The phenomenon known as ischemic preconditioning (IPC) was defined as 1 or several brief episodes of myocardial ischemia that render the heart resistant to infarction from a subsequent lethal episode of sustained ischemia.1 This original characterization was subsequently extended not only to many species, including humans,2,3 but also to endpoints other than infarct size, such as contractile dysfunction, arrhythmia, and endothelial dysfunction.4,5 IPC-induced protection against ischemia/reperfusion injuries is intensively studied in heart, but it has been observed in a number of other organs, including brain,6 liver,7 and skeletal muscles.8
In 1993, Przyklenk et al9 reported a mechanism of protection where a region of a dog heart that was preconditioned caused protection to a remote virgin myocardium subject to subsequent sustained coronary occlusion. Similarly, brief occlusions of the mesenteric artery have been observed to result in protection of the rat heart.10 In 1999, Dickson et al11 found that the cardioprotection induced by IPC could be transferred inter-individuals by transfusion of the coronary effluent from a preconditioned donor heart to a non-preconditioned receptor heart. Afterward, the same group found that the transfusion of whole blood from preconditioned donor to non-preconditioned receptor rabbits also confers cardioprotection.12 These observations are strong evidence that one or more factors released in response to the brief episodes of ischemia during IPC can interact with receptors, locally or at a distance, triggering the protective response. Identification of such factors has great potential benefit for the development of pharmacological therapies that could protect the myocardium from severe injury after an acute myocardium infarct.
Numerous studies have focused on characterizing the signaling mechanisms involved in IPC, and some of these studies suggest as putative cellular mechanisms for IPC the activation of adenosine, bradykinin, opioid, or other receptors.13 Nonetheless, the precise adaptive response triggered by agents released during the short preconditioning episodes of ischemia/reperfusion remains unresolved. Moreover, studies of the cardioprotective mechanism by IPC have identified the activation of several important cellular mediators, among which is protein kinase C (PKC).14
PKC is a key component in the myocardial-signaling cascade that can be activated by ischemia.15 PKC, in turn, alters the function of downstream effectors, including calcium channels,16 contractile proteins,17 and mitochondrial KATP channels.18
In IPC, multiple pathways, including receptor-mediated and reactive oxygen species generation, converge on PKC activation.19 The potential role of intracellular mediators, such as PKC, in the cardioprotective mechanism by IPC is not completely understood. In the present study, we aimed at establishing a preliminary characterization of the cardioprotective factors released in the coronary effluent collected from isolated rat hearts during IPC stimulus and at testing the hypothesis that activation of PKC contributes to myocardial protection by the transferred preconditioning factors.
Perfused Heart Preparation
Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996), and the work was approved by the Institutional Animal Care and Use committee.
Male Wistar rats weighing 200 to 250 g were used throughout the experiments. Animals were anesthetized with diethyl ether and heparinized (sodium heparin, 250 IU/100 g IP). The hearts were rapidly excised, and the aortas were cannulated for retrograde perfusion at constant flow of 10 mL/min with a non-recirculating Krebs-Henseleit buffer solution [KHB solution containing (in mmol/L) NaCl 118.0, NaHCO3 25.0, KCl 4.7, KH2PO4 1.2, MgSO4.7H2O 1.2, CaCl2 1.25, and glucose 11.0]. The KHB solution was saturated with a gas mixture of 95% O2 and 5% CO2 (pH = 7.4) and maintained at 37°C.
Ischemia/Reperfusion and IPC Protocols
All protocols used are shown in Figure 1. All hearts were subject to the ischemia/reperfusion (I/R) protocol, consisting of 30 minutes of global normothermic ischemia before 60 minutes of reperfusion. Global ischemia was initiated by stopping the coronary perfusion after switching off the peristaltic pump, and reperfusion was achieved by restarting the peristaltic pump to reestablish the coronary flow.
The IPC protocol consisted of 3 consecutive cycles of 5 min ischemia plus 5 min reperfusion immediately before the I/R protocol. Coronary effluent (150 mL) was collected from hearts during the 15 minutes immediately before the I/R protocol (non-preconditioned coronary effluent) or during the three 5 min episodes of reperfusion in the IPC protocol (preconditioned coronary effluent). Hearts were maintained immersed in KHB and warmed to 37°C, except during the collection of coronary effluent.
To assess the potential cardioprotective activity of fresh (used until 1 hour after collection) preconditioned coronary effluent, hearts were assigned to 4 treatment groups: (1) donor control group (DC, n = 5) underwent only the I/R protocol; (2) receptor control group (RC, n = 5) was perfused with 150 mL of fresh non-preconditioned coronary effluent from DC hearts before the I/R protocol; (3) donor preconditioned group (DPC, n = 5) was subjected to IPC, and (4) receptor preconditioned group (RPC, n = 5) was perfused with 150 mL of fresh preconditioned coronary effluent collected from DPC group, immediately before the I/R protocol.
To verify if the protective factors were of proteic nature, we determined their sensitivity to heat and proteolysis. To assess the thermolability of the cardioprotective activity in the preconditioned coronary effluent, hearts were perfused with 150 mL of IPC coronary effluent, previously heated to 37°C (PCE37), 70°C (PCE70), or 100°C (PCE100) for 5 minutes (n = 4 for PCE37 and PCE70 and n = 5 for PCE100). To avoid the boiling effect on ion content of KHB solution, the preconditioned effluent was lyophilized and dialyzed (3,500 MW cutoff) against distilled water before heating to 70°C and 100°C. After that, it was reconstituted in KHB solution, passed through a qualitative filter paper (Klabin 80 g/m2, Maringa, PR, Brazil) under gravity, and gassed with 95% O2 and 5% CO2 (pH 7.4, at 37°C).
Dry Extract of Preconditioned Coronary Effluent
Samples of coronary effluent collected during IPC (150 mL) were immediately frozen in liquid nitrogen and lyophilized. The samples were solubilized in distilled water, dialyzed against distilled water for 24 hours at 4°C using a dialysis membrane with 3500 molecular weight cutoff (Spectrum Laboratories, Inc., CA), and centrifuged at 2500 × g for 20 minutes. The resulting pellet extract (about 7.5 mg/heart) was used immediately or stored at 8°C. To assess the stability of this material, 7.5 mg of pellet extract was resuspended in KHB solution and perfused in receptor hearts before the I/R protocol during the first (PS1), tenth (PS10), twentieth (PS20), and thirtieth (PS30) days since the extraction (n = 5 in each group). To obtain a preliminary purification of the cardioprotective substances, the resulting pellet extract was resuspended in water and applied to an adsorbent cartridge (Sep-Pak C-18, 20 mg, Waters Corp., Milford, MA) at a constant flow of 3 mL/min (Gilson Minipuls3 peristaltic pump). The cartridge was previously conditioned by flushing 30 mL of 100% acetonitrile and 10 mL H2O before the addition of the sample. After applying the sample, the cartridge was washed with 30 mL of water to elute unbound hydrophilic compounds. The hydrophobic compounds bound to the C-18 silica were eluted using 30 mL of 20% acetonitrile. The organic phase was removed, evaporated under a stream of nitrogen at 37°C and lyophilized. The hydrophilic eluted fraction (E, n = 6) and the hydrophobic analyte fraction (A, n = 6) were resuspended in 150 mL of KHB solution and perfused in receptor hearts before the I/R protocol.
To determine whether the protective effects were mediated by PKC activation, we used a specific PKC inhibitor, chelerythrine (Sigma, St. Louis, MO) dissolved in buffer and perfused for 10 minutes before IPC (CHE + PC, n = 5) or analyte (CHE + A, n = 5).
The cardioprotective effect of fresh or pre-processed preconditioned coronary effluent was assessed by evaluation of post-ischemic recovery of left ventricular developed pressure (LVDP) and determination of infarct size after 60 minutes of reperfusion.
Measurement of Left Ventricular Developed Pressure
For continuous measurement of left ventricular pressure, an incision was made in the left atrium, and a fluid-filled latex balloon was passed through the mitral orifice and placed in the left ventricle. The balloon was connected to a pressure transducer (MLT 0380 BP Transducer, ADInstruments Inc), and its volume was adjusted to obtain an initial end-diastolic pressure of 10 mm Hg. The pressure signal was amplified by a bridge amplifier (ML110, ADInstruments Inc) and digitized (Powerlab/400, ADInstruments Inc) for off-line analysis using Chart 5.0.1 data recording software (ADInstruments Inc). LVDP was calculated as the difference between peak systolic pressure and end-diastolic pressure. Baseline values for LVDP were obtained after at least 20 minutes of stabilization and 1 minute before ischemia.
At the end of each experiment, the heart was removed from the perfusion apparatus and sectioned transversely from apex to base into five 2-mm-thick slices. These slices were incubated in 1% triphenyltetrazolium chloride (Sigma, St. Louis, MO) in pH 7.4 phosphate buffer at 37°C for 5 minutes. After staining, the hearts were fixed in a 10% formalin solution for 24 hours. The slices were placed between 2 microscope slides and digitally imaged using a scanner (Hewlett-Packard Scanjet 5p). The measurements of the infarct size were quantified by computerized planimetry (ImageJ, NIH). For global ischemia, we assumed the area at risk of ischemic injuries as total ventricular area minus the cavity spaces. The infarct size for each heart was expressed as a percentage of risk area not stained.
Results are expressed as mean ± SEM. Infarct size, heart rate, LVDP, and end-diastolic pressure were compared intragroup and intergroup using 2-way repeated measures of analysis of variance (ANOVA), followed by Bonferroni post hoc test (Prism 4.0, Graphpad Software, Inc., San Diego, CA). Differences were regarded as statistically significant at P < 0.05.
Fresh Preconditioned Coronary Effluent
The IPC markedly attenuated the infarct size measured at 60-min reperfusion after 30-min ischemia in rat hearts subject to the preconditioning protocol. As shown in Figure 2A, mean infarct size in the DPC group was 17 ± 2% compared with 37 ± 1% in control DC group. The pretreatment of non-preconditioned rat hearts with the fresh coronary effluent collected from hearts subjected to IPC evoked a cardioprotective effect similar to the classical IPC protocol, reducing significantly the infarct size to 16 ± 3% in the RPC group. In contrast, the pretreatment with fresh coronary effluent collected from control DC hearts had no effect on ischemic tolerance of non-preconditioned receptor hearts, with infarct size averaging 36 ± 1% in RC group.
We observed that the transfer of fresh preconditioned effluent elicited cardioprotection when maintained at room temperature and subsequently transferred to receptor hearts within 1 hour after collection. Nevertheless, as shown in the Figure 2B, when maintained at room temperature for 24 hours before perfusion in receptor hearts, the preconditioned coronary effluent failed to evoke ischemic tolerance in the receptor myocardium (infarct size of 40 ± 3% in -PI group). In contrast, when preconditioned effluent was maintained for 24 hours at room temperature but in the presence of protease inhibitors, its perfusion in receptor hearts evoked a partial but significant infarct size limitation (27 ± 3% in +PI group). The protease inhibitors resuspended in control effluent did not evoke cardioprotection when perfused before ischemia and reperfusion in PIC group (42 ± 4%).
Concentrate of the Preconditioned Coronary Effluent
The lyophilized concentrate generated by dialysis and centrifugation of fresh preconditioned coronary effluent presented cardioprotective activity for at least 30 days. Perfusion of non-preconditioned receptor hearts with this extract reconstituted in KHB solution reduced the infarct size to 13 ± 4% (PS1), 15 ± 3% (PS10), 14 ± 2% (PS20), and 17 ± 2% (PS30 group), when using extracts reconstituted after 1, 10, 20, and 30 days, respectively (Figure 3A).
The effect of the lyophilized material after passing through Sep-Pak C18 cartridges to extract hydrophobic compounds is shown in Figure 3B. The hydrophilic compounds present in the sample did not influence infarct size (38 ± 2% in E group). On the other hand, hydrophobic compounds adsorbed by Sep-Pak C18 and eluted with 20% acetonitrile were able to induce cardioprotection with reduction of infarct size to 17 ± 2% in A group. When chelerythrine was perfused before the preconditioning stimulus or analyte perfusion, the drug was able to abrogate the cardioprotective effects as shown in Figure 3B by inhibition of the reduction in infarct size in CHE + PC group (35 ± 4%) and CHE + A group (37 ± 3%).
Functional parameters measured at baseline immediately before the onset of 30-min ischemia and at 60 min reperfusion are shown in Table 1. There were no significant differences among the groups concerning baseline values. During the reperfusion period, however, the increase in end-diastolic pressure was substantially lower in preconditioned hearts than in control, which resulted in a significantly higher developed pressure at 10 min and 60 min of reperfusion in this group. Hearts receiving hydrophobic analyte for 15 min before ischemia expressed a similar improvement in postischemic functional recovery and attenuated the increment of end-diastolic pressure compared with hydrophilic eluate. As shown in Figure 4A, LVDP recovery at the end of reperfusion was significantly higher in DPC (74 ± 6% of baseline levels versus DC group with 17 ± 7%, P < 0.001, n = 5 in each group) and A (66 ± 7% of baseline levels versus E group with 8 ± 4%, P < 0.001, n = 6 in each group) groups compared with their respective controls.
The protective effect of IPC or hydrophobic analyte perfusion on functional recovery of the ischemic myocardium was completely abolished by chelerythrine, a PKC blocker (Table 1 and Figure 4A). The LVDP recovery at the end of reperfusion was 12 ± 6% of baseline in the CHE + PC group (n = 5, P < 0.01 versus DPC group) and 19 ± 7% in the CHE + A group (n = 5, P < 0.01 versus A group). The perfusion of 100 μM chelerythrine during 10 minutes in the absence of ischemia and reperfusion protocol did not generate any detectable myocardial injury (data not shown).
As shown in Figure 4B, the thermolability of the cardioprotective activity in the preconditioned coronary effluent was demonstrated by the reduction of post-ischemic recovery of LV developed pressure in non-preconditioned receptor hearts perfused with preconditioned coronary effluent previously heated to 70°C (PCE70 group) or 100°C (PCE100 group) compared with hearts perfused with preconditioned effluent heated to 37°C (PCE37 group).
The present study investigated the cardioprotective properties of the coronary effluent collected from isolated buffer-perfused rat hearts during the IPC. Our results indicate that the coronary effluent from preconditioned rat hearts contains substances released during the IPC that decrease the infarct size when previously perfused in non-preconditioned hearts subjected to 30 min global ischemia and 60 min reperfusion. Our results in rat hearts are in agreement with findings previously reported by Dickson et al11 in rabbit hearts. This group demonstrated inter-heart transfer of cardioprotection in isolated buffer-perfused rabbit hearts transfused with coronary effluent obtained from donor hearts subjected to IPC. Moreover, the same group showed in vivo inter-heart cardioprotection transfer by transfusion to a virgin receptor cohort of whole blood from rabbit preconditioned by 5 short episodes of coronary or renal artery occlusion.12
Several studies have suggested that the release of endogenous substances during preconditioning episodes plays an essential role in the protective effect of preconditioning. Adenosine,20 bradykinin,21 catecholamine,22 and opioids23 are some of the substances released locally during the IPC that are of primary importance in mediating cardioprotection. A paracrine/endocrine mechanism for cardioprotection by IPC was suggested by Przyklenk et al,24 who demonstrated in dogs that preconditioning of a limited region of the heart not only evoked a local cardioprotective response but also protected a remote region, suggesting that the IPC could be mediated by factor(s) produced and distributed throughout the whole heart during preconditioning episodes. Other studies expanded the concept of remote preconditioning introduced in the study of Przyklenk et al,24 showing that the IPC of non-cardiac tissues could protect the myocardium against ischemia/reperfusion injuries,25 further supporting the hypothesis that endogenous substances released by the preconditioned tissue might be circulating as a mediator that triggers protective effects in remote region.
In our study, the crude preconditioned effluent lost its cardioprotective effect when stored at room temperature for 24 hours. Incubation of this effluent with protease inhibitors significantly reduced the in vitro degradation of cardioprotective activity. These results suggest that the substance(s) involved with the cardioprotective effects of the preconditioned effluent are sensitive to proteolysis. A direct effect of protease inhibitors in the reduction of infarct size could be excluded because the perfusion of control effluent in presence of protease inhibitors did not evoke any protection in our experiments. Moreover, our results show that heating the effluent to 70°C or 100°C for 5 minutes reduces the activity of the substance(s) involved in the cardioprotective effect of the preconditioned effluent. This sensitivity to heat and proteolysis could suggest a protein nature for the cardioprotective factor(s) present in the coronary effluent from preconditioned rat hearts.
Purification by reverse phase chromatography revealed that the cardioprotective factors present in the coronary effluent from preconditioned rat hearts are hydrophobic compounds. This result confirms previous observations that hydrophobic substances from coronary effluent of preconditioned rabbit hearts could induce a cardioprotective effect in non-preconditioned hearts.12 However, in the present study we showed that the factors involved in cardioprotection by preconditioning are not only hydrophobic but also have molecular weight higher than 3.5 kDa, since the effluent was dialyzed by a 3.5 kDa cutoff dialysis membrane before extraction by Sep-Pak C-18 cartridge. This result excludes the participation of adenosine (267.24 Da), opioids (500-800 Da), bradykinin (1060.22 Da), and other substances with molecular weights below the dialysis cutoff as putative mediators of preconditioning induced by the analyte fraction of Sep-Pack C-18 purification of the preconditioned coronary effluent. But, it does not exclude the participation of these endogenous substances in the transferred preconditioning in rat hearts; it is well established that adenosine, opioids, bradykinin, and other substances might synergistically mediate cardioprotection induced by IPC.13 Lang et al26 using proteomic methods were not able to detect a cardioprotective factor in the blood of rats subjected to regional myocardial or remote renal preconditioning. However, the methods used by the authors are only suitable to detect substances with a molecular weight of more than 8 to 10 kDa; therefore, they could not exclude a factor with a lower molecular weight. Hence, we could suggest a cardioprotective factor with a molecular weight above of 3.5 kDa but smaller than 8 kDa.
The participation of PKC in the signaling pathways of IPC is well established.27-29 In our present study, the infarct size reduction and improvement of postischemic LVDP recovery caused by hydrophobic compounds present in the analyte of Sep-Pack C-18 cartridge were completely abolished by the isoform nonselective PKC inhibitor chelerythrine. Our results are in agreement with previous studies suggesting activation of different PKC isoforms on IPC, as PKCε and PKCη in rabbits30 and PKCε and PKCδ in rats.30,31 Moreover, it was identified that the PKCε isoform is important in mediating remote preconditioning in rats.32
In conclusion, the current study provides insight into the characterization of the protective factor(s) released in coronary effluent during IPC stimulus in rat hearts. Our data support a humoral mechanism involved in remote preconditioning mediated by thermolabile hydrophobic substances with molecular weights higher than 3.5 kDa and acting through PKC activation.
1. Murry CE, Jennings RB, Reimer KA. Preconditioning
: a delay of lethal cell injury in ischemic myocardium. Circulation
2. Yellon DM, Dana A. The preconditioning
phenomenon: a tool for the scientist or a clinical reality? Circ Res
3. Heusch G. Nitroglycerin and delayed preconditioning
in humans. Yet another new mechanism for an old drug? Circulation
4. Raeburn CD, Zimmerman MA, Arya J, et al. Ischemic preconditioning
: fact or fantasy? J Card Surg
5. Crisostomo PR, Wairiuko GM, Wang M, et al. Preconditioning
versus postconditioning: mechanisms and therapeutic potentials. J Am Coll Surg
6. Kitagawa K, Matsumoto M, Tagaya M, et al. ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res
7. Peralta C, Closa D, Xaus C, et al. Hepatic preconditioning
in rats is defined by a balance of adenosine and xanthine. Hepatology
8. Lee HT, Schroeder CA Jr, Shah PM, et al. Preconditioning
or adenosine protects skeletal muscle from ischemic tissue reperfusion injury. J Surg Res
9. Przyklenk K, Bauer B, Ovize M, et al. Regional ischemic ‘preconditioning
’ protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation
10. Takano H, Manchikalapudi S, Tang X-L, et al. Nitric oxide synthase is the mediator of late preconditioning
against myocardial infarction in conscious rabbits. Circulation
11. Dickson EW, Lorbar M, Porcaro WA, et al. Rabbit heart can be ‘preconditioned’ via transfer of coronary effluent. Am J Physiol Heart Circ Physiol
12. Dickson EW, Reinhardt CP, Renzi FP, et al. Ischemic preconditioning
may be transferable via whole blood transfusion: preliminary evidence. J Thromb Thrombolysis
13. Cohen MV, Baines CP, Downey JM. Ischemic preconditioning
: from adenosine receptor to KATP channel. Annu Rev Physiol
14. Fryer RM, Schultz E, Hsu AK, et al. Importance of PKC and tyrosine kinase in single or multiple cycles of preconditioning
in rat hearts. Am J Physiol Heart Circ Physiol
15. Sharma A, Singh M. Protein kinase C activation and cardioprotective effect of preconditioning
with oxidative stress in isolated rat heart. Mol Cell Biochem
16. Stokke M, Aksnes G, Lande K, et al. Density of L-type calcium channels in ischaemically preconditioned porcine heart regions. Acta Physiol Scand
17. Puceat M, Vassort G. Signalling by protein kinase C isoforms in the heart. Mol Cell Biochem
18. Uchiyama Y, Otani H, Wakeno M, et al. Role of mitochondrial KATP channels and protein kinase C in ischaemic preconditioning
. Clin Exp Pharmacol Physiol
19. Costa AD, Garlid KD, West IC, et al. Protein kinase G transmits the cardioprotective signal from cytosol to mitochondria. Circ Res
20. Mubagwa K, Flameng W. Adenosine, adenosine receptors and myocardial protection: an updated overview. Cardiovasc Res
21. Parratt JR, Vegh A, Zeitlin IJ, et al. Bradykinin and endothelial-cardiac myocyte interactions in ischemic preconditioning
. Am J Cardiol
22. Hearse DJ, Sutherland FJ. Catecholamines and preconditioning
: studies of contraction and function in isolated rat hearts. Am J Physiol Heart Circ Physiol
23. Gross GJ. Role of opioids in acute and delayed preconditioning
. J Mol Cell Cardiol
24. Przyklenk K, Bauer B, Ovize M, et al. Regional ischemic ‘preconditioning
’ protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation
25. Gho BC, Schoemaker RG, van den Doel MA, et al. Myocardial protection by brief ischemia
in noncardiac tissue. Circulation
26. Lang SC, Elsasser A, Scheler C, et al. Myocardial preconditioning
and remote renal preconditioning
Identifying a protective factor using proteomic methods? Basic Res Cardiol
27. Liu Y, Ytrehus K, Downey JM. Evidence that translocation of protein kinase C is a key event during ischemic preconditioning
of rabbit myocardium. J Mol Cell Cardiol
28. Downey JM, Cohen MV. Arguments in favor of protein kinase C playing an important role in ischemic preconditioning
. Basic Res Cardiol
29. Wolfrum S, Schneider K, Heidbreder M, et al. Remote preconditioning
protects the heart by activating myocardial PKCepsilon-isoform. Cardiovasc Res
30. Weinbrenner C, Nelles M, Herzog N, et al. Remote preconditioning
by infrarenal occlusion of the aorta protects the heart from infarction: a newly identified non-neuronal but PKC-dependent pathway. Cardiovasc Res
31. Ping P, Zhang J, Qiu Y, et al. Ischemic preconditioning
induces selective translocation of protein kinase C isoforms epsilon and eta in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res
32. Gray MO, Karliner JS, Mochly-Rosen D. A selective epsilon-protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem