Laboratory Investigation
Induction of Early Bio markers in a ThrombusInduced Sheep Model of Ischemic Heart Failure
Aluganti N. Chandrakala, PhD Pawel Kwiatkowski, MD Chittoor B. Sai-Sudhakar, MD Benjamin Sun, MD Angela Phillips, RVT Sampath Parthasarathy, PhD, MBA
The levels of brain natriuretic peptide (BNP) and monocyte chemoattractant protein-1 (MCP-1) are known to be increased in the sera of subjects with heart failure. Existing models do not account for the biomass of thrombus that occurs in patients undergoing myocardial infarction. In this study, we compared the expressions of sheep-derived genes for BNP, MCP-1, and atrial natriuretic peptide in a new large-animal model of thrombusinduced heart failure. Thrombus of autologous platelets was injected directly into the left circumflex coronary arteries of sheep. Cardiac ischemic injury was evaluated by troponin I levels, and heart failure progression was monitored with the aid of echocardiogram-based evaluation. After outlined time intervals, the sheep were killed and their hearts excised for tissue sampling. Reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay (ELISA) tests were performed to establish gene and protein expressions. At 72 hours after embolization, myocardial BNP and MCP-1 expressions had increased significantly in the ischemic region, compared either with the nonischemic region or with tissue from healthy sheep. As heart failure progressed to 90 days after embolization, the expression of BNP in the ischemic region decreased, whereas its expression in the nonischemic region increased several fold. In contrast, MCP-1 gene expression showed no changes in either tissue after 90 days of embolization. Plasma levels of BNP, determined by Western blot and ELISA, also correlated with the gene-expression studies. Our results show regional changes in BNP and MCP-1, as well as differences in the expression of these 2 genes. (Tex Heart Inst J 2013;40(5):511-20)
Drs. Chandrakala and Kwiatkowski contributed equally to this report. Key words: Atrial natriuretic peptide; biological markers/ blood; blood platelets; C-reactive protein; chemokines/ blood; disease models/animal; heart failure; monocyte chemoattractant protein-1/ blood; myocardial ischemia; natriuretic peptide, brain; sheep; stroke volume; ventricular dysfunction, left/ etiology From: Division of Cardiac Surgery, Department of Surgery, The Ohio State University Medical Center, Columbus, Ohio 43210 Address for reprints: Pawel Kwiatkowski, MD, Division of Cardiac Surgery, Department of Surgery, N. Doan Hall, Rm. 847, 410 W. 10th Ave., The Ohio State University Medical Center, Columbus, OH 43210 E-mail: [email protected] © 2013 by the Texas Heart ® Institute, Houston
Texas Heart Institute Journal
M
onocyte chemoattractant protein-1 (MCP-1) and brain natriuretic peptide (BNP) levels are simple and objective measures of cardiac function in patients who are undergoing ischemic injury. These measurements can be used to diagnose heart failure, including diastolic dysfunction 1 and inflammation.2 Plasma levels of MCP-1,3 BNP, and atrial natriuretic peptide (ANP)4 are noticeably elevated in heart failure, especially after myocardial infarction (MI). Furthermore, within heart tissue, gene expressions of MCP-1, BNP, and ANP are reportedly upregulated in animal models with MI and heart failure,5,6 and in human heart disease.7,8 It is generally believed that ventricular wall stress and volume might contribute to increased BNP levels.9,10 Heart failure is also characterized by increased circulating levels of inflammatory cytokines.1 This phenomenon is associated and correlated with progression and severity of the disease. In addition to myocardium, other cells and tissues, including endothelial cells, tissue macrophages, platelets, and leukocytes, are also involved in inflammation. Activation of the immune system might also play a role in the pathogenesis of heart failure.11 Monocyte chemoattractant protein-1 belongs to the C-C chemokine family and has a significant role in the recruitment and activation of monocytes and macrophages.12 Usually, myocardial ischemia is induced by ligation of the appropriate arteries. However, considering that atherothrombotic mechanisms might play a major role in myocardial ischemia in vivo, we recently developed a sheep model of heart failure via the injection of autologous thrombus. Using this model in the current study, we report a significant and early induction of MCP-1 and BNP gene expressions when little or no volume or pressure overload was indicated, which suggests that early recruitment of leukocytes, and the inflammatory changes themselves, could contribute to ischemic remodeling. In addition, we used 2 existing models of ischemic heart failure in order to Early Biomarkers in Thrombus-Induced Model of Ischemic Heart Failure
511
compare early and late expression of MCP-1, BNP, and ANP in these models with early and late expression in our novel model of thrombus-based coronary obstruction. The first of these scientifically established models uses catheter-based direct injection of polystyrene beads into the coronary circulatory system.13 The 2nd model uses suture-based ligation of the left anterior descending coronary artery (LAD) via open-chest surgical access. Ligation of the LAD is routinely used in small animals such as rodents, in which the catheter-based approach is very restricted due to the small size of the carotid artery. These 2 models do not entirely replicate the clinical situation wherein thrombus forms inside coronary arteries and restricts blood flow to cardiac tissue. In addition, the presence of thrombus can lead to the release of substances such as thrombomodulin, thromboplastin, and matrix metalloproteinases that subsequently can alter the inflammatory response and induce chronic cardiac ischemia. Finally, these 3 methods for the development of ischemic heart failure—surgical obstruction, polystyrene-bead blockage, and thrombus-based obstruction—enabled us to compare parallel biological changes in cardiac tissue. This specific approach also validated the new model of coronary artery obstruction with autologous platelet-derived thrombus.
Materials and Methods All animals in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. The protocol was approved by The Ohio State University Institutional Animal Care and Use Committee. Isolation of Platelets and Preparation of Thrombus
Platelets were isolated from freshly collected blood of adult Suffolk sheep (Ovis aries) by a modified procedure of Kiktenko and colleagues.14 Blood (200 mL) was collected from the sheep’s left jugular vein into anticoagulant (EDTA and sodium chloride; pH, 7.5) tubes for isolation of autologous platelets. Labeling of Platelets with Mepacrine. In order to determine the presence of platelets in cardiac tissue, we labeled them with mepacrine (final concentration, 10 µM mepacrine in 1× phosphate-buffered saline solution [PBS]; pH, 7.5) and incubated them for 1 hour at room temperature. Mepacrine is rapidly taken up and localized in dense granules of platelets. After incubation, the cells were centrifuged at 1,500 ×g for 5 min. The thrombus for selective intracoronary injection and embolization was prepared by collecting the platelet suspension into a syringe (approximately 2 × 108 cells/ mL), adding 25 U of thrombin, and allowing the mixture to sit for 2 minutes. 512
After the terminal procedure (death of the sheep and sampling of tissue), freshly cut snap-frozen cardiac tissue samples were examined under fluorescent microscopy (Olympus IX51® inverted microscope; Olympus Corporation; Tokyo, Japan) to determine the presence or absence of labeled aggregated platelets. Animal Selection and Preparation
Thirty animals for this experimental purpose were divided into acute heart failure, chronic heart failure, and control groups, for comparison studies. The acute group consisted of 6 animals that underwent LAD ligation, 6 that underwent polystyrene bead embolization, and 6 that underwent thrombus embolization. The chronic group of 6 animals underwent multiple (3–6) microembolizations at biweekly intervals, in order to achieve a permanent drop in left ventricular ejection fraction (LVEF) below 0.35, consistent with the development of chronic heart failure. The control group (another 6 animals) consisted of 3 healthy sheep and 3 sheep injected with nonaggregated platelets directly into the coronary circulation. The sheep were sedated with an intramuscular injection of Telazol® (Zoetis; Florham Park, NJ). A catheter was placed in the right external jugular vein, and anesthesia was induced with an intravenous injection of etomidate. Orotracheal intubation was performed, and anesthesia was maintained with a 1% to 3% isoflurane and 100% oxygen mixture for the duration of the procedure. Positive-pressure ventilation (10–15 mL/ kg) and intravenous fluids (10 mL/kg/hr) were used in all animals. The left neck of each animal was clipped and aseptically draped. Local anesthesia with 2% lidocaine hydrochloride mixed 1:1 with 0.5% bupivacaine hydrochloride (5 cc) was injected into the skin and subcutaneous tissues for local analgesia at the site of the 3-cm incision for left carotid artery access. The artery was exposed, and a 5-0 Prolene purse-string suture was placed to facilitate passage of a 6F or 7F arterial introducer (11 cm) by means of the Seldinger technique (needle access, wire-guided placement). The animal was heparinized (4,000 U heparin), and lidocaine (40 mg) and magnesium sulfate (2 g) were administered intravenously to reduce the risk of arrhythmias. For delivery of the platelet aggregates, left circumflex coronary artery (LCx) access was achieved with a variety of coronary angiographic catheters and guidewires. After selective catheterization under fluoroscopic guidance, the autologous platelet aggregates were injected directly into the LCx. Subsequently, these sheep were humanely killed at 4, 24, 48, 72 hours, and 90 days after thrombus embolization, and the hearts were excised for tissue sampling. To provide control data, cardiac tissue was collected from the corresponding regions of healthy sheep that had not undergone surgery. The details of the model are described
Early Biomarkers in Thrombus-Induced Model of Ischemic Heart Failure
Volume 40, Number 5, 2013
in a separate publication.15 Commercially available polystyrene beads, with an average diameter of 90 µm, were delivered in a similar fashion. The LAD-ligation group did not undergo embolization, but instead underwent thoracotomy to facilitate the creation of heart failure via sequential ligation of the LAD and the diagonal branch at a point approximately 40% of the distance from the apex of the heart to the branching of the diagonal coronary artery. After anesthetic induction in the same fashion as described above, the animal was placed in lateral recumbency and an incision was made at the 4th intercostal space. The pericardium was opened and the identified diagonal branch was ligated. The pericardium and thoracotomy were closed in standard fashion, and the recovered animal returned to routine husbandry. Evaluation of Cardiac Injury
Cardiac injury was monitored by measuring cardiac troponin I levels from blood samples collected at baseline (before the embolization procedure) and at 3 days after embolization. The development of cardiac insufficiency was evaluated by means of a Vivid 7 ® cardiac ultrasonography system (GE VingMed Ultrasound AS; Horten, Norway). Embolizations were continued until echocardiography conf irmed an LVEF lower than 0.35. Tissue Sample Collection
At different time intervals after embolization, all animals were humanely killed and tissue samples taken in order to evaluate early or chronic changes. Different situations were evaluated: 1) normal/control, 2) nonaggregated platelet-embolized, 3) bead-embolized, 4) thrombus-embolized via the LCx, and 5) LAD-ligation–induced heart failure. Cardiac tissue from either healthy sheep or sheep with heart failure induced by thrombus was obtained at time intervals of 4, 24, 48, and 72 hr, and 90 days. To compare the gene expressions from different heart failure models, we collected tissue samples from sheep exposed to bead embolization and LAD ligation. During the terminal procedure, all sheep were anesthetized (as described above for the embolization procedures) and positioned in right lateral recumbency. After the collection of blood specimens, left lateral thoracotomy was performed to harvest the heart. Excised hearts were examined for features consistent with pathologic changes related to ischemic conditions. Ischemic and nonischemic regions of the left ventricle (LV) were identified on the basis of blood-flow distribution and apparent changes arising from acute and chronic ischemia (acute: wall thickness, discoloration, pallor and paleness of ischemic region; chronic: wall thickness, fibrotic lesions). Ischemic and nonischemic tissue sections were collected and snap-frozen in liquid nitrogen or placed in 10% formaldehyde. Texas Heart Institute Journal
Expressions of MCP-1, BNP, and ANP Messenger RNA
Total RNA from 25 mg of control, ischemic, and nonischemic regions of cardiac tissue (healthy and thrombus embolized) was isolated by the method of Chomczynski and Sacchi16 with use of Invitrogen TRIzol® reagent (Life Technologies Corporation; Grand Island, NY). Quality and concentration of RNA were measured by the NanoDrop method with use of a NanoDrop ® 1000 Spectrophotometer (Thermo Fisher Scientific; Wilmington, Del). The total RNA of 1 µg was then reverse-transcribed into complementary DNA with use of the SuperScript III First-Strand Synthesis system (Life Technologies). A complementary DNA (50-ng) sample was used to perform quantitative real-time polymerase chain reaction (PCR) with use of an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.; Hercules, Calif ) with SYBR® Green (Life Technologies). Invitrogen sheep oligonucleotide primers for reverse transcription polymerase chain reaction (RT-PCR) were purchased from Life Technologies. Polymerase chain reaction was carried out with MCP-1–specific primers (forward: 5′-CAGAAGAGTCACCACCAGCSA-3′; reverse: 5′-AGATGGTTTATGGCGTCCTG-3′), resulting in 170-base pair (bp) fragment and BNP-specif ic primers (forward: 5′-TGGAAGAAACCTGGGACTC-3′; reverse: 5′AGTACCTCCTCAGCACGTTG-3′), resulting in a 195-bp fragment. The ANP-specific primers (forward: 5′-CAGTGAAAGGCCAAAGAAGC-3′; reverse: 5′-CAGAGCCATACACGGGATTT-3′) resulted in a 200-bp fragment. As a reference gene, we used sheep GAPDH primers (forward: 5′-AATGCCTCCTGCACCACCAA-3′; reverse: 5′-TTGGCAGCGCCAGTAGAAGC-3′), resulting in a 198-bp fragment. The PCR was performed with an initial step of denaturation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 20 s. Melt curves were established for the reactions. Normalized fold expression was calculated by using the 2–∆∆Ct method. Entire reaction products underwent electrophoresis on 2% agarose gels. Each experiment was repeated 3 times. Western Blot Analysis
Plasma protein concentration was determined with use of DC protein assay (Bio-Rad). Protein of 8 to 10 µg was separated by 15% to 20% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The gels were transblotted onto a 0.2-µm nitrocellulose membrane with use of the Mini Trans-Blot ® Electrophoretic Transfer Cell (Bio-Rad) for 1 hr at 100V in a cold room. Membranes were blocked with 5% nonfat milk in Tris-buffered saline solution (TBS-T; pH, 7.5) with 0.1% Tween-20 at room temperature for 1 hr and then incubated with primary antibody to a BNPderived synthetic peptide (Biocentra LLC and Alpha
Early Biomarkers in Thrombus-Induced Model of Ischemic Heart Failure
513
Diagnostics Intl., Inc.; both of San Antonio, Texas) (1:5,000 v/v dilution) overnight at 4 °C. The blots were then incubated with secondary antibody (chicken anti-rabbit immunoglobulin G [IgG] conjugated to horseradish peroxidase 1:5,000 v/v dilution) for 1 hr. Finally, signals were detected with an enhanced chemiluminescence kit (Thermo Fisher). For MCP-1, commercial antibodies were used. ELISA for BNP
The BNP concentrations were measured in plasma samples of healthy and heart failure sheep. Several 96-well ELISA plates were coated with 50 µL of BNP peptide (0–2 ng diluted with ELISA coating buffer)— both healthy and heart failure plasma samples—and were kept overnight at 4 °C. As a blocking buffer, 5% nonfat milk in TBS-T was used. One hundred µL of primary antibody to BNP was added to each well in triplicate. The plates were incubated for 3 hr at room temperature. Unbound materials were washed out with TBS with 0.1% Tween. One hundred µL of the secondary antibody (chicken anti-rabbit IgG conjugate to horseradish peroxidase) was added to each well. The plates were incubated for 1 hr at room temperature. After the washing, the activity of horseradish peroxidase was estimated by adding 100 µL of 3,3,5,5-tetramethyl benzidine (TMB) to the wells, and incubation was continued for 30 min at room temperature. The reaction was stopped by using 1N HCl. Optical density at 450 nm was determined by a microplate reader (Bio-Rad). Standard curves were generated by Sigma plot, and the concentrations of different samples were determined from the standard curves. Hematoxylin and Eosin Staining
Formalin-fixed, paraffin-embedded tissue samples of sheep from healthy and experimentally induced failing hearts were used. A section of desired thickness (7–20 µm) was cut with use of a microtome. The tissue sections were deparaffinized and hydrated by using ethanol. Sections were stained with hematoxylin and eosin. Tissue sections were dehydrated by using ethanol, acetone, and xylene. Finally, sections were fixed with mounting fluid and were analyzed under a microscope. Statistical Analysis
Each benchtop-based experiment was performed in triplicate. Data are presented as mean ± SD, with the acceptance of P
Induction of Early Bio markers in a ThrombusInduced Sheep Model of Ischemic Heart Failure
Aluganti N. Chandrakala, PhD Pawel Kwiatkowski, MD Chittoor B. Sai-Sudhakar, MD Benjamin Sun, MD Angela Phillips, RVT Sampath Parthasarathy, PhD, MBA
The levels of brain natriuretic peptide (BNP) and monocyte chemoattractant protein-1 (MCP-1) are known to be increased in the sera of subjects with heart failure. Existing models do not account for the biomass of thrombus that occurs in patients undergoing myocardial infarction. In this study, we compared the expressions of sheep-derived genes for BNP, MCP-1, and atrial natriuretic peptide in a new large-animal model of thrombusinduced heart failure. Thrombus of autologous platelets was injected directly into the left circumflex coronary arteries of sheep. Cardiac ischemic injury was evaluated by troponin I levels, and heart failure progression was monitored with the aid of echocardiogram-based evaluation. After outlined time intervals, the sheep were killed and their hearts excised for tissue sampling. Reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay (ELISA) tests were performed to establish gene and protein expressions. At 72 hours after embolization, myocardial BNP and MCP-1 expressions had increased significantly in the ischemic region, compared either with the nonischemic region or with tissue from healthy sheep. As heart failure progressed to 90 days after embolization, the expression of BNP in the ischemic region decreased, whereas its expression in the nonischemic region increased several fold. In contrast, MCP-1 gene expression showed no changes in either tissue after 90 days of embolization. Plasma levels of BNP, determined by Western blot and ELISA, also correlated with the gene-expression studies. Our results show regional changes in BNP and MCP-1, as well as differences in the expression of these 2 genes. (Tex Heart Inst J 2013;40(5):511-20)
Drs. Chandrakala and Kwiatkowski contributed equally to this report. Key words: Atrial natriuretic peptide; biological markers/ blood; blood platelets; C-reactive protein; chemokines/ blood; disease models/animal; heart failure; monocyte chemoattractant protein-1/ blood; myocardial ischemia; natriuretic peptide, brain; sheep; stroke volume; ventricular dysfunction, left/ etiology From: Division of Cardiac Surgery, Department of Surgery, The Ohio State University Medical Center, Columbus, Ohio 43210 Address for reprints: Pawel Kwiatkowski, MD, Division of Cardiac Surgery, Department of Surgery, N. Doan Hall, Rm. 847, 410 W. 10th Ave., The Ohio State University Medical Center, Columbus, OH 43210 E-mail: [email protected] © 2013 by the Texas Heart ® Institute, Houston
Texas Heart Institute Journal
M
onocyte chemoattractant protein-1 (MCP-1) and brain natriuretic peptide (BNP) levels are simple and objective measures of cardiac function in patients who are undergoing ischemic injury. These measurements can be used to diagnose heart failure, including diastolic dysfunction 1 and inflammation.2 Plasma levels of MCP-1,3 BNP, and atrial natriuretic peptide (ANP)4 are noticeably elevated in heart failure, especially after myocardial infarction (MI). Furthermore, within heart tissue, gene expressions of MCP-1, BNP, and ANP are reportedly upregulated in animal models with MI and heart failure,5,6 and in human heart disease.7,8 It is generally believed that ventricular wall stress and volume might contribute to increased BNP levels.9,10 Heart failure is also characterized by increased circulating levels of inflammatory cytokines.1 This phenomenon is associated and correlated with progression and severity of the disease. In addition to myocardium, other cells and tissues, including endothelial cells, tissue macrophages, platelets, and leukocytes, are also involved in inflammation. Activation of the immune system might also play a role in the pathogenesis of heart failure.11 Monocyte chemoattractant protein-1 belongs to the C-C chemokine family and has a significant role in the recruitment and activation of monocytes and macrophages.12 Usually, myocardial ischemia is induced by ligation of the appropriate arteries. However, considering that atherothrombotic mechanisms might play a major role in myocardial ischemia in vivo, we recently developed a sheep model of heart failure via the injection of autologous thrombus. Using this model in the current study, we report a significant and early induction of MCP-1 and BNP gene expressions when little or no volume or pressure overload was indicated, which suggests that early recruitment of leukocytes, and the inflammatory changes themselves, could contribute to ischemic remodeling. In addition, we used 2 existing models of ischemic heart failure in order to Early Biomarkers in Thrombus-Induced Model of Ischemic Heart Failure
511
compare early and late expression of MCP-1, BNP, and ANP in these models with early and late expression in our novel model of thrombus-based coronary obstruction. The first of these scientifically established models uses catheter-based direct injection of polystyrene beads into the coronary circulatory system.13 The 2nd model uses suture-based ligation of the left anterior descending coronary artery (LAD) via open-chest surgical access. Ligation of the LAD is routinely used in small animals such as rodents, in which the catheter-based approach is very restricted due to the small size of the carotid artery. These 2 models do not entirely replicate the clinical situation wherein thrombus forms inside coronary arteries and restricts blood flow to cardiac tissue. In addition, the presence of thrombus can lead to the release of substances such as thrombomodulin, thromboplastin, and matrix metalloproteinases that subsequently can alter the inflammatory response and induce chronic cardiac ischemia. Finally, these 3 methods for the development of ischemic heart failure—surgical obstruction, polystyrene-bead blockage, and thrombus-based obstruction—enabled us to compare parallel biological changes in cardiac tissue. This specific approach also validated the new model of coronary artery obstruction with autologous platelet-derived thrombus.
Materials and Methods All animals in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. The protocol was approved by The Ohio State University Institutional Animal Care and Use Committee. Isolation of Platelets and Preparation of Thrombus
Platelets were isolated from freshly collected blood of adult Suffolk sheep (Ovis aries) by a modified procedure of Kiktenko and colleagues.14 Blood (200 mL) was collected from the sheep’s left jugular vein into anticoagulant (EDTA and sodium chloride; pH, 7.5) tubes for isolation of autologous platelets. Labeling of Platelets with Mepacrine. In order to determine the presence of platelets in cardiac tissue, we labeled them with mepacrine (final concentration, 10 µM mepacrine in 1× phosphate-buffered saline solution [PBS]; pH, 7.5) and incubated them for 1 hour at room temperature. Mepacrine is rapidly taken up and localized in dense granules of platelets. After incubation, the cells were centrifuged at 1,500 ×g for 5 min. The thrombus for selective intracoronary injection and embolization was prepared by collecting the platelet suspension into a syringe (approximately 2 × 108 cells/ mL), adding 25 U of thrombin, and allowing the mixture to sit for 2 minutes. 512
After the terminal procedure (death of the sheep and sampling of tissue), freshly cut snap-frozen cardiac tissue samples were examined under fluorescent microscopy (Olympus IX51® inverted microscope; Olympus Corporation; Tokyo, Japan) to determine the presence or absence of labeled aggregated platelets. Animal Selection and Preparation
Thirty animals for this experimental purpose were divided into acute heart failure, chronic heart failure, and control groups, for comparison studies. The acute group consisted of 6 animals that underwent LAD ligation, 6 that underwent polystyrene bead embolization, and 6 that underwent thrombus embolization. The chronic group of 6 animals underwent multiple (3–6) microembolizations at biweekly intervals, in order to achieve a permanent drop in left ventricular ejection fraction (LVEF) below 0.35, consistent with the development of chronic heart failure. The control group (another 6 animals) consisted of 3 healthy sheep and 3 sheep injected with nonaggregated platelets directly into the coronary circulation. The sheep were sedated with an intramuscular injection of Telazol® (Zoetis; Florham Park, NJ). A catheter was placed in the right external jugular vein, and anesthesia was induced with an intravenous injection of etomidate. Orotracheal intubation was performed, and anesthesia was maintained with a 1% to 3% isoflurane and 100% oxygen mixture for the duration of the procedure. Positive-pressure ventilation (10–15 mL/ kg) and intravenous fluids (10 mL/kg/hr) were used in all animals. The left neck of each animal was clipped and aseptically draped. Local anesthesia with 2% lidocaine hydrochloride mixed 1:1 with 0.5% bupivacaine hydrochloride (5 cc) was injected into the skin and subcutaneous tissues for local analgesia at the site of the 3-cm incision for left carotid artery access. The artery was exposed, and a 5-0 Prolene purse-string suture was placed to facilitate passage of a 6F or 7F arterial introducer (11 cm) by means of the Seldinger technique (needle access, wire-guided placement). The animal was heparinized (4,000 U heparin), and lidocaine (40 mg) and magnesium sulfate (2 g) were administered intravenously to reduce the risk of arrhythmias. For delivery of the platelet aggregates, left circumflex coronary artery (LCx) access was achieved with a variety of coronary angiographic catheters and guidewires. After selective catheterization under fluoroscopic guidance, the autologous platelet aggregates were injected directly into the LCx. Subsequently, these sheep were humanely killed at 4, 24, 48, 72 hours, and 90 days after thrombus embolization, and the hearts were excised for tissue sampling. To provide control data, cardiac tissue was collected from the corresponding regions of healthy sheep that had not undergone surgery. The details of the model are described
Early Biomarkers in Thrombus-Induced Model of Ischemic Heart Failure
Volume 40, Number 5, 2013
in a separate publication.15 Commercially available polystyrene beads, with an average diameter of 90 µm, were delivered in a similar fashion. The LAD-ligation group did not undergo embolization, but instead underwent thoracotomy to facilitate the creation of heart failure via sequential ligation of the LAD and the diagonal branch at a point approximately 40% of the distance from the apex of the heart to the branching of the diagonal coronary artery. After anesthetic induction in the same fashion as described above, the animal was placed in lateral recumbency and an incision was made at the 4th intercostal space. The pericardium was opened and the identified diagonal branch was ligated. The pericardium and thoracotomy were closed in standard fashion, and the recovered animal returned to routine husbandry. Evaluation of Cardiac Injury
Cardiac injury was monitored by measuring cardiac troponin I levels from blood samples collected at baseline (before the embolization procedure) and at 3 days after embolization. The development of cardiac insufficiency was evaluated by means of a Vivid 7 ® cardiac ultrasonography system (GE VingMed Ultrasound AS; Horten, Norway). Embolizations were continued until echocardiography conf irmed an LVEF lower than 0.35. Tissue Sample Collection
At different time intervals after embolization, all animals were humanely killed and tissue samples taken in order to evaluate early or chronic changes. Different situations were evaluated: 1) normal/control, 2) nonaggregated platelet-embolized, 3) bead-embolized, 4) thrombus-embolized via the LCx, and 5) LAD-ligation–induced heart failure. Cardiac tissue from either healthy sheep or sheep with heart failure induced by thrombus was obtained at time intervals of 4, 24, 48, and 72 hr, and 90 days. To compare the gene expressions from different heart failure models, we collected tissue samples from sheep exposed to bead embolization and LAD ligation. During the terminal procedure, all sheep were anesthetized (as described above for the embolization procedures) and positioned in right lateral recumbency. After the collection of blood specimens, left lateral thoracotomy was performed to harvest the heart. Excised hearts were examined for features consistent with pathologic changes related to ischemic conditions. Ischemic and nonischemic regions of the left ventricle (LV) were identified on the basis of blood-flow distribution and apparent changes arising from acute and chronic ischemia (acute: wall thickness, discoloration, pallor and paleness of ischemic region; chronic: wall thickness, fibrotic lesions). Ischemic and nonischemic tissue sections were collected and snap-frozen in liquid nitrogen or placed in 10% formaldehyde. Texas Heart Institute Journal
Expressions of MCP-1, BNP, and ANP Messenger RNA
Total RNA from 25 mg of control, ischemic, and nonischemic regions of cardiac tissue (healthy and thrombus embolized) was isolated by the method of Chomczynski and Sacchi16 with use of Invitrogen TRIzol® reagent (Life Technologies Corporation; Grand Island, NY). Quality and concentration of RNA were measured by the NanoDrop method with use of a NanoDrop ® 1000 Spectrophotometer (Thermo Fisher Scientific; Wilmington, Del). The total RNA of 1 µg was then reverse-transcribed into complementary DNA with use of the SuperScript III First-Strand Synthesis system (Life Technologies). A complementary DNA (50-ng) sample was used to perform quantitative real-time polymerase chain reaction (PCR) with use of an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.; Hercules, Calif ) with SYBR® Green (Life Technologies). Invitrogen sheep oligonucleotide primers for reverse transcription polymerase chain reaction (RT-PCR) were purchased from Life Technologies. Polymerase chain reaction was carried out with MCP-1–specific primers (forward: 5′-CAGAAGAGTCACCACCAGCSA-3′; reverse: 5′-AGATGGTTTATGGCGTCCTG-3′), resulting in 170-base pair (bp) fragment and BNP-specif ic primers (forward: 5′-TGGAAGAAACCTGGGACTC-3′; reverse: 5′AGTACCTCCTCAGCACGTTG-3′), resulting in a 195-bp fragment. The ANP-specific primers (forward: 5′-CAGTGAAAGGCCAAAGAAGC-3′; reverse: 5′-CAGAGCCATACACGGGATTT-3′) resulted in a 200-bp fragment. As a reference gene, we used sheep GAPDH primers (forward: 5′-AATGCCTCCTGCACCACCAA-3′; reverse: 5′-TTGGCAGCGCCAGTAGAAGC-3′), resulting in a 198-bp fragment. The PCR was performed with an initial step of denaturation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 20 s. Melt curves were established for the reactions. Normalized fold expression was calculated by using the 2–∆∆Ct method. Entire reaction products underwent electrophoresis on 2% agarose gels. Each experiment was repeated 3 times. Western Blot Analysis
Plasma protein concentration was determined with use of DC protein assay (Bio-Rad). Protein of 8 to 10 µg was separated by 15% to 20% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The gels were transblotted onto a 0.2-µm nitrocellulose membrane with use of the Mini Trans-Blot ® Electrophoretic Transfer Cell (Bio-Rad) for 1 hr at 100V in a cold room. Membranes were blocked with 5% nonfat milk in Tris-buffered saline solution (TBS-T; pH, 7.5) with 0.1% Tween-20 at room temperature for 1 hr and then incubated with primary antibody to a BNPderived synthetic peptide (Biocentra LLC and Alpha
Early Biomarkers in Thrombus-Induced Model of Ischemic Heart Failure
513
Diagnostics Intl., Inc.; both of San Antonio, Texas) (1:5,000 v/v dilution) overnight at 4 °C. The blots were then incubated with secondary antibody (chicken anti-rabbit immunoglobulin G [IgG] conjugated to horseradish peroxidase 1:5,000 v/v dilution) for 1 hr. Finally, signals were detected with an enhanced chemiluminescence kit (Thermo Fisher). For MCP-1, commercial antibodies were used. ELISA for BNP
The BNP concentrations were measured in plasma samples of healthy and heart failure sheep. Several 96-well ELISA plates were coated with 50 µL of BNP peptide (0–2 ng diluted with ELISA coating buffer)— both healthy and heart failure plasma samples—and were kept overnight at 4 °C. As a blocking buffer, 5% nonfat milk in TBS-T was used. One hundred µL of primary antibody to BNP was added to each well in triplicate. The plates were incubated for 3 hr at room temperature. Unbound materials were washed out with TBS with 0.1% Tween. One hundred µL of the secondary antibody (chicken anti-rabbit IgG conjugate to horseradish peroxidase) was added to each well. The plates were incubated for 1 hr at room temperature. After the washing, the activity of horseradish peroxidase was estimated by adding 100 µL of 3,3,5,5-tetramethyl benzidine (TMB) to the wells, and incubation was continued for 30 min at room temperature. The reaction was stopped by using 1N HCl. Optical density at 450 nm was determined by a microplate reader (Bio-Rad). Standard curves were generated by Sigma plot, and the concentrations of different samples were determined from the standard curves. Hematoxylin and Eosin Staining
Formalin-fixed, paraffin-embedded tissue samples of sheep from healthy and experimentally induced failing hearts were used. A section of desired thickness (7–20 µm) was cut with use of a microtome. The tissue sections were deparaffinized and hydrated by using ethanol. Sections were stained with hematoxylin and eosin. Tissue sections were dehydrated by using ethanol, acetone, and xylene. Finally, sections were fixed with mounting fluid and were analyzed under a microscope. Statistical Analysis
Each benchtop-based experiment was performed in triplicate. Data are presented as mean ± SD, with the acceptance of P
