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Am J Physiol Heart Circ Physiol 292: H2459-H2466, 2007. First published January 19, 2007; doi:10.1152/ajpheart.00459.2006
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beta1-Adrenoreceptor activation contributes to ischemia-reperfusion damage as well as playing a role in ischemic preconditioning

Joseph F. Spear, Subbuswamy K. Prabu, Domenico Galati, Haider Raza, Hindupur K. Anandatheerthavarada, and Narayan G. Avadhani

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Submitted 5 May 2006 ; accepted in final form 10 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protein kinase A (PKA) activation has been implicated in early-phase ischemic preconditioning. We recently found that during ischemia PKA activation causes inactivation of cytochrome-c oxidase (CcO) and contributes to myocardial damage due to ischemia-reperfusion. It may be that beta-adrenergic stimulation during ischemia via endogenous catecholamine release activates PKA. Thus beta-adrenergic stimulation may mediate both myocardial protection and damage during ischemia. The present studies were designed to determine the role of the beta1-adrenergic receptor (beta1-AR) in myocardial ischemic damage and ischemic preconditioning. Langendorff-perfused rabbit hearts underwent 30-min ischemia by anterior coronary artery ligation followed by 2-h reperfusion. Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk and volume of myocardial necrosis after 2-h reperfusion. In some hearts ischemic preconditioning was accomplished by two 5-min episodes of global low-flow ischemia separated by 10 min before coronary occlusion-reperfusion. Orthogonal electrocardiograms were recorded, and coronary flow was monitored by a drip count. Three hearts from each experimental group were used to determine mitochondrial CcO and aconitase activities. Two-hour reperfusion after occlusion caused an additional decrease in CcO activity vs. that after 30-min occlusion alone. Blocking the beta1-AR during occlusion-reperfusion reversed CcO activity depression and preserved myocardium at risk for necrosis. Similarly, mitochondrial aconitase activity exhibited a parallel response after occlusion-reperfusion as well as for the other interventions. Furthermore, classic ischemic preconditioning had no effect on CcO depression. However, blocking the beta1-AR during preconditioning eliminated the cardioprotection. If the beta1-AR was blocked after preconditioning, the myocardium was preserved. Interestingly, in both of the latter cases the depression in CcO activity was reversed. Thus the beta1-AR plays a dual role in myocardial ischemic damage. Our findings may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in coronary reperfusion intervention.

cytochrome-c oxidase; aconitase; protein kinase A; Langendorff-perfused rabbit heart

EARLY-PHASE ISCHEMIC PRECONDITIONING that lasts from minutes to hours was first described by Murry et al. (32) in 1986. It refers to the "salvaging" of myocardium at risk for necrosis by prior brief periods of ischemia. Since its first description ischemic preconditioning has been extensively studied and has been reported to occur in a variety of species including humans (48). Several current reviews cover the signal transduction pathways involved in preconditioning and its clinical relevance (33–36, 48, 49). Protein kinase A (PKA) activation has been implicated in contributing to early-phase ischemic preconditioning (27, 43, 44). Activation and translocation of PKC are also undoubtedly involved (7, 19, 26, 29). There is good evidence that the final mediators of protection are the ischemia-induced opening of the mitochondrial ATP-sensitive K+ (KATP) channel and the production of free radicals. These preserve myocardial function by attenuating mitochondrial matrix calcium overload, attenuating apoptosis, and attenuating additional reactive oxygen species (ROS) production at reoxygenation (1, 12, 31, 37). In addition, it was recently shown that preconditioning activates the kinases ERKs and Akt during the reperfusion phase and these act to inhibit opening of the mitochondrial permeability transition pore by desensitizing it to calcium overload (2, 18, 21).

Cytochrome-c oxidase (CcO) activity is substantially altered in mammalian cells exposed to acute hypoxia and tissues subjected to ischemia and reperfusion (10, 11, 24, 25). Similarly, aconitase enzyme has been shown as a sensitive marker of mitochondrial oxidative stress (16, 41). We recently found that during ischemia PKA activation contributes to inactivation of CcO by way of hyperphosphorylation of subunits I, IVi1, and Vb; if PKA is blocked during ischemia, CcO inactivation is prevented and myocardial damage due to coronary occlusion and reperfusion is attenuated (40). Myocardial ischemia is accompanied by a large release of endogenous catecholamines (23). Thus it may be beta-adrenergic stimulation via these catecholamines that activates PKA and contributes to myocardial damage. It is clear that beta-adrenergic stimulation plays an important role in ischemic cell death, and recent evidence indicates that the two beta-adrenoreceptor subtypes (beta1-AR and beta2-AR) have opposing effects (13, 45, 51). The beta2-AR is linked to the Gi-mediated ERK activation and is antiapoptotic. In contrast, the beta1-AR activates PKA and is a potent apoptotic trigger in rat ventricular myocytes (20, 45). Importantly, PKA that is activated by the beta2-receptor is compartmentalized to a subsarcolemmal space and is not involved in mitochondrial phosphorylation (22). Also, it was shown in norepinephrine-depleted rat hearts that perfusion for 5 min with isoproterenol before ischemia-reperfusion provided protection against myocardial damage similar to that of ischemic preconditioning; this protection was prevented by beta1-AR blockade but not by beta2-AR blockade (15). Together these data suggest that activation of the beta1-AR by ischemia may be the triggering mechanism for ischemic preconditioning mediated by PKA as well as the mechanism by which CcO function is altered during ischemia. Thus the beta1-AR may mediate both myocardial protection and damage during ischemia.

The present studies were designed to determine the role of the beta1-AR in myocardial ischemic damage and ischemic preconditioning with a classic rabbit heart model. We found that the beta1-AR plays a dual role in myocardial ischemic damage. Blocking the beta1-AR during occlusion-reperfusion is protective in that it preserves CcO activity and attenuates the resulting myocardial damage comparable to ischemic preconditioning. However, blocking the beta1-AR during ischemic preconditioning attenuates the ischemic preconditioning response. This does not happen if the beta1-AR is blocked after the preconditioning episodes occur.


   METHODS
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 METHODS
RESULTS
 DISCUSSION
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All animals were treated according to the "Guiding Principles in the Care and Use of Animals" approved by the Council of the American Physiological Society, the "Principles for the Use of Animals," and the Guide for the Care and Use of Laboratory Animals adopted by the National Institutes of Health. The University of Pennsylvania Institutional Animal Care and Use Committee approved the protocol for these studies. Sixty-five New Zealand White male rabbits weighing 3.0–4.0 kg were sedated with a combination of ketamine hydrochloride (40 mg/kg) and xylazine (4.4 mg/kg) administered intramuscularly. After sedation the rabbits were heparinized (500 U iv) and euthanized with propofol (100 mg iv). Our preparation was published previously (46). The hearts were removed via a midsternal thoracotomy and rinsed in cool, oxygenated Tyrode solution. The aorta was cannulated, and the heart was hung on a Langendorff perfusion apparatus by which the coronary arteries were perfused with nonrecirculated, oxygenated (95% O2-5% CO2), temperature-controlled Tyrode solution at a constant pressure of 80 mmHg. The heart was surrounded by a temperature-controlled 50-ml chamber with an overflow such that the heart was submerged in its effluent. Coronary flow was measured by a timed drip count from the overflow. Myocardial temperature was monitored with a thermistor probe placed within the 50-ml chamber surrounding the heart and maintained at 36°C. The composition of the Tyrode solution was (mM) 137.0 NaCl, 24.0 NaHCO3, 5.5 dextrose, 2.7 KCl, 2.7 CaCl2, 0.9 NaH2PO4, and 0.5 MgCl2.

Experimental protocols. To monitor contractility, a water-filled latex balloon connected to a pressure gauge was inserted into the left ventricular cavity. The preload was adjusted to ~5–10 mmHg. The pulse pressure was measured to the nearest 0.5 mmHg. The changes in left ventricular balloon pressure were used to provide an index of contractility. The bath chamber that surrounded the heart allowed us to record simulated orthogonal electrocardiograms. Two bipolar electrocardiograms (X and Y leads) were recorded by two pairs of 1-cm-diameter gold-plated electrodes that were affixed to the walls of the chamber and positioned left (positive) to right (negative) and anterior (positive) to posterior (negative) relative to the heart. We used changes in the ST segment as one measure of the extent of ischemia during the coronary ligation. The deviation of the ST segment from baseline was measured to the nearest 0.025 mV at the same point in time near the midpoint of the ST segment for both orthogonal X and Y leads. The X and Y values were converted to a single vector magnitude, and the difference in millivolts from similar baseline measurement was calculated.

After 30 min of baseline equilibration, coronary occlusion was performed by ligating the anterior division of the left coronary artery near its origin (38) with a snare made with 3-0 gauge silk suture passed under the artery and sheathed in a 20-gauge polyethylene tube. After 30 min of occlusion the snare was released and reperfusion was allowed for 2 h. Six experimental groups were evaluated (see Fig. 1). Three hearts underwent 30 min of coronary occlusion without reperfusion. These were used only in the biochemical analyses. A control experimental group of 13 hearts underwent 30 min of coronary occlusion followed by 2 h of reperfusion (Control). Another group of 10 hearts had the beta1-AR inhibitor CGP-20712A (0.3 µM; Sigma) added to the perfusate 30 min before occlusion and maintained throughout reperfusion (CGP-20712A). Ten additional hearts underwent classic ischemic preconditioning (Precon). Hearts were preconditioned by two global ischemic episodes of 5 min in duration at a perfusion rate of 2.5 ml/min and separated by 10 min (46). Ten minutes after the second preconditioning episode, coronary occlusion was performed for 30 min, followed by 2 h of reperfusion. A group of 10 hearts had the beta1-AR inhibitor CGP-20712A added to the perfusate 30 min before ischemic preconditioning and maintained throughout coronary occlusion and reperfusion (Precon + CGP-20712A). In 10 hearts the beta1-AR inhibitor CGP-20712A was added after the preconditioning ischemic episodes 5 min before coronary occlusion and maintained throughout coronary occlusion and reperfusion (CGP-20712A after Precon). Nine additional hearts served as sham-treated controls. These had the occlusive snare placed but were perfused for 3 h without coronary occlusion (Sham).


Figure 1
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  		Fig. 1. Schematic drawing showing the protocol used for each group. The moment of coronary occlusion for the experimental groups is set as zero on the time axis. See text for details. Precon, preconditioning; Ant Occl, anterior coronary occlusion.

 
Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk during the occlusion and the volume of myocardial necrosis after 2 h of reperfusion (50). After 2 h of reperfusion, the snare was reapplied and the heart perfused with India ink. The region not stained indicated the nonperfused volume at risk. The heart was removed from the apparatus, sectioned transversely from apex to base in ~4-mm-thick sections, and incubated for 20 min at 36°C in 1% triphenyltetrazolium chloride solution to delineate the necrotic zone (14). After incubation the tissues were sandwiched between clear plastic plates that had spacers between them to fix the thickness of the tissues at 3.5 mm. Both sides of the sections were photographed and enlarged by 200%. The areas of the necrotic region and the areas at risk for both sides of each tissue were measured from photographs with computer-based planimetry. The volumes of the necrotic regions and the risk regions were calculated for each section, using the average areas of both sides of the tissue section and the thickness of the slice. Separate hearts were used for the biochemical analyses, and these were not perfused with India ink to avoid contaminating the reactions.

Results are presented as means and SD. Differences between paired variables were determined with two-tailed Student's t-tests for paired data. Differences among nonpaired variables were evaluated with a single-factor analysis of variance followed by individual comparisons with two-tailed Student's t-test for nonpaired data. P values <0.05 were considered statistically significant.

Analysis of cytochrome-c oxidase and aconitase activities. Three hearts from each experimental group were used for CcO and aconitase assays. With a scalpel, a small transmural section of tissue was removed from the region at risk on the anterior left ventricle.

The CcO assay was described previously (6, 40). Briefly, mitochondria were isolated from myocardial tissue by differential centrifugation of tissue homogenates in mitochondria isolation buffer (mM: 70 sucrose, 220 mannitol, 2.5 HEPES, pH 7.4, and 2 EDTA), and the CcO activity of mitochondria was assayed by measuring the rate of oxidation of ferrocytochrome c (0.2–0.8 µM) at 550 nm, using 5 µg of mitochondrial protein in a final reaction volume of 1 ml with a Cary-1E spectrophotometer (Varian Instruments, Walnut Creek, CA).

Mitochondrial aconitase activity was determined essentially as described by Gardner et al. (16). Briefly, the assay indirectly determines the rate of formation of isocitrate from citrate, using isocitrate dehydrogenase that utilizes NADP as a cofactor. The rate of formation of NADPH was measured in a 1.0-ml assay system consisting of 100 µg of mitochondrial protein, 50 mM Tris pH 7.4, 0.2 mM NADP, 0.6 mM MnCl2, and 30 mM sodium citrate by following the linear absorbance change at 340 nm after the initial lag. We report aconitase activity as micromoles per liter of NADPH produced per minute per milligram of protein.


   RESULTS
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DISCUSSION
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Electrophysiological and mechanical responses. The spontaneous heart rates among all of the groups including the Sham group at baseline ranged from 154.4 to 170.5 beats/min and were not significantly different. In all groups the heart rates tended to slow over time. After 2 h of reperfusion the heart rates ranged from 131.0 to 152.5 beats/min. PR intervals, QRS durations, and QT intervals were similar among all groups at baseline and did not change significantly during occlusion or reperfusion, although the QT interval tended to prolong slightly over time as the heart rate slowed. These data verify the overall stability of the preparation during the course of the experiments, and also verify that the local coronary occlusion was not producing global alterations in electrophysiology.

Table 1 shows the coronary flows, pulse pressures, and ST segment values for all groups. These parameters were used as indexes of the degree of ischemic injury during coronary occlusion. Coronary flow was similar among the groups during baseline. As can be seen in the Sham group, coronary flow tended to decrease over time. After 3 h of perfusion it decreased from control values by –29.8%. As expected, anterior coronary occlusion significantly reduced coronary flow for all experimental groups.


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  		Table 1. Coronary flow, pulse pressure, and ST segment before and after 30 min of coronary occlusion and 2 h of reperfusion

 
The Sham group shows that pulse pressure remained relatively constant over time. During coronary occlusion the pulse pressure significantly decreased in all experimental groups, and the pulse pressures remained significantly less than baseline values at the end of reperfusion. This depression could be a reflection of myocardial stunning due to the ischemic interventions (17) and/or due to loss of viable myocardium.

The ST segment was not different among the groups at baseline and, as can be seen for the Sham group, did not change with time. The ST segment significantly increased in all experimental groups during coronary occlusion. The ST segment returned toward baseline values after reperfusion and, except for the control experimental group, was not significantly different from baseline.

Effects on myocardial survival. The data of Table 2 show that block of the beta1-AR (CGP-20712A) during occlusion was protective and support our previous study in which PKA blockade was protective (40). The mean necrotic volume-to-risk volume ratio was significantly reduced by 38% compared with control. Less myocardial tissue became necrotic after reperfusion. Ischemic preconditioning (Precon) reduced the mean necrotic volume-to-risk volume ratio by a similar amount, 37%. Interestingly, ischemic preconditioning in the presence of CGP-20712A (Precon + CGP-20712A) was not protective. However, if CGP-20712A was given after ischemic preconditioning (CGP-20712A after Precon) protection against occlusion-reperfusion damage was seen compared with control. However, the necrotic-to-risk volume ratio was not significantly different from the Precon + CGP-20712A group (P = 0.161). Presumably, this was due to the latter's relatively large SD. Since the size of the necrotic volume varies with the size of the risk volume (50), we applied another statistical test to the data. Linear regression was performed on the pooled data from the experimental groups relating the necrotic volume to the volume at risk (Fig. 2A). The residuals or the vertical distances of the individual data points from the pooled regression line were calculated for each group (Fig. 2B). Analysis of variance showed the residuals for the experimental groups to be significantly different with P = 0.002. The residuals of the beta1-AR blockade group, the preconditioned group, and the group with beta1-AR blockade after preconditioning, but not the group preconditioned in the presence of CGP-22071A, were significantly different from control. These data verify the findings of Table 2.


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  		Table 2. Myocardial ventricular volumes

 

Figure 2
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  		Fig. 2. Relationship of the necrotic volume to the volume at risk for 5 experimental groups (A) and the residuals of the data points from the linear regression line of the pooled data (B). In A the solid lines are the regression lines for the individual groups. The dashed line is the regression line for the pooled data of all the groups. The equation for this line is at top. In B are the residuals of the data points from this pooled line for each group. They were significantly different from one another by analysis of variance (P = 0.002). The residuals were significantly less by nonpaired t-test in the CGP-20712A group, the preconditioned group, and the group given CGP-20712A after preconditioning than in the control group. The group preconditioned in the presence of CGP-20712A was not different from control. NS, no significant difference.

 
Effects on CcO and aconitase activities. In Fig. 3 are shown the effects of occlusion and reperfusion on CcO activity with and without beta1-AR blockade and/or ischemic preconditioning. CcO specific activity in micromoles per minute per milligrams of protein was significantly less (P = 0.001) after 30 min of occlusion (6.3) than in the Sham hearts (7.7). Two hours of reperfusion reduced CcO activity significantly further to 5.2 (Control). Thus reperfusion had additional effects on altering CcO activity. As can be seen, beta1-AR blockade (CGP-20712A) completely reversed the reperfusion-induced attenuation of CcO activity. Interestingly, preconditioning (Precon) had no effect on CcO activity. With ischemic preconditioning in the presence of beta1-AR blockade (Precon + CGP-20712A) the attenuation of CcO activity was also reversed, even though myocardial protection was suppressed (Table 2). When beta1-AR blockade occurred after ischemic preconditioning (CGP-20712A after Precon) the attenuation of CcO activity was preserved similarly, but in this case the myocardium was also protected (Table 2). In all cases when beta1-AR blockade was present the attenuation of CcO activity was reversed. These findings support our hypothesis that occlusion-reperfusion-induced activation of the beta1-AR attenuates CcO activity, which is presumably due to PKA mediated CcO hyperphosphorylation (40). However, we did not see a myocardial protective effect with beta1-AR blockade during preconditioning, even though CcO activity was preserved. This indicates that other factors were involved.


Figure 3
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  		Fig. 3. Mitochondrial cytochrome-c oxidase (CcO) activity was assayed in triplicate in tissues removed from 3 hearts in each group. In 1 group CcO activity was determined after 30 min of occlusion (Occlusion). The other hearts underwent 30 min of coronary occlusion and were reperfused for 2 h before tissues were taken. One group had no further intervention (Control), 1 had undergone ischemic preconditioning (Precon), 1 was given the beta1-adrenergic receptor (beta1-AR) blocker CGP-20712A 30 min before occlusion (CGP-20712A), 1 was given CGP-20712A 30 min before preconditioning (Precon + CGP-20712A), and 1 was given CGP-20712A 5 min after preconditioning (CGP-20712A after Precon). The Sham control hearts were perfused for 3 h before tissues were removed. The y-axis shows the CcO activity measured as micromoles per liter of ferrocytochrome c oxidized per minute per milligram of protein. Error bars indicate SD. P values are by nonpaired t-test.

 
In Fig. 4 are shown the effects of occlusion and reperfusion on aconitase activity with and without beta1-AR blockade and/or ischemic preconditioning. The changes in aconitase activity with the various interventions parallel those of CcO activity (Fig. 3).


Figure 4
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  		Fig. 4. Mitochondrial aconitase activity was assayed in triplicate in tissues removed from 3 hearts in each group. The groups are the same as those described in Fig. 3. The y-axis shows the aconitase activity measured as micromoles per liter of NADPH produced per minute per milligram of protein.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of our study are that reperfusion following ischemia caused an additional decrease in CcO activity and that blocking the beta1-AR during occlusion-reperfusion both attenuated the depression in CcO activity and preserved myocardium at risk for necrosis. Furthermore, we found that blocking the beta1-AR during ischemic preconditioning eliminated cardioprotection. If the beta1-AR was blocked after ischemic preconditioning, myocardium was preserved compared with control (Fig. 3 and Table 2).

Occlusion-reperfusion in isolated heart preparation. The preparation remained stable over the duration of the experiment, with the sinus rate being the only parameter that tended to decrease with time. As expected, since coronary occlusion-reperfusion produces only regional left ventricular damage and the electrocardiographic parameters reflect global behavior, the PR intervals, QRS durations, and QT intervals remained stable throughout.

In Table 1 it can be seen for the Sham group that coronary flow tended to decrease slightly over time. Coronary occlusion in the experimental groups produced an additional significant decrease in overall flow. This was accompanied by decreases in pulse pressure and changes in the electrocardiographic ST segments reflecting the resulting ventricular ischemic injury. An analysis of variance indicated no significant difference among the experimental groups. Therefore, the degree of ischemia was comparable for all groups.

Dual role of beta1-adrenergic receptor in cardioprotection. Our finding that beta1-AR blockade alone is cardioprotective and reduces the necrotic volume-to-risk volume ratio comparable to preconditioning (Table 2) is consistent with our previous work. We previously found (40) that PKA blockade with H89 or myristoylated peptide inhibitor is also cardioprotective. This together with the present study indicates that the protective effect of beta1-AR blockade relies on the PKA pathway.

Multiple triggers of preconditioning have been found, and it is likely that there are multiple signal transduction pathways involved (33–36, 48, 49). One of the most studied is the one involving PKC. Blocking PKC prevents preconditioning (7, 19, 26, 29). Recent studies have also implicated the beta1-AR, PKA, cAMP pathway in preconditioning (15, 27, 43, 44). Asimakis and coworkers (3, 4) found that brief beta-adrenergic stimulation preconditions the rat heart against postischemic contractile dysfunction. Saltman et al. (42) showed in the rabbit heart that brief inhibition of phosphodiesterase with amrinone before coronary occlusion is more potent than ischemic preconditioning in protecting the heart. Others found that blocking cAMP activity during ischemic preconditioning eliminates its protective effect. Importantly, if PKA blockade is applied only during the preconditioning ischemic episodes protection is effectively blocked, indicating that a protective cascade involving PKA is set in motion by preconditioning (27, 44). This is consistent with our finding that beta1-AR blockade inhibits the protective effect of preconditioning only if it occurs during the preconditioning episodes (Table 2). However, in our study the beta1-AR blockade was present throughout ischemia and reperfusion as well (Table 1). This group (Precon + CGP-20712A) showed the greatest heart-to-heart variability in the necrotic-to-risk volume ratio. This indicates that the persistent presence of beta1-AR blockade was having inconsistent effects during ischemia and reperfusion. This is considered further below.

Our results showing that beta1-AR blockade can inhibit preconditioning and also protect the myocardium (Table 2) can be explained depending on the duration of ischemia and the relative timing of the blockade. The beta1-AR stimulation initiated by the brief ischemic preconditioning episodes sets in motion a cascade that provides a short-term window of protection. This protects against a later, more prolonged ischemic episode. beta-Blockade during the brief ischemic episodes blocks this pathway, preventing it from contributing to the ischemic preconditioning response. In the case of persistent prolonged ischemia without preconditioning, the protective cascade initiated by beta1-AR stimulation would not be able to fully develop before irreversible ischemic damage begins to take place. In the latter case of prolonged ischemia, beta1-AR blockade acts to protect the heart by blocking the effects of beta1-AR stimulation that lead to PKA activation and myocardial damage.

The fact that we did not see a myocardial protective effect when beta1-AR blockade was present throughout preconditioning and occlusion-reperfusion is surprising and difficult to reconcile in light of the fact that beta1-AR blockade without preconditioning is cardioprotective (Table 2). Perhaps the two 5-min ischemic episodes with preconditioning being inhibited by beta1-AR blockade produced additional myocardial damage that was variably able to be reversed by the persistent presence of beta1-AR blockade during occlusion and reperfusion. These two conflicting possible actions of beta1-AR blockade during preconditioning seem to be borne out by the large SD of the necrotic-to-risk volume ratios for the Precon + CGP-20712A group of hearts versus those in the group given only CGP-20712A or given CGP-20712A after preconditioning, as seen in Table 2. Two of the seven hearts in the Precon + CGP-20712A group had necrotic-to-risk ratios that fell below the means for the preconditioned and beta1-AR blockade groups.

Role of CcO in ischemic injury. We used aconitase activity as an additional biomarker of mitochondrial genetic stability, respiratory function, and oxidative damage (16, 41). In comparing Figs. 3 and 4 it can be seen that the changes in CcO activity and aconitase activity in response to our interventions parallel each other. This supports the validity of our CcO findings and further highlights the role of these enzymes in occlusion-reperfusion-dependent oxidative damage.

The importance of CcO in mitochondrial function and cellular metabolism makes it a prime candidate for directly contributing to cell survival. There is good evidence that CcO functions as an O2 sensor in the mitochondrial compartment, where >90% of the cellular O2 is reduced to H2O (8–10, 28, 39). The CcO activity is substantially altered in mammalian cells exposed to acute hypoxia and tissues subjected to ischemia-reperfusion (10, 11, 24, 25, 40). We (40) and others (5, 28, 47) have previously shown that cAMP-mediated phosphorylation plays a role in the regulation of CcO activity. In a previous study (40) we subjected our Langendorff-perfused rabbit hearts to varying degrees of global and regional ischemia. Exposure of the hearts to progressive durations of anterior division coronary occlusion (0.5, 1.5, and 3.0 h) produced progressive inhibition of CcO activity in mitochondria of left ventricular tissues taken from within the ischemic zone. This was not seen in the left ventricular tissues from the normally perfused zone. The specific inhibitors of PKA, H89 or myristoylated peptide (40 nM), had protective effects in that the ischemia-induced decrease in CcO activity was reversed. However, Go-6850 (50 nM), a specific inhibitor of PKC, did not prevent the decrease in CcO activity. The present study suggests that ischemia-induced stimulation of the beta1-AR via the release of endogenous catecholamines is the triggering mechanism that leads to a decrease in CcO activity due to cAMP-mediated phosphorylation of CcO subunits. Thus part of the protective effect of beta1-AR blockade may involve the prevention of CcO phosphorylation.

There are likely multiple factors that contribute to occlusion-reperfusion myocardial damage. In all groups in which beta1-AR blockade was present the depression of CcO activity was attenuated (Fig. 3), and in two of the three groups with beta1-AR blockade, myocardium was preserved. The fact that occlusion-reperfusion myocardial damage was not attenuated in the group that had beta1-AR blockade before the ischemic preconditioning episodes (Table 2) while CcO activity was preserved (Fig. 3) indicates that there are factors other than CcO activity that contribute to myocardial damage. The finding that classic ischemic preconditioning does not reverse the reperfusion-induced depression of CcO activity (Fig. 3) suggests that ischemic preconditioning, while protecting myocardium, does not rely on preserving CcO activity for its myocardial protective effects. However, the present data together with our previous work (40) strongly support our contention that depression of CcO activity is a cause rather than an effect of ischemic damage. CcO determines the rate of mitochondrial respiration and ATP synthesis and directly contributes to ROS production as shown by our recent data (40). As proposed in our recent study, altered CcO function during myocardial ischemia causing increased ROS production and decreased ADP recycling are important contributing factors in myocardial damage. A recent study by Meyer et al. (30) also supports our observation that a reduced ADP recycling in mitochondria will enhance ROS formation and ischemic damage. In our present study, although we did not measure ROS directly, we measured aconitase activity (Fig. 4), which is inversely related to ROS production and, as indicated in Fig. 3, directly correlates with the pattern of CcO activity. For these reasons, we believe that CcO inhibition is a cause of increased cardiac damage. Thus preservation of CcO activity (and preservation of aconitase activity) by beta1-AR blockade most likely represents one possible mode of its cardioprotective influence.

These data further support not only the idea that the beta1-AR is involved in ischemic preconditioning but also the idea that CcO phosphorylation contributes to ischemic damage. These studies may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in the setting of coronary reperfusion intervention.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported in part by National Institute of General Medical Sciences Grant GM-49683 to N. G. Avadhani and an award from the American Heart Association to J. F. Spear.


   ACKNOWLEDGMENTS
 
We thank Kenneth Fitzgerald for expert technical help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. F. Spear, Dept. of Animal Biology, School of Veterinary Medicine, Univ. of Pennsylvania, 3800 Spruce St., Philadelphia PA 19104-6046 (e-mail: spearj{at}vet.upenn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 DISCUSSION
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