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Am J Physiol Heart Circ Physiol 279: H1157-H1165, 2000;
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Vol. 279, Issue 3, H1157-H1165, September 2000

Preconditioning reduces myocardial complement gene expression in vivo

Elaine J. Tanhehco1, Koji Yasojima2, Patrick L. McGeer2, Edith G. McGeer2, and Benedict R. Lucchesi1

1 Department of Pharmacology, University of Michigan Medical School Ann Arbor, Michigan 48109-0632; and 2 Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver, British Columbia, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This investigation examined the effect of preconditioning in an in vivo model of ischemia-reperfusion injury. Anesthetized New Zealand White rabbits underwent 30 min of regional myocardial ischemia followed by 2 h of reperfusion. Hearts preconditioned with two cycles of 5 min ischemia-10 min reperfusion (IPC) or with the ATP-sensitive K (KATP) channel opener, diazoxide (10 mg/kg), exhibited significantly (P < 0.05) smaller infarcts compared with control. These treatments also significantly (P < 0.001 to P < 0.05) reduced C1q, C1r, C3, C8, and C9 mRNA in the areas at risk (AAR). The KATP channel blocker 5-hydroxydecanoate (5-HD; 10 mg/kg) attenuated infarct size reduction elicited by IPC and diazoxide treatment. 5-HD partially reversed the decrease in complement expression caused by IPC but not diazoxide. There were no significant differences in complement gene expression in the nonrisk regions and livers of all groups. Western blot analysis revealed that IPC also reduced membrane attack complex expression in the AAR. The data demonstrate that preconditioning significantly decreases reperfusion-induced myocardial complement expression in vivo.

ischemia-reperfusion injury; ATP-sensitive potassium channels; membrane attack complex


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PRECONDITIONING refers to the ability of brief episodes of ischemia to reduce the infarct size in the myocardium subjected to a subsequent, prolonged ischemic insult (32). Potential mediators of preconditioning include adenosine (24), protein kinase C (PKC) (50), and activation of ATP-sensitive K channels (KATP) channels (14, 15). Though much work has been devoted to the cellular mechanisms governing preconditioning, the effects of preconditioning on complement activation remain to be explored in greater detail.

Pharmacological blockade of KATP channels abolishes the protective effect of ischemic preconditioning (14, 39), whereas KATP channel openers ameliorate ischemia-reperfusion injury (16, 17). Recent attention has focused more specifically on the mitochondrial KATP channel as the end effector of preconditioning (11, 25). Diazoxide, a mitochondrial-specific KATP channel opener, effectively preserves myocytes from an ischemia-induced injury (11, 25), whereas 5-hydroxydecanoate (5-HD), a mitochondrial KATP channel blocker, reverses the cardioprotection of ischemic preconditioning (19, 38). Although previous studies have focused on the antiischemic actions of KATP channel openers (5, 6, 9, 17), the link between modulation of mitochondrial KATP channel function and ischemia-reperfusion-induced complement activation has not been established.

Complement mediates a portion of the tissue damage associated with ischemia-reperfusion injury (21). Previously, McGeer and colleagues (48) demonstrated that myocardial tissue locally expresses complement, and that this expression significantly increases in response to ischemia and reperfusion. Lucchesi and colleagues (41) also determined that ischemic and chemical preconditioning attenuate myocardial complement production by the isolated heart. Together, these observations suggest that tissue-derived complement may play a role in advancing reperfusion injury and that reduction of complement expression may represent one mechanism by which preconditioning salvages reperfused myocardial tissue.

The present study sought to determine whether ischemic and chemical preconditioning reduce myocardial complement production in vivo. This work extends our previous findings and examines the correlation between infarct size and local complement generation. We also establish a role for the mitochondrial KATP channel in modulating complement gene expression in the ischemic myocardium.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Guidelines for Animal Research. The procedures used in this study were in accordance with the guidelines of the University of Michigan Unit for Laboratory Animal Medicine. The University of Michigan is accredited by the American Association of Accreditation of Laboratory Animal Health Care, and the animal care use program conforms to the standards in The Guide for the Care and Use of Laboratory Animals [DHEW Publ. No.(NIH) 86-23].

In vivo myocardial ischemia-reperfusion studies. Male New Zealand White rabbits (2.2-2.5 kg) were anesthetized with a mixture of xylazine (3.0 mg/kg) and ketamine (35 mg/kg) intramuscularly, followed by an intramuscular injection of pentobarbital sodium (90 mg/kg). Anesthesia was maintained with intramuscular injections of 6% pentobarbital sodium as needed. After insertion of a cuffed endotracheal tube, the animals were placed on positive-pressure ventilation with room air. The left jugular vein was isolated and cannulated for drug administration. The left carotid artery was isolated, cannulated, and connected to a Statham P23 ID transducer (Gould Instrument Systems, Valley View, OH) to monitor blood pressure. A lead II electrocardiogram was monitored throughout the experiment. A left thoracotomy and pericardiotomy were performed, followed by identification of a major branch of the coronary artery. A silk suture (3-0; Deknatel, Fall River, MA) was passed behind the artery and secured against a length of polyethylene tubing for 30 min to occlude the artery. Because the major branch of the coronary artery was not always in the same location, the infarct was not always in the exact same location of the heart. However, the infarct was always in between the apex and lateral center of the left ventricle. Ischemia was confirmed by cyanosis distal to the occlusion site. Reperfusion was initiated by removing the polyethylene tubing. Animals were equilibrated for 10 min before ischemic preconditioning (IPC) or drug administration.

Experimental protocol. Five treatment groups were studied. Group 1 (control) animals were open chested for 30 min before experiencing 30 min of regional ischemia followed by 2 h of reperfusion. This period of ischemia and reperfusion was used for all groups. Group 2 hearts were ischemically preconditioned with two cycles of 5 min of ischemia followed by 10 min of reperfusion before ischemia-reperfusion. Group 3 animals were treated with 10 mg/kg iv diazoxide (Hyperstat IV, Schering-Plough, Kenilworth, NJ) 10 min before ischemia-reperfusion. Group 4 hearts underwent the exact same protocol as group 3 except the animals received 10 mg/kg iv 5-hydroxydecanoate (5-HD; RBI, Natick, MA) 5 min before diazoxide treatment and 15 min before ischemia-reperfusion. The 5-HD was dissolved in 0.9% sterile saline. Group 5 animals underwent the exact same protocol as group 2 hearts but were administered 10 mg/kg 5-HD 5 min before IPC and 35 min before ischemia-reperfusion.

Two additional control groups were also examined. Sham-operated animals (n = 2) were instrumented and ventilated for 30 min (to mimic the 30-min pretreatment period of the preconditioned animals) before removal of the heart and liver. The time control group (n = 3) was instrumented and ventilated for 3 h before heart and liver removal. Neither control group was subjected to ischemia.

Determination of infarct size and area at risk. At the completion of the 2-h reperfusion period, hearts were removed, the aortas were cannulated, and the coronary vascular bed was perfused with saline for 1 min on the Langendorff perfusion apparatus to remove blood.

The previously occluded coronary artery was once again ligated at the same location used during induction of regional myocardial ischemia. The hearts were perfused with 0.9% saline to remove plasma and blood cellular components from the vascular compartment. Perfusion was stopped, and 0.5 ml of an india ink colloidal suspension (Higgins, Bellwood, IL) was injected slowly through a sidearm port cannula connected to the aortic cannula. Presence of the black colloidal suspension was used to demarcate the left ventricular tissue that was not subjected to regional ischemia, as opposed to the risk region. Hearts were removed from the perfusion apparatus and rinsed to remove excess ink. The hearts were cut into six transverse sections at right angles to the vertical axis. The right ventricle and atria were discarded. Sections of the left ventricle were incubated, without agitation, in 0.4% triphenyltetrazolium chloride (TTC) solution for 15 min at 37°C.

TTC demarcates the noninfarcted myocardium within the area at risk (AAR) with a brick red color, indicating the presence of formazan precipitate resulting from the reduction of TTC by coenzymes present in the viable myocardial tissue. Infarcted tissue, lacking coenzymes, is unable to form the formazan precipitate and appears pale yellow. Both surfaces of each heart slice were traced onto clear acetate sheets. The images were photocopied and enlarged. The photocopies were photographed by a digital camera (Olympus D600L, Melville, NY) and downloaded into Adobe Photoshop (Adobe Systems, Seattle, WA). The areas of the normal left ventricle (NLV) nonrisk regions, AAR, and infarcted region were determined by calculating the number of pixels occupying each area. Total AAR is expressed as the percentage of the left ventricle. Infarct size is expressed as the percentage of the AAR.

RNA preparation and RT-PCR. Separate experiments were performed to obtain tissue samples for mRNA analysis. These tissues were not stained with TTC but stained with india ink in the same manner described previously to delineate the AAR from the nonrisk region. The india ink did not interfere with RNA isolation. Total RNA from ~500 mg of each tissue sample was extracted by the acid guanidinium thiocyanate-phenol-chloroform method (8). Because the AAR and nonrisk regions were separated, 500 mg comprised the majority of the AAR. Because the RT-PCR and infarct size data were very reproducible, we do not believe the differences in the amounts of necrotic tissue within each group would be significant enough to alter the conclusion that preconditioning reduces myocardial complement expression. The extracted RNA was quantified by scanning spectrophotometry. The A260-to-A280 ratio of all preparations was >1.8. The RNA was then reverse transcribed and specific cDNAs amplified by the PCR technique as previously described in detail (48, 49).

Briefly, in preliminary studies, we found that the amount of PCR product increased exponentially from 20 to 29 cycles for cyclophilin and from 25 to 37 cycles for the complement cDNAs. A plateau phase was reached after 29 and 37 cycles, respectively, due to the plateau effect (22). Accordingly, each cDNA sample was treated by the PCR procedure with the cyclophilin product being amplified for 27 cycles and the complement products amplified for 35 cycles. Each PCR reaction product was electrophoresed through a 6% polyacrylamide gel and the product visualized by incubation for 10 min in a solution containing 10 ng/ml of ethidium bromide. Resulting gel bands were imaged using a GDS 6700 image analyzer (Ultra Violet Products, Uplands, CA). The relative intensities of the bands, expressed as optical density units, were quantitatively analyzed using NIH image software 1.61. Each complement mRNA amplification was run in parallel with a cyclophilin mRNA amplification to provide an internal standard. Direct optical density values were analyzed, as well as values relative to cyclophilin. Polaroid photographs of the gels were taken.

Choice of specific primers. The DNA sequences of rabbit C1q, C1r, C3, C8, C9, and cyclophilin were obtained from the GenBank data base (accession numbers AF089083, AF105768, M32434, U20055, L26980, and YO0052, respectively). Cyclophilin mRNA was chosen as the internal standard because it is expressed at a relatively constant level in virtually all tissues. We previously reported the primer sequences used to detect mRNAs for rabbit C1q, C1r, C3, C8, C9, and cyclophilin (41, 42).

Restriction digest analysis. The PCR products were purified by the ethanol precipitation procedure (49). Unique restriction sites and restriction enzymes were selected using the DNA strider computer program. The restriction enzymes chosen were as follows: Sac I for C1q, Sau3A I for C1r, Hinc II for C3, Mse I for C8, and BamH I for C9. The restriction digestion reaction was carried out for 2 h at 37°C. The digested PCR products were analyzed by electrophoresis on a 6% nondenaturing polyacrylamide gel.

Western blot analysis. Western blots for the membrane attack complex were performed as previously described (41, 49). Samples were taken from the NLV and AAR from control and IPC hearts. These were the same hearts from which RNA was extracted. Western blots were performed on the solubilized fraction of homogenates of rabbit heart. Heart samples were homogenized in ×5 vol/protein extraction buffer (0.02 M Tris · HCl, pH 7.5) containing the protease inhibitors phenylmethylsulfonyl fluoride (10 µg/ml) and aprotinin (10 µg/ml) and also containing 1 mM EDTA. Homogenates were centrifuged at 18,000 g at 4°C for 30 min. The protein content of the supernatants was determined, and the samples were diluted in SDS sample buffer (60 mM Tris, pH 6.8, 2.5% SDS, 5% beta -mercaptoethanol) to a final protein content of 1 mg/ml and boiled for 3 min. Because of the high molecular weight of the membrane attack complex (MAC), modifications of the electrophoresis and protein transfer steps were required. Samples containing 10 µg of protein were loaded onto a 3% polyacrylamide gel, and separation was carried out for 2.5 h at 100 V in a cold room with the apparatus surrounded by ice. The transfer to the membranes was then carried out at 100 V for 5 h in the cold. Membranes were blocked in 5% low-fat milk for 2 h. The immunoblots were treated for 4 h at room temperature with a chicken-anti-rabbit MAC antibody (1:500 dilution). The anti-rabbit MAC antibody was developed in conjunction with Lampire Biological Laboratories (Pipersville, PA) using rabbit C5b-9 antigen supplied by Dr. S. Bhakdi (Institute of Medical Microbiology and Hygiene, Johannes Gutenberg University, Mainz, Germany). The membranes were washed and treated for 3 h with a goat anti-chicken IgG (1:8,000; Sera Lab). Immunoreactivity was visualized by incubation with Supersignal CL-HRO chemiluminescent substrate (Pierce Chemical, Rockford, IL). After draining was completed, the membranes were covered in clear plastic wrapping and exposed to X-ray film (Hyper film ECL, Amersham Life Science) for 20 s.

Statistical analysis. Data are expressed as means ± SE. For the RT-PCR data, values were normalized to the housekeeping gene cyclophilin. Differences between control and experimental groups were checked for statistical significance (P < 0.05) by ANOVA followed by the Student's t-test for unpaired observations, corrected by Holm's stepdown procedure (20). These analyses were performed on both uncorrected data and those normalized to the cyclophilin value obtained in parallel amplification of each experiment. Because cyclophilin values typically varied <1%, the statistical significances were the same by the two methods.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic parameters. The rate-pressure product (RPP) was used as an index of myocardial oxygen consumption and is defined as the product of the systolic blood pressure and heart rate divided by 100. The effects of the treatments on RPP are illustrated in Fig. 1. Only the 5-HD/diazoxide-treated animals (10 mg/kg, each drug, n = 13) exhibited a significantly higher RPP than controls (n = 12) during the protocol. Diazoxide alone (n = 11) and in combination with 5-HD significantly, but transiently (P < 0.01), decreased RPP. The decrease in RPP caused by diazoxide was not attenuated by 5-HD.


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  		Fig. 1.   Rate-pressure product (RPP) (calculated as HR × MAP/100) for groups 1-5 (see METHODS). T indicates end of treatment period. There were no significant differences in the baseline RPP between any groups. 5-Hydroxydecanoate (5-HD)-diazoxide-treated animals exhibited a significantly higher RPP than controls. Diazoxide alone and in combination with 5-HD significantly, but transiently (P < 0.01), decreased RPP. RPP of animals administered diazoxide was restored to control values by 1 h of reperfusion. Data are expressed as means ± SE. IPC, ischemic preconditioning. Student's t-test, P < 0.05 considered significant.

The combination of 5-HD (10 mg/kg) and diazoxide was the only treatment that significantly (P < 0.01) increased heart rate compared with each group throughout the protocol. This effect has been reported previously (6). Diazoxide slightly but significantly (P < 0.05) increased heart rate after 1 h of reperfusion; however, heart rate in these animals returned to control values by 2 h of reperfusion. Diazoxide alone and in combination with 5-HD significantly, but transiently, decreased mean arterial pressure (MAP) immediately after administration.

Infarct size. Figure 2 illustrates the AAR and infarct area percentages in hearts subjected to various treatments. There were no significant differences for the AAR expressed as a percentage of the left ventricle among groups. IPC (n = 6) and treatment with diazoxide (10 mg/kg, n = 6) significantly (P < 0.05) reduced infarct size expressed as percentage of AAR compared with control (n = 6) (18.53 ± 4.2% and 14.63 ± 1.8% vs. 37.0 ± 3.1%, respectively). Hearts treated with 5-HD before IPC (10 mg/kg, n = 5) exhibited infarct sizes 34.8 ± 6.9% of the AAR (P > 0.05 compared with control). The effects of diazoxide were partially blocked by 5-HD with 5-HD/diazoxide-treated hearts (n = 7) developing infarcts in 28.7 ± 2.3% of the AAR. This was significantly (P < 0.05) smaller than control hearts but significantly (P < 0.05) larger than hearts treated with diazoxide alone.


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  		Fig. 2.   Effect of various treatments on infarct size in groups 1-5 (see METHODS) after 30 min of regional ischemia and 2 h of reperfusion, as expressed as a percentage of area of risk (AAR). There were no significant differences between the size of the AAR among groups. IPC (n = 6), diazoxide (Diaz) treatment (n = 6), and 5-HD plus diazoxide (5-HD/Diaz; n = 7) significantly (P < 0.05) decreased infarct size compared with controls. Hearts treated with 5-HD before IPC (5-HD/IPC; n = 5) exhibited similar infarct sizes to controls. Hearts treated with 5-HD before Diaz treatment developed infarcts significantly (P < 0.05) larger than hearts treated with Diaz alone. Student's t-test, P < 0.05 considered significant.

RT-PCR analysis. The complement factors C1q, C1r, C3, C8, and C9 were studied because these are the only components for which the rabbit DNA sequences are known. Therefore, these are the only complement genes that can be examined for changes in expression levels using RT-PCR. ANOVA analysis revealed no significant differences in C1q, C1r, C3, C8, and C9 mRNA levels in the liver (data not shown) or nonrisk region (NLV) between treatment groups (Fig. 3). Therefore, no further statistical analysis was undertaken for these data. However, significant differences in complement expression within the AAR of the various groups were observed (Fig. 4). Cyclophilin levels were essentially constant from sample to sample. The values reported in Fig. 4 are based on values normalized to cyclophilin levels.


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  		Fig. 3.   Relative expression of complement mRNA values for normal left ventricles (NLV) in groups 1-5 (see METHODS) expressed in arbitrary optical density units normalized to cyclophilin values (means ± SE). No significant differences were noted among groups for C1q, C1r, C3, C8, and C9 mRNA levels. ANOVA followed by the Student's t-test for unpaired observations, corrected by Holm's stepdown procedure (19), was used for determining significant differences. n = 6, all groups.



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  		Fig. 4.   Relative expression of complement mRNA values for the AAR in groups 1-5 (see METHODS) expressed in arbitrary optical density units normalized to cyclophilin values (means ± SE). The values for C1q, C1r, C3, C8, and C9 mRNA of the IPC, diazoxide, and 5-HD plus diazoxide-treated groups are significantly lower than the values for control (P < 0.001 to P < 0.05). Treatment with 5-HD before IPC reversed (P > 0.05) the decrease in C1q and C9 transcription caused by preconditioning. ANOVA followed by the Student's t-test for unpaired observations, corrected by Holm's stepdown procedure (19), was used for determining significant differences. n = 6 for all groups.

Figure 4 depicts the effects of the different treatments on complement mRNA synthesis in the AAR. IPC (n = 6), diazoxide treatment (10 mg/kg, n = 6), and diazoxide in combination with 5-HD (10 mg/kg, n = 6) significantly (P < 0.001 to P < 0.05) decreased C1q, C1r, C3, C8, and C9 mRNA expression compared with controls (n = 6) (Fig. 4). The effect of diazoxide was not blocked by 5-HD, but 5-HD did partially block the reduction of complement transcription caused by IPC (n = 6). Treatment with 5-HD before IPC abolished the significance (P > 0.05) of the decrease in C1q and C9 transcription caused by preconditioning and reduced the significance of the decrease of the other complement mRNA levels.

Though the C1q, C1r, C3, C8, and C9 mRNA levels were not significantly different between the NLV and AAR of control hearts, the NLV transcripts of the treated groups were higher than their corresponding AAR complement mRNAs. It was determined that complement was not activated in the NLV of preconditioned hearts (see Western blot analysis). The liver complement transcripts were greater than the corresponding mRNAs in the AAR for the IPC, diazoxide-treated, and 5-HD/diazoxide-treated hearts.

In addition, the myocardial and liver complement mRNA levels of nonischemic sham-operated animals were determined after 30 min (sham group; to mimic the pretreatment time before induction of ischemia) and 3 h (time control group) (data not shown). Compared with sham and time control animals, the NLV and AAR of controls subjected to ischemia and reperfusion exhibited significantly (P < 0.05) higher C3 and C8 mRNA levels. Sham-operated and time control animals also expressed significantly lower C3, C8, and C9 liver mRNA levels compared with control groups. This suggests that myocardial ischemia increases complement transcription in the NLV and liver. Because the time control group also displayed significantly (P < 0.05) higher myocardial C3 and C8 and liver C3, C8, and C9 mRNA levels than sham animals, the increase in complement expression may also be in part attributable to the anesthetic, pentobarbital sodium, or a reaction to instrumentation.

Western blot analysis. Figure 5 depicts representative Western blot data for the MAC from the NLV and AAR of IPC heart tissue (lanes 1 and 2, respectively) and AAR and NLV from control heart tissue (lanes 3 and 4, respectively). Antibodies against rabbit C1q, C1r, C8, and C9 were not available, and therefore Western blot analysis could not be performed for these proteins. However, the presence of the MAC indicates that the heart is expressing all of the proteins necessary for its assembly, including C1q, C1r, C3, C8, and C9. The data from the NLV of these groups reveal an absence of MAC expression, whereas an intense MAC band is observed for the control AAR. A weak MAC band is seen for the AAR of the IPC group, which is consistent with the RT-PCR data.


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  		Fig. 5.   Representative Western blot analysis of heart ventricular tissue (see METHODS for details) for membrane attack complex (MAC). Lane 1: NLV from IPC heart; lane 2: AAR from IPC heart; lane 3: AAR from control heart; lane 4: NLV from control heart.

Figure 6A depicts representative Western blot data for the MAC from the AAR of control (lane 1), IPC (lane 2), diazoxide-treated (lane 3), 5-HD/IPC (lane 4), and 5-HD/diazoxide-treated hearts (lane 5). Lanes 6 and 7 are from the left ventricles of a time control and sham-operated heart, respectively. The strongest band was obtained from the control group. Weaker bands were observed for the IPC, 5-HD/IPC, and 5-HD/diazoxide-treated hearts, whereas the weakest band was obtained from diazoxide-treated hearts. Complement bands were absent from sham-operated and time control heart samples. Western blot analysis also revealed an absence of MAC bands for the NLV of all groups (data not shown). Figure 6B represents Western blot data for the MAC from liver. Lanes 1-7 are from the same groups as shown in Fig. 6A. Complement bands were not detected in liver samples from any group, suggesting that the effects of ischemia-reperfusion and preconditioning were limited to the myocardium (namely, the AAR) rather than reflective of an overall change in complement synthesis by the whole animal.


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  		Fig. 6.   A: representative Western blot analysis of the AAR of each group (see METHODS for details) for MAC. Lane 1: control; lane 2: IPC; lane 3: diazoxide treated; lane 4: 5-HD/IPC; lane 5: 5-HD/diazoxide; lane 6: time control; lane 7: sham. B: representative Western blot analysis of liver samples of each group (see METHODS for details) for MAC. Lane 1: control; lane 2: IPC; lane 3: diazoxide treated; lane 4: 5-HD/IPC; lane 5: 5-HD/diazoxide; lane 6: time control; lane 7: sham. Arrow demarcates the row showing the bands representing the membrane attack complex (MAC).

Previous immunohistochemical analysis of isolated rabbit hearts subjected to various treatments, including ischemia-reperfusion, suggest that complement (namely, C3 and MAC) proteins are expressed along the endothelium as well as in myocytes (41, 42). However, the data were inconclusive because the immunohistochemistry was performed on cryostat sections. Freezing tissue distorts its morphology and makes it difficult to differentiate between cell types.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study expands on our previous work by demonstrating that ischemia-reperfusion increases myocardial complement expression in vivo, and that IPC significantly attenuates this increase. We show that the mitochondrial KATP channel opener diazoxide also limits complement gene transcription, suggesting that the mitochondrial KATP channel influences local complement production. The mitochondrial KATP channel blocker 5-HD reversed the infarct-limiting effects of IPC and diazoxide treatment but only partially reversed the complement reduction due to IPC. The data imply that local complement production does not entirely account for the irreversible tissue injury associated with reperfusion. Tissue complement may also play a greater role in vitro than in vivo as Yasojima et al. (48) demonstrated that increases in complement expression parallelled the decrease of function in reperfused isolated hearts. Though hearts treated with 5-HD in combination with diazoxide exhibited larger infarcts than hearts treated with diazoxide alone, they developed significantly smaller infarcts compared with controls, suggesting that tissue-derived complement may partially contribute to tissue damage. Interestingly, complement gene expression in NLV did not correlate with MAC expression. This suggests that preconditioning affects local complement production at both the transcriptional and translational levels and stresses the importance of examining both mRNA and protein expression when studying tissue complement.

Studies from our laboratory show that infarct size increases on average from 40% AAR at 2 h of reperfusion to 60% after 5 h (23, 33, 40). However, other groups report that significant infarct expansion does not occur between early (1-2 h) and late (up to 48 h) reperfusion (10, 37). The models for these studies employed animals other than rabbits, which may account for the discrepancy. The 2-h reperfusion time point was chosen for the present study because this has been used in several investigations examining the effects of preconditioning in rabbits (19, 29, 34, 43). Yasojima et al. (49) determined that complement expression occurs in new and aged human myocardial infarcts and does not occur in normal tissue. They demonstrated that myocardial complement expression continues long after reperfusion is initiated and presumably after infarct expansion has ceased. This work, in conjunction with our findings, strongly suggests that ischemia and reperfusion stimulate complement production by myocardial tissue in vivo and may be independent of infarct expansion. In addition, inhibition of local complement expression does not necessarily lead to cardioprotection as demonstrated by the 5-HD/diazoxide-treated group of the present study. We used TTC to determine infarct size because this agent provides a reliable index of tissue necrosis and is a well-accepted method of infarct size determination (35). Infarct determination can also be made by direct histological examination or using immunohistochemical techniques to stain for damaged tissue. Though these are also valid methods, they are not easily quantifiable in terms of overall assessment of infarct size. The staining achieved with TTC allows us to measure infarct size using a computer graphics program.

A significant portion of the tissue injury that accrues during ischemia-reperfusion can be attributed to complement activation within the AAR (21). Inhibitors of complement activation ameliorate myocardial reperfusion injury in vivo (21, 40). The finding from our laboratories that the myocardium increases complement production in response to reperfusion has led us to reexamine the effects of various treatments on myocardial tissue. We, in Lucchesi's lab (41), previously demonstrated that ischemic and chemical preconditioning significantly reduce reperfusion-induced myocardial complement generation in vitro, in the absence of plasma components. Whereas this in vitro global ischemia model diminished sampling problems (because the whole heart was ischemic), our hypothesis also needed to be validated in vivo. Because of the use of global ischemia in our previous investigations, we could not make a correlation between infarct size and the extent of complement expression.

Murohara et al. (31) demonstrated that both the classic and alternative pathways of complement activation participate in reperfusion injury. Upregulation of C1q and C1r transcription was detected in hearts subjected to ischemia and reperfusion in the absence of treatment, implicating activation of the classic pathway in the ischemic myocardium. A similar observation was noted in Lucchesi's lab in vitro study (41) as well as in the human heart (49). In addition, in the in vivo experiments, full activation of the complement cascade, as evidenced by Western blot analysis for the MAC, was found to occur in the AAR but not in normal, uninjured myocardial tissue or in sham-operated controls. The level of MAC production correlates with infarct size and indicates that regulation at the transcriptional and translational levels is important. Upregulation of C1q and C1r mRNAs suggest that the classic pathway is activated in the experimental model. However, activation of the alternative pathway of complement activation cannot be ruled out because free radicals, which are known to be generated during ischemia-reperfusion, also initiate the alternative pathway (4).

Though the mechanisms of preconditioning vary among species, KATP channel openers are regarded to be cardioprotective in practically all animal models examined to date (5, 9, 30, 45). Blockade of KATP channels also attenuates the effects of IPC in various species (16, 19, 38, 39) . Mitochondrial, rather than sarcolemmal, KATP channels have been proposed as to be the primary mediators of preconditioning (11, 25). In the present study, diazoxide, a specific opener of the mitochondrial KATP channel (12), also decreased complement expression. Surprisingly, the mitochondrial KATP channel blocker 5-HD did not reverse this effect, even though it diminished the infarct reduction afforded by diazoxide. The effects of diazoxide on complement regulation may be distinct from its actions on the KATP channel; however, Lucchesi's laboratory (41) demonstrated that pinacidil, which opens mitochondrial and sarcolemmal KATP channels, also significantly reduces reperfusion-induced complement expression in the isolated myocardium. Because 5-HD was also not able to attenuate the effects of diazoxide on peripheral vascular resistance, this modification of systemic blood pressure may also explain the ability of diazoxide to decrease complement expression.

Though several studies use 5 mg/kg 5-HD to block the protection afforded by preconditioning (6, 38), we did not observe a reversal of the infarct-reducing effects of diazoxide at this dose in our preparation. Because we wanted to determine whether there was a correlation between infarct size and complement expression, we used the 10 mg/kg dose of 5-HD for this study (which did reverse diazoxide's effects). The effect of 10 mg/kg 5-HD on infarct size has not been determined in the rabbit in vivo. Miura et al. (28) reported that 30 µM 5-HD alone did not alter infarct size in rabbit isolated hearts and that this dose attenuated the infarct-reducing effect of diazoxide. They also reported that 100 µM 5-HD alone increased infarct size but attributed this to differences in temperature at which the experiments were performed. Even if complement expression was found to be upregulated in hearts treated with 5-HD alone, it would not be clear whether this was due to ischemia or the drug. It is doubtful that 5-HD alone has an effect in the nonischemic heart because it has been determined to be an ischemia-selective KATP channel (27). In addition, it is assumed that the KATP channels of normal, nonischemic myocardium are already blocked.

Glyburide, a sarcolemmal KATP channel blocker, counteracted the effects of pinacidil, suggesting that these agents may be altering complement transcription via modulation of sarcolemmal KATP channels. This seems unlikely, however, from the fact that diazoxide, which also reduced complement expression, specifically targets mitochondrial KATP channels (11, 12, 18). The effects of glyburide and 5-HD on mitochondrial KATP channels are still under investigation (7, 18). Glyburide has also been shown to inhibit nitric oxide synthase (47), which participates in reperfusion injury (46). The data indicate that 5-HD was able to partially reverse the complement reduction elicited by IPC, and 5-HD may have inhibited a process that leads to KATP channel opening during IPC. Thus diazoxide may have acted downstream from this pathway. Another explanation for the failure of 5-HD to reverse the reduction in complement expression by diazoxide may be that the dose of 5-HD was not sufficient to achieve complete inhibition. The discordance between complement gene expression and infarct size suggest that a decrease in myocardial complement production is not secondary to tissue sparing.

The exact stimulus for the upregulation of myocardial complement expression during ischemia-reperfusion remains unknown. Free radicals represent a possible candidate for mediating complement production in this setting. Reactive oxygen species are produced and contribute to tissue destruction during ischemia-reperfusion (26, 51). Neutrophils aggregating within the AAR may also release free radicals (26). Free radicals initiate the transcription of a variety of genes, as well as activate the ubiquitous transcription factor, nuclear factor-kappa B (1, 36). We have determined that reactive oxygen species also increase complement expression by the myocardium (42). Preconditioning suppresses the formation of free radicals in the ischemic myocardium (44), and diazoxide has been shown to protect against free radical-mediated toxicity (13). The antioxidant attributes of preconditioning may explain the manner by which it abrogates local complement generation.

It is feasible that local complement generation also contributes to myocardial injury in the chronically reperfused heart. Yasojima et al. (49) determined that complement production and activation continues in aged, infarcted myocardium as well as in viable tissue within the AAR. Because the MAC is composed of independent subunits, tissue expressed complement components may interact with plasma-derived complement and facilitate MAC formation. The complement cleavage products C3a and C5a also act as chemoattractants, recruiting neutrophils into the risk region (21). Neutrophil accumulation exacerbates the inflammatory response and advances reperfusion injury (26). Certain complement activation products also initiate a variety of intracellular signaling pathways, which can lead to potentially deleterious responses, including increased vascular permeability, expression of adhesion molecules, and smooth muscle contraction [for a review see Ashgar et al. (3)].

In conclusion, we demonstrate that preconditioning significantly reduces reperfusion-induced myocardial complement expression and activation in vivo. The modulation of tissue complement generation in response to ischemia-reperfusion points to a potential role for locally produced complement in advancing the pathogenesis of reperfusion injury. Further investigation into the function of myocardial complement in a chronic setting and its effects on adhesion molecule expression and leukocyte recruitment are worthy of future study.


    ACKNOWLEDGEMENTS

This work was supported by a Midwest Affiliate Predoctoral Fellowship from the American Heart Association, Midwest Affiliate (to E. J. Tanhehco) and donations from individual British Columbians.


    FOOTNOTES

Address for reprint requests and other correspondence: B. R. Lucchesi, Dept. of Pharmacology, Univ. of Michigan, 1301 MSRB III, A220C, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0632 (E-mail: benluc{at}umich.edu).

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. Section 1734 solely to indicate this fact.

Received 28 October 1999; accepted in final form 28 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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