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CARDIOVASCULAR
Departments of Emergency Medicine (K.P., M.M., C.E.D., P.W.) and Anesthesiology (K.P., P.W.), University of Massachusetts Medical School, Worcester Massachusetts
Received April 8, 2005; accepted May 23, 2005.
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Abstract |
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). However, this observation is enigmatic; i.e., whereas IP3 (and presumably D-myo-IP3) signaling is conventionally initiated by receptor binding, IP3 receptors have historically been considered to be intracellular (specifically located on the endoplasmic reticulum or in the perinuclear region), and D-myo-IP3 is well known to be membrane-impermeable (Berridge, 1993, 2002; Taylor and Broad, 1998; Wilcox et al., 1998; Gysembergh et al., 1999; Ibarra et al., 2004; Vermassen et al., 2004).
One potential explanation for this apparent paradox is that reduction of infarct size triggered by exogenous D-myo-IP3 is a nonspecific effect that occurs independently of IP3 receptor binding. In this regard, there is one report of an apparent increase in the fluidity of in vitro liposomal membrane preparations with D-myo-IP3 (Brailoiu et al., 1998), the physiologic relevance and mechanisms of which are unknown. However, we propose that D-myo-IP3-induced cardioprotection is receptor-mediated and, in particular, may be explained by the existence of as-yet poorly characterized external IP3 receptors identified in proximity to cardiac gap junctions and at the cell periphery (Kijima et al., 1993; Mackenzie et al., 2002; Vermassen et al., 2004). Accordingly, our aims in the current study were to test the hypotheses that: 1) infarct size reduction initiated by prophylactic administration of exogenous D-myo-IP3 is receptor-mediated; and 2) communication via gap junctions or hemichannels may play a role in triggering D-myo-IP3-induced cardioprotection.
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Materials and Methods |
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Surgical Preparation
Experiments were conducted using the isolated buffer-perfused rabbit heart model, a well characterized preparation utilized routinely by our group and others (Ytrehus et al., 1994; Bauer et al., 1999; Gysembergh et al., 1999, 2001; Krieg et al., 2004). In brief, 84 New Zealand White rabbits weighing 2.5 to 3.5 kg were anesthetized with an intramuscular injection of ketamine + xylazine (150 and 100 mg, respectively). A tracheostomy was performed, the animals were ventilated with room air, the hearts were exposed via a left lateral thoracotomy, and the pericardium was incised. For all animals enrolled in protocols 1 and 2, a dominant anterior branch of the left circumflex coronary artery was ensnared with a 2-0 silk suture for later occlusion/reperfusion.
The hearts were then excised and placed in an ice bath, and after rapid cannulation of the aortic root, retrograde perfusion (nonrecirculating) was initiated at a pressure of 85 mm Hg. The buffer was composed of 118 mM NaCl, 4.7 mM KCl, 24 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4-7H2O, 11 mM glucose, and 2.5 mM CaCl2 anhydrous in distilled water at a pH of 7.4 and was continuously oxygenated with 95% O2, 5% CO2. The perfusate was warmed to 37°C, and heart temperature was maintained at 37°C by immersion in a water-jacketed chamber. An incision was made in the left atrium, and a latex balloon connected to a pressure transducer was positioned in the left ventricular (LV) cavity for continuous assessment of hemodynamic function. The balloon was initially inflated to an end-diastolic pressure of 5 to 10 mm Hg, and thereafter, the balloon volume was held constant. All hearts were paced at 210 beats/min via electrodes positioned on the right ventricle. After a 15-min equilibration period, baseline hemodynamic data (described below) were obtained and baseline coronary flow was measured by the timed collection of coronary effluent.
Protocol 1: Role of IP3 Receptors in D-myo-IP3-Induced Cardioprotection
Study Design. If infarct size reduction seen with exogenous administration of D-myo-IP3 is receptor-mediated, we first reasoned that L-myo-IP3, a negative enantiomer of D-myo-IP3 not recognized by the IP3 receptor (Polokoff et al., 1988), would fail to evoke cardioprotection. To test this concept, hearts enrolled in protocol 1 (n = 28) underwent 30 min of coronary artery occlusion (CO) followed by 2 h of reperfusion, achieved by tightening and releasing the coronary snare (Fig. 1). The sustained test occlusion was preceded by an intervention period, during which hearts were randomly assigned to receive 6 µM D-myo-IP3 [final concentration in perfusate (Gysembergh et al., 1999)], 6 µM L-myo-IP3, brief preconditioning (PC) ischemia, or uninterrupted buffer perfusion (controls) (n = 6–8/group). Both D-myo-IP3 and L-myo-IP3 (Calbiochem, San Diego, CA) were dissolved in 5 ml of buffer and administered over 1 min, beginning 25 min before the onset of coronary artery occlusion, via a side arm located immediately proximal to the heart. A 6 µM concentration of D-myo-IP3 given in this manner was shown in initial pilot studies to provide optimum cardioprotection. The lowest doses that evoked protection were on the order of 0.1 to 0.5 µM, whereas, interestingly, higher concentrations of D-myo-IP3 (i.e., 20 µM) failed to limit infarct size and may even have modestly exacerbated necrosis (data not shown). The preconditioned group was included, because this represents the current "gold standard" of experimental cardioprotection, and was initiated by the standard stimulus of a 5-min brief coronary artery occlusion followed by 10 min of reflow (Fig. 1).
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As a second test of this hypothesis, we further proposed that if D-myo-IP3-induced cardioprotection is receptor-mediated, then coadministration of an IP3 receptor inhibitor should attenuate the benefits of D-myo-IP3 treatment. Accordingly, additional post hoc cohorts of control (n = 3) and D-myo-IP3-treated hearts (n = 6) received the IP3 receptor antagonist xestospongin C (XeC) (Miyamoto et al., 2000; Ibarra et al., 2004). XeC (Calbiochem) was dissolved in DMSO, diluted in 10-ml buffer, and infused via a second proximal side arm for a total of 10 min, beginning 9 min before D-myo-IP3 treatment (final concentration of XeC in perfusate, 3 µM; final concentration of DMSO in perfusate, <0.01%) (Fig. 1). Our choice of the 3 µM dose of XeC was based on previous studies investigating IP3-mediated signaling in guinea pig papillary muscle (Miyamoto et al., 2000). Infusion of XeC was terminated immediately after D-myo-IP3 treatment (thereby allowing a lengthy 24-min period without XeC treatment before the onset of coronary occlusion) in an effort to limit the action of the inhibitor to the "trigger phase" of D-myo-IP3-induced cardioprotection, rather than influencing the sustained ischemic period per se.
For all hearts, hemodynamics (i.e., maximum LV systolic pressure, end-diastolic pressure, and peak-positive and peak-negative LV dP/dt) were recorded at 1-min intervals throughout the protocol on a computerized data acquisition system (Micro-Med, Louisville, KY), and coronary flow was measured by timed collection of effluent at 10 min into coronary occlusion and at 10 min and 2 h postreflow.
At the conclusion of the 2-h reperfusion period, the coronary branch was briefly reoccluded and fluorescent polymer beads (2–9 µm: Duke Scientific, Palo Alto, CA) were injected into the coronary circulation to delineate the extent of the occluded vascular bed or area at risk of infarction (AR). The heart was immediately removed from the apparatus, sliced into five to seven transverse sections, illuminated under ultraviolet light, and digitally photographed. To distinguish necrotic from viable myocardium, the heart sections were then incubated in triphenyltetrazolium chloride for 15 min at 37°C, rephotographed, and stored in formalin (Vivaldi et al., 1985; Ytrehus et al., 1994; Bauer et al., 1999; Gysembergh et al., 1999, 2001).
Endpoints. The primary endpoint of protocol 1 was infarct size. For all hearts, right ventricular tissue was trimmed and each LV heart slice was weighed. AR and area of necrosis (AN) in each heart slice were quantified from the digital photographs using image analysis software (SigmaScan Pro; Systat, Point Richmond, CA), corrected for tissue weight, and summed for each heart. AR was then expressed as a percentage of the total LV weight, and AN was expressed as a percentage of the AR (Ytrehus et al., 1994; Bauer et al., 1999; Gysembergh et al., 1999, 2001).
Secondary endpoints of the study were hemodynamics and coronary flow. LV pressures and LV dP/dt were tabulated for each heart at baseline (before randomization), immediately before CO, at 5 and 30 min into CO, and at 15 min, 30 min, 1 h, and 2 h following relief of ischemia. For each time point, LV-developed pressure was calculated as the difference between maximum LV systolic pressure and end-diastolic pressure.
Protocol 2: Effect of Gap Junction Blockers on D-myo-IP3-Induced Cardioprotection
The goal of protocol 2 was to investigate the possible contribution of gap junctions to the reduction of infarct size initiated by D-myo-IP3. Accordingly, three additional pairs of control and D-myo-IP3-treated hearts (n = 38) were pretreated with heptanol (Sigma-Aldrich, St. Louis, MO), Gap 27 (Tocris Cookson Inc., Ellisville, MO), or no inhibitor (buffer alone) (n = 6–7/group). Heptanol is a classic and reversible, albeit nonselective, gap junction inhibitor (Evans and Boitano, 2001), whereas Gap 27 is a novel peptide homolog to extra-cellular loop of connexin 43, the primary cardiac gap junction protein (Chaytor et al., 1997; Boitano and Evans, 2000; Evans and Boitano, 2001). The inhibitors were infused over 5 min via a proximal side port beginning 4 min before D-myo-IP3 treatment with final concentrations of heptanol and Gap 27 in the perfusate of 0.5 mM and 6 µM, respectively. Doses of heptanol in the range from 0.5 to 2 mM have been used previously in isolated buffer-perfused heart models to assess the role of gap junction-mediated communication in the setting of ischemia-reperfusion (Garcia-Dorado et al., 1997; Gysembergh et al., 2001; Li et al., 2002; Saltman et al., 2002; Miura et al., 2004) with concentrations <1 mM considered relatively selective for gap junction uncoupling (Christ et al., 1999; Gysembergh et al., 2001). In contrast, Gap 27, although selective for connexin 43, has not, to our knowledge, been administered to the intact heart; thus, we made an empiric choice in the low micromolar range and validated its efficacy in protocol 3. As described for the use of XeC in protocol 1, infusions of both heptanol and Gap 27 were terminated immediately upon administration of D-myo-IP3 in an effort to have the agents present only during the trigger phase of D-myo-IP3-induced cardioprotection.
All of the hearts underwent 30 min of sustained coronary artery occlusion and 2 h of reperfusion (Fig. 1). Hemodynamics and coronary flow were assessed repeatedly throughout the protocol, and infarct size, the primary endpoint, was quantified as described for protocol 1.
Protocol 3: Confirmation of Gap Junction Inhibition
To confirm that the concentrations of 0.5 mM heptanol and, importantly, 6 µM Gap 27 administered in protocol 2 inhibited gap junctions/hemichannels in normoxic myocardium (i.e., the conditions under which D-myo-IP3 was administered and initiated protection), we assessed the intercellular transfer and tissue penetration of Lucifer yellow, a gap junction-permeable (but membrane-impermeable) fluorescent tracer dye (Ruiz-Meana et al., 2001; Miura et al., 2004), in nine additional hearts. After stabilization, hearts received a 5-min infusion of heptanol or Gap 27 as described for protocol 2 or buffer alone (n = 3/group). The hearts were then rapidly removed from the apparatus and cut into five transverse slices. We used previously published methods (Ruiz-Meana et al., 2001; Miura et al., 2004) with minor modifications to introduce the dye into the myocardial slices and quantify fluorescence. Specifically, in the three slices obtained from the mid-myocardial region (apex and base discarded), shallow incisions were made on the epicardial surface at a uniform calibrated depth of 1 mm, thereby disrupting sarcolemmal membranes and allowing initial uptake of the membrane-impermeable dye. The slices were then incubated for 20 min in oxygenated buffer containing 2.5 ml/min Lucifer yellow (Sigma-Aldrich). Transmural cuts were made at the sites of the shallow incisions, and the cut surfaces (three per heart) were photographed under ultraviolet light. To ensure that samples from different treatment groups were imaged under the same conditions, one control, one heptanol-, and one Gap 27-treated sample were included in each photograph. Digital images of the cut surface were planimetered, and average intensity of fluorescence within each cut surface was quantified (SigmaScan Pro).
Statistical Analyses
For protocols 1 and 2, AN/AR and AR/LV were compared among groups by analysis of variance (ANOVA), whereas for variables measured repeatedly throughout the protocols (hemodynamics, coronary flow), two-factor ANOVA (for group and time) was applied. If significant F values were obtained, post hoc pairwise comparisons were made using the Newman-Keuls test. Comparisons of hemodynamics and coronary flow were made using both absolute and relative (normalized to baseline) values; however, because both analyses yielded identical results, all data are reported for simplicity as percentage of baseline. Data obtained in protocol 3 (intensity of Lucifer yellow fluorescence for control, heptanol-, and Gap 27-treated hearts) were compared by ANOVA + the Newman-Keuls post test. All of the values are reported as the means ± S.E.M., and p values 
0.05 were considered statistically significant.
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Results |
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Administration of D-myo-IP3, L-myo-IP3, or XeC had no effect on hemodynamics with LV-developed pressure maintained at 97 to 99% of baseline values. However, as expected, hearts that received brief PC ischemia were modestly stunned before the onset of sustained coronary artery occlusion (i.e., developed pressure reduced to 79 ± 3% of baseline values; p < 0.05 versus baseline and p < 0.05 versus controls). LV-developed pressure was reduced in all hearts during coronary occlusion and remained depressed with no differences among the six cohorts throughout reperfusion (Table 1). These data support the concept that preconditioning has no independent beneficial effect on the acute recovery of viable myocardium salvaged by reperfusion (Colantonio et al., 2004) and suggest that, similarly, D-myo-IP3 does not attenuate postischemic contractile dysfunction. Results obtained for peak-positive and peak-negative LV dP/dt were similar to those observed for LV-developed pressure (data not shown).
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Infarct Size. The area at risk was 41 ± 3, 49 ± 4, 36 ± 3, 36 ± 1, 42 ± 2, and 42 ± 3% of the total LV weight in the control, PC, D-myo-IP3-treated, L-myo-IP3-treated, XeC-treated, and XeC + D-myo-IP3-treated groups, respectively, and although by chance AR/LV tended to be larger in hearts that received PC ischemia (p = 0.09), this difference was not significant.
In control hearts, the area of necrosis averaged 52 ± 6% of the risk region (Fig. 2). Infarct size was reduced with both PC ischemia and D-myo-IP3 treatment to a mean of 29 and 31%, respectively (p < 0.05 versus controls). In contrast, the negative enantiomer, L-myo-IP3, failed to trigger cardioprotection (mean AN/AR of 58%; p = N.S. versus controls), and coadministration of XeC + D-myo-IP3 blocked the benefits of D-myo-IP3 treatment (mean AN/AR of 49%; p < 0.05 versus D-myo-IP3 and p = N.S. versus controls) (Fig. 2).
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; Li et al., 2002; Miura et al., 2004), averaging 75 to 87% of baseline values immediately before the onset of coronary occlusion. However, there were no significant differences among groups in either LV-developed pressure or coronary flow throughout the protocol (Table 2).
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Infarct Size. AR/LV ranged from 33 to 41% and did not differ among the six treatment groups. Infarct size in control and D-myo-IP3-treated cohorts in protocol 2 averaged 54 and 30% of the risk region, comparable to the values of 52 and 31% observed in protocol 1. Administration of heptanol had no effect on the development of necrosis in control hearts but blocked the reduction of infarct size achieved with D-myo-IP3; i.e., mean AN/AR was 55% in the heptanol + D-myo-IP3-treated group (p = N.S. versus heptanol-treated controls; Fig. 3). Infarct size in gaptreated controls was 39% of the risk region, a value that interestingly tended to be smaller than that seen in the no-inhibitor controls. Nonetheless, there was no evidence of D-myo-IP3-induced cardioprotection in the presence of Gap 27 (mean AN/AR of 44%; p = N.S. versus gap-treated controls; Fig. 3).
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Discussion |
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Infarct Size Reduction with D-myo-IP3. IP3 is a ubiquitous second messenger generated in parallel with diacylglycerol via activation of G-protein-coupled receptors and subsequent phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The established classical role of IP3 lies in the control of calcium homeostasis (specifically mobilizing the release of calcium from intracellular stores) initiated by the binding of IP3 to IP3 receptors (Berridge, 1993, 2002; Vermassen et al., 2004). Moreover, data from our group and others suggest that alterations in calcium homeostasis (and endogenous IP3 signaling, in particular) may contribute to cardioprotection initiated by both brief preconditioning ischemia and opioid receptor stimulation (Node et al., 1997; Przyklenk et al., 1997, 1999; Bauer et al., 1999; Gysembergh et al., 1999; Barrere-Lemaire et al., 2005).
We further found that pretreatment of isolated buffer-perfused rabbit hearts with exogenous D-myo-IP3 was cardioprotective. That is, the analog delivered as a slow bolus 25 min before the onset of coronary artery occlusion triggered a 35% reduction of infarct size that was comparable in magnitude to that obtained with brief preconditioning ischemia (Gysembergh et al., 1999), an observation corroborated by the 40% reduction of infarct size seen with D-myo-IP3 in protocols 1 and 2. However, this finding is intriguing given that D-myo-IP3 is well recognized to be membrane-impermeable and that IP3 receptors are historically considered to be intracellular (Wilcox et al., 1998; Gysembergh et al., 1999).
To gain insight into this apparent paradox, we first exploited the stereospecific requirements of IP3 receptor stimulation and reasoned that, if infarct size reduction with D-myo-IP3 is receptor-mediated, then time- and concentration-matched pretreatment with the negative enantiomer, L-myo-IP3, should fail to elicit protection. Results obtained in protocol 1 demonstrated that, whereas D-myo-IP3 limited infarct size, the extent of necrosis was indeed comparable in L-myo-IP3-treated hearts versus controls. Although we cannot definitively exclude the possibility that, as in the in vitro liposomal preparation (Brailoiu et al., 1998), the benefits of D-myo-IP3 may involve nonspecific actions such as alterations in membrane fluidity, these data are consistent with the concept that D-myo-IP3-induced cardioprotection is receptor-mediated.
If IP3 receptor activation is involved, then coadministration of an IP3 receptor inhibitor together with D-myo-IP3 should negate the protective effects of the analog. However, this antagonist approach is complicated by the small number of available tool drugs targeting the IP3 receptor and, for the agents of choice [XeC and 2-aminoethoxydiphenyl borate (2-APB)], their limited use and poor characterization in intact tissues and organs (Taylor and Broad, 1998; Wilcox et al., 1998; Gysembergh et al., 1999). In fact, we found in previous studies that the temporal profile of 2-APB (i.e., prolonged 
1-h time to onset of action followed by sustained inhibition) made it unsuitable for the selective blockade of a brief antecedent protective stimulus in the isolated rabbit heart (Gysembergh et al., 1999). Protocol 1 revealed that XeC blocked the reduction of infarct size seen with D-myo-IP3 but had no effect on the development of necrosis in control hearts. Despite the caveats involved in the use of XeC [i.e., the agent is membrane-permeable and thus does not discern the site of IP3 receptor stimulation; both XeC and 2-APB may also block store-operated calcium entry, inhibit sarco/endoplasmic reticulum calcium ATPase activity, and, paradoxically, potentiate release of calcium from intracellular stores, thus raising questions regarding selectivity (Miyamoto et al., 2000; Bootman et al., 2002)], these data, together with the lack of benefit of the negative enantiomer L-myo-IP3, support the concept that D-myo-IP3-induced cardioprotection is receptor-mediated.
Role of Gap Junctions and/or Hemichannels in D-myo-IP3-Induced Cardioprotection. If infarct size reduction with D-myo-IP3 is initiated by receptor binding and if intracellular receptors are most probably not involved, these data imply the presence of additional populations of presumably external IP3 receptors. Although it is well recognized that IP3 receptors are expressed on the endoplasmic reticulum, there is increasing evidence obtained from multiple cell types for the presence of IP3 receptors on other organelles, in close association with cytoskeletal and scaffolding proteins, and, most notably, on the plasma membrane (Khan et al., 1992; Feng and Kraus-Friedmann, 1993; Barrera et al., 2004; Vermassen et al., 2004). Moreover, despite the paucity of data obtained in cardiac cells, IP3 receptors have been identified in the subsarcolemmal region, at the cell periphery, and at the intercalated disks (Kijima et al., 1993; Mackenzie et al., 2002; Vermassen et al., 2004).
This latter observation, together with preliminary findings from our laboratory implicating the possible presence of IP3 receptors at the periphery of rabbit cardiomyocytes, prompted us to propose that the reduction of infarct size achieved with exogenous D-myo-IP3 may be triggered by binding to external IP3 receptors associated with cardiac gap junctions or hemichannels and, most importantly, that communication via connexin-formed channels may play a role in D-myo-IP3-induced cardioprotection. In this regard, evidence in support of a functional coupling between IP3 receptors and gap junctions has been described in confluent monolayers of rat kidney cells where 2-APB was shown to block intercellular gap junction-mediated communication (Harks et al., 2003).
We tested this hypothesis by using two structurally distinct gap junction inhibitors, the classic but nonselective agent heptanol, as well as the novel and selective connexinmimetic peptide, Gap 27, at concentrations confirmed by our quantitative assessment of Lucifer yellow fluorescence to inhibit intercellular gap junction-mediated communication in normoxic rabbit heart. D-myo-IP3 failed to limit infarct size when administered in the presence of either inhibitor; area of necrosis was comparable in heptanol + D-myo-IP3 and Gap + D-myo-IP3-treated groups versus matched inhibitor-treated controls. This lack of D-myo-IP3-induced cardioprotection in the presence of heptanol and Gap 27 is consistent with the concept that communication via gap junctions is involved in initiating the infarct-sparing effect of D-myo-IP3.
The goal of our study was to investigate the role of connexin-formed channels in infarct size reduction triggered by D-myo-IP3. However, gap junction-mediated intercellular communication has also been proposed to contribute to lethal myocardial ischemia-reperfusion injury per se, a concept based in part on reports that heptanol at concentrations of 1 to 2 mM is cardioprotective (Garcia-Dorado et al., 1997; Saltman et al., 2002; Miura et al., 2004). These data are in apparent contrast to previous results from our group (Gysembergh et al., 2001; Li et al., 2002) and current results obtained in protocol 2 in which heptanol (concentration of 0.5 mM infused for 5 min followed by 24 min of washout) had no effect on infarct size in control hearts. In addition to variations in dose [and thus possibly the loss of selectivity at concentrations >1 mM (Christ et al., 1999)], this discrepancy may be due to differences in the timing of treatment. Heptanol is known to rapidly and reversibly disrupt gap junction-mediated communication. Thus, perhaps not surprisingly, in all studies showing reduction of ischemia- or reperfusion-induced injury with heptanol, the agent was administered either during sustained ischemia (Miura et al., 2004), during reoxygenation (Garcia-Dorado et al., 1997), or with only a brief 5-min washout period (Saltman et al., 2002).
Interestingly, we did observe a trend toward smaller infarcts in gap-treated control hearts when compared with no-inhibitor controls; in fact, there was no statistical difference in infarct size between Gap 27-controls and D-myo-IP3-treated hearts. Although both heptanol and Gap 27 are reversible inhibitors of gap junction-mediated communication, this seemingly disparate effect of Gap 27 versus heptanol may reflect the reportedly more prolonged inhibition of cell-cell communication with Gap 27 [i.e., 20–60 min (Chaytor et al., 1997; Boitano and Evans, 2000; Evans and Boitano, 2001)], which in contrast to the effects of heptanol would persist into the 30-min sustained ischemic insult. Although further studies are required to establish the specific temporal profile of Gap 27 in the intact heart, the tendency toward smaller infarcts in gap-treated controls would be consistent with the hypothesis that accelerated the closure of gap junctions during sustained myocardial ischemia is cardioprotective (Miura et al., 2004). Moreover, the results of protocol 2 may be interpreted to suggest that Gap 27 does not block infarct size reduction with D-myo-IP3 as such but, rather, that D-myo-IP3 fails to confer greater protection in the presence of Gap 27. Additional experiments are required to discern between these two possibilities.
Future Directions. Results obtained in the current study implicate the involvement of IP3 receptor stimulation and intercellular communication via gap junctions as initial steps in D-myo-IP3-induced cardioprotection. However, multiple questions regarding the infarct-sparing effect of D-myo-IP3 remain unexplored. First, on a fundamental level, although D-myo-IP3 administered 25 min before the onset of sustained occlusion limits infarct size, the precise temporal characteristics of D-myo-IP3-induced cardioprotection are unknown. Second, all of current data were obtained using IP3 analogs and pharmacologic antagonists in the rabbit heart model, and arguably, more definitive insight may be provided by interrogating the effects of D-myo-IP3 in genetically modified mice. However, this approach is undermined by the fact that mice in which the IP3 receptor gene has been disrupted, as well as connexin 43 null mice, die shortly after birth (Matsumoto and Nagata, 1999; Suadicani et al., 2000). Third, we have focused on the trigger phase of infarct size reduction with D-myo-IP3, and although pilot experiments suggest the involvement of phosphatidylinositol 3-kinase signaling (Przyklenk et al., 2004), the distal mediators and signaling pathways activated by administration of the analog are largely undefined. Finally, the identity of the IP3 receptor isoform(s) involved in the infarct-sparing effect of D-myo-IP3 (Thrower et al., 2001) as well as details concerning the nature of the inter-relationship between the relevant population of IP3 receptors and connexin-formed channels await further study.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: IP3, inositol 1,4,5-trisphosphate; myo-IP3, myo-inositol-1,4,5-trisphosphate; LV, left ventricular; ANOVA, analysis of variance; AR, area at risk of infarction; AN, area of necrosis; CO, coronary artery occlusion; PC, preconditioning; XeC, xestospongin C; 2-APB, 2-aminoethoxydiphenyl borate.
Address correspondence to: Dr. Karin Przyklenk, Department of Emergency Medicine, University of Massachusetts Medical School, 55 Lake Avenue N., Worcester, MA 01655. E-mail: karin.przyklenk{at}umassmed.edu
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, p = N.S. versus matched inhibitor-treated control.