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Am J Physiol Heart Circ Physiol 277: H2458-H2469, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 6, H2458-H2469, December 1999

SPECIAL TOPIC
Pharmacological manipulation of Ins(1,4,5)P3 signaling mimics preconditioning in rabbit heart

Anne Gysembergh1, Stéphanie Lemaire2, Christophe Piot2, Catherine Sportouch2, Sylvain Richard2, Robert A. Kloner1, and Karin Przyklenk1

1 Heart Institute, Good Samaritan Hospital and University of Southern California, Los Angeles, California 90017-2395; and 2 Institut de Génétique Humaine, Centre National de la Recherche Scientifique, UPR 1142, Montpellier, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recent evidence revealed biphasic alterations in myocardial concentrations of the second messenger inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] with ischemic preconditioning (PC), i.e., increase during brief PC ischemia and decrease early during sustained test occlusion. Our aim was to determine whether an agonist and an antagonist of Ins(1,4,5)P3 signaling {D-myo-inositol-1,4,5-trisphosphate hexasodium salt [D-myo-Ins(1,4,5)P3] and 2-aminoethoxydiphenyl borate (2-APB), respectively}, given such that they mimic this biphasic profile, would mimic infarct size reduction with PC. To test this concept, isolated, buffer-perfused rabbit hearts received no intervention (control), ischemic PC, D-myo-Ins(1,4,5)P3, D-myo-Ins(1,4,5)P3 + PC, 2-APB, or 2-APB + PC. All hearts then underwent 30-min coronary occlusion and 2 h reflow, and infarct size was delineated by tetrazolium staining. In addition, the effects of D-myo-Ins(1,4,5)P3 and 2-APB on Ins(1,4,5)P3 signaling were evaluated in isolated fura 2-loaded rat cardiomyocytes. Mean infarct size was reduced with PC and in all D-myo-Ins(1,4,5)P3- and 2-APB-treated groups versus control (59 and 42-55%, respectively, vs. 80% of myocardium at risk, P < 0.05). Thus pharmacological manipulation of Ins(1,4,5)P3 signaling mimics the cardioprotection achieved with ischemic PC in rabbit heart.

myocardial ischemia; myocardial infarction; signal transduction; 2-aminoethoxydiphenyl borate; calcium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ESTABLISHED that one or more brief episodes of ischemia protect or "precondition" the heart and render it more tolerant to a subsequent ischemic insult. However, despite intensive investigation, the signal transduction pathways contributing to infarct size reduction with preconditioning remain incompletely resolved.

Recent evidence from our laboratory has revealed complex, biphasic alterations in myocardial concentrations of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with ischemic preconditioning. Specifically, there was a twofold increase in Ins(1,4,5)P3 content of rabbit myocardium in response to 5 min of brief preconditioning ischemia (5), whereas, in contrast, Ins(1,4,5)P3 levels were ~25% lower during the initial minutes of the sustained test occlusion in preconditioned rabbit hearts versus controls (30). These data raise the intriguing possibility that one or both of these biphasic alterations in Ins(1,4,5)P3---upregulation of Ins(1,4,5)P3 signaling during the preconditioning stimulus, i.e., release of Ins(1,4,5)P3, stimulation of Ins(1,4,5)P3 receptors on the sarcoplasmic reticulum and/or intercalated disks, and resultant increase in intracellular calcium concentration ([Ca2+]i), or downregulation in Ins(1,4,5)P3 signaling during sustained ischemia---may play a role in eliciting cardioprotection. If so, these data further suggest that pharmacological agonists and antagonists of Ins(1,4,5)P3 signal transduction may provide a novel therapeutic approach for protecting the heart against infarction.

Our primary aim was to determine, using the isolated, buffer-perfused rabbit heart model of regional ischemia, whether pharmacological manipulation of Ins(1,4,5)P3 signaling, either before or during sustained coronary artery occlusion, could mimic the reduction in infarct size achieved with ischemic preconditioning. Evaluation (and therapeutic application) of the first component of the biphasic response is, in theory, hampered by the notable lack of selective and cell-permeant Ins(1,4,5)P3 agonists (22). Thus we specifically sought to assess whether brief antecedent infusion of the synthetic analog of Ins(1,4,5)P3, D-myo-inositol-1,4,5-trisphosphate hexasodium salt [D-myo-Ins(1,4,5)P3] would, despite being cell impermeant (46), elicit cardioprotection via stimulation of extracellular Ins(1,4,5)P3 receptors. The second corollary, the consequences of inhibition of Ins(1,4,5)P3 signaling during prolonged ischemia, was explored by administration of 2-aminoethoxydiphenyl borate (2-APB), a recently identified and purportedly selective, cell-permeant modulator of the Ins(1,4,5)P3 receptors (22). Finally, using isolated fura 2-loaded neonatal rat ventricular cardiomyocytes, we sought to confirm via direct measurement of [Ca2+]i that D-myo-Ins(1,4,5)P3 is indeed cell impermeant and that 2-APB is an inhibitor of Ins(1,4,5)P3 signaling.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Model and Study Design: Isolated Rabbit Heart Preparation

Studies conducted using the isolated rabbit heart model were approved by the Institutional Animal Care and Use Committee of Good Samaritan Hospital, Los Angeles, CA (an American Association for Accreditation of Laboratory Animal Care-accredited institution), and conform with the guiding principles for research involving experimental animals endorsed by the American Physiological Society.

Isolated, perfused rabbit heart model. Forty-two male New Zealand White rabbits weighing between 2.0 and 3.2 kg were anesthetized with intramuscular injections of 100 mg of xylazine and 200 mg of ketamine. A tracheostomy was performed, and the animals were ventilated with room air. After a left thoracotomy via the fourth left intercostal space, the pericardium was excised, the heart was exposed, and a large marginal branch of the left circumflex artery that perfuses the antero-apical-lateral wall of the left ventricle (LV) was encircled for later occlusion, using a 4-0 suture attached to a small curved needle. Through a medial incision of the thorax, the heart was rapidly removed, placed in an ice bath, and, after rapid cannulation of the aortic root, mounted on a Langendorff apparatus.

All hearts were perfused retrogradely (nonrecirculating) at a constant pressure of 75 mmHg with Krebs-Henseleit buffer (in mM: 17 NaHCO3, 126 NaCl, 4.7 KCl, 2.52 CaCl2, 1.22 MgSO4, and 1.17 KH2PO4 in deionized water, pH 7.4) at 37°C, supplemented with 10 mM glucose and equilibrated with 95% O2-5% CO2. The temperature of the hearts was maintained at 37°C by the use of a water-jacketed apparatus. A collapsed latex balloon attached to a stainless steel catheter was inserted into the LV through the left atrium. The cannula was connected to a pressure transducer (Spectramed, Oxnard, CA) and a Gould recorder (Gould, Cleveland, OH). Diastolic pressure was set to 5-10 mmHg by filling the balloon with saline, and heart rate was maintained at 200 beats/min by atrial pacing (Grass Medical Instruments, Quincy, MA). The hearts were allowed to stabilize for 25-30 min before baseline measurements were obtained and group assignments were made. LV pressure was monitored throughout the experiment. Pulmonary artery effluent was collected at discrete time points (see protocol 1) and used as an index of coronary flow.

Protocol 1: Effect of D-myo-Ins(1,4,5)P3 and 2-APB on infarct size. STUDY DESIGN. All hearts underwent 30 min of sustained coronary occlusion (CO) followed by 2 h of reflow. The sustained test occlusion was preceded by a 32-min treatment period during which hearts received either uninterrupted perfusion (control groups) or two 5-min periods of preconditioning (PC) occlusion interspersed with 5 min of reflow (PC groups) (Fig. 1).


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  		Fig. 1.   Study design for protocol 1 (infarct size) and protocol 2 [time course of 2-aminoethoxydiphenyl borate (2-APB)] performed in isolated rabbit heart model. For protocol 1, all hearts underwent 30-min coronary artery occlusion and 2-h reflow. For protocol 2, all hearts remained normally perfused and were not rendered ischemic at any time. Cont, control; PC, preconditioning; veh., vehicle; Ins(1,4,5)P3, D-myo-Ins(1,4,5)P3; NE, norepinephrine; NE1-NE4, bolus injections of NE. n, no. of hearts.

Hearts were randomly assigned into one of seven study groups (Fig. 1). To determine whether antecedent exposure to exogenous Ins(1,4,5)P3 can limit infarct size, two groups received D-myo-Ins(1,4,5)P3 (100 µM dissolved in buffer; RBI, Natick MA) administered over 1 min into a sidearm immediately proximal to the heart (final concentration in perfusate ~5-8 µM), beginning 2 min before the onset of the control or PC regimen [Ins(1,4,5)P3 + control, n = 6; Ins(1,4,5)P3 + PC, n = 5]. To determine whether inhibition of Ins(1,4,5)P3 signaling during the test occlusion can initiate cardioprotection, two additional groups received 2-APB (35 µM dissolved in buffer; Aldrich, St. Louis, MO; Ref. 22), infused over 5 min via the proximal side branch (final concentration in perfusate ~1-3 µM), beginning 7 min before the start of the control or PC period (2-APB + Control, n = 9; 2-APB + PC, n = 8). One group (PC, n = 5) received PC ischemia with no prior pharmacological intervention, while two control cohorts received either no pharmacological intervention (n = 5) or buffer via the sidearm [vehicle for D-myo-Ins(1,4,5)P3 and 2-APB, n = 4].

LV pressure and coronary flow were measured at baseline (before randomization), immediately before CO (i.e., after treatment), 30 min into CO, and at 5 min and 2 h after reflow. At the end of the experiment the coronary artery was religated at the site of the previous occlusion, and hearts were perfused with blue pigment to delineate the area at risk (AR). Hearts were cut into six to eight transverse slices and photographed. Slices were then incubated for 10 min in a 37°C, pH 7.4 phosphate buffer of 1% triphenyltetrazolium chloride (TTC) to distinguish necrotic from viable myocardium, rephotographed for later measurement of area of necrosis (AN), and finally stored in Formalin.

ANALYSIS OF RISK REGION AND INFARCT SIZE. Photographic images of tissue slices were projected and traced. The extent of AR and AN was then quantified by computerized planimetry and corrected for the weight of the LV slices. AR was expressed as a percentage of total LV weight, and AN was expressed as a percentage of AR.

Protocol 2: Temporal profile of 2-APB. To confirm that 2-APB, as administered in protocol 1, was effective during sustained coronary occlusion rather than during the control or PC period, seven additional hearts were isolated and buffer-perfused as described for protocol 1 (Fig. 1). All hearts received a bolus injection (NE1; 30 µM) of norepinephrine (NE), an alpha 1-adrenergic agent well known to elicit a transient increase in LV pressure due in part to stimulation of the phosphatidylinositol pathway, release of Ins(1,4,5)P3, and increase in [Ca2+]i (41, 47). Ten minutes later, each heart was randomly assigned to a five-minute infusion of 2-APB (n = 5) or buffer (n = 2) as in protocol 1. Identical bolus injection of NE was then repeated in all hearts at 7, 32, and 42 min after 2-APB or vehicle infusion (NE2, NE3, and NE4, respectively), i.e., at times corresponding to the end of the first PC episode (NE2) and at 5 (NE3) and 15 (NE4) min into sustained CO in protocol 1 (Fig. 1). [Note, however, that all hearts in this time-course study remained normally perfused (i.e., were not rendered ischemic) throughout the protocol.] LV pressure was continuously monitored, and the maximum LV systolic pressure in response to each of the four repeated NE challenges was measured.

Model and Study Design: Isolated Rat Myocyte Preparation

All studies involving isolated rat myocytes were conducted in accordance with the guidelines of the Institut de Génétique Humaine, Montpellier, France, and the French Ministry of Agriculture and conform with the guiding principles endorsed by the American Physiological Society.

Isolated neonatal rat cardiomyocyte model. CELL PREPARATION. Ventricles harvested from eight batches of 12-17 newborn (1-4 days after birth) Wistar-Kyoto male rats were rapidly placed in a solution containing (in mM) 116 NaCl, 20 HEPES, 0.77 NaH2PO4, 5.5 glucose, 5.4 KCl, and 0.83 MgSO4 at pH 7.35 ± 0.05. The tissues were minced and thereafter incubated for 6 min at 37°C in an enzymatic solution containing 160 U/ml collagenase (Worthington) and 0.8 mg/ml pancreatin (Gibco BRL). Three milliliters of enzymatic solution were then added, and tissues were incubated for 20 min at 37°C with gentle, continuous shaking. The obtained dispersed cells (supernatant) were centrifuged (100g, 6 min), resuspended in 2 ml of newborn calf serum (NCS), centrifuged again, subsequently resuspended in 4 ml of NCS, and stored at 37°C in an air-CO2 incubator. This sequence was repeated five times. Finally, the resting cells were centrifuged for 6 min and submitted to a double Percoll gradient to separate the myocardial cells. Cells were plated in disposable borosilicate recording chambers (Lab-tek) at 500,000 cells/well. Cultures were always subconfluent, with most cells being physically isolated. Cells were maintained at 37°C in an air-CO2 incubator. They started to demonstrate spontaneous contractile activity 12 h after plating. The culture medium [50% DMEM-50% M199 with 10% horse serum, 5% fetal calf serum, glutamine (2 mM), penicillin-streptomycin (1%)] was changed every 2 days.

MEASUREMENTS OF [CA2+]I. The dual-excitation ratiometric Ca2+-sensitive dye fura 2 was used for [Ca2+]i-imaging studies in single cells with an Olympus-LSR system (MERLIN; Life Science Resources, Cambridge, UK). The variations in [Ca2+]i were detected with the sensing fluorescent probe by means of a digital charge-coupled device camera. Briefly, at 6-15 days after isolation, the cells were loaded by incubation with 2.5 µM of the acetoxymethyl ester of fura 2 (fura 2-AM) and 0.02% Pluronic F-127 (Molecular Probes, Eugene, OR) for 20 min at 37°C in Locke buffer containing in (mM) 140 NaCl, 5 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.8 CaCl2, 10 glucose, and 10 HEPES (pH 7.2). Cells were then rinsed thoroughly in Locke buffer and mounted on the stage of an inverted microscope (Olympus IX70) equipped for epifluorescence. Illumination was via a LSR SpectraMASTER monochromator coupled to the microscope fitted with an ultraviolet transparent oil objective (Uapo/340 ×40, 1.35). The image was detected with an LSR Astrocam 12/14-bit frame transfer digital camera. The MERLIN system controlled the illuminator and camera and performed image ratioing and analysis. The intensity of fluorescent light emission at lambda  = 510 nm, using excitation at 340 and 380 nm, was monitored from each single fura 2-loaded cell. The ratio of fluorescence emission when excited at 340 nm (absorbance peak of fura bound to Ca2+) to that at 380 nm (absorbance peak of free fura) was used as an index of [Ca2+]i, with an increase in the ratio signifying an increase in intracellular free calcium.

Acute (1-2 min) extracellular application of the test agents [NE, D-myo-Ins(1,4,5)P3, 2-APB] was achieved using a multiple-capillary perfusion system (200-µm inner diameter; 100 µl/min) placed in close proximity (<0.5 mm) to each cell evaluated, whereas more prolonged incubations were carried out in a 500-µl Locke buffer-containing bath. After each application, cells were washed with Locke buffer.

Protocol 3: Effect of D-myo-Ins(1,4,5)P3 on [Ca2+]i. After confirming, in 181 individual myocytes, that brief (1-2 min) superfusion with NE (1 µM) elicits the expected significant increase in [Ca2+]i, we then, in an additional 51 myocytes, measured and compared [Ca2+]i at baseline and after 1- to 2-min exposure to D-myo-Ins(1,4,5)P3 (1 µM). To ensure that cells unresponsive to D-myo-Ins(1,4,5)P3 were capable of Ins(1,4,5)P3-mediated signaling, an additional 14 cells were sequentially exposed to D-myo-Ins(1,4,5)P3 and, after washout, to the standard NE test stimulus (1 µM).

Protocol 4: Effect of 2-APB on [Ca2+]i. The acute effect of 2-APB on Ins(1,4,5)P3-mediated signaling was evaluated in 20 myocytes by exposing the cells to 1 µM 2-APB, 10 µM 2-APB, and then, after washout, to the NE test stimulus. Consequences of prolonged exposure to 2-APB (1 µM) were assessed in 74 myocytes by comparing the change in [Ca2+]i in response to NE, at 1 h after incubation in the inhibitor, to baseline values of [Ca2+]i. Finally, to ensure that loss of responsiveness to NE at 1 h after application of 2-APB was not a nonspecific temporal effect, time-matched control experiments were conducted in which the effects of the NE test stimulus were evaluated after 1-h exposure of myocytes (n = 70) to buffer alone.

Statistical Analysis

For protocol 1 (isolated rabbit heart), comparisons of risk region and infarct size were made by ANOVA followed by the Student-Newman-Keuls post hoc test. In addition, analysis of covariance (ANCOVA) was used to compare the relationship between AN and AR among groups. LV developed pressure and coronary flow were analyzed by two-factor ANOVA (for treatment and time) with repeated measures across the second factor, and if significant F ratios were obtained, subsequent pairwise comparisons were made by Student-Newman-Keuls test. Statistical comparisons of developed pressure and coronary flow were conducted using both absolute (mmHg and ml/min, respectively) and relative (% of baseline) values. Because both analyses yielded identical results, all hemodynamic data are, for simplicity, reported as percentage of baseline. Finally, because both hemodynamics and infarct size were comparable in controls that received no intervention (n = 5) and vehicle for Ins(1,4,5)P3 (n = 4), all data from these two control cohorts have been pooled for presentation.

For protocol 2 (isolated rabbit heart), the responses to repeated NE challenge (expressed as maximum LV systolic pressure in both mmHg and % of baseline) were compared using two-factor ANOVA (for group and time) with replication followed by Student-Newman-Keuls test.

For each cohort of cells used in protocols 3 and 4 (isolated myocyte preparation), changes in F340-to-F380 ratios after exposure to the test agent(s) were compared with the matched baseline values by either paired Student's t-test (when 1 test agent was evaluated) or one-way ANOVA with replication (for repeated assessment of 1 or more test compounds).

All values are reported as means ± SE, and P values <0.05 were considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Protocol 1: Effect of D-myo-Ins(1,4,5)P3 and 2-APB on Infarct Size

Hemodynamics. LV developed pressure averaged ~85 mmHg at baseline, with no significant differences among groups (Fig. 2). In all hearts, contractility declined by ~10-20% during the treatment period, decreased markedly during the 30-min sustained test occlusion, rebounded significantly during the initial minutes after resumption of flow, and, as expected from previous studies using this model (5, 20, 48), deteriorated during the subsequent 2-h reflow. Importantly, however, there were no significant differences in LV developed pressure among treatment groups versus controls at any time during the protocol (Fig. 2A). Identical results were obtained when absolute values of LV developed pressure were compared (not shown).


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  		Fig. 2.   Left ventricular (LV) developed pressure (A) and pulmonary artery effluent (index of coronary flow; B) in isolated rabbit heart model. Data are expressed as % of baseline, and points represent group means ± SE. CO, sustained coronary artery occlusion.

Baseline release of pulmonary artery effluent (assessed as an index of coronary flow) averaged ~55 ml/min, with no significant differences among groups. Changes in flow during the protocol mirrored those seen in LV contractility, i.e., although both variables showed the expected significant temporal variations, no differences among groups were observed whether flow was expressed as a percentage of baseline (Fig. 2B) or in absolute values (not shown).

Risk region and infarct size. AR ranged from 33 to 42% of the LV weight among the six groups (P = not significant by ANOVA; Fig. 3). In control hearts, AN/AR averaged 80 ± 5% (81 ± 4% for the 5 controls that received no intervention; 79 ± 10% for the 4 controls that received buffer via the side branch). As expected, ischemic PC elicited a significant reduction of infarct size to 59 ± 8% of AR (P < 0.05 vs control; Fig. 3). This was further confirmed by ANCOVA, demonstrating that the mass of necrosis was significantly decreased in PC hearts (irrespective of the mass of myocardium at risk) versus controls (P < 0.001; Fig. 4A).


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  		Fig. 3.   Area at risk (AR) expressed as % of total LV weight (AR/LV) and area of necrosis (AN) expressed as % of AR (AN/AR) for the 6 study groups. Data are expressed as means ± SE. * P < 0.05 vs. controls.



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  		Fig. 4.   Infarct size (necrosis, in grams) plotted as a function of AR (grams). Each point represents 1 individual heart each in control (), PC (triangle ), Ins(1,4,5)P3 + Cont (), Ins(1,4,5)P3+PC (black-triangle), 2-APB + Cont (), and 2-APB + PC (+) groups. A: Cont vs. PC. B: Cont vs. Ins(1,4,5)P3 + Cont. C: Cont vs. Ins(1,4,5)P3 + PC. D: Cont vs. 2-APB + Cont. E: Cont vs. 2-APB + PC. F: Cont + 2-APB vs. PC + 2-APB. G: PC vs. 2-APB + PC.

Infarct size in both control and PC groups that received antecedent bolus administration of Ins(1,4,5)P3 averaged 52 ± 8% and 53 ± 6% of the myocardium at risk, respectively, significantly smaller than the value of 80% observed in controls (P < 0.05 by ANOVA, Fig. 3; P < 0.01 by ANCOVA, Fig. 4, B and C) and similar to the value of 59% seen in hearts that received PC alone.

Administration of 2-APB also limited infarct size in both control and PC groups (Fig. 3 and Fig. 4, D and E). The extent of necrosis in the 2-APB + control group (AN/AR = 55 ± 5%) showed a significant reduction versus controls by both ANOVA (P < 0.05, Fig. 3) and ANCOVA (P < 0.01, Fig. 4D). Moreover, reduction of infarct size tended to be augmented in the 2-APB + PC group (AN/AR = 42 ± 5%) versus either PC-alone or 2-APB-alone groups, but these trends did not achieve statistical significance (P = 0.10 by ANCOVA, Fig. 4, F and G).

Protocol 2: Temporal Profile of 2-APB

Maximum LV systolic pressure averaged ~93 mmHg at baseline (before NE1) in the seven hearts enrolled in protocol 2 (Fig. 5). As expected, the first NE challenge elicited a consistent increase in maximum LV pressure, to a mean of 161 mmHg (or 176% of baseline) in hearts later randomized to receive 2-APB and 160 mmHg (or 179% of baseline) in hearts that would later receive vehicle.


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  		Fig. 5.   Effect of 2-APB (left) and vehicle (Krebs-Henseleit buffer, right) on maximal LV systolic pressure in response to 30 µM NE stimulation in isolated, perfused rabbit heart. NE1 reflects response to NE before vehicle/2-APB treatment, whereas NE2, NE3, and NE4 correspond to end of 1st preconditioning episode and 5 and 15 min into sustained coronary occlusion, respectively. Data are expressed as means ± SE. * P < 0.05 vs. baseline.

In the two hearts that received vehicle, the increase in maximum LV systolic pressure was maintained at a mean of ~167% of baseline in response to the following three NE challenges (Fig. 5). In hearts treated with 2-APB, there was no acute difference in the response to NE between the second versus the first NE tests. However, a significant temporal difference was manifest with the third and fourth injections (i.e., NE3 and NE4 at 32 and 42 min after 2-APB treatment, respectively), with the maximum increase in LV systolic pressure elicited in response to the final NE bolus averaging only 147% of baseline (P < 0.05 vs. NE1; Fig. 5). Identical results were obtained when absolute values of maximum LV systolic pressure were compared (not shown). Thus, despite early administration of 2-APB, these data suggest that significant inhibitory effects of 2-APB occurred at times corresponding to the initial half of the sustained test occlusion in protocol 1 rather than during the control or PC period.

Protocol 3: Effect of D-myo-Ins(1,4,5)P3 on [Ca2+]i

As expected (9, 38), exposure of isolated neonatal myocytes to 1 µM NE elicited a consistent and highly significant transient increase in [Ca2+]i versus baseline (P < 0.0001; Fig 6). This effect of NE was reproducible on repeated successive applications, with no sign of desensitization (not shown).


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  		Fig. 6.   Original recording showing NE-induced rise in F340/F380 [index of intracellular Ca2+ concentration ([Ca2+]i)] in a single neonatal rat cardiomyocyte. Inset, average effect observed in 181 cells (* P < 0.0001 vs. baseline).

External application of 1 µM D-myo-Ins(1,4,5)P3 to single myocytes had no effect on resting [Ca2+]i versus baseline (Fig. 7, A and B), thus confirming the reported inability of D-myo-Ins(1,4,5)P3 to cross the sarcolemma (39, 45). Cells unresponsive to D-myo-Ins(1,4,5)P3 were, however, capable of Ins(1,4,5)P3 signaling, because the typical significant increase in [Ca2+]i was manifest with subsequent NE stimulation (Fig. 7C).


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  		Fig. 7.   A: original recording showing no effect of external application of 1 µM D-myo-Ins(1,4,5)P3 on [Ca2+]i in a single neonatal rat cardiomyocyte. B: average effect of D-myo-Ins(1,4,5)P3 measured in 51 cells [P = not significant (ns) vs. baseline]. C: average effects in 14 cells in which D-myo-Ins(1,4,5)P3 and NE were tested successively. * P < 0.05 vs. baseline.

Protocol 4: Effect of 2-APB on [Ca2+]i

Short-term exposure (1-2 min) to successive 1 and 10 µM concentrations of 2-APB failed to block the increase in [Ca2+]i obtained with application of NE (Fig. 8A), i.e., significant Ins(1,4,5)P3-induced release of calcium was preserved in the initial minutes after acute 2-APB administration. In fact, brief exposure to 1 and 10 µM 2-APB triggered an unexpected acute and dose-dependent increase in the resting [Ca2+]i, the magnitude of which averaged ~10 and 20% of that achieved with NE (P < 0.05 vs. baseline; Fig. 8A).


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  		Fig. 8.   A: original recording showing, in a single neonatal rat cardiomyocyte (left), that 1 and 10 µM 2-APB had no acute effect on the rise in [Ca2+]i induced by 1 µM NE. However, acute exposure to 2-APB was associated with a dose-dependent increase in [Ca2+]i. Data were obtained in 20 cells [*P < 0.01 (1 and 10 µM 2-APB) and < 0.0001 (NE vs. baseline); right]. B: effect of 1 µM NE in 74 2-APB-exposed cells (P = ns vs. baseline; left) and 70 buffer-exposed cells (* P < 0.0001 vs. baseline; right). Experiments were performed in parallel, in cells taken from same cultures and incubated for 1 h with 2-APB and buffer, respectively.

Although 2-APB failed to inhibit Ins(1,4,5)P3-induced calcium release during the initial minutes after acute exposure to the antagonist (Fig. 8A), two pieces of evidence indicated that Ins(1,4,5)P3 signaling was, indeed, later inhibited with this agent. First, [Ca2+]i measurements obtained in a small number of cells suggested that, as in our isolated heart preparation, Ins(1,4,5)P3-induced calcium release was inhibited when NE was applied 30-45 min after acute exposure to 2-APB (not shown). Moreover, prolonged 1-h exposure to 2-APB (10 µM) completely abrogated the response to NE challenge: no increase in [Ca2+]i was observed (Fig. 8B). This was not caused by cells being rendered unresponsive during the lengthy 1-h observation period, because time-matched control myocytes superfused with buffer alone continued to exhibit the standard response to NE at 1 h after baseline measurements were obtained (Fig. 8B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we make two novel observations. First, we report that D-myo-Ins(1,4,5)P3, administered before a sustained episode of coronary artery occlusion, mimics the infarct size-limiting effect obtained with ischemic preconditioning in isolated, buffer-perfused rabbit heart. Interestingly, cardioprotection with D-myo-Ins(1,4,5)P3 was achieved despite the inability of this synthetic Ins(1,4,5)P3 analog to cross the sarcolemma. Second, we found that 2-APB, a compound demonstrated to both inhibit Ins(1,4,5)P3 receptor-mediated calcium release at times corresponding to the sustained test occlusion and, surprisingly, trigger a modest, short-term increase in [Ca2+]i, also reduced infarct size in our model. These data are consistent with the concept that agonists and antagonists of Ins(1,4,5)P3 signaling, administered such that they mimic the biphasic profile of Ins(1,4,5)P3 seen with brief preconditioning ischemia, may provide a novel strategy for protecting the heart against infarction.

Reduction of Infarct Size With Ischemic Preconditioning

We confirmed, as expected, that ischemic preconditioning reduces infarct size in the isolated, buffer-perfused rabbit heart subjected to regional ischemia (5, 20, 48). Despite the large volumes of myocardium rendered ischemic by proximal coronary occlusion in our model [40% of LV; mass of ~ 2 g; cf. risk regions of ~0.65-0.95 g in many other studies (e.g., Ref. 48)], preconditioning initiated by two 5-min episodes of ischemia nonetheless evoked a significant ~25% reduction in the extent of myocardial necrosis when compared with controls.

Although ischemic preconditioning is without question cardioprotective, the cellular mechanisms by which brief antecedent ischemia elicits this protection remain a topic of intensive ongoing investigation. The theory that, to date, has received the greatest attention is that preconditioning is initiated by stimulation of G protein-coupled receptors during brief antecedent ischemia-reflow and mediated by the subsequent activation and translocation of one or more isoforms of protein kinase C (PKC) from the cytosol to the sarcolemmal membrane (21, 30, 48) and/or other sites including, for example, the nucleus (24). Numerous studies using PKC agonists and/or antagonists or direct indexes of PKC activity and subcellular location (21, 24, 28, 30, 48) have supported the concept that PKC plays a pivotal role in infarct size reduction with preconditioning. Not all studies, however, have confirmed the "PKC hypothesis" (12, 19, 32, 36, 42, 44). Indeed, there is growing evidence that multiple mediators and signaling pathways, acting redundantly, synergistically, or possibly in some models instead of PKC, contribute to preconditioning-induced cardioprotection (2, 23, 43, 44).

It is well recognized that stimulation of G protein-coupled receptors not only generates diacylglycerol (the physiological substrate for PKC activation) but also initiates the parallel production of the second messenger Ins(1,4,5)P3 (6). The established role of this ubiquitous cellular messenger is to activate Ins(1,4,5)P3 receptors located intracellularly (i.e., on the sarcoplasmic reticulum), on the intercalated disk (17), and in some cell types (pancreatic cells, hepatocytes, T lymphocytes) on the plasma membrane (10, 15, 16, 35). Activation of Ins(1,4,5)P3 receptors results in a well-described mobilization of calcium from intracellular stores that, in turn, serves to trigger downstream cellular events (7). To date, however, the possible contribution of Ins(1,4,5)P3 signaling to ischemic preconditioning has remained largely unexplored (5, 31).

Role of Ins(1,4,5)P3 in Cardioprotection?

Of the handful of published studies addressing the consequences of Ins(1,4,5)P3 signaling in the setting of myocardial ischemia-reperfusion, all have been conducted in isolated rat heart and most have focused on its role in the genesis of reperfusion-induced arrhythmias. Specifically, Ins(1,4,5)P3 content reportedly increases immediately on relief of a prolonged (20-30 min) global ischemic insult (8, 25, 26, 47). This sudden release of Ins(1,4,5)P3 has been proposed to enhance calcium oscillations associated with membrane potential variations, leading to delayed afterdepolarizations and rhythm disturbances (8, 47). In contrast, alterations in Ins(1,4,5)P3 during ischemia are controversial, with both reduced (26, 47) and unchanged (25) concentrations of Ins(1,4,5)P3 observed at the end of a sustained ischemic episode. Interestingly, there is one report, using recovery of LV function as the end point, that inhibition of Ins(1,4,5)P3 during prolonged ischemia-reperfusion achieved indirectly via pretreatment with L-arginine was cardioprotective (25).

Reduction of infarct size with D-myo-Ins(1,4,5)P3. Recent results from our laboratory (5) provided the first indication that, in isolated, buffer-perfused rabbit hearts subjected to coronary artery occlusion-reperfusion, myocardial concentrations of Ins(1,4,5)P3 are altered with brief preconditioning ischemia-reflow. Specifically, we observed an approximately twofold increase in Ins(1,4,5)P3 content (by competitive binding assay) with 5 min of brief preconditioning ischemia when compared with nonischemic sham-operated hearts (5). Further evidence suggested that these changes in Ins(1,4,5)P3 may contribute to infarct size reduction, because administration of neomycin [known to inhibit Ins(1,4,5)P3 formation by binding to its precursor, phosphatidylinositol 4,5-bisphosphate] blocked both the increase in myocardial Ins(1,4,5)P3 levels seen with preconditioning ischemia and the reduction in infarct size achieved with preconditioning (5). Interpretation of these data must, however, be tempered by the fact that neomycin does not exclusively inhibit Ins(1,4,5)P3: neomycin has been reported to inhibit mechanogated ion channels (14) and, at high concentrations, can inhibit phospholipase C (1). Our current results showing reduction of infarct size in the D-myo-Ins(1,4,5)P3 + control group reinforce our earlier findings and provide more substantive support for the concept that increased myocardial concentrations of Ins(1,4,5)P3 before the onset of sustained coronary artery occlusion, achieved by either brief preconditioning ischemia (5) or brief exogenous administration of a synthetic Ins(1,4,5)P3 analog, may render the heart resistant to infarction. Moreover, combined administration of D-myo-Ins(1,4,5)P3 together with brief PC ischemia yielded no additive protection, consistent with (but not proof of) a common or redundant mechanism for the reduction of infarct size in both groups.

It is well established that D-myo-Ins(1,4,5)P3 is membrane impermeant, an observation confirmed in the present study using isolated neonatal rat cardiomyocytes. For this reason, cellular studies using D-myo-Ins(1,4,5)P3 [and, indeed, other Ins(1,4,5)P3 agonists such as adenophostins] permeabilize the cell membranes, deliver the agents into intact cells via direct microinjection, or administer chemically modified (i.e., photolabile "caged") forms of the agents into isolated cells (45, 46). It is equally well recognized that Ins(1,4,5)P3 receptors are typically present on the sarcoplasmic reticulum, where, on binding and stimulation, they serve to trigger a release of calcium from this intracellular store (7). Our data therefore raise the intriguing question, How did D-myo-Ins(1,4,5)P3 reduce infarct size in our rabbit model?

We do not have a definitive answer to this question. The data do, however, imply the presence of "extracellular" Ins(1,4,5)P3 receptors in the rabbit heart. There is evidence that, in some tissues and cell types (including pancreatic homogenates, T cells, and hepatocytes), there is a population of Ins(1,4,5)P3 receptors on the plasma membrane (10, 15, 16, 35). More importantly, in rat heart, immunohistochemical studies have revealed the presence of Ins(1,4,5)P3 receptors on intercalated disks (17). Although their physiological purpose is, at present, unknown, it has been speculated that these Ins(1,4,5)P3 receptors located at cell junctions (and thus not manifest in our isolated myocyte preparation) may modulate calcium entry from the extracellular space and/or regulate communication and exchange of calcium, or even Ins(1,4,5)P3 per se, between adjacent cells (17), perhaps via gap junctions (11, 33, 34). In this regard, it is interesting to note that, in confluent cell monolayers, microinjection of Ins(1,4,5)P3 or one of its analogs into one cell can result in a transfer of Ins(1,4,5)P3, and a resultant increase in [Ca2+]i, in neighboring cells (45). It is therefore tempting to speculate that stimulation of Ins(1,4,5)P3 receptors on intercalated disks by "superfused," cell-impermeant D-myo-Ins(1,4,5)P3 in our isolated rabbit heart model was responsible for initiating the reduction in infarct size. Resolution of this complex issue will, however, require further extensive study.

Reduction of infarct size with 2-APB. Our previous biochemical studies (31) revealed that, although myocardial Ins(1,4,5)P3 concentrations were increased in response to brief preconditioning ischemia, Ins(1,4,5)P3 content was reduced during the early minutes of the subsequent sustained test occlusion in preconditioned rabbit myocardium versus time-matched controls. The importance of this observation was not, however, explored. Thus our second major goal in the current study was to attempt to determine whether a decrease or downregulation of Ins(1,4,5)P3 signaling during sustained occlusion may contribute to cardioprotection.

This objective---and, in fact, the objective of all studies aimed at modulation of Ins(1,4,5)P3 signaling---has, as mentioned previously, been confounded by the lack of selective antagonists of the Ins(1,4,5)P3 receptor (39, 46). For example, heparin, although recognized to be a potent Ins(1,4,5)P3 receptor antagonist, also results in receptor-G protein uncoupling and, perhaps of even greater concern, attenuates calcium release from intracellular stores via inhibition of ryanodine receptors in the same micromolar range (22). 2-APB is a recently identified membrane-permeant agent that reportedly inhibits Ins(1,4,5)P3-mediated calcium release by structural modification of Ins(1,4,5)P3 receptors with no apparent effect on ryanodine-mediated calcium release (22, 46) and was first used to study Ins(1,4,5)P3 signaling in rat brain (13). Thus 2-APB was our antagonist of choice in the present investigation.

2-APB did, despite some variability in the infarct size data, significantly reduce the extent of necrosis in our rabbit heart model. Moreover, we confirmed, both in the isolated heart and in isolated myocytes, that 2-APB inhibited Ins(1,4,5)P3-mediated calcium release. Importantly, however, 2-APB did not have an acute, inhibitory effect; rather, evidence from both models revealed that pretreatment with the agent was required to attenuate Ins(1,4,5)P3 signaling during sustained occlusion. The reasons for this "delayed" response are not clear but may reflect that time and/or prolonged exposure are needed for 2-APB to effect the purported structural modification of the Ins(1,4,5)P3 receptor (22) in cardiomyocytes.

Although 2-APB did not acutely inhibit Ins(1,4,5)P3 signaling, the agent did elicit a modest, acute increase in [Ca2+]i in isolated rat myocytes. This observation is not without precedent because, in rat cerebral microsomal preparations, a paradoxical release of calcium with 2-APB has also been described (22). However, in contrast to our observations with 1 and 10 µM 2-APB, much higher concentrations (>90 µM) were required (22), raising the possibility that tissues may vary in their sensitivity to 2-APB. Indeed, it is also possible that sensitivity and response to 2-APB may differ between the two models (normoxic neonatal rat cardiomyocytes and intact rabbit myocardium subjected to ischemia) used in our protocols. In any case, in contrast to our initial expectations, these data introduce the possibility (discussed below) that the reduction in infarct size obtained with 2-APB may not only be due to the anticipated inhibition of Ins(1,4,5)P3-mediated signaling during sustained occlusion. Rather, the surprising small increase in [Ca2+]i acutely during drug administration may also play a role.

How Do D-myo-Ins(1,4,5)P3 and 2-APB Limit Infarct Size: Role of Calcium?

Our major conclusion is that D-myo-Ins(1,4,5)P3 and 2-APB mimic the cardioprotection achieved with ischemic preconditioning. The obvious question is, How do these agents limit infarct size? The recognized role of Ins(1,4,5)P3 is to modulate the release of calcium from intracellular stores (7). Moreover, alterations in calcium homeostasis have been implicated to play a role in infarct size reduction with preconditioning, i.e., brief exogenous infusions of CaCl2 or calcium gluconate, presumably resulting in a brief, modest, and transient increase in [Ca2+]i, have been demonstrated to render the heart resistant to a subsequent sustained ischemic insult (18, 27, 29, 31), whereas other studies have reported a reduction or downregulation of [Ca2+]i during sustained ischemia in preconditioned hearts versus controls (37). Thus the data intuitively implicate calcium as a pivotal mediator in the results obtained with our test agents.

We propose that an increase in myocardial concentrations of Ins(1,4,5)P3 before the onset of a sustained test occlusion, achieved by brief preconditioning ischemia (5), by exogenous administration of NE and other alpha 1-receptor agonists (3, 4, 40), or, in the present study, by D-myo-Ins(1,4,5)P3, may initiate protection via a resultant modulation of calcium release. The "biphasic" action of 2-APB is more complex and difficult to interpret but, as with ischemic preconditioning per se, may involve the modest, acute increase in [Ca2+]i during antecedent drug treatment and/or the inhibition of Ins(1,4,5)P3 signaling (i.e., decrease or downregulation of [Ca2+]i) during sustained occlusion. Indeed, it could be postulated that an increase in [Ca2+]i during antecedent drug infusion, even if modest in magnitude, may underlie the nonsignificant trend toward additive protection observed in hearts that received 2-APB + PC.

As with any study using pharmacological agonists and antagonists, it could be argued that the reduction in infarct size obtained with D-myo-Ins(1,4,5)P3 and 2-APB was caused by other, nonspecific cellular effects rather than alterations in Ins(1,4,5)P3-mediated signaling and calcium release. In this regard, we reasoned that our choice of an analog of Ins(1,4,5)P3 per se, rather than other potent agonists such as adenophostins, might serve to minimize this concern. Similarly, 2-APB was selected on the basis of its reported lack of effect on calcium release via ryanodine channels (46). However, the possibility of other, as yet unrecognized, confounding effects cannot be excluded.

Finally, although ischemic preconditioning, D-myo-Ins(1,4,5)P3, and 2-APB limited infarct size to a similar extent and alteration in Ins(1,4,5)P3-mediated calcium release is a logical common theme among all three interventions, there are differences between the pharmacological and ischemic triggers. For example, although our results obtained with D-myo-Ins(1,4,5)P3 suggest that stimulation of "external" Ins(1,4,5)P3 receptors, presumably located on the intercalated disk, is sufficient to limit infarct size, the site of receptor stimulation in the setting of brief ischemia-reperfusion (i.e., on the intercalated disk, the sarcoplasmic reticulum, or both) is unknown. Similarly, it is not known whether the sustained (possibly irreversible) functional inhibition of Ins(1,4,5)P3 receptors by 2-APB is also manifest in the setting of prolonged coronary artery occlusion.

Future Directions

The current results, providing the first evidence that pharmacological manipulation of Ins(1,4,5)P3 signaling can protect the heart against infarction, have yielded numerous intriguing observations and questions that merit further prospective study. For example, the data imply (but do not conclusively document) the presence of "extracellular" Ins(1,4,5)P3 receptors on rabbit myocytes. Confirmation of this concept, as well as identification of the specific sites of these receptors on the intercalated disks and/or on the plasma membrane, will assist in resolving the relative contributions and efficacies of intracellular versus extracellular Ins(1,4,5)P3-receptor stimulation in limiting infarct size with conventional ischemic preconditioning and with drug treatment. A second, related issue is whether the temporal profile of protection achieved with D-myo-Ins(1,4,5)P3 and 2-APB is comparable to that seen with brief antecedent ischemia, i.e., it is well recognized that the benefits of ischemic preconditioning wane if the time interval between the preconditioning stimulus and the onset of the sustained test occlusion is prolonged to 1-2 h (30). To date, we have no information on the consequences of altering either the timing of D-myo-Ins(1,4,5)P3 and 2-APB treatment relative to the onset of the index ischemia or the sequence of drug treatment relative to the preconditioning stimulus [i.e., preconditioning ischemia followed by D-myo-Ins(1,4,5)P3 or 2-APB, rather than D-myo-Ins(1,4,5)P3 or 2-APB + PC, as used in the current study]. The final and perhaps most compelling question to arise from our data is, What are the downstream mediators and pathways that contribute to the reduction of infarct size with D-myo-Ins(1,4,5)P3 and 2-APB? Is there even potential downstream "cross-talk" between Ins(1,4,5)P3 and PKC? The current results provide no insight as to the kinases, phosphatases, and/or ion channels that may participate in eliciting the ultimate reduction in infarct size with drug treatment and do not establish whether conventional ischemic preconditioning and our pharmacological agents act via common downstream signaling pathways. Resolution of all these important issues awaits further investigation.

Conclusions and Implications

Does the significant reduction in infarct size obtained with D-myo-Ins(1,4,5)P3 and 2-APB reveal a new potential therapeutic approach to protect the human heart against infarction? Although any extrapolation of these data can only be made with caution, the apparently ubiquitous nature of Ins(1,4,5)P3 signaling and homology among species in the structure of Ins(1,4,5)P3 receptors (46) may support this concept. Our results do, however, demonstrate that pharmacological manipulation of Ins(1,4,5)P3 signaling effectively mimics the reduction in infarct size achieved with ischemic preconditioning in rabbit heart.


    ACKNOWLEDGEMENTS

This work was supported in part by the Drown Foundation, by in-house funds from Good Samaritan Hospital, Los Angeles, CA, and by grants from La Fédération Française de Cardiologie (C. Piot) and La Fondation de France (S. Richard). A. Gysembergh was supported by the Pasarow Fellowship for the Heart Institute, Good Samaritan Hospital.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: K. Przyklenk, Heart Institute/Research, Good Samaritan Hospital, 1225 Wilshire Boulevard, Los Angeles, CA 90017-2395 (E-mail: karinp{at}dnamail.com).

Received 2 August 1999; accepted in final form 26 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 277(6):H2458-H2469
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