50488H and phenylephrine plus propranolol increased the phosphorylation levels of C protein and TnI. These findings suggest that -opioid- and -adrenergic receptor-dependent improvement of LVDP in the rat heart may be due to a slowing of cross-bridge cycling and ATP use through a change in the phosphorylation of the myofibrillar proteins.

It has previously been shown that administration of phenylephrine before global ischemia improves postischemic LVDP by ~30% compared with control hearts (2, 20, 21). Thus we included a phenylephrine plus propranolol group in these studies as positive controls. The results of this study are consistent with these previous findings and, furthermore, demonstrate that pretreatment with U-50488H also improves postischemic LVDP.

LVDP and EDP were used in the present studies as indicators of postischemic myocardial damage. Whereas postischemic LVDP was significantly higher in hearts treated with -opioid or -adrenergic agonists, the postischemic increase in EDP was attenuated by -opioid receptor activation in only one of these groups. This discrepancy might be explained by the relatively high variability in EDP seen in the first set of experiments. Previous studies have used preischemic EDP of 5-6 mmHg (2, 20), whereas our study allowed for the inclusion of hearts that had preischemic EDP of 5-15 mmHg. The high preischemic variability in EDP may have led to the higher intragroup variability seen in the first set of experiments. This high intragroup variability may have obscured any apparent differences in the means. As such, although we saw a trend of a decrease in postischemic EDP in agonist-treated hearts, this observation did not reach statistical significance.

We have previously observed that U-50488H increases the Ca²⁺ sensitivity of cardiac myofilaments (unpublished observation). Others have noted that U-50488H causes a transient increase in twitch amplitude in isolated cardiac myocytes, followed by a negative inotropy (44). In the present study, an increase in LVDP was observed after 2 min of U-50488H exposure, with LVDP returning to baseline values before the ischemic period (Fig. 1). The initial positive inotropy has been attributed to an increase in the release of Ca²⁺ from the sarcoplasmic reticulum (44), which reduces intracellular Ca²⁺ stores and leads to the subsequent negative inotropy. We believe that the observed increase in preischemic LVDP with U-50488H treatment is due to an increase in intracellular Ca²⁺ levels as well as an increase in the Ca²⁺ sensitivity of the myofilaments.

Using myofibrils isolated from postischemic hearts, we demonstrated that hearts that had been pretreated with phenylephrine, phenylephrine plus propranolol, or U-50488H had an ~20% decrease in Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity compared with myofibrils isolated from control hearts. These findings are consistent with those of Noland and Kuo (26), who demonstrated that PKC exposure leads to a 30% reduction in myofibrillar ATPase activity. Moreover, we found that treatment of nonischemic, isolated ventricular myocytes with U-50488H slowed actin-myosin cycling, as demonstrated by the observed 17% reduction in V_max. Previous work by Strang and Moss (39) demonstrated that phenylephrine also decreases V_max by 33% in cardiac myocytes isolated from rats.

The present studies, which demonstrate U-50488H-dependent decreases in V_max and Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity, are consistent with a decrease in actin-myosin ATP consumption. However, it should be noted that V_max and ATPase values were obtained under different conditions. The use of isolated myofibrils to measure Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity allowed for a postischemic determination of actin-myosin ATPase activity. The determination of single-cell nonischemic V_max after U-50488H treatment allowed for determination of actin-myosin ATPase activity in a preparation with an intact myofilament lattice capable of bearing loads. The combined results of these experiments suggest that in an intact heart, -opioid receptor stimulation would lead to a decrease in actin-myosin ATPase activity.

The release of endogenous opioid peptides has been shown to occur during myocardial ischemia (19). Several previous studies have noted that the cardioprotective effects of ischemic preconditioning are mediated in part by endogenous opioids (12, 32-34). Schultz et al. (32) demonstrated that antagonists of ₁-opioid but not -opioid receptors decrease the protective effects of preconditioning in rat heart. Although the work of Schultz et al. (32) fails to support the cardioprotective ability of -opioids suggested by our present study, these findings are not necessarily contradictory. It is possible that endogenous -opioid peptides are not released in sufficient quantities in ischemic preconditioning to affect protection against myocardial ischemia. The activation of -opioid receptors with the exogenous administration of U-50488H has previously been shown to increase cellular viability and twitch amplitude after 5 min of severe metabolic inhibition in isolated cardiomyocytes (45), a finding that is consistent with the results of our present study.

The intracellular cascade that produces the beneficial effects of cardioprotection has yet to be elucidated. A number of studies have implicated PKC as a key second messenger involved in improved postischemic function of the rat heart (15, 20, 37). In the adult rat heart, there are four PKC isoforms: PKC-, -, -, and - (30, 38). Activation of ₁-adrenergic receptors by phenylephrine has been reported to result in the activation of PKC- (20) and PKC- (17), whereas phorbol esters appear to activate all PKC isoforms. Ventura et al. (44) have shown that U-50488H increases inositol 1,4,5-trisphosphate production, which may be associated with an increase in PKC activation. Exogenous PKC has been shown to phosphorylate the myofilament proteins TnI, TnT, and C protein, which subsequently decreases Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity in a reconstituted actin-myosin system (26, 43). In the present study, treatment of ventricular myocytes with phenylephrine or U-50488H led to increases in the phosphorylation of TnI and C protein. We also observed a decrease in Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity with -adrenergic receptor activation. In addition, -opioid receptor activation decreased actomyosin ATPase activity, and PMA treatment and -opioid receptor activation both decreased V_max. These findings are consistent with the hypothesis that phorbol esters, -adrenergic receptors, and -opioid receptors all activate an identical second-messenger pathway. This pathway most likely involves the activation of PKC.

In addition to metabolic slowing and ATP conservation, other mechanisms have been proposed to explain receptor agonist-induced improved postischemic myocardial function. Although our results suggest that metabolic slowing may be involved, this does not necessarily preclude other mechanisms. In summary, we propose that a reduction in Ca²⁺-dependent actomyosin Mg²⁺-ATPase activity and slowed cross-bridge cycling rate improve postischemic myocardial function by conserving ATP. This reserve of ATP can then be used for ATP-dependent ion channels and pumps, which act to maintain Ca²⁺ homeostasis and decrease ischemia-induced Ca²⁺ overload. Ca²⁺ overload is damaging to the heart because Ca²⁺ has been shown to activate proteases, which catabolize myocardial proteins (13).