INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PRECONDITIONING THE HEART with brief periods of sublethal ischemia-reperfusion protects it from a subsequent prolonged ischemic event. Preconditioning is the most potent cardioprotective adaptation known with a profound ability to reduce the adverse effects of ischemia in a number of experimental models (18, 20, 28, 31, 45). With the description of clinical correlates of preconditioning (11, 38, 44) and the possibility of harnessing this powerful adaptive response in the management of ischemic heart disease, earnest investigation continues in pursuit of its physiological mechanisms. Potential clinical interventions directed at inducing, mimicking, or prolonging the protective effects of preconditioning will require a thorough understanding of the cellular events mediating its effects.

In its classical description, the primary beneficial effect of preconditioning is a delay in the development of myocardial necrosis (28, 31). Although this end point has been observed in a number of experimental models, other end points of protection have been described including improved postischemic recovery of contractile function (8, 40) and reduction in the release of cardiac enzymes (9, 42). Furthermore, it is now apparent that the benefits of preconditioning are manifest during (at least) two distinct time periods following sustained ischemia. The first reports of preconditioning documented an early protective effect that was lost within a few hours following prolonged ischemia (31). Growing evidence supports a delayed protection in which the beneficial effects of preconditioning are manifest as late as 72 h after a prolonged ischemic event (3, 4, 17, 23, 24). Recent reports also raise the interesting possibility that delayed cardioprotection conferred by preconditioning can be sustained through repeated A₁ adenosine receptor activation (10).

Since its earliest observations (28, 31), a large body of evidence has accumulated implicating a number of experimental triggers of the preconditioning response. These include but are not limited to adenosine and A₁ adenosine receptor activation (1, 12, 18, 22, 26), bradykinin stimulation (16, 43), opioid receptor activation (21, 33), nitric oxide (6, 36), and both - and -adrenergic receptor activation (2, 39). Although this diversity suggests a natural redundancy in the process of initiating preconditioning, substantial evidence exists supporting the activation of A₁ adenosine receptors as both a key initiator and a mediator of its cardioprotection (1, 18, 22, 37; and reviewed in 12, 26, 45). The capacity for adenosine to mediate the preconditioning response is consistent with its well-documented role as an endogenous cardioprotectant (5, 13, 27). Nevertheless, the role of A₁ adenosine receptor activation in mediating preconditioning remains only partially elucidated.

With the recent characterization of ischemic preconditioning in the mouse heart (17, 35, 41), there exists a new potential to examine its cellular mechanisms through the use of genetically engineered animals. Matherne et al. (25) have previously developed a murine model of transgenic A₁ adenosine receptor overexpression in the heart demonstrating improved tolerance to ischemia-reperfusion (19, 25). To further investigate this enhanced ischemic tolerance, the present study examines the preconditioning response in wild-type and transgenic hearts overexpressing A₁ receptors, documenting its effects on functional recovery, cardiac enzyme release, and infarct size. Additionally, we sought to determine whether A₁ receptor activation is involved in murine ischemic preconditioning through use of selective A₁ receptor antagonism. We hypothesized that ischemic preconditioning in mice is mediated by A₁ receptor activation and that the response to preconditioning would be increased in hearts overexpressing A₁ adenosine receptors.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transgenic mice with overexpressed cardiac A₁ adenosine receptors. The construct design and initial characterization of the transgenic model of A₁ adenosine receptor overexpression in the mouse heart has been previously described (14, 25). Briefly, the rat A₁ adenosine receptor cDNA was inserted in the mouse genome under the control of the cardiac-specific -myosin heavy chain promoter using standard transgenic techniques. Transgene detection was performed by Southern analysis as previously reported (25).

Langendorff heart model. Isolated heart experiments were performed as previously described (25). Animals were anesthetized with 50 mg/kg ip pentobarbital sodium, a thoracotomy was performed, and the hearts were excised into ice-cold perfusate. The aorta was rapidly cannulated with a 20-gauge needle, and retrograde coronary perfusion was initiated at a constant pressure of 80 mmHg with a modified Krebs bicarbonate buffer containing (in mM) 120 NaCl, 25 NaHCO₃, 4.7 KCl, 1.2 KH₂PO₄, 2.5 CaCl₂, 1.2 Mg₂SO₄, 15 glucose, and 0.05 EDTA. The perfusate was equilibrated with 95% O₂-5% CO₂ at 37°C, giving a pH of 7.4. Hearts were bathed in perfusate within a water-jacketed bath maintained at 37°C. The pulmonary artery was cannulated for collection of coronary effluent. The left ventricle was vented with a polyethylene apical drain. Coronary perfusion was monitored using an ultrasonic flow probe (Transonic Systems, Ithaca, NY) in the aortic perfusion line. For most experiments, developed tension was examined as a gross indicator of contractile function, and this was assessed using a small stainless steel hook attached to the cardiac apex and connected to a tension transducer (model FT03C, Grass). Transducer position was adjusted to yield a diastolic tension of 1.0 g. Apicobasal displacement was continuously recorded with a MacLab four-channel data acquisition unit (ADInstruments, Castle Hill, Australia) to yield heart rate and developed tension.

Preconditioning and ischemia-reperfusion protocol. All hearts were equilibrated for 40 min. To correct for a lower basal rate, transgenic hearts were paced at 340 beats/min (12-ms square wave, voltage 20% above threshold) beginning 10 min into equilibration and continuing until the onset of sustained global ischemia. After equilibration, preconditioned groups underwent two cycles of 30 s of global ischemia each followed by 150 s of reperfusion, and control groups underwent an additional 6 min of extended equilibration. All hearts then underwent 20 min of global normothermic ischemia during which pacing was discontinued. Global ischemia was produced by clamping the aortic cannula and simultaneously bubbling 95% N₂-5% CO₂ through the bathing perfusate to reduce PO₂. Reperfusion was achieved by unclamping the aortic cannula and discontinuing the nitrogen bubbling. All hearts were paced at 6 Hz during reperfusion beginning 1 min after unclamping the aortic cannula. A 30-min reperfusion period was used to assess recovery of contractile function. Two subsets of hearts underwent longer reperfusion periods to adequately examine other end points of protection [35 min for collection of coronary effluent to determine lactate dehydrogenase (LDH) concentration and 60 min for infarct size determination by the triphenyltetrazolium chloride (TTC) method].

A₁ adenosine receptor blockade. To determine whether activation of A₁ adenosine receptors is involved in murine ischemic preconditioning, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was used as a selective A₁ receptor antagonist. For these experiments, isolated mouse hearts were perfused using an intraventricular isovolumic balloon. Control and preconditioning protocols were performed in wild-type hearts as described above with the exception that 100 nM DPCPX was added to the coronary perfusate 10 min before preconditioning (10 min before sustained ischemia in the control group). In transgenic hearts, 500 nM DPCPX was used to ensure blockade of markedly overexpressed A₁ adenosine receptors.

Determination of coronary effluent LDH content. Coronary venous effluent was collected from a subset of hearts in each group for quantitation of LDH release. To determine whether the preconditioning stimuli alone resulted in enzyme release, a sample was collected immediately before the onset of sustained global ischemia. After 20 min of global ischemia, aliquots of coronary effluent were continuously collected during reperfusion. Because our preliminary observations demonstrated peak efflux of LDH at 25 to 30 min, we extended the reperfusion period to 35 min in this subset of hearts to assure capture of peak LDH release. Upon reperfusion, an initial 5-min aliquot was collected followed by three successive 10-min aliquots. Samples were stored at 20°C until analysis. LDH concentration was determined by an enzymatic assay (Sigma, St. Louis, MO).

Infarct size determination with TTC staining. A subset of hearts in each group was reperfused for 60 min after which 15 ml of 1% wt/vol TTC (Sigma) in phosphate-buffered saline was infused into the coronary circulation at a rate of 1.5 ml/min. Hearts were then removed from the cannula, weighed, and fixed overnight in 10% Formalin. Hearts were removed from Formalin and stored frozen at 20°C until sectioning for analysis of infarct size. Hearts were sectioned along the atrioventricular plane into ~0.5-mm sections. Sections were placed between two microscope slides and digitally photographed (Leaf Lumina). To eliminate infarct artifacts caused by the apical drain and tension hook, planimetry was performed on five sections from the middle portion of each heart. Computerized area analysis was performed automatically (Image Pro), and the infarct size of each section was expressed as a fraction of the total area at risk. Infarct size for each heart was determined by averaging the infarct area of the five sections.

Data analysis and statistical comparisons. Baseline functional data were analyzed by two-way ANOVA. To examine differences between groups at multiple time points, three comparisons were made in each of the analyses of developed tension, coronary flow, and efflux of LDH: 1) wild-type control versus wild-type preconditioned groups, 2) transgenic control versus transgenic preconditioned groups, and 3) wild-type control versus transgenic control groups. These analyses were performed using multivariate ANOVA for repeated measures with Bonferroni's correction for multiple comparisons and Student-Newman-Keuls post hoc test for individual comparisons. Comparisons between groups for total LDH efflux, infarct size, and effect of DPCPX were made using two-way ANOVA with Student-Newman-Keuls post hoc test. Statistical significance was accepted for P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolated Langendorff Perfused Hearts

Baseline function. Baseline functional data for wild-type hearts (n = 34, 22 ± 2.0 wk, heart wt 141 ± 4 mg) and transgenic hearts (n = 18, 28 ± 1.5 wk, heart wt 155 ± 7 mg) are shown in Table 1. Transgenic hearts were paced to correct for their lower intrinsic rates (14, 15, 25). No differences in baseline functional parameters were observed between wild-type and transgenic hearts. These data are consistent with previous observations that transgenic overexpression of A₁ adenosine receptors does not modulate baseline function in isolated working hearts (14).

Functional effects of ischemia-reperfusion. Myocardial function (expressed as percent change from baseline developed tension) during ischemia-reperfusion is shown in Fig. 1. With the onset of global ischemia, all hearts demonstrated an immediate decline in developed tension and full arrest within 2-3 min. Upon reperfusion, hearts resumed spontaneous contraction within 30-60 s. After an initial increased recovery lasting 2-3 min, all hearts demonstrated a rapid decline in function followed by a progressive recovery of developed tension for the remainder of reperfusion. In the control groups (Fig. 1, open symbols) transgenic hearts demonstrated an improved recovery of developed tension compared with wild-type hearts at all times during reperfusion. This difference was particularly marked at 2 min of reperfusion when transgenic hearts recovered to 56.4 ± 5.4% of baseline compared with only 27.3 ± 4.0% recovery in wild-type hearts (P < 0.05).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Developed tension (expressed as percentage of baseline) during 20 min of global ischemia and 30 min reperfusion. Time 0 marks onset of ischemia. Values for wild-type control (n = 18, ), wild-type preconditioned (n = 16, ), transgenic control (n = 10, ), and transgenic preconditioned (n = 8, ) are shown. Values are means ± SE. *Significant differences between wild-type control and wild-type preconditioned groups; **significant differences between wild-type control and transgenic control groups. P < 0.05.

Effects of preconditioning. In wild-type hearts (Fig. 1, squares) preconditioning significantly improved recovery of developed tension throughout reperfusion except at the initial 2-min time point. Final functional recovery was improved from 25.6 ± 1.7% of baseline developed tension in control hearts to 37.4 ± 2.2% in preconditioned hearts. Despite marked overexpression of A₁ adenosine receptors, preconditioning did not alter the functional recovery in transgenic hearts (Fig. 1, circles). Coronary flow changes during ischemia-reperfusion and preconditioning are shown in Fig. 2. The brief periods of preconditioning ischemia resulted in a mild, although not statistically significant, increase in coronary flow immediately before sustained ischemia. After 20 min of global ischemia, all groups demonstrated a marked increase in coronary flow up to 450% of baseline. With continued reperfusion, coronary flow returned to near steady state at ~200% of baseline. For all time points, there were no statistically significant differences between the groups.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Coronary flow (expressed as percentage of baseline) through preconditioning, sustained global ischemia, and reperfusion. Time 0 marks onset of global ischemia. Values for wild-type control (n = 18, ), wild-type preconditioned (n = 16, ), transgenic control (n = 10, ), and transgenic preconditioned (n = 8, ) are shown. Values are means ± SE. No significant differences were noted between groups.

A₁ adenosine receptor antagonism. The effect of A₁ adenosine receptor blockade on final functional recovery in isovolumically contracting isolated hearts is shown in Fig. 3. Treatment with DPCPX before ischemia-reperfusion slightly decreased contractile recovery (expressed as percent change from preischemic +dP/dt) in wild-type hearts from 37.9 ± 2.4% to 25.2 ± 8.4%. Preconditioning improved functional recovery in wild-type hearts from 37.9 ± 2.4% to 74.2 ± 7.3%, and treatment with DPCPX completely eliminated the beneficial effect of preconditioning on functional recovery (Fig. 3). Transgenic A₁ adenosine receptor overexpression resulted in markedly improved functional recovery (84.1 ± 12.9% vs. 37.9 ± 2.4% in wild-type hearts), and treatment with DPCPX eliminated this functional advantage (Fig. 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine on final functional recovery in isovolumically contracting isolated mouse hearts: WT, wild-type control (n = 8 untreated, n = 7 treated; open bars); PC, wild-type preconditioned (n = 8 untreated, n = 8 treated; gray bars); and trans, transgenic (n = 7 untreated, n = 8 treated; black bars). Values are means ±SE. *Differs from untreated, P < 0.05; differs from wild-type treated and untreated groups, P < 0.05.

Determinants of Tissue Viability

LDH release. Preconditioning slightly but significantly increased LDH release before sustained global ischemia in both wild-type (22.4 ± 9.2 vs. 50.5 ± 8.0 U · l¹ · g¹) and transgenic groups (28.7 ± 7.2 vs. 48.7 ± 8.6 U · l¹ · g¹). LDH concentration was similar in all groups during the first 5 min of reperfusion after sustained ischemia (Fig. 4). Subsequently, LDH release was greatest in the wild-type control group until the last time point. By the end of reperfusion, LDH concentration in the coronary effluent was decreasing and did not reach statistical significance (Fig. 4). Total efflux of LDH for all groups is shown in Fig. 5A (wild-type control, n = 6; wild-type preconditioned, n = 8; transgenic control, n = 10; transgenic preconditioned, n = 7). Preconditioning reduced the release of LDH in wild-type hearts from 7,012 ± 1,451 to 1,691 ± 1,256 U · l¹ · g¹ · min¹, P < 0.05 (Fig. 5A). Efflux of LDH in transgenic control hearts was markedly less than that observed for wild-type control hearts (917 ± 1,123 vs. 7,012 ± 1,451 U · l¹ · g¹ · min¹, respectively, P < 0.05). The minimal release of LDH in transgenic hearts was unaffected by preconditioning (Fig. 5A).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Lactate dehydrogenase (LDH) concentration in coronary venous effluent during reperfusion after 20 min sustained global ischemia. Values for wild-type control (n = 6, ), wild-type preconditioned (n = 8, ), transgenic control (n = 10, ), and transgenic preconditioned (n = 7, ) are shown. Values are means ± SE. *Different from all other groups, P < 0.05.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Determinants of tissue viability assessed by total efflux of LDH (A) and infarct size (B). CTL, control groups; PRE, preconditioned groups. Values are means ± SE. *Different from all other groups; **different from transgenic groups. P < 0.05.

Infarct size. Coronary injection with TTC demonstrated a distinctive staining pattern. Viable tissue appeared deep red, and regions of infarction remained pale. Images representative of hearts in each experimental group are shown in Fig. 6. Ischemic preconditioning reduced the infarct size in wild-type hearts from 62.6 ± 5.1% (n = 5) to 32.3 ± 11.5% (n = 5), P < 0.05 (Fig. 5B). Overexpression of A₁ adenosine receptors reduced infarct size from 62.6 ± 5.1% in wild-type control hearts to 6.5 ± 2.1% in transgenic control hearts (n = 4), P < 0.05 (Fig. 5B). This minimal degree of infarction noted in transgenic control hearts was unaffected by preconditioning (8.0 ± 2.3%, n = 4). In addition, the reduced efflux of LDH in transgenic hearts was similar to the reduction of infarct size as determined by TTC staining (Fig. 5, A and B).

View larger version (80K): [in this window] [in a new window]   Fig. 6.   Representative images of heart sections stained with triphenyltetrazolium chloride. With this method, viable tissue stains red and infarcted tissue remains pale. Photos are representative of wild-type control (A), wild-type preconditioned (B), transgenic control (C), and transgenic preconditioned (D) groups.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The dual purpose of this study was to determine whether activation of A₁ adenosine receptors is involved in ischemic preconditioning in murine hearts and to examine the effect of preconditioning in transgenic hearts overexpressing A₁ adenosine receptors. A protocol of ischemic preconditioning is presented that improves postischemic contractile function, decreases release of LDH, and reduces infarct size in isolated wild-type mouse hearts. Selective A₁ receptor antagonism demonstrates that the improved postischemic contractile function conferred by preconditioning in wild-type hearts is mediated by endogenous activation of A₁ adenosine receptors. Although preconditioning enhances tolerance to ischemia in wild-type hearts, it does not augment functional and tissue-protective advantages of A₁ adenosine receptor overexpression. Indeed, the greater functional recovery and tissue viability resulting from transgenic A₁ receptor overexpression surpasses the cardioprotection offered by ischemic preconditioning alone (Figs. 1 and 5B). Importantly, the effect of preconditioning wild-type hearts and the protection afforded by A₁ receptor overexpression are equally attenuated by selective A₁ receptor blockade with DPCPX (Fig. 3).

The current observation of improved postischemic functional recovery in transgenic hearts overexpressing A₁ adenosine receptors (Figs. 1 and 3) is consistent with previous reports from our laboratory demonstrating increased tolerance to ischemia (19, 25). The ability to eliminate the postischemic functional advantage of A₁ receptor overexpression by DPCPX treatment confirms that this effect is mediated through endogenous activation of A₁ adenosine receptors (Fig. 3). By documenting decreased LDH release and reduced infarct size in transgenic hearts, we further characterize the favorable effects of A₁ adenosine receptor overexpression to include enhanced tissue viability following global ischemia (Figs. 4 and 5).

Preconditioning in the isolated mouse heart. Since the initial reports of myocardial infarct-size reduction in dogs (28, 31), the beneficial effects of preconditioning have been observed in a variety of species including humans (11, 38), swine (32), rabbits (16, 22), and rats (18). With recent reports of ischemic preconditioning in isolated and in vivo mouse hearts (17, 34, 35, 41), it is now possible to take advantage of targeted genetic modification to investigate the cellular mechanisms of its protection. In addition to infarct-size reduction, a number of studies have shown that preconditioning reduces postischemic contractile dysfunction (7, 8, 35, 40) and release of cardiac enzymes (9, 42). The current study examines the effect of preconditioning on each of these relevant end points in both wild-type and transgenic mouse hearts overexpressing A₁ adenosine receptors.

Myocardial protection in transgenic hearts with increased A₁ adenosine receptors. Considerable evidence exists for the cardioprotective role of endogenous adenosine during ischemia-reperfusion (12, 18, 22, 26). Because adenosine is rapidly released during ischemia in amounts sufficient to saturate available receptors, it may be difficult to augment this intrinsic cardioprotection (5, 18). That is, the limiting factor in harnessing adenosine-mediated cardioprotection during ischemia may be receptor number (or availability) rather than the concentration of endogenous agonist. Taking this approach, our initial characterization of transgenic hearts overexpressing A₁ adenosine receptors demonstrated improved recovery after ischemia-reperfusion (25).

Preconditioning and transgenic overexpression of A₁ adenosine receptors. Although the data demonstrate clear beneficial effects of preconditioning in wild-type mouse hearts, they indicate that preconditioning does not augment the protective effects of A₁ receptor overexpression. Hearts overexpressing A₁ receptors are intrinsically more tolerant to ischemia (Figs. 1 and 3; Refs. 15, 19, 25), and preconditioning does not improve recovery beyond the advantage conferred solely by A₁ receptor overexpression. Thus the cardioprotective effects of ischemic preconditioning and A₁ receptor overexpression are not additive. This suggests that preconditioning and A₁ receptor overexpression affect cardioprotection through common mechanisms or pathways, or alternatively, that A₁ adenosine receptor overexpression confers the maximal tolerance to ischemia, superceding even that resulting from preconditioning (Figs. 1 and 5B). However, because selective A₁ blockade effectively abolishes the protection resulting from both preconditioning and A₁ receptor overexpression (Fig. 3), we conclude that the two interventions operate through common pathways (i.e., both involving A₁ adenosine receptor activation).

Study limitations. It is recognized that measurement of apicobasal displacement, as utilized by previous investigators (25, 29, 30, 34, 35, 41) and in the present study, is a gross indicator of myocardial contractile function. Although this technique sufficiently discriminates differences in contractile recovery between experimental groups (Fig. 1), we have abandoned it in favor of isovolumic measurement of contractile function using an intraventricular balloon (Fig. 3 and MATERIALS AND METHODS). It is important to note, however, that the improved postischemic functional recovery conferred by both preconditioning and A₁ receptor overexpression remained consistent between the two models (Figs. 1 and 3). A second limitation in all studies using isolated crystalloid-perfused hearts is the absence of blood-borne factors, including neutrophils and platelets. However, this may represent an advantage because it permits the clear separation of the contributions of such elements to ischemic injury and cardioprotection, permitting assessment of intrinsic myocardial responses.

Conclusions and future directions. This study provides evidence that ischemic preconditioning in the mouse heart involves endogenous activation of A₁ adenosine receptors. The cardioprotection conferred by transgenic overexpression of A₁ adenosine receptors mimics that of ischemic preconditioning. Preconditioning does not further enhance the intrinsic tissue-protective and functional advantages of A₁ receptor overexpression. Thus although each is individually advantageous, the cardioprotection conferred by transgenic overexpression of A₁ adenosine receptors and ischemic preconditioning is not additive. Coupled with the observation that A₁ receptor blockade equally reduces the protective effects of both preconditioning and A₁ receptor overexpression, these data support the possibility that the two interventions affect cardioprotection via common mechanisms. Further work to elucidate the cellular mechanisms involved in ischemic preconditioning can now take advantage of this and other models of targeted gene modification.