This protocol presents a surgical large animal model of chronic, single vessel ischemia that results in regional abnormalities but does not create infarct, known as hibernating myocardium. Following establishment of chronic ischemia, animals are treated with off-pump LIMA-LAD coronary artery bypass graft surgery to revascularize the ischemic tissue.
Chronic cardiac ischemia that impairs cardiac function, but does not result in infarct, is termed hibernating myocardium (HM). A large clinical subset of coronary artery disease (CAD) patients have HM, which in addition to causing impaired function, puts them at higher risk for arrhythmia and future cardiac events. The standard treatment for this condition is revascularization, but this has been shown to be an imperfect therapy. The majority of pre-clinical cardiac research focuses on infarct models of cardiac ischemia, leaving this subset of chronic ischemia patients largely underserved. To address this gap in research, we have developed a well-characterized and highly reproducible model of hibernating myocardium in swine, as swine are ideal translational models for human heart disease. In addition to creating this unique disease model, we have optimized a clinically relevant treatment model of coronary artery bypass surgery in swine. This allows us to accurately study the effects of bypass surgery on heart disease, as well as investigate additional or alternate therapies. This model surgically induces single vessel stenosis by implanting a constrictor on the left anterior descending (LAD) artery in a young pig. As the pig grows, the constrictor creates a gradual stenosis, resulting in chronic ischemia with impaired regional function, but preserving tissue viability. Following the establishment of the hibernating myocardium phenotype, we perform off-pump coronary artery bypass graft surgery to revascularize the ischemic region, mimicking the gold-standard treatment for patients in the clinic.
Coronary heart disease (CHD) affects an estimated 15.5 million people in the United States 1 and is one of the leading causes of death globally. While the mortality rate associated with CHD has gone down in recent years, the incidence and burden on patients and the health care system remain high 1. Primary treatment of severe CAD is revascularization, which improves survival and reduces angina2,3,4. However, cardiac function often remains depressed, especially under increased workload and can progress to heart failure5,6. Clinical trials of coronary artery bypass surgery (CABG) for chronic ischemia demonstrate improvement in survival and symptoms, but ejection fraction shows only modest improvements of 8-10% 7,8. Our innovative and well-characterized swine model of chronic myocardial ischemia is a model of clinical CAD with progressive vascular stenosis. We have demonstrated reduced myocardial contractility resulting from gradual reduction in blood flow 9. The myocardium does not infarct and can remain viable in this scenario. Recovery is possible, though outcomes are variable even with timely revascularization. Chronically ischemic myocardium that remains viable has been characterized by reduced blood flow and function at rest with retained contractile reserve has been termed HM and treatment requires CABG.Although the revascularization of HM should restore contractile function, experimental and clinical observations demonstrate that the recovery is incomplete8,10.
HM is characterized by the presence of viable yet dysfunctional myocardium in the presence of reduced regional blood flow11. Despite impaired contractility and metabolic activity at rest, HM is able to demonstrate functional and metabolic reserve under inotropic stimulation12. HM is suspected in a majority of patients with CAD, and encompasses a broad spectrum of disease. In this protocol, we demonstrate our established swine model of HM bypassed with left internal mammary artery (LIMA) to LAD artery that mimics the clinical scenario. The swine provide an excellent model of heart disease over other large animals as they do not have epicardial bridging collaterals. This allows the stenosis of the LAD alone to result in regional ischemia13.
Here, we describe the surgical method of inducing hibernating myocardium in swine by creating single-vessel stenosis at the LAD artery. Once chronic ischemia has been established (8 weeks post implant of LAD constrictor), we describe the method of recreating the clinical treatment for HM in our swine model: an off-pump coronary artery bypass graft. These surgical methods can be used to not only study a clinically relevant model of chronic cardiac ischemia, but also to investigate the effects of bypass surgery on cardiac ischemia as well as test potential alternate or adjunctive therapies for cardiac ischemia.
All animal studies were approved by the Institutional Animal Care and Use Committees of the Minneapolis VA Medical Center and the University of Minnesota. Follow current National Institutes of Health guidelines for the use and care of laboratory animals.
1. Hibernation Surgery
2. Revascularization or Off Pump Bypass
Following the initial hibernation surgery, stenosis greater than 70% should be able to be observed by clinical imaging techniques such as angiography or cardiac MRI (Figure 1A). 8 weeks following the surgical application of the LAD constrictor, analysis of regional function by ECHO or cardiac MRI reveals reduced function under increased workload (Figure 2). This can be tested by stimulating the heart with dobutamine (5mg/kg/min), and measuring circumferential strain and regional wall thickening. PET imaging demonstrates decreased blood flow and increased glucose uptake in the hibernating region as compared to the non-LAD territory within the same heart, indicating that the ischemic tissue remains viable. This enhancement in glucose uptake relative to blood perfusion is known as the "flow-metabolism mismatch" and mimics clinical findings of chronically hibernating myocardium4. There is no evidence of infarct by any imaging technique. If an infarct in the LAD region is present, the constrictor was too tight and created a full occlusion of the artery. If no regional abnormalities are evident, a hibernating phenotype has not been achieved.
Following successful coronary artery bypass surgery, animals may show incremental improvements in regional cardiac function both at rest and under inotropic stimulation with dobutamine, though these improvements will not restore function back to normal levels (Figure 3). Successful bypass surgery will eliminate the mortality risk associated with HM (Figure 4). The patent graft can be visualized by either angiography or cardiac MRI (Figure 1B). At necropsy, the LAD stenosis and LIMA patency is confirmed using sized coronary dilators. Myocardium is inspected to confirm viable tissue is present in all regions with infarction.
Figure 1. Cardiac MRI images of stenosis and bypass graft. Cardiac MRI visualizes A) the LAD stenosis following constrictor placement, and B) the LIMA-LAD graft following coronary artery bypass surgery. This figure has been adapted from Hocum Stone, et al.9. Please click here to view a larger version of this figure.
Figure 2. Cardiac MRI measurement of percent wall thickening. Measurement of wall thickening % by cardiac MRI shows impairment of regional function in hibernating regions of the left ventricle. Wall thickening % is significantly decreased in hibernating animals both at rest and under dobutamine infusion as compared to remote regions. Bypassed animals showed improvement in wall thickening % both at rest and under dobutamine infusion. (*=p< 0.05;**=p<0.01 ***=p<0.001) (Hibernation n=12; 1 month revasc n=4; 3 months revasvc n=5) This figure has been adapted from Hocum Stone, et al.9. Data are presented as mean ± SEM. Please click here to view a larger version of this figure.
Figure 3. Regional functional deficit remains present following bypass surgery. Echocardiogram performed 4 weeks following revascularization of hibernating myocardium shows regional functional deficit under inotropic stimulation following bypass surgery. (n=5) Data are presented as mean ± SEM. Please click here to view a larger version of this figure.
Figure 4. Effect of bypass surgery on mortality in HM. Surgical revascularization eliminates mortality risk associated with HM. (Bypass n=18; hibernation n=48). This figure has been adapted from Holley, et al.15 Please click here to view a larger version of this figure.
Here, we show that our swine model of HM accurately mimics the clinical experience of patients with single vessel disease and preserved left ventricular function. Prior to the revascularization, animals with single vessel HM exhibit minimal impairment in global function as measured by ejection fraction, but show significant reduction in regional wall thickening. Following the revascularization, CMR imaging at one or three months of recovery demonstrates preserved viability and graft patency but persistent regional dysfunction, as noted by the estimates of contractile reserve with low dose dobutamine stress testing.
There are several critical steps for the initial hibernation operation. Entering the chest at the third intercostal space allows for the easiest access to the proximal LAD. The retraction of the left atrial appendage with a moist gauze facilitates visualization of the vessel without inducing arrhythmias. The assistance to expose the LAD minimizes the bleeding and secures the constrictor. Chest closure with the Valsalva maneuver to evacuate the air prevents pneumothorax.
There are several critical steps for a successful revascularization procedure. Appropriate depth of anesthesia and use of paralytic agent ensured no movement during the anastomosis portion of the procedure. Use of lidocaine and 200p units/kg Heparin eliminates arrhythmia and thrombosis events. Using a femoral arterial line to maintain appropriate blood pressure monitoring is critical to the hemodynamic stability of the animal. Use a flo-thru device to improve animal stability during the anastomosis and alleviate the need for retraction stabilizing tapes. While sewing the graft, an O2 blower is helpful to visualize the anastomosis.
During the revascularization procedure, if arrhythmia is observed, the animal may require a second dose of lidocaine. If the LAD is difficult to visualize, place the stabilizer after dissection of epicardial fat or fibrinous tissue. Anesthetically, once the stabilizer is placed and the heart is lifted, mild decreases in arterial blood pressure will be noted in addition to ST depression on the ECG. These abnormalities are usually tolerable and do not require intervention. If changes in cardiovascular stability are more dramatic, a dose of phenylephrine (5-20 µg/kg IV) may be given IV to increase arterial blood pressure. Epinephrine (0.1 µg/kg IV; diluted 1:10,000) would be used as an emergency rescue drug if changes are life threatening. Blood loss is replaced with a crystalloid solution IV. A bolus of 100-300 mL of normal saline is used for additional blood pressure support. The LIMA is most easily taken down as a near-skeletonized vessel, but it may be necessary to have papaverine available if a spasm occurs.
Our model uses off-pump surgery rather than on-pump for the revascularization, as this allows us to minimize the operative time and avoid the cannulation of the aorta and right atrium with full heparinization. It also reduces the risk of post-operative bleeding and/or cardiac tamponade, simplifying the animal's recovery. Of note, there is no similar model of coronary bypass surgery in a model of HM in an animal that is then allowed to recover for 30-120 days. These are presumed advantages based on clinical experience in patients undergoing CABG both on and off pump.
This technique can be expanded to involve multiple coronary artery disease by the placement of a constrictor on the circumflex artery at the same time as the LAD or as an alternative vessel. This two-vessel disease model would result in more rapid development of ischemic cardiomyopathy and greater understanding of resultant myocardial adaptions. It is a model that would still allow for ongoing adjunctive interventions including pharmacologic, cell based or mechanical options.
This complex model of the revascularization of HM reflects the clinical difficulty of managing patients with viable but chronically ischemic, dysfunctional myocardium. There is a high prevalence of patients with HM, often presenting with various comorbidities and cardiovascular structural diseases16, and are at risk for sudden cardiac death (SCD)6. The revascularization of viable, impaired myocardium is associated with a 79% reduction in annual death rate3. In fact, we have shown that revascularization of patients with viable hibernating myocardium, as defined by PET imaging, is associated with a greater degree of improvement in LV ejection fraction at 6 weeks following CABG17. In animals with HM, circumferential strain is impaired at baseline, but there is evidence of contractile reserve under inotropic stimulation with a low dose of dobutamine. The presence of contractile reserve is one of the most specific indicators of myocardial viability 18, and the presence of such viability is a predictor of the potential benefit of bypass surgery when present.
Our model is limited by the necessity of using young, healthy animals to create the model of HM. It is necessary to use a young animal to implant the constrictor on the LAD artery as juvenile animals have arteries that are small enough to place the constrictor around without creating immediate stenosis. This model cannot be achieved by beginning with adult pigs, though that would more closely simulate the clinical experience, due to size limitations of both the constrictor, as well as the size of standard surgical and MRI equipment.
An additional limitation is that this animal model of HM only allows the analysis of the effects of a single territory of chronic ischemia, whereas clinical cases are typically far more complex, and may respond differently to the revascularization.
The authors have nothing to disclose.
This work was supported by the VA Merit Review #I01 BX000760 (RFK) from the United States (U.S.) Department of Veterans Affairs BLR&D. The contents of this work do not represent the views of the U.S. Department of Veterans Affairs of the United States Government.
Bair Hugger | 3M | Model 505 | Patient Warming system |
SR Buprenorphine 10 mg/mL | Abbott Labs | NADA 141-434 | Post operative Analgesic |
Surgical Spring Clip | Applied Medical | A1801 | Clamp end of LIMA after takedown |
Arterial Line Kit | Arrow | ASK-04510-HF | Femoral catheter for blood pressure monitoring |
1000mL 0.9% Sodium chloride | Baxter | 2B1324X | IV replacement fluid |
250 mL 0.9% saline | Baxter | UE1322D | Replacement IV Fluid |
500mL 0.9% Sodium chloride | Baxter | 2B1323Q | Drug delivery, Provide mist for Blower Mister |
Flo-thru 1.0 | Baxter | FT-12100 | used to anastomos LIMA to L |
Flo-thru 1.25 | Baxter | FT-12125 | |
Flo-thru 1.5 | Baxter | FT-12150 | |
Flo-thru 2.0 | Baxter | FT-12200 | |
Cloroprep | Becton Dickenson | 260815 | Surgical skin prep |
Meloxicam | Boehringer Ingelheim Vetmedica, Inc. | 0010-6013-01 | NSAID for analgesia |
Hypafix | BSN Medical | 4210 | Secure wound dressing and IV catheters |
IV Tubing for Blower Mister | Carefusion | 42493E | Adapts to IV Fluids for Blower/Mister |
Bovie Cautery hand piece | Covidien | E2516 | Hemostasis |
Chest Tube | Covidien | 8888561043 | Evacuates air from chest cavity |
Monopolar Cautery | Covidien | Valleylab FT10 | Hemostasis |
Telpha pad | Covidien | 2132 | Sterile wound dressing |
4-0 Tevdek II Strands | Deknatel | 7-922 | Suture to secure constrictor around LAD |
Propofol | Diprivan | 269-29 | Induction agent |
long blade for laryngoscope | DRE | 12521 | Allows for visualization of trachea for intubation |
ECG Pads | DRE | 1496 | Monitor heart rhythm |
laryngoscope | DRE | 12515 | |
Anesthesia Machine + ventilator | DRE Drager- Fabius Tiro | DRE0603FT | Deliver Oxygen and inhalant to patient |
5 Ethibond | Ethicon | MG46G | Suture |
0 Vicryl | Ethicon | J208H | Suture |
2-0 Vicryl | Ethicon | J317H | Suture |
3-0 Vicryl | Ethicon | VCP824G | Suture |
7-0 Prolene | Ethicon | M8702 | Suture |
Dermabond | Ethicon | DNX12 | Skin adhesive |
Ligaclips | Ethicon | MSC20 | Surgical Staples for LIMA takedown |
Sterile Saline 20 mL | Fisher Scientific | 20T700220 | Flush for IV catheters |
Telazol 100 mg/mL | Fort Dodge | 01L60030 | Pre operative Sedative |
Triple Antibiotic Ointment | Johnson & Johnson | 23734 | Topical over wound |
6.0 mm ID endotracheal tube | Mallinckrodt | 86049 | Establish airway for Revasc,MRI and Termination |
1" medical tape | Medline | MMM15271Z | Secure wound dressing and IV catheters |
4.0 mm ID endotracheal tube | Medline | DYND43040 | Establish airway for Hibernation |
4.5 mm ID endotracheal tube | Medline | DYND43045 | Establish airway for Hibernation |
5.0 mm ID endotracheal tube | Medline | DYND43050 | Establish airway for Hibernation |
6.5 mm ID endotracheal tube | Medline | DYND43065 | Establish airway for Revasc,MRI and Termination |
7.0 mm ID endotracheal tube | Medline | DYND43070 | Establish airway for Revasc,MRI and Termination |
Bair Hugger Blanket - Large size, underbody | Medline | AUG55501 | Patient Warming system |
Basic pack | Medline | DYNJP1000 | Sterile drapes and table cover |
Bone Wax | Medline | ETHW31G | Hemostasis of cut bone |
Suction tubing | Medline | DYND50223 | |
Suction Container | Medline | DYNDCL03000 | |
1 mL Syringe | Medtronic/Covidien | 1188100777 | Administer injectable agents |
12 mL Syringe | Medtronic/Covidien | 8881512878 | Administer injectable agents |
20 mL Syringe | Medtronic/Covidien | 8881520657 | Administer injectable agents |
3 mL Syinge | Medtronic/Covidien | 1180300555 | Administer injectable agents |
6 mL Syringe | Medtronic/Covidien | 1180600777 | Administer injectable agents |
60 mL Syringe | Medtronic/Covidien | 8881560125 | Administer injectable agents |
Blower Mister Kit | Medtronic/Covidien | 22120 | Clears surgical field for vessel anastomosis |
Roncuronium | Mylan | 67457-228-05 | Neuromuscular blocking agent |
# 40 clipper blade | Oster | 078919-016-701 | Remove hair from surgery sites |
Hair Clipper | Oster | 078566-011-002 | Remove hair from surgery sites |
Bupivicaine | Pfizer | 00409-1161-01 | Local Anesthetic |
Cephazolin | Pfizer | 00409-0805-01 | Antibiotic |
Heparin | Pfizer | 0409-2720-03 | anticoaggulant |
Lidocaine 2% | Pfizer | 00409-4277-01 | Local Anesthetic/ antiarrthymic |
Succinylcholine 20 mg/mL | Pfizer | 00409-6629-02 | Neuromuscular blocking agent |
Anesthesia Monitor | Phillips Intellivue | MP70 | Supports ventilation with inhalant |
Artificial Tears | Rugby | 0536-1086-91 | Lubricate eyes to prevent corneal drying |
Buprenorphine 0.3 mg/mL | Sigma Aldrich | B9275 | Pre operative Analgesic for survivial procedures |
Isoflurane | Sigma Aldrich | CDS019936 | General Anesthestic- Inhalant |
36” Pressure monitoring tubing | Smith’s Medical | MX563 | Connect art. Line to transducer |
48” Pressure monitoring tubing | Smith’s Medical | MX564 | Connect art. Line to transducer |
Jelco 18 ga IV catheter | Smiths medical | 4054 | IV access in Revasc, MRI and Term |
Jelco 20 ga IV catheter | Smiths medical | 4059 | IV access in the MRI |
Jelco 22 ga IV catheter | Smiths medical | 4050 | IV access in Hibernation Procedure |
OPVAC Synergy II | Terumo Cardiovascular System | 401-230 | Heart positioner and Stabilizer |
Sternal Saw/ Necropsy Saw | Thermo Fisher | 812822 | Used to open chest cavity |
Delrin Constrictor | U of MN | Custom made | Creates stenosis of LAD |
Oxygen Tank E cylinder | various | various | Used for Blower Mister if anesthesia machine doesn't have auxiliary flow meter |
Pressure Transducer | various | Must adapt to anesthesia monitor | Monitor direct arterial pressures |
Xylazine 100 mg/mL | Vedco | 468RX | Pre operative Sedative/ analgesic |