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Ann Thorac Surg 2002;74:1213-1218
© 2002 The Society of Thoracic Surgeons
a Department of Thoracic and Cardiovascular Surgery, Osaka Medical College, Takatsuki, Osaka, Japan
b Department of Chemistry, Osaka Medical College, Takatsuki, Osaka, Japan
Accepted for publication May 29, 2002.
* Address reprint requests to Dr Horimoto, Department of Thoracic and Cardiovascular Surgery, 2-7 Daigakucho, Takatsuki, Osaka 569-8686, Japan
e-mail: tho043{at}poh.osaka-med.ac.jp
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Abstract |
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Methods. Rats were subjected to either ligation of the left anterior descending coronary artery (LAD) resulting in MI, or a sham operation. Two weeks later, hearts were isolated and perfused. Six groups (n = 7 each) were studied: group 1, control (sham operation); group 2, sham operation + IPC; group 3, post-MI; group 4, post-MI + IPC; group 5, post-MI + forskolin; group 6, post-MI + forskolin + IPC. IPC consisted of two cycles of 5 minutes of global ischemia. The adenylate cyclase agonist forskolin (1.0 x 10-8M) was administered in post-MI hearts either alone (group 5) or for 5 minutes before IPC (group 6). All hearts were then subjected to 20 minutes of global ischemia followed by 120 minutes of reperfusion, after which infarct size was measured. Concentrations of endogenous catecholamines and myocardial mRNA expression of beta 2 adrenergic receptor were measured in the post-MI model.
Results. (1) IPC reduced infarct size in shams, from 34.7 ± 5.2% in group 1 to 21.4 ± 3.8% in group 2, but did not affect infarct size in post-MI hearts (group 3 versus group 4). (2) Forskolin combined with IPC reduced infarct size in post-MI hearts to 29.3 ± 3.4% (group 6), but not in group 5 where the value was 39.3 ± 4.8%. (3) Beta 2 adrenergic receptor mRNA expression in post-MI hearts was significantly decreased as compared with sham-operated animals.
Conclusions. The results indicate that downregulation of beta adrenergic receptors in post-MI hearts may be associated with ineffectiveness of IPC, and that beta adrenergic signaling, especially in relation to adenylate cyclase activation, may be required to generate the IPC response in post-MI hearts.
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Introduction |
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Several reports have suggested that beta adrenergic stimulation is involved in IPC effects in ischemia by causing an increase in cAMP [6–8]. In failing hearts, however, profound alterations in the expression of beta adrenergic receptors (BAR) may occur, including downregulation of beta 1 adrenergic receptors (B1AR) and uncoupling of beta 2 adrenergic receptors (B2AR) [9, 10]. These alterations may attenuate downstream signaling pathways, leading to increased levels of guanosine triphosphate-binding regulatory proteins, inactivation of adenylate cyclase (AC), decreased levels of cAMP, and upregulation of BAR kinase 1 [11]. This attenuation of beta signaling in failing hearts may prevent IPC from exerting myoprotective effects, a phenomenon which is not noted in intact hearts.
In this study, we investigated: (1) effects of IPC on hearts after myocardial infarction (MI) compared with hearts of sham-operated animals; (2) the relationship between B2AR expression and IPC response in post-MI hearts; (3) the role of AC in the IPC response, as assessed by infusing the AC agonist forskolin in post-MI hearts.
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Material and methods |
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Two weeks after surgery, rats were brought into the laboratory and reanesthetized. A catheter (Millar, SPR-249) was placed into the carotid artery and connected to a transducer (Millar, TCB-500, Houston, TX). After blood pressure had been measured, blood was sampled for catecholamines.
Langendorff perfusion protocol
After 300 U heparin infusion via a femoral vein, the heart was rapidly excised and placed in an iced bath (4°C) of Krebs-Henseleit solution (Na+ 135 mM, K+ 4.7 mM, Ca++ 1.7 mM, PO4- 1.1 mM, Mg++ 1.2 mM, HCO3- 25 mM, glucose 11.5 mM, pyruvate 4.9 mM, fumarate 5.4 mM). The aorta was cannulated, and the heart was suspended from the cannula within a heated glass chamber. Then the aorta was perfused with oxygenated (95% O2/5% CO2) Krebs-Henseleit solution at 37°C and 75 mmHg root pressure, and the pulmonary artery was incised. During equilibration, the left atrium was excised and a water-filled balloon was placed through the mitral valve into the left ventricle (LV). The initial LV end-diastolic pressure was set at 5 to 10 mmHg by balloon inflation, and the volume remained constant throughout the experiment. LV peak developed pressure (LVPDP) was calculated from the balloon tracing as the difference between end-systolic pressure and end-diastolic pressure for each beat. The experimental protocol is shown in Figure 1.
Six groups, each consisting of seven hearts, were studied: group 1, sham-operated control hearts; group 2, sham hearts received IPC; group 3, post-MI hearts had coronary ligation; group 4, post-MI hearts received IPC; group 5, post-MI hearts received forskolin infusion; group 6, post-MI hearts received forskolin infusion and IPC. Groups 1 and 3 serve as controls without IPC or forskolin. In groups 2, 4, and 6, IPC was accomplished with two episodes of 5 minutes of complete aortic occlusion and 5 minutes of reperfusion. Group 5 received 5 minutes of pretreatment with 1.0 x 10-8 M forskolin, which was washed out during a subsequent 20 minutes of stabilization. Group 6 received 5 minutes of pretreatment with 1.0 x 10-8 M forskolin prior to IPC. As shown in Figure 1, all groups were then subjected to an ischemia-reperfusion protocol, which consisted of 20 minutes of global ischemia and 120 minutes of reperfusion.
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Real-time quantitative reverse transcriptase-polymerase chain reaction
B2AR mRNA was quantified in myocardium of sham (n = 6) and post-MI hearts (n = 7). The hearts were divided into right and left ventricles and frozen immediately, excluding the region of experimental infarction. Total RNA was purified from each sample with guanidium thiocyanate [13]. First-strand cDNA was synthesized from 1 µg of total RNA with Super-Script II reverse transcriptase (RT) (Gibco-BRL, Tokyo, Japan) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was carried out in a total volume of 50 µl with 1 x PCR Master Mix Buffer, 200 nM of the Taq-Man Probe (Perkin-Elmer Applied Biosystems, Osaka, Japan), 1 µM of appropriate sense and antisense primers, and 1 µl of the cDNA. The following primers chosen with the assistance of the computer program Primer Express (Perkin-Elmer Applied Biosystems) were used for the application:
Sense primer: 5'-GCCACGACATCACTCAGGAAC-3';
Antisense primer: 5'-CGATAACCGACATGAGGATGG3'; and
Taq-Man probe: 5'-FAM-CGAAGCGTGGGTGGTGGGCAT-TAMRA-3'.
The samples were placed in an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems) and then subjected to 30 cycles consisting of a 30-second denaturation period at 94°C, a 30-second annealing period at 60°C, and a 120-second period at 72°C for extension of the annealed primers. The ABI Prism 7700 Sequence Detection System detected the signal from the fluorescent probe during PCR. Linearized cDNA of B2AR was diluted and used as a standard.
Statistics
Statistical analysis was performed using Stat View (version 5) software (SAS Institute Inc, Cary, NC). All results are expressed as mean ± standard deviation. Comparisons between groups of sham and post-MI hearts in Table 1
were made with the unpaired Student’s t test. Comparisons within groups of the ischemia-reperfusion protocol at different time points were made with one-way ANOVA using the Scheffe correction. Significant changes were considered present when p values were less than 0.05.
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Results |
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Forty-eight post-MI rats were initially entered into this study. A total of 13 rats died in their cages after surgery, possibly due to ventricular fibrillation or severe heart failure accompanied by pulmonary congestion. Therefore, the mortality was 27.1% and the 35 surviving rats contributed to the study. All 20 sham-operated rats survived, and were also entered into this study.
Left ventricular peak developed pressure
The time course of change in LVPDP is shown in Figure 2.
The pretreatment value of the LVPDP in post-MI hearts was significantly decreased as compared with control (group 1 = 78.7 ± 5.2 mmHg, group 3 = 50.7 ± 6.4 mmHg p = 0.0026). The LVPDP in group 3 was significantly decreased after reperfusion as compared with group 1 (group 1 = 51.4 ± 9.9 mmHg, group 3 = 33.9 ± 5.6 mmHg, p = 0.0051 at 120 minutes of reperfusion). No significant differences in LVPDP were observed among groups 3, 4, 5, and 6 throughout the experiment. There were also no significant differences between Groups 1 and 2.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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In this study, real-time quantitative RT-PCR revealed that B2AR mRNA expression in post-MI hearts was significantly decreased compared with sham hearts. It is generally accepted that myocardial cellular derangements due to MI, valvular disease, or dilated cardiomyopathy sometimes alter expression of contractile proteins and metabolic enzymes [10]. Since the original description of BAR desensitization in failing human hearts, various abnormalities of its signal transduction pathway have been demonstrated in different models of heart failure. In failing hearts it has been shown that the density of B1AR was reduced to 50% of normal values, while the density of B2AR remained unchanged [15]. Also, it has been demonstrated that there is a marked increase in B1AR and B2AR uncoupling in heart failure. Altered BAR expression is known to be associated with increased catecholamine levels. In our study, we also observed that endogenous cathecholamines were increased.
In the present study, we noted that B2AR was downregulated, although it cannot be excluded that B1AR was also downregulated. Downregulation of B2AR in hearts with MI suggests the possibility that the downstream cascade of beta signaling including the BAR/AC pathway may be also attenuated. To determine whether AC plays a role in the IPC response, we administered the AC agonist forskolin to MI hearts prior to IPC. As shown in Figure 3, forskolin restored the myoprotective effects of IPC in terms of the infarct size reduction. Forskolin alone, however, failed to provide a myoprotective effect. These results may indicate that AC activation is requisite to trigger the protective effect of IPC. Recent evidence demonstrated that AC activity in the sarcolemma and the junctional sarcoplasmic reticulum in preconditioned hearts is significantly higher compared with nonpreconditioned hearts [16] and that decreases in AC activity after ischemia are attenuated by IPC [17]. These lines of evidence may imply that the AC system is involved in mechanisms underlying IPC.
BARs are thought to be coupled primarily to the stimulatory GTP regulatory protein (Gs). The stimulatory subunit of Gs binds and activates AC, causing production of cAMP and activation of protein kinase A [18]. Recent findings, however, have revealed that B2AR is coupled to the inhibitory GTP regulatory protein (Gi), in addition to Gs. In failing hearts, increases in both Gi mRNA and Gi activity have been reported. Elevated plasma cathecholamine levels are also thought to increase Gi expression or Gi activity [19]. Since elevated levels of cathecholamines were detected in the present study, Gi may be predominantly activated. Gi is also reported to negatively regulate the Gs-mediated contractile response. Therefore, it is possible that increased Gi activity attenuates downstream effects from Gs including AC activation. Although we did not measure Gi mRNA or changes in Gi activity in this study, it is suggested that attenuated responses to IPC in the post-MI hearts may be associated with increased Gi activity due to elevated levels of cathecholamines. Interestingly, preischemic infusion of forskolin alone failed to offer myoprotection against reperfusion injury, as opposed to the myoprotective effect noted when forskolin infusion was combined with IPC. These findings suggest that AC may be an essential component in the IPC response, but that AC itself is not protective.
Since the IPC response has also been shown to exist in human hearts, the results of the present study may provide intriguing knowledge in a clinical sphere. With the current increasing interest in off-pump coronary artery bypass grafting, preconditioning may be helpful in protecting an area of the heart which is undergoing revascularization without cardiopulmonary bypass and cardioplegia. Especially in the failing hearts after myocardial infarction, preischemic infusion of forskolin preceding IPC may prove useful in augmenting myoprotective effects of IPC.
Although this study has established that AC is an important component in IPC, it does not provide precise information concerning the underlying mechanism. Because we did not directly measure intracellular AC, we cannot determine the time course over which the concentration of AC might have been altered. In addition, forskolin is known to exert vasodilatory activity [20]. Since isolated Langendorff perfused hearts were employed in this study, it is impossible to predict what effect this dose of forskolin would have on systemic vascular tone and sympathetic activity, and how those changes might influence the heart. The same protocol used in this study should be tested in whole-animal models.
In this communication, we have demonstrated that IPC failed to exert myoprotective effects in post-MI hearts associated with decreased expression of B2AR mRNA, and that the AC agonist forskolin restored IPC effects in post-MI hearts. It is therefore concluded that AC is a requisite factor to generate the IPC response.
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Acknowledgments |
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References |
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