| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 31, 2005 as doi:10.1096/fj.04-3067fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Pathology, Duke University Medical Center, Durham, North Carolina, USA;
* Department of Pediatrics, Stanford University, Stanford, California, USA;
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, USA
1 Correspondence: Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA. E-mail: steen001{at}mc.duke.edu
SPECIFIC AIMS
The overall goal of the present study was to evaluate the role of activation of ß-adrenergic receptor (ß-AR) signaling in protection against myocardial ischemia/reperfusion injury. The specific aims of the present study were to 1) explore the role of ß-AR coupling to Gi in cardioprotection by testing whether ISO was protective in pertussis toxin (PTX) pretreated hearts, 2) examine by using transgenic mice with targeted disruption of the ß2-AR gene (ß2-AR–/–) whether the protection of ischemic preconditioning (PC) is through the ß2-AR, and 3) determine by using PKA inhibitors H-89 and PKI whether the protective effect of PC is dependent on PKA-mediated switching of ß2-AR coupling from Gs to Gi.
PRINCIPAL FINDINGS
1. Transient isoproterenol mimics PC in the mouse heart
Brief intermittent periods of ischemia and reperfusion, termed ischemic preconditioning (PC), can increase myocardial resistance to ischemic injury. Considerable data suggest that the protective effect of PC is mediated by G-protein-coupled receptors (GPCRs). ß-ARs are members of the GPCR superfamily and ß-AR activation has been reported to be protective against ischemic injury in isolated rat hearts. To test whether ß-AR activation results in cardioprotection, Langendorff perfused mouse hearts were perfused for a 20 min stabilization period, followed by 10 min of control perfusion (control group) or treated with the ß-AR agonist ISO (ISO group), 10 nmol/L, for 5 min followed by 5 min washout before 20 min of no-flow ischemia and 2 h of reperfusion. Recovery of left ventricular developed pressure (LVDP), expressed as a percentage of the initial preischemic LVDP, before PC or other treatment was measured at 1 h of reperfusion. Infarct size expressed as a percentage of whole heart was measured after 2 h of reperfusion by TTC staining. Treatment with ISO produced positive inotropic and chronotropic effects in the hearts. Transient brief ISO treatment induced cardioprotection in mouse hearts. ISO-treated hearts showed improved recovery of postischemic LVDP compared with control hearts (58.5±4.8% for ISO vs. 22.0±6.3% for control; P<0.001) (Fig. 1
A) and reduced infarct size (31.0±2.4% for ISO vs. 53.0±4.6% for control; P<0.01) (Fig. 1B
). These data show that transient activation of ß-ARs can elicit protection in the mouse heart, similar to PC.
|
2. Transient isoproterenol-induced protection requires Gi
ß-AR coupling to Gs produces a proapoptotic effect and coupling to Gi exerts an anti-apoptotic effect. We tested whether ISO was protective in PTX pretreated hearts. Mice were given an i.p. injection of PTX 24 h prior to isolation and perfusion of the hearts. Blockade of Gi signaling was documented by lack of a chronotropic response to carbachol. Figure 1A
shows that pretreatment with PTX blocked the ISO-induced improvement in postischemic LVDP (26.0±6.6%, P<0.01 compared with ISO alone). In the ISO/PTX group, PTX pretreated hearts were treated with the same protocol as ISO. Postischemic function in the group treated with PTX alone (17.5±5.4%) was not significantly different from that of the control group. We also examined the effect of PTX pretreatment on infarct size. As shown in Fig. 1B
, pretreatment with PTX blocked the ISO-induced reduction in infarct size (47.2±5.1%, P<0.05 compared with ISO). Infarct size in the group treated with PTX alone (57.0±5.2%) was not significantly different from that of the control and ISO/PTX groups. Taken together, these data suggest that pretreatment with PTX blocks the protective effect of ISO, implying the protection is through coupling to Gi.
3. ß2-Adrenergic receptor mediates PC
Figure 1
suggests that ISO-induced cardioprotection is mediated by signaling through receptors coupling to Gi. Since ß2-AR couples to Gi, we tested the role of ß2-AR in PC by using transgenic mice with targeted disruption of the ß2-AR gene (ß2-AR–/–). After stabilization, hearts were perfused an additional 10 min (control) or subjected to the PC protocol (4 cycles of 5 min of ischemia and 5 min of reperfusion), followed by 20 min of no-flow ischemia and 2 h of reperfusion. As shown in Fig. 2
A, PC resulted in improved recovery of postischemic LVDP in WT mice (44.2±7.1% for PC vs. 24.8±7.5% for non-PC control; P<0.05). However, PC did not improve recovery of postischemic LVDP in ß2-AR–/– hearts (20.6±4.5% for PC vs. 22.4±6.1% for non-PC). As shown in Fig. 2B
, in WT hearts PC results in significantly less necrosis than in non-PC hearts (51.0±3.0% in non-PC hearts vs. 40.6±2.7% in PC hearts; P<0.05). However, PC did not reduce infarct size in ß2-AR–/– mice (50.2±1.8% in non-PC hearts vs. 53.6±5.3% in PC hearts). These data suggest that disruption of the ß2-AR eliminated the protection afforded by PC.
|
4. PKA activation is required for PC and isoproterenol-induced cardioprotection
Phosphorylation of the ß2-AR by cAMP-dependent PKA is a prerequisite for the switching mechanism regulating the G-protein coupling specificity of the receptor. To test whether the protective effect of PC and ISO is dependent on PKA-mediated switching of ß2-AR coupling from Gs to Gi, we used two PKA inhibitors, H-89 and PKI. Nine groups of hearts (control, PC, PC/H-89, PC/PKI, ISO, ISO/H-89, ISO/PKI, H-89, and PKI) were perfused for 20 min before being subjected to PC or drug treatments. In the PC/H-89 or PC/PKI groups, hearts were treated with 10 µmol/L H-89 or 1 µmol/L PKI 5 min before and throughout PC. In the ISO/H-89 or ISO/PKI group, H-89 or PKI was administrated 5 min before, during, and after ISO treatment. In the H-89- or PKI-alone group, hearts were treated with H-89 or PKI for 25 min. PC resulted in a significant improvement in recovery of postischemic LVDP compared with control (59.7±8.4% vs. 25.4±5.3%; P<0.0001). Treatment with H-89 or PKI blocked the PC-induced improvement in postischemic LVDP (13.6±2.8% for PC/H-89 and 32.4±7.1% for PC/PKI, both P<0.05 compared with PC). Postischemic function in the group treated with H-89 alone (15.0±5.0%) or PKI alone (19.0±0.6%) was not significantly different from the control non-PC group. PC hearts have significantly smaller infarcts than control non-PC hearts (52.3±3.7% infarct in control vs. 34.8±5.2% in PC hearts; P<0.01). Treatment with H-89 or PKI blocked the PC-induced reduction in infarct size (55.6±1.9% for PC/H-89 and 55.0±4.8% for PC/PKI, P<0.05 compared with PC). Infarct size in the non-preconditioned groups treated with H-89 (50.0±6.0%) or PKI (57.6±1.9%) was not significantly different from that of the control group. H-89 or PKI also blocked the ISO-induced improvement in postischemic LVDP (58.5±4.8% for ISO vs. 19.0±6.1% for ISO/H-89 or 33.7±5.8% for ISO/PKI; P<0.001) and the ISO-induced reduction in infarct size (31.0±2.4% for ISO vs. 51.0±2.1% for ISO/H-89 or 44.0±5.3% for ISO/PKI; P<0.05). These data suggest that the protective effect of ISO and PC is dependent on PKA activation.
SIGNIFICANCE
Cardioprotection is mediated by ß2-AR-Gi activation
ISO resulted in a cardioprotective effect that mimics PC, suggesting that ß-AR is capable of mediating cardioprotection. Coupling to Gs elicits a proapoptotic effect whereas coupling to Gi elicits an anti-apoptotic effect. We thus speculated that the cardioprotection induced by ß-AR stimulation is likely to be mediated by the ß2-AR through coupling to Gi. We hypothesize that PC or transient ISO pretreatment results in enhanced coupling of ß2-AR to Gi, thereby mediating protection when these receptors are activated by catecholamines released during sustained ischemia. This hypothesis would account for the "memory" observed in PC. In agreement with our hypothesis, preconditioning does not protect the ß2-AR–/– heart from ischemia/reperfusion injury as assessed using recovery of function or infarct size, implying that the protective effect of PC likely involves ß2-AR-Gi signaling.
Next, we tested the hypothesis that the protective effect of the ß-AR is through Gi activation. We found that PTX-treated hearts were not protected by ISO, suggesting that the protective effect of ISO is mediated through the Gi protein. We conclude that the protective effect of ß2-ARs is mediated through coupling to the Gi protein.
Switching of ß-adrenergic receptor coupling from Gs to Gi
ß2-AR initially couples to Gs, leading to activation of PKA. Activated PKA phosphorylates the ß2-AR, leading to switching of coupling of this receptor from Gs to Gi. We found that PKA inhibitors H-89 and PKI blocked cardioprotection of PC and ISO, suggesting that PKA activation is an essential element in the cardioprotective effect of PC and ISO. Several groups have found that PKA is involved in PC, but the mechanism of PKA-induced cardioprotection is unclear. Previous studies have reported that cAMP increases during PC, but the rise in cAMP is attenuated in PC hearts compared with non-PC hearts during the sustained ischemia. These data are consistent with the reduced coupling to Gs during sustained ischemia in PC heart. Others have found that ischemia/reperfusion increases PKA activity but decreases PLB phosphorylation, which is opposite to what would be expected due to activation of Gs pathways alone. We speculate that the reason for this reduced phosphorylation of PLB could be due to PKA-induced switching of ß2-AR coupling to Gi, resulting in inhibition of Gs-mediated adenylyl cyclase signaling. Here we show the novel finding that the protective effect of PC is mediated via the ß2-AR, which is initially coupled to the Gs protein, resulting in an increase in PKA activation that can phosphorylate the receptor and switch coupling to the Gi protein (Fig. 3
).
|
CONCLUSIONS
Taken together, our results indicate that the protective effect of PC can be induced by ß-AR stimulation and that this effect is mediated through the ß2-AR subtype. The ß2-AR can couple to Gs and Gi; through a switch in coupling mediated by PKA, ß2-AR binds to Gi protein, whereby the cardioprotective effect is achieved.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3067fje; doi: 10.1096/fj.04-3067fje
This article has been cited by other articles:
|
|
 |
|
  H. Nishida, T. Sato, M. Miyazaki, and H. Nakaya Infarct size limitation by adrenomedullin: protein kinase A but not PI3-kinase is linked to mitochondrial KCa channels Cardiovasc Res, January 15, 2008; 77(2): 398 - 405. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Licker, J. Diaper, and C. Ellenberger Perioperative {beta}-Blockade: Still Not Enough for Adequate Cardioprotection! Anesth. Analg., July 1, 2007; 105(1): 278 - 279. [Full Text] [PDF]
|
|
|
|
 |
|
 M.-H. Huang, H.-Q. Wang, W. R. Roeske, Y. Birnbaum, Y. Wu, N.-P. Yang, Y. Lin, Y. Ye, D. J. McAdoo, M. G. Hughes, et al. Mediating {delta}-opioid-initiated heart protection via the beta2-adrenergic receptor: role of the intrinsic cardiac adrenergic cell Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H376 - H384. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sadayappan, H. Osinska, R. Klevitsky, J. N. Lorenz, M. Sargent, J. D. Molkentin, C. E. Seidman, J. G. Seidman, and J. Robbins Cardiac myosin binding protein c phosphorylation is cardioprotective PNAS, November 7, 2006; 103(45): 16918 - 16923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Peart and G. J. Gross Cardioprotective effects of acute and chronic opioid treatment are mediated via different signaling pathways Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1746 - H1753. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Gross and G. J. Gross Ligand triggers of classical preconditioning and postconditioning Cardiovasc Res, May 1, 2006; 70(2): 212 - 221. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
