Effect of postconditioning on dynamic expression of tenascin-C and left ventricular remodeling after myocardial ischemia and reperfusion

Background Tenascin-C (TNC), an extracellular matrix glycoprotein, is expressed transiently in distinct areas in association with active tissue remodeling. This study aimed to explore how ischemic postconditioning (PC) affects myocardial expression of TNC and ventricular remodeling using 125I-labeled anti-TNC antibody (125I-TNC-Ab) in a rat model of ischemia and reperfusion. Methods In control rats (n = 27), the left coronary artery (LCA) was occluded for 30 min followed by reperfusion for 1, 3, 7, and 14 days. PC (n = 27) was performed just after the reperfusion. At the time of the study, 125I-TNC-Ab (1.0 to 2.5 MBq) was injected. Six to 9 h later, to verify the area at risk, 99mTc-MIBI (100 to 200 MBq) was injected intravenously just after the LCA reocclusion, with the rats sacrificed 1 min later. Dual tracer autoradiography was performed to assess 125I-TNC-Ab uptake and area at risk. To examine the ventricular remodeling, echocardiography was performed 2 M after reperfusion in both groups. Results In control rats, 125I-TNC-Ab uptake ratio at 1 day after reperfusion was 3.73 ± 0.71 and increased at 3 days (4.65 ± 0.87), followed by a significant reduction at 7 days (2.91 ± 0.55, P < 0.005 vs 3 days) and14 days (2.01 ± 0.17, P < 0.005 vs 1 and 3 days). PC attenuated the 125I-TNC-Ab uptake throughout the reperfusion time from 1 to 14 days; 2.59 ± 0.59 at 1 day, P < 0.05: 3.10 ± 0.42 at 3 days, P < 0.005: 1.93 ± 0.37 at 7 days, P < 0.05: 1.40 ± 0.07 at 14 days, P < 0.001. In echocardiography, PC reduced the ventricular end-diastolic and systolic dimensions (1.00 ± 0.06 cm to 0.83 ± 0.14 cm (P < 0.05) and 0.90 ± 0.15 cm to 0.62 ± 0.19 cm (P < 0.05), respectively) and prevented a decline of ventricular percentage fractional shortening (10.5 ± 3.7 to 28.2 ± 10.7, P < 0.005). Conclusions These data indicate that 125I-TNC-Ab imaging may be a way to monitor myocardial injury, the subsequent repair process, and its response to novel therapeutic interventions like PC by visualizing TNC expression.


Background
Extracellular matrix, in addition to cardiac cells, is essential to maintain the integrity of cardiac tissue and plays an important role not only in providing structural and mechanical support but also in modulating cell function. Cardiac repair after myocardial infarction is regulated through activation and suppression of the acute inflammatory process. Necrosis of cardiomyocytes provokes acute inflammation to remove dead cells and matrix debris, followed by timely infiltration of the infarcted myocardium with myofibroblasts. These secrete large amounts of extracellular matrix proteins, leading to replacement of myocardial dropout with mature collagen-based scar tissue in the infarct. These inflammatory and fibrotic processes are critically involved in the pathogenesis of ventricular remodeling, which is accompanied by changes in the structure and composition of the extracellular matrix and is a significant predictor of left ventricular dysfunction and an adverse prognosis [1].
Tenascin-C (TNC), an extracellular matrix glycoprotein, could provide important biological signaling that influences cell motility, proliferation, differentiation, survival, or apoptosis via cell-extracellular matrix interaction during tissue remodeling of various tissues [2,3]. TNC appears in the heart only in the early stages of embryonic development and is not normally expressed in the adult. However, it reappears transiently associated with myocardial injury such as myocardial infarction [4,5], hibernating myocardium [6], myocarditis [7][8][9], and dilated cardiomyopathy [10,11]. Its site-specific expression suggests that TNC plays important roles during tissue remodeling but also that it can serve as an indicator of myocardial disease activity.
Recently, other groups and we have reported increased serum TNC in heart failure patients, reflecting the severity and progression of ventricular remodeling [12][13][14][15].
Of particular interest is that serum levels of TNC within 1 week of infarction can be a possible predictor of ventricular remodeling and poor prognosis [16,17]. Also, our previous study implied that TNC expression might be altered if the ischemic insults were modified by cardioprotective interventions [18].
Postconditioning, defined as several repeated cycles of intermittent reperfusion and reocclusion after an index ischemia, is cardioprotective in a canine model of ischemia and reperfusion [19]. However, the effect of postconditioning on the TNC expression and left ventricular remodeling is unspecified. This prompted us to explore the effect of postconditioning on the spatial and temporal changes occurring in the expression of TNC after myocardial ischemia and reperfusion using 125 I-labeled anti-tenascin-C antibody ( 125 I-TNC-Ab) and left ventricular remodeling in a rat model of acute ischemia and reperfusion.

Animal model of acute ischemia and reperfusion
All experimental procedures involving animals were conducted in accordance with the institutional guidelines set by the Institute for Experimental Animals, Kanazawa University Advanced Science Research Center. Eight to 11-week-old male Wistar rats (n = 54) were anesthetized with an intraperitoneal administration of 40 mg of pentobarbital per kilogram and ventilated mechanically with room air. After left thoracotomy and exposure of the heart, a 7-0 polypropylene suture on a small curved needle was passed through the myocardium beneath the proximal portion of the left coronary artery (LCA), and both ends of the suture were passed through a small vinyl tube to make a snare. The suture material was pulled tight against the vinyl tube to occlude the LCA. Myocardial ischemia was confirmed by regional cyanosis of the myocardial surface and ST-segment elevation on the ECG. In control rats, LCA was occluded for 30 min and reperfusion was obtained by release of the snare and confirmed by the finding of a myocardial blush over the area at risk. In the group of rats with postconditioning, after the 30 min of LCA occlusion, 10 s of reperfusion followed by 10 s of LCA reocclusion was repeated six times at the beginning of the reperfusion. The snare was left loose on the surface of the heart for reocclusion of the LCA just before sacrifice to identify the area at risk (18). Groups of animals with and without postconditioning were studied at 1 (n = 5, 5), 3 (n = 6, 5), 7 (n = 4, 6), and 14 days (n = 6, 5, respectively) after reperfusion. At the time of the study, 125 I-TNC-Ab (1.0 to 2.5 MBq) was administered via a tail vein. Six to 9 h afterward, 100 to 200 MBq of 99m Tc-MIBI was injected via a tail vein just after the reocclusion of the proximal portion of the LCA for delineation of the area at risk. One minute later, the rat was euthanized and the heart was removed for analysis. The excised heart was rinsed in saline, frozen in isopentane, cooled in dry ice, and embedded in methyl cellulose. Serial short-axis heart sections 20 μm thick were obtained by sectioning on a cryostat to create a series of rings for autoradiography.
To evaluate the left ventricular remodeling 2 months after reperfusion, 30 min of LCA occlusion followed by reperfusion with (n = 6) and without postconditioning (n = 6) was performed. Two months later, echocardiography was performed to evaluate the left ventricular dimensions and function. After echocardiography, 100 to 200 MBq of 99m Tc-MIBI was injected via a tail vein. One minute later, the rat was euthanized and the heart was removed for autoradiography.

Radiolabeling of anti-tenascin-C antibody
Anti-TNC mouse IgG Fab' , a mouse monoclonal antibody against TNC, clone 4F10TT, was raised by immunization of a TNC-null mouse with purified human TNC as described previously [7].
Radiolabeling of anti-TNC antibody with 125 I was achieved by the chloramine-T method [20]. Briefly, [ 125 I] sodium iodide solution (37 MBq/10 μL, Perkin Elmer, Waltham, MA) was added to 60 μL of antibody in PBS (0.32 mg/mL). Following mixing, 4 μL of chloramine-T aqueous solution (1 mg/mL) was added. After 15 min at room temperature, the reaction was quenched with 20 μL of Na 2 H 2 SO 5 . The crude product was purified with a PD-10 column (GE Healthcare UK Ltd., Buckinghamshire, England) with saline as the eluate. The radiochemical purity of 125 I-TNC-Ab was determined by thin layer chromatography (TLC). TLC analyses were performed with silica plates (Art 5553, Merck, Darmstadt, Germany) with saline as the developing solvent. Radiochemical purity was defined as the percent protein-bound activity, which was assessed by dividing the counts at the TLC origin by the total TLC counts. Specificity of the 125 I-TNC-Ab was approved by the autoradiography with 125 I-labeled nonspecific antibody in the previous study [18].

Dual-tracer autoradiography
Dual tracer autoradiography of the left ventricular short axis slices was performed for the assessment of 125 I-TNC-Ab uptake and ischemic area (or area at risk) demonstrated by 99m Tc-MIBI image. The first autoradiographic exposure on an imaging plate (BAS-MS, Fuji Film) was performed for 15 to 20 min to visualize the area at risk expressed by 99m Tc-MIBI distribution at 1 to 2 h after sacrifice. Three days later (12 half-lives of 99m Tc), the second exposure was made for 7 days to image the distribution of 125 I-TNC-Ab.

Data analysis
125 I-TNC-Ab accumulation was evaluated in three myocardial slices at the mid-ventricular level spaced 1 mm apart. The distribution of the tracers was determined by analysis of the digitized autoradiographs. The photostimulated luminescence in each pixel (100 × 100 μm) was determined using a bioimaging analyzer (BAS-5000, Fuji Film). For quantitative analysis, the uptake values of each region of interest (ROI) were expressed as the background-corrected photostimulated luminescence per unit area (1 mm 2 ). A background ROI was set adjacent to the left ventricle. Ischemic and normally perfused areas were defined from the 99m Tc-MIBI image, and these ROIs were applied to the 125 I-TNC-Ab images to evaluate the uptake of 125 I-TNC-Ab. 125 I-TNC-Ab uptake area was also set manually as a ROI by circumscribing the increased activity of the 125 I-TNC-Ab. 125 I-TNC-Ab uptake ratio was calculated by dividing the uptake value in an ischemic area by that of a normally perfused area. The ratio of 125 I-TNC-Ab uptake ROI area to an ischemic ROI area was defined as the percentage of the 125 I-TNC-Ab uptake area. All parameters in each rat were expressed as the average value obtained from the analysis of three representative slices.
The autoradiography in rats just administered 99m Tc-MIBI after echocardiography 2 months after ischemia, and reperfusion was analyzed for myocardial viability. Area of infarction was defined arbitrarily with less than 60% of maximal 99m Tc-MIBI uptakes in the three representative slices.

Immunohistochemical staining
Short axis frozen sections adjacent to the slices for autoradiography were mounted on slides. These short axial heart sections were washed with PBS and stained with mouse monoclonal anti-TNC mouse 4F10TT, the same antibody used for autoradiography, with direct immunoperoxidase technique. For paraffin sections, the heart was fixed in 4% paraformaldehyde, embedded in paraffin, and immunostained as previously described [5]. Briefly, after treatment with pepsin for 10 min for antigen retrieval, sections were incubated with anti-TNC antibody 4F10TT and subsequently with peroxidase-conjugated anti-mouse IgG Fab' (Medical and Biological Lab Co. Ltd., Nagoya, Japan). After washing, diaminobenzidine/ H 2 O 2 solution was used to demonstrate antibody binding. The site and distribution of the expression of the TNC was examined with the aid of the hematoxylineosin stained slice adjacent to the immunohistochemical stained slice.

Echocardiography
Echocardiography was performed in rats with (n = 6) and without (n = 6) postconditioning at 2 months after ischemia and reperfusion under anesthesia with 1% to 2% isoflurane using Vivid 7 (GE Healthcare, Milwaukee, WI, USA) with a 14-MHz i13L linear array transducer. Three cardiac cycles of the parasternal short axis view at papillary muscle level were recorded at a frame rate of 265 per second. The images were processed offline using commercially available software (EchoPac 6.1, GE Healthcare, Milwaukee, WI, USA). The left ventricular internal dimensions at the end of diastole (LVEDD) and at the end of systole (LVESD) were measured digitally using anatomical M-mode feature. LV fractional shortening (%FS) was calculated as [(LVEDD-LVESD)/LVEDD] × 100.

Statistical analysis
All results were expressed as mean ± 1 SD. Statistical analyses were performed using a Macintosh computer with software JMP 5.0.1 J. Group comparisons were performed using Tukey-Kramer method to identify differences among groups. A value of P < 0.05 was considered statistically significant.

I-TNC-Ab Uptake
In visual analysis, strong 125 I-TNC-Ab uptake was observed in the area at risk at 1 and 3 days after reperfusion, followed by moderate uptake at 7 and 14 days after reperfusion. Postconditioning attenuated the 125 I-TNC-Ab uptake throughout the course of reperfusion time (Figure 2).
In the quantitative analysis, 125 I-TNC-Ab uptake ratio of the rats without postconditioning at 1 day after reperfusion was 3.73 ± 0.71 and increased at 3 days (4.65 ± 0.87), followed by a significant reduction at 7 days after reperfusion (2.91 ± 0.55, P < 0.005 vs 3 days) and followed by further reduction at 14 days (2.01 ± 0.17, P < 0.005 vs 1 and 3 days).

Histopathological findings
At 1 to 3 days after ischemia and reperfusion, muscle lysis, muscle destruction, and coagulation necrosis of cardiomyocytes with severe inflammatory cell infiltration were observed in the infarcted area. TNC deposition was observed throughout the infarcted area with inflammation, though strong staining was limited to the border zone between infracted and intact areas. No TNC expression was detected in the intact area. This TNC expression pattern was not changed significantly by postconditioning, but staining of TNC tended to be weaker in both border zone and infarcted area. Then, necrotic tissue was gradually replaced by granulation tissue with attenuation of inflammation. TNC deposition gradually decreased until day 14. With postconditioning, the spatiotemporal pattern of TNC deposition came to resemble that of control animals, while the degree of staining became weaker (Figure 4).

Discussion
The present study revealed that ischemic postconditioning significantly attenuated TNC expression as demonstrated by the accumulation of 125 I-TNC-Ab throughout the entire period until 2 weeks after ischemia and reperfusion.
Postconditioning also attenuated left ventricular remodeling and reduced infarct size at 2 months after ischemia and reperfusion. These data suggest that attenuation of TNC expression during the acute to subacute phase of tissue healing after infarction by postconditioning might be related to the attenuation of ventricular remodeling at 2 months after myocardial infarction, although whether the relationship is cause and effect or simple association remained to be elucidated.  Figure 4 Immunostaining for TNC at 3, 7, and 14 days after reperfusion with and without postconditioning is presented. Strong TNC brown staining was seen in border zone between intact and infarcted area, and also scattered brown staining was seen throughout the infarcted area at 3 days after reperfusion. With postconditioning, TNC staining tended to be weaker in both border zone and infarcted area. At 7 and 14 days after reperfusion, TNC staining became weaker at both border zone and infarcted area. With postconditioning, TNC staining at both border zone and infarcted area became weaker. Intact, intact area; Inf, infarcted area; Scale bars, 100 μm. Since the discovery of the cardio-protective effect of postconditioning defined as rapid sequential intermittent interruption of blood flow applied during early moments of reperfusion in a canine model of 60 min of myocardial ischemia and reperfusion [19], the cardio-protective effect induced by postconditioning has been extensively investigated and confirmed in many species, including man [21]. The attenuation of cardiac injury afforded by postconditioning has been attributed to suppression of reperfusion injury with consequent attenuation of myocardial apoptosis and necrosis. Many of the primary physiological endpoints of protection during reperfusion have been investigated including delay in washout of endogeneous autacoids, reduction of inflammatory response, protection of vascular endothelium, stimulation of survival kinase and transmitter, inhibition of death kinase, preservation of mitochondrial function, and reduction of cardiomyocyte loss [21]. Although a number of protective mechanisms targeted by postconditioning have been explored, it is generally recognized that inhibition of mitochondrial permeability transition pore opening by postconditioning is the final step in a complicated series of cellular events responsible for cell protection [21][22][23]. After 30-m ischemia of the isolated rat heart, mitochondrial permeability transition pore opening does not occur during ischemia but does within the first 5 m of reflow [24]. Therefore, it is important to induce postconditioning just after reperfusion to inhibit mitochondrial permeability transition pore opening, at which time its effect on the reduction of tissue damage has been demonstrated.

Postconditioning
On the other hand, the effect of postconditioning on tissue repair including interstitial signaling and remodeling has been scarcely investigated. After myocardial infarction in the permanent ligation model, TNC is markedly but transiently upregulated during the early phase of tissue repair, is predominantly produced by interstitial fibroblasts in the vicinity of the injured cardiomyocytes but not by cardiomyocytes themselves [25], and is localized in the border zone between infarcted and viable myocardium [4,5] while in a reperfusion model it is scattered throughout the infarcted area in addition to border zone [18]. TNC may exert conflicting effects on cardiac tissue repair, healing, and ventricular remodeling [26,27]. TNC may loosen the strong adhesion of cardiomyocytes from the matrix and upregulate the expression and activity of matrix metalloproteinases [28,29], promoting degradation of the extracellular matrix and slippage of myocytes and facilitate inflammatory cell infiltration within the ventricular wall, resulting in an increased risk of ventricular thinning and dilatation. However, these functions promote cell rearrangement and allow myofibroblasts and capillary vessels to spread into tissue during the restoration process. Because of the limited regenerative ability of cardiomyocytes, the interstitial cells become a major player in myocardial tissue repair, with especially myofibroblasts playing an important role in wound healing by synthesizing collagens and exerting strong contractile forces to promote wound healing [30]. It is suggested that TNC produced by interstitial cells at an early phase of myocardial infarction promotes recruitment of myofibroblasts to injured sites by accelerating migration and differentiation and enhancing traction forces [25]. Therefore, these functions of TNC may facilitate tissue healing, reinforcement of the cardiac matrix, and fibrosis and prevent ventricular dilatation by generating traction forces [5,7,25].
Although it is difficult to conclude whether TNC is more beneficial or detrimental for tissue healing and reconstruction after myocardial infarction, a timely and proper degree of TNC expression might be beneficial for myocardial tissue repair, whereas prolonged overexpression of TNC might interfere with sound tissue repair and cause inappropriate reconstruction of infarcted ventricle, resulting in so-called ventricular remodeling.
In the mice model of permanent ligation of the coronary artery, ventricular remodeling in the TNC knockout mouse was significantly reduced and cardiac function was improved compared with the wild type at day 28 after myocardial infarction [31]. In patients with acute myocardial infarction, peak serum TNC level at 5 days after infarction is an important independent predictor of prognosis, and higher TNC level is associated with a greater risk of left ventricular remodeling at 6 months after infarction, indicating that overexpression of TNC may aggravate ventricular remodeling [16]. The results of the present study were consistent with those of these animal and human studies in that less TNC expression due to postconditioning was found to be associated with less ventricular remodeling.
The application of target radionuclide imaging of regional TNC expression as proposed in the present study holds the potential to quantify the extent, amount, and localization of TNC expression and relate the pathophysiological events to the tissue repair and remodeling occurring after severe myocardial ischemia. The current study also demonstrated that the 125 I-TNC-Ab imaging approach after myocardial ischemia and reperfusion provides an opportunity to monitor the effect of postconditioning, potentially leading to novel therapeutic interventions, directed at the reducing the extracellular matrix remodeling occurring after myocardial infarction.
One of the limitations of this study was that the left ventricular function was assessed on only short axis slices in echocardiography, because remodeling and dilation mainly occur on the apical part of the ventricle after anterior myocardial infarction.
For future clinical application as a SPECT agent, 123 I-TNC-Ab is required. Labeling with 123 I can easily be performed in the same way as for 125 I. In vivo SPECT imaging by 111 In-TNC-Ab has already been obtained successfully at 6 h after administration of the tracer in a rat model of 3 days after permanent coronary artery ligation. The biodistribution study of 111 In-TNC-Ab showed that target-to-blood, target-to-lung, and targetto-liver ratios were 3.3, 2.0, and 0.35, respectively [32]. On the other hand, our previous study demonstrated that rapid blood clearance and retention of 125 I-TNC-Ab in the infarcted area, resulting in a decent target-toblood ratio (1.85 ± 0.14 at 5 h and 4.8 ± 1.0 at 24 h after injection) and good target-to-lung ratio (1.97 ± 0.19 at 5 h and 4.9 ± 1.2 at 24 h after injection), indicate the feasibility of in vivo imaging at 5 h or later after tracer injection [18]. However, relatively high liver uptake resulted in a low target-to-liver ratio (0.66 at 5 h and 0.82 at 24 h), suggesting that careful interpretation might be required for myocardial uptake adjacent to the liver, although the ratio was improved compared to that of 111 In-TNC-Ab.

Conclusions
The present study demonstrated that postconditioning attenuates the whole process of TNC expression demonstrated by 125 I-TNC-Ab after myocardial ischemia and reperfusion. Postconditioning also attenuated left ventricular remodeling at 2 months after ischemia and reperfusion. The obtained data also implied that radiolabeled TNC-Ab imaging may be useful as a noninvasive way to monitor the changes occurring during the process of myocardial injury and its repair by various therapeutic interventions after ischemia and reperfusion by visualizing TNC expression.