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Summary

This study presents a surgical porcine model of chronic myocardial ischemia due to progressive coronary artery stenosis, resulting in impaired cardiac function without infarction. Following ischemia, animals undergo off-pump coronary artery bypass graft with epicardial placement of stem cells-derived exosomes-laden collagen patch. This adjunctive therapy improves myocardial function and recovery.

Abstract

Chronic myocardial ischemia resulting from progressive coronary artery stenosis leads to hibernating myocardium (HIB), defined as myocardium that adapts to reduced oxygen availability by reducing metabolic activity, thereby preventing irreversible cardiomyocyte injury and infarction. This is distinct from myocardial infarction, as HIB has the potential for recovery with revascularization. Patients with significant coronary artery disease (CAD) experience chronic ischemia, which puts them at risk for heart failure and sudden death. The standard surgical intervention for severe CAD is coronary artery bypass graft surgery (CABG), but it has been shown to be an imperfect therapy, yet no adjunctive therapies exist to recover myocytes adapted to chronic ischemia. To address this gap, a surgical model of HIB using porcine that is amenable to CABG and mimics the clinical scenario was used. The model involves two surgeries. The first operation involves implanting a 1.5 mm rigid constrictor on the left anterior descending (LAD) artery. As the animal grows, the constrictor gradually causes significant stenosis resulting in reduced regional systolic function. Once the stenosis reaches 80%, the myocardial flow and function are impaired, creating HIB. An off-pump CABG is then performed with the left internal mammary artery (LIMA) to revascularize the ischemic region. The animal recovers for one month to allow for optimal myocardial improvement prior to sacrifice. This allows for physiologic and tissue studies of different treatment groups. This animal model demonstrates that cardiac function remains impaired despite CABG, suggesting the need for novel adjunctive interventions. In this study, a collagen patch embedded with mesenchymal stem cell (MSC)-derived exosomes was developed, which can be surgically applied to the epicardial surface distal to LIMA anastomosis. The material conforms to the epicardium, is absorbable, and provides the scaffold for the sustained release of signaling factors. This regenerative therapy can stimulate myocardial recovery that does not respond to revascularization alone. This model translates to the clinical arena by providing means of physiological and mechanistic explorations regarding recovery in HIB.

Introduction

Globally, severe CAD affects over a hundred million patients, and although the mortality rate has decreased, it remains one of the leading causes of death^1,2. CAD has a wide clinical spectrum from myocardial infarction (MI) to ischemia with preserved viability. Most pre-clinical research focuses on MI, characterized by the presence of infarcted tissue as it is possible to study in small and large animal models. However, that model does not address patients with preserved viability and amenable to revascularization. Most patients undergoing CABG have decreased blood supply and limited function while maintaining variability in contractile reserve and viability³. Without treatment, these patients can progress to advanced heart failure and sudden death, especially during increased workload⁴. Among these patients, coronary artery bypass graft (CABG) is an effective therapy but may not result in complete functional recovery⁵. Importantly, diastolic dysfunction, which is a marker for worse clinical outcomes, fails to recover after revascularization suggesting the need for novel adjuvant therapies during CABG^6,7. Currently, there are no clinically available adjuvant interventions used with CABG to restore cardiomyocytes to full functional capacity. This is a major therapeutic gap given that many patients progress to advanced heart failure despite appropriate revascularization⁸.

An innovative porcine model of chronic myocardial ischemia that is amenable to CABG, to mimic clinical CAD experience was created⁹. Swine provide a good model of heart disease over other large animals as they do not have epicardial bridging collaterals so stenosis of the LAD alone results in regional ischemia¹⁰. In this study, 16-week-old female Yorkshire-Landrace pigs were used. In this model, the LAD was revascularized with off-pump CABG using the left internal mammary artery (LIMA) graft (Supplementary Table 1). Percutaneous coronary intervention (PCI) is not possible to open the stenosis as the constrictor is a rigid device. Cardiac magnetic resonance imaging (MRI) is used to assess global and regional function, coronary anatomy, and tissue viability. Cardiac MRI analysis showed diastolic function, characterized by peak filling rate (PFR) remains impaired despite CABG⁶. The mechanism of diastolic dysfunction likely relates to impaired mitochondrial bioenergetics and collagen formation in HIB that persist following CABG¹¹.

Mesenchymal stem cells (MSC) provide therapeutic signaling through exosomes to improve myocardial recovery when applied during CABG. In this swine model and parallel in vitro studies, it was shown that placement of an epicardial MSC vicryl patch during CABG recovers contractile function with increase in key mitochondrial proteins namely PGC-1α¹², an important regulator of mitochondrial energy metabolism¹³. The in vitro model allowed us to investigate the signaling mechanism of MSCs on impaired mitochondrial function. Exosomes are secreted stable microvesicles (50-150 nm) that contain protein or nucleic acids including microRNA (miRNA)¹⁴. Recent in vitro data suggest that MSC-derived exosomes are an important signaling mechanism necessary for recovery of mitochondrial respiration.

Stem cell derived exosomes are promising adjunctive therapeutics as they are readily accessible, can be commercially produced, and lack ethical conflicts. In consideration of clinical translation, a collagen patch embedded with MSC-derived exosomes was created that can be surgically sutured to the hibernating region of myocardium. It was demonstrated that there is sustained delivery of exosomes using this patch and it provides a cell-free regenerative therapy with paracrine signaling mechanism that targets mitochondrial recovery and enhance mitochondrial biogenesis¹⁵. This procedure provides the pre-clinical model to study the impact of MSC-derived therapies to improve cardiac function by means of enhancing mitochondrial function and reducing inflammation at the time of revascularization and reverse the myocyte adaptations to chronic ischemia.

In this study, a surgical method of off-pump CABG using LIMA to LAD anastomosis to bypass the area of proximal LAD stenosis mimicking the standard treatment for patients with CAD is shown. As an adjunctive therapy with CABG, the surgical application of MSC-derived exosome embedded collagen patch on the ischemic region of the myocardium was demonstrated. This surgical model can be used to study the physiologic responses to the paracrine effect seen with use of an exosome patch as well as the molecular mechanisms of recovery.

Protocol

The Institutional Animal Care and Use Committees (IACUC) of the Minneapolis VA Medical Center and the University of Minnesota have approved all of the animal studies. The current National Institutes of Health (NIH) guidelines for the use and care of laboratory animals were followed.

1. Isolation of mesenchymal stem cells and preparation and characterization of exosomes

1. Isolation of bone marrow derived mesenchymal stem cells (MSCs)
   1. Obtain 30-50 mL of sterile bone marrow from the sternum or tibia of a 20-week-old female Yorkshire-Landrace swine. To do this, introduce a 25 mm 15G interosseous needle into the sternum or tibia and draw the sample into a 60 mL syringe with 10 mL of heparin.
      NOTE: For further details on the collection of bone marrow refer to Pittenger et al. and Hocum-Stone et al.^12,16.
   2. In brief, pass the bone marrow specimen through a Vacutainer CPT tube with heparin for 30 min at 1800 x g.
   3. Remove the buffy coat containing the mononuclear cells and wash with Hank's balanced salt solution. Pellet mononuclear cells by centrifugation and resuspend in growth medium (10% fetal bovine serum [FBS]).
   4. Transfer the mononuclear cells to cell culture flasks for adherent growth. Isolate the MSCs from the mononuclear fraction by their adherent nature.
   5. Wash all non-MSCs within 24 h, leaving a monolayer of MSCs in the tissue culture flask. Confirm the MSC phenotype by flow cytometry, ensuring negativity for CD45, a hematopoietic marker, and positivity for CD90 and CD105, markers of MSCs.
2. Preparation and characterization of exosomes from porcine mesenchymal stem cells
   1. Seed 1 x 10⁴ H9C2 rat cardiomyocytes and culture in 1x DMEM+ 10% FBS and 1x Pen/strep. Seed 2 x 10⁴ porcine MSCs in advanced DMEM + 5% FBS and 1x Pen/strep.
   2. Once both cell lines are at least 80% confluent, change the media to exosome depleted H9C2 and MSC media.
   3. Expose H9C2 cardiomyocytes to mild hypoxia (1% O₂ for 24 h). Remove flasks from hypoxia after 24 h and pipette out H9C2 media.
   4. Remove and discard the MSC media from MSC flask. Add purified H9C2 media to MSC flask. Incubate the flask for 6 h in normoxic conditions (5% CO₂, 20% O₂, and 37 °C).
   5. Extract the exosomes from the co-cultured conditioned media using the total exosome isolation reagent following manufacturer's instructions.
   6. Verify the identification of exosomes by western blot detection of common exosomal proteins with antibodies against CD-63 (1:1000)¹⁷.
   7. Perform nanoparticle tracking analysis (NTA) to quantify the exosomes and the assessment of nanoparticle size and its distribution. To do this, dissolve total protein (50 μg) of exosomes in 500 μL of PBS to determine the concentration and size distribution of exosomes by using nanoparticle tracking analyzer.
   8. Analyze the data using nanoparticle tracking software.

2. Off pump coronary artery bypass graft surgery

1. Animal preparation
   1. Weigh the animal (16-week-old female Yorkshire-Landrace pigs) 3 days before scheduled for surgery. Fast the animal for 12 h before surgery while having access to water during fasting.
   2. Give buprenorphine 0.18 mg/kg via intramuscular route 2-4 h before surgery.
2. Induction of the animal
   1. Sedate the animal by giving intramuscular injection of 6.6 mg/kg tiletamine-zolazepam/xylazine.
   2. Wait for 15 min to ensure adequate sedation by assessing the jaw tone followed by 22G catheter placement in the central ear vein.
      NOTE: Another peripheral vein may be considered (i.e., cephalic vein) if ear vein is inadequate.
   3. Administer ophthalmic ointment topically to each eye. Administer 1-2 mg/kg of propofol via intravenous route to induce general anesthesia. Jaw tone most reliably reflects the depth of anesthesia and should be assessed throughout the procedure.
   4. Intubate the animal with an endotracheal tube of appropriate size.
3. Surgery
   1. Shave the sternum and groin of the animal in preparation for surgical procedure.
   2. Set mechanical ventilation at 10-15 breaths per min, oxygen 1-4 L/min, and isoflurane 1.0-3.0% as needed to maintain deep anesthesia for surgery. Check for absent eye or jaw reflex to confirm deep anesthesia.
   3. Position monitoring equipment (Electrocardiogram, end tidal CO₂, heart rate, Oxygen saturation, blood pressure, and temperature) on the animal.
   4. Connect the IV catheter to a bag of normal saline or lactate ringers' solution to administer maintenance fluids continuously.
   5. Prepare the skin using aseptic technique with povidone Iodine scrub and solution 3x for adequate sterility and to minimize the risk of surgical site infection.
   6. Give Lidocaine via intravascular route (loading dose of 2 mg/kg or continuous infusion at dose of 50 mcg/kg/min) to prevent arrhythmias.
   7. Position the animal dorsally and drape with sterile towels.
   8. Perform either left or right femoral artery cut down for arterial line placement by Seldinger technique followed by connecting the catheter to the transducer for continuous blood pressure monitoring at the time of surgery.
   9. Use monopolar electrocautery to make a 20 cm incision extending from the sternal notch proximally down to the xyphoid process distally, and to incise layers of muscles, subcutaneous fat, and connective tissue down to the sternum.
   10. Perform median sternotomy by using oscillating saw.
       NOTE: Standard saw is avoided for repeat sternotomy as it carries higher risk for myocardial injury from previous pericardial adhesions from left thoracotomy procedure done to place the LAD constrictor.
   11. Divide the posterior sternal plate using a pair of scissors. Use a specialized chest retractor for adequate visualization of the mediastinum.
   12. Dissect adhesions using either monopolar electrocautery or the Metzenbaum scissors. Carefully dissect the peristernal muscle and fat to expose the left internal mammary artery (LIMA).
   13. Once LIMA is exposed lateral to the sternal edge, gently separate it from the chest wall using blunt dissection with electrocautery tip. Use the LIMA as a skeletonized graft.
   14. Start dissection at the level of 3^rd intercostal space. Gently elevate the left sternal border for optimal visualization.
   15. Use gentle traction on the adventitia to expose the arterial and venous branches of LIMA. Clip the LIMA side of the branches using hemoclips and cauterize the chest wall side of the branches.
       NOTE: Care must be taken not to cauterize the clip on the LIMA, because this may cause conduit narrowing.
   16. Once an initial segment of LIMA has been mobilized, continue the dissection proximally toward the level of subclavian vein and distally until the LIMA bifurcation.
   17. Once dissection is finished, administer heparin via intravenous route at a dose of 100-300 U/kg. Wait for 3 min after the heparin is administered.
   18. After 3 min, clip the distal end of the LIMA, just before the level of the LIMA bifurcation, and divide the conduit. Sew the distal end with a free 2-0 silk suture tie.
   19. Prepare the proximal end for grafting. Inspect the flow quality visually by letting the graft bleed for a few seconds.
   20. Gently clamp the distal end of LIMA conduit with an atraumatic bulldog clamp to avoid bleeding. Open the pericardium with an inverted-T making an approximately 5-6 cm incision. Place 3-0 size sutures on the pericardium for traction at both sides of the slit.
   21. Stabilize the LAD with silicone retraction tapes and tissue stabilizer, which is secured to the sternal retractor. Make an arteriotomy in LAD artery distal to the stenosis (caused by constrictor band) with an 11-blade and extend with an iris scissors.
   22. Place an appropriately sized coronary shunt in the LAD. Perform the LIMA to LAD anastomosis with 7-0 running non-absorbable suture using an off-pump bypass technique. Release the bulldog occluder on the LIMA and confirm the hemostasis.
4. Preparation of Mesenchymal Stem Cell (MSC)-derived exosome patch
   1. Following successful isolation of exosomes from MSCs, suspend roughly 3 x 10⁸ exosomes in 3 mL of normal saline and add to collagen sponge.
   2. Bring 3 mL of exosome suspension to room temperature at around 22 °C for 10 min. Place 2 absorbable collage sponges (each 1.27 cm x 2.54 cm) into a medium Petri dish.
   3. Use a 5 mL syringe with an 18G needle to gently mix exosome suspension. Slowly pipette 1.5 mL suspension onto each collagen sponge and wait for 5 min for full absorption.
5. Placement of exosome patch
   1. Place the Exosome-laden sponge upside down onto the hibernating region of the heart, which is the epicardium of the anterior septal region in the distribution of the LAD.
   2. Gently place two sponges to cover the hibernating region of the heart. Use one 3.5 cm x 1.0 cm polyglactin mesh to cover each collagen sponge.
   3. Sew the mesh onto the epicardium with fine 7-0 interrupted sutures.
6. Chest tube placement
   1. Place a chest tube through separate stab incision, near the inferior aspect of the sternotomy incision. Place the chest tube cautiously over the anterior aspect of the heart.
   2. Once the tube is in place, place a purse string suture with 3-0 suture using a horizontal mattress stitch to allow for closure of the wound upon removal of the tube.
   3. The chest tube is maintained until complete chest closure.
7. Chest closure
   1. Approximate the sternum with non-absorbable sutures using a figure eight pattern. Administer 1 mg/kg bupivacaine via intramuscular route along the entire length of the incision.
      NOTE: Suture is used rather than wires to avoid interference with MRI imaging.
   2. Close layers of muscle and skin in the standard fashion using 2-0 and 3-0 absorbable suture, respectively.
   3. Perform a breath hold and suction to evacuate all the air out of the thoracic cavity. Monitor the airway pressure on the ventilator cautiously and maintain the pressure between 15-22 mmHg and release when complete.
   4. Once all the air is evacuated, remove the chest tube while closing the wound using the purse string suture. Apply adhesive glue topically to cover the sternal incision.
8. Post operative care after surgery
   1. Gradually wean the animal off the ventilator as skin incision is being closed. Ensure that the animal is able to spontaneously breathe and protect reflexes before disconnecting the animal from anesthesia equipment.
   2. Remove the endotracheal tube after confirming that animal is able to protect its airway. Cover the skin incision with sterile and non-adherent dressing embedded with antibiotic ointment to minimize surgical site infection.
   3. Continue to monitor vital signs including heart rate, respiratory rate, body temperature every 15 min until animal is able to hold its position without assistance.
   4. Ensure animal is not left unattended until able to lift and hold its head up and can stand without assistance. Administer meloxicam at a dose of 0.2 mg/kg via subcutaneous route before transporting the animal to the recovery unit.
   5. Transport the animal to the recovery unit when animal is stable. Keep the surgical site dressing on the incision until postoperative day 3. Replace the dressing if it becomes soiled.
   6. Continue to monitor the level of the pain, skin incision and overall well-being of the animal for the first 5 days after surgery. Administer half a dose of meloxicam (0.1 mg/kg) as needed once daily for breakthrough pain.
   7. Single house the animal for first 5 days after surgery while the incision(s) heal to reduce the risk of surgical site infection by another animal. Return the animal to group housing after 5 days.
   8. Report any complications or changes in the animal's condition (fever, ascites, weight loss, inappetence etc.) to the veterinarian or appropriate staff.

3. Coronary angiography using femoral access

1. Secure the animal on the operating table in the dorsal recumbency. Initiate mechanical ventilation at 10-15 breaths per min. Set oxygen at 2-4 L/min, isoflurane at 1% and 4%, as needed to maintain a deep plane of anesthesia.
2. Place ECG leads on the animal's limb to monitor for heart rhythm. Evaluate the animal for the depth of anesthesia. Consider the animal deeply anesthetized when the eye or jaw reflex is absent.
3. Clean the chest and neck area with povidone iodine scrub and then drape the animal with towels.
4. Access the femoral artery via surgical cut-down and expose the femoral artery and vein. Make a 1-2 mm longitudinal incision with a no. 11 blade in femoral artery and cannulate the artery using an 11 Fr introducer sheath in the vessel lumen.
5. After obtaining access, advance the catheter to perform coronary angiography to assess the anatomy patency of LIMA-LAD graft.

Representative Results

Following revascularization, coronary angiography is performed to assess for LAD stenosis (greater than 80%) and patency of the LIMA-LAD graft (Figure 1). Four weeks following the revascularization surgery and placement of the exosome-laden collagen patch, cardiac MRI is performed to assess for systolic and diastolic function of the heart at rest and under stress using low-dose dobutamine infusion at 5µg/kg/min. Systolic function is analyzed by measuring wall thickness percentage (wall thickness at end systole – wall thickness at end diastole). Diastolic function is analyzed by measuring peak filling rate over end diastolic volume (PFR/EDV; Figure 2). Delayed contrast imaging was performed to confirm lack of myocardial infarction in the LAD territory. If an infarct in the LAD region is present, it is likely due to the occluded artery secondary to thrombosis caused by the constrictor. Absence of regional wall motion abnormalities demonstrate lack of hibernating phenotype.

At low dose of dobutamine infusion, HIB animals demonstrate significant decrease in diastolic function, measured by PFR/EDV, compared to control group (5.5 ± 0.8 vs. 6.9 ± 1.5, respectively, p < 0.05). CABG group demonstrates a trend towards improvement in PFR/EDV compared to HIB group (6.3 ± 0.9 vs. 5.5 ± 0.8, respectively, p = 0.06). However, CABG + MSC group demonstrate a significant increase in PFR/EDV when compared to HIB group (6.6 ± 1.1 vs. 5.5± 0.8, respectively, p = 0.03; Figure 3). Cardiac MRI was used to confirm lack of necrosis and patency of the Left internal mammary artery (LIMA) to left anterior descending artery (LAD) bypass graft distal to the area of stenosis¹⁸.

At rest, CABG + MSC group does not alter regional systolic function (measured by percentage wall thickness) compared with CABG alone (26.3% ± 7.0% vs. 34.9% ± 6.3%; p = 0.19). Under stress, CABG + MSC group shows significant improvement in regional systolic function compared with CABG alone (78.3% ± 19.6% vs. 39.2% ± 5.6%; p = 0.05)¹² (Figure 4).

At necropsy, appropriately sized coronary dilators were used to ensure the LAD stenosis and LIMA patency. Myocardium was grossly inspected to ensure the tissue viability is present in all regions especially in the ischemic region. Triphenyltetrazolium chloride (TTC) stain confirmed absence of scar.

Figure 1. Cardiac angiogram demonstrating the anatomy. Coronary angiography demonstrates >80% stenosis of proximal LAD artery and patent LIMA-LAD graft anastomosis. Abbreviations: LIMA= Left internal mammary artery, LAD= Left anterior descending Please click here to view a larger version of this figure.

Figure 2. Assessment of diastolic relaxation, global contractile function, and viability using cardiac MRI. (A) Diastolic relaxation: Relationship of left ventricular (LV) volume during a cardiac cycle. The x-axis is time in s; y-axis is volume of left ventricle in mL. The red line indicates peak filling rate (fastest rate at which the LV increases volume). PFR is normalized to the animal's end-diastolic volume (PFR/EDV) to account for size variance across animals. (B) Global contractile function: Segmental circumferential strain (Circ strain) during the cardiac cycle (x-axis: time in ms; y-axis: percentage change in circumferential length of left ventricle segment compared with end-diastolic measurement). Peak circumferential strain is represented by the most negative value of cycle. (C) Representative cardiac MRI image of LAD distribution: LAD distribution is highlighted in red represents the anteroseptal wall. There was no evidence of infarction based on enhanced gadolinium contrast on 4 chamber (D) long axis and (E) short axis views. Abbreviations: LV= left ventricle/ventricular; LAD= left anterior descending; MRI= magnetic resonance imaging. This figure has been modified from ⁶. Please click here to view a larger version of this figure.

Figure 3. MRI assessment of Peak filling rate/End diastolic volume. Diastolic function, measured by PFR/EDV, was compared among four groups (Control, HIB, CABG, and CABG + MSC). At rest, PFR/EDV is comparable among four animal groups. However, under stress using low dose dobutamine infusion (5µg/kg/min), HIB group showed a significant decrease in PFR/EDV when compared to control (p < 0.05) with trend towards improvement in CABG group (p = 0.06) and significant increase in CABG + MSC group (p < 0.05). Statistical analyses were performed using one-way analysis of variance (ANOVA) test. Data are presented as means ± SD. Abbreviations: CABG= Coronary artery bypass graft, PFR= Peak filling rate, EDV= end diastolic volume; MRI= Magnetic resonance imaging, MSC= Mesenchymal Stem Cells, SD= Standard deviation. Please click here to view a larger version of this figure.

Figure 4. MRI assessment of regional systolic function by wall thickening %. Treatment with MSC patch shows improvement in regional cardiac function compared to sham patch. (A) Regional systolic function, measured by wall thickening percentage, does not significantly improve at rest with MSC patch treatment (n = 6) compared with sham (n = 6). (B) Under stress using low dose dobutamine infusion (5µg/kg/min), there is significant improvement in regional systolic function following treatment with the MSC patch compared with sham animals (P<.05). statistical analyses were performed using Mann-Whitney test. Horizontal bars indicate mean standard deviation. *P<.05. Abbreviations: MSC= Mesenchymal stem cells. This figure has been modified from ¹². Please click here to view a larger version of this figure.

Supplementary table 1. Overview of procedures and timeline of each procedure. Please click here to download this File.

Discussion

This study presents the first porcine model of chronically ischemic myocardium, in which it was shown that treatment with an MSC-derived exosome laden collagen patch during surgical revascularization recovers diastolic and systolic function upon inotropic stimulation potentially by targeting mitochondrial recovery. Previously, it was demonstrated that in a large animal model of HIB the diastolic and systolic function, as measured by cardiac MRI, remains impaired and only slightly improves with revascularization without complete recovery^6,19. The dysfunction occurred despite preserved left ventricular ejection fraction. These findings accurately mimic the clinical experience seen in patients with chronic ischemic myocardium in a single vessel territory with preserved left ventricular function.

There are several critical and technical challenges during revascularization surgery especially in the setting of previous thoracotomy. Cardiovascular injury on sternal entry is a risk as the pericardium has already been breached and adhesions may be present. Sternotomy may cause cardiac injury due to proximity or adherence to the sternum. This risk can be mitigated by use of an oscillating saw, which has been shown to promote an uneventful sternal re-entry.

A key aspect to obtain successful CABG is graft quality. Meticulous LIMA harvesting is an important technical aspect in successfully performing high-quality CABG and is associated with improved graft patency. LIMA can be harvested using two techniques: pedicled and skeletonized. Pedicled technique includes dissecting the LIMA from the sternum along with its veins, fascia, fat, and lymphatics. Skeletonized technique includes dissecting the LIMA free of all surrounding tissue, and therefore yielding the artery only²⁰. In this model, the skeletonization technique was implemented as it can minimize sternal ischemia and the graft is longer than a pedicled LIMA²⁰. LIMA is a delicate structure, any undue stretching, clamping, or misplaced clips can result in vascular injury and unsatisfactory results. During dissection, the cautery tip should be used with caution and at low voltage. When separating the artery from its perforating branches, the LIMA side of the branches are clipped using hemoclips. Care must be taken not to cauterize the clips as it can result in narrowing of the conduit. Confirm pulsatile flow prior to grafting.

Ensure that reasonable amounts of anesthesia and paralytics are maintained to minimize movement during the surgery especially while sewing the anastomosis. It is crucial to use the appropriate doses of lidocaine and heparin (200-300 units/kg) to eliminate the risk of arrhythmia and thrombosis, respectively. A second dose of lidocaine might be indicated if the animal experiences any arrhythmias during surgery. Use of femoral arterial line allows continuous hemodynamic monitoring. When performing the anastomosis, it is useful to either put 1-2 surgical sponges behind the heart or place sutures on either side of pericardium to lift the left ventricle upward. In this model, we use silastic tapes and the tissue stabilizer that utilizes suction pressure to effectively immobilize the target site. A mild drop in arterial blood pressure in addition to ST depression on the EKG may be noted once the stabilizer is placed and the heart is lifted. These hemodynamic derangements are usually well tolerated without requiring any interventions. In situations where hemodynamic instability is significant, a dose of phenylephrine (5-20 µg/kg) via intravenous route may be administered to raise the arterial blood pressure. If hemodynamic instability is life threatening, a dose of Epinephrine (0.1 µg/kg; diluted 1:10,000) can be administered via intravenous route as an emergency rescue drug. Once the LAD is exposed with a beaver blade, an arteriotomy is performed with an 11-blade and completed with microsurgical scissors. One must be careful not to injure the posterior wall of the LAD during this maneuver. It is critical to keep the arteriotomy site bloodless during off-pump CABG to allow for accurate suturing and several techniques have been described including intermittent irrigation with saline solution, use of CO₂ blower, and intraluminal coronary shunts²¹. In this study, the CO₂ blower along with an appropriately sized intraluminal coronary shunt were used as both are routinely used in the off-pump CABG operations. A potentially lethal complication of the CO₂ mister blower is an air embolism. However, the risk of air embolism can be negated by using coronary shunt, which can act as a physical barrier within the arteriotomy. Additionally, the use of coronary shunt helps to keep the surgical field bloodless, which permits the use of a lower gas flow and further minimizes the risk of air embolism. Shunts also improve the technical precision for anastomosis and prevent the inadvertent injury to back wall of the artery while suturing²².

In this well-established porcine model, an off-pump rather than on-pump technique during CABG surgery was employed. The advantages of using this technique, instead of the on-pump, is to minimize the operative time and avoid the central cannulation of the aorta and right atrium with full heparinization. In addition, it helps in faster recovery of the animal after surgery by reducing the risk of post-operative bleeding and/or cardiac temponade. These are presumed advantages based on clinical experience in patients undergoing CABG on both on and off pump.

This exosome-laden collagen patch is novel in that it can be quantified and surgically secured to the region of ischemia that has been revascularized. This allows sustained release of exosomes from the patch over the course of several days resulting in continuous and direct treatment of the ischemic region. Histopathology of the hibernating tissue 4 weeks following treatment with CABG and exosome demonstrated lack of inflammatory response of myocardium to the patch itself, although some inflammation was noted at the site of sutures as evident by staining for inflammatory cells. While a variety of methods has been suggested for exosome delivery into the myocardium, common techniques such as direct injection of exosome result in low retention of therapeutic product in the injured area, as up to 90% of exosomes wash away or disperse after injection²³. Analysis of exosomes retention following injection has been completed for up to 3 h post injection and has shown significant decreases in the exosome content²⁴. Exosomes are easy to isolate and have more flexibility in storage conditions over long periods of time, presenting an opportunity for off-the-shelf products that can be used in the acute setting making it more translatable to patients.

This study has several limitations including age and sex of animals. Given surgical and logistic limitations, considerations around animal welfare regulations and staff safety, only juvenile female swine were studied. Although, CABG surgery adds to the complexity of the model, it was a required intervention as other less invasive interventions (percutaneous coronary intervention or PCI) would not allow to open the stenotic region of LAD due to the rigid nature of the constrictor¹⁸. Furthermore, this model of single vessel stenosis without any comorbidities does not fully simulate the extent and effects of long-standing coronary atherosclerosis as seen in human population. Future studies will focus on using a multivessel disease model of hibernating myocardium by surgically placing the constrictor on the circumflex artery and LAD. However, this two-vessel disease model would result in hibernating myocardium with reduced ejection fraction. Animal mortality would likely increase, and revascularization surgically is more complex and requires on-pump bypass support. In future, if there were problems with the practical applications of patch type, other scaffold material options will be explored, such as decellularized extracellular matrix, or alternate form of hydrogels.

Materials

5 Ethibond	Ethicon	MG46G	Suture
# 40 clipper blade	Oster	078919-016-701	Remove hair from surgery sites
0 Vicryl	Ethicon	J208H	Suture
1 mL Syringe	Medtronic/Covidien	1188100777	Administer injectable agents
1" medical tape	Medline	MMM15271Z	Secure wound dressing and IV catheters
1000mL 0.9% Sodium chloride	Baxter	2B1324X	IV replacement fluid
12 mL Syringe	Medtronic/Covidien	8881512878	Administer injectable agents
18 ga needles	BD	305185	Administration of injectable agents
20 ga needles	BD	305175	Administration of injectable agents
20 mL Syringe	Medtronic/Covidien	8881520657	Administer injectable agents
2-0 Vicryl	Ethicon	J317H	Suture
250 mL 0.9% saline	Baxter	UE1322D	Replacement IV Fluid
3 mL Syinge	Medtronic/Covidien	1180300555	Administer injectable agents
3-0 Vicryl	Ethicon	VCP824G	Suture
36” Pressure monitoring tubing	Smith’s Medical	MX563	Connect art. Line  to transducer
4.0 mm ID endotracheal tube	Medline	DYND43040	Establish airway for Hibernation
4-0 Tevdek II Strands	Deknatel	7-922	Suture to secure constrictor around LAD
48” Pressure monitoring tubing	Smith’s Medical	MX564	Connect art. Line  to transducer
500mL 0.9% Sodium chloride	Baxter	2B1323Q	Drug delivery, Provide mist for Blower Mister
6  mL Syringe	Medtronic/Covidien	1180600777	Administer injectable agents
6.0 mm ID endotracheal tube	Mallinckrodt	86049	Establish airway for Revasc,MRI and Termination
6.5 mm ID endotracheal tube	Medline	DYND43065	Establish airway for Revasc,MRI and Termination
6” pressure tubing line	Smith’s Medical	MX560	Collect bone marrow
60 mL Syringe	Medtronic/Covidien	8881560125	Administer injectable agents
7.0 mm ID endotracheal tube	Medline	DYND43070	Establish airway for Revasc,MRI and Termination
7-0 Prolene	Ethicon	M8702	Suture
Advanced DMEM (1X)	ThermoFisher Scientific	12491023
Alcohol Prep pads	MedSource	MS-17402	Skin disinfectant
Amicon Ultra-15 Centrifugal Filter Unit	Millipore Sigma	UFC910024
Anesthesia Machine	Drager	Fabious Trio	maintains general anesthesia
Anesthesia Machine + ventilator	DRE Drager- Fabius Tiro	DRE0603FT	Deliver Oxygen and inhalant to patient
Anesthesia Monitor	Phillips  Intellivue	MP70	Multiparameter for patient safety
Arterial Line Kit	Arrow	ASK-04510-HF	Femoral catheter for blood pressure monitoring
Artificial Tears	Rugby	0536-1086-91	Lubricate eyes to prevent corneal drying
Bair Hugger	3M	Model 505	Patient Warming system
Basic pack	Medline	DYNJP1000	Sterile drapes and table cover
Blood Collection Tubes- green top	Fisher Scientific	02-689-7	Collect microsphere blood samples
Blower Mister Kit	Medtronic/Covidien	22120	Clears surgical field for vessel anastomosis
BODIPY TR Ceramide	ThermoFisher Scientific	D7540
Bone marrow needle- 25mm 15 ga IO needle	Vidacare	9001-VC-005	Collect bone marrow
Bone Wax	Medline	ETHW31G	Hemostasis of cut bone
Bovie Cautery hand piece	Covidien	E2516	Hemostasis
Bupivicaine	Pfizer	00409-1161-01	Local Anesthetic
Buprenorphine 0.3 mg/mL	Sigma Aldrich	B9275	Pre operative Analgesic for survivial procedures
Cell Scrapers	Corning	353085
Cephazolin 1 gr	Pfizer	00409-0805-01	Antibiotic
Chest Tube	Covidien	8888561043	Evacuates air from chest cavity
Cloroprep	Becton Dickenson	260815	Surgical skin prep
Corning bottle-top vacuum Filter System (500mL)	Millipore Sigma	430758
CPT tube	BD	362753	MSC isolation from bone marrow
Delrin Constrictor	U of MN	Custom made	Creates stenosis of LAD
Dermabond	Ethicon	DNX12	Skin adhesive
DMEM (1X) Dulbecco's Modified Eagle Medium, HEPES	ThermoFisher Scientific	12430062
Dobutamine 12.5 mg/mL	Pfizer	00409-2344-01	Increases blood pressure and heart rate during the second microsphere blood collection
ECG Pads	DRE	1496	Monitor heart rhythm
Exosome-Depleted FBS	ThermoFisher Scientific	A2720801
Falcon Disposable Polystyrene Serological Pipets, Sterile, 10mL	Fisher Scientific	13-675-20
Femoral and carotid introducer	Cordis- J&J	504606P	femoral and carotis cannulas
Fetal Bovine Serum, Heat Inactivated, Gibco FBS	ThermoFisher Scientific	16140089
Flo-thru 1.0	Baxter	FT-12100	used to anastomos LIMA to L
Flo-thru 1.25	Baxter	FT-12125	FT-12125
Flo-thru 1.5	Baxter	FT-12150	FT-12150
Flo-thru 2.0	Baxter	FT-12200	FT-12200
GlutaMAX Supplement	ThermoFisher Scientific	35050061
Hair Clipper	Oster	078566-011-002	Remove hair from surgery sites
Helistat collagen sponge	McKesson	570973 1690ZZ	Sponge for embedding exosomes
Heparin	Pfizer	0409-2720-03	anticoaggulant
Histology Jars	Fisher Scientific	316-154	Formalin for tissue samples
HyClone Characterized Fetal Bovine Serum (FBS)	Cytiva	SH30071.03
Hypafix	BSN Medical	4210	Secure wound dressing and IV catheters
Isoflurane	Sigma Aldrich	CDS019936	General Anesthestic- Inhalant
IV Tubing for Blower Mister	Carefusion	42493E	Adapts to IV Fluids for Blower/Mister
Jelco 18 ga IV catheter	Smiths medical	4054	IV access in Revasc, MRI and Term
Lidocaine 2%	Pfizer	00409-4277-01	Local Anesthetic/ antiarrthymic
Ligaclips	Ethicon	MSC20	Surgical Staples for LIMA takedown
Long blade for laryngoscope	DRE	12521	Allows for visualization of trachea for intubation
Meloxicam 5 mg/mL	Boehringer Ingelheim	141-219	Post operative Analgesic
Microsphere pump			Collect blood samples from femoral introducer
Monopolar Cautery	Covidien	Valleylab™ FT10	Hemostasis
Nanosight NS 300	Malvern Panalytical	MAN0541-03-EN
NTA 3.1.54 software	Malvern Panalytical	MAN0520-01-EN-00
OPVAC Synergy II	Terumo Cardiovascular System	401-230	Heart positioner and Stabilizer
Oxygen Tank E cylinder	various	various	Used for Blower Mister if anesthesia machine doesn't have auxiliary flow meter
PBS, pH 7.2	ThermoFisher Scientific	20012050
Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture	ThermoFisher Scientific	15640055
Pigtail 145 catheter 6 French	Boston Scientific	08641-41	Measure LV pressures
Pressure Transducer	various	Must adapt to anesthesia monitor	Monitor direct arterial pressures
Propofol	Diprivan	269-29	Induction agent
Roncuronium	Mylan	67457-228-05	Neuromuscular blocking agent
SR Buprenorphine 10 mg/mL	Abbott Labs	NADA 141-434	Post operative Analgesic
Sterile Saline 20 mL	Fisher Scientific	20T700220	Flush for IV catheters
Sternal Saw/ Necropsy Saw	Thermo Fisher	812822	Used to open chest cavity
Stop Cocks	Smith Medical	MX5311L	2 to connect to pig tail
Succinylcholine 20 mg/mL	Pfizer	00409-6629-02	Neuromuscular blocking agent
Suction  tubing	Medline	DYND50223
Suction Container	Medline	DYNDCL03000
Surgery pack with chest retractor	various	See pack list	Femoral cut down and median sternotomy
Surgical Instruments	various	See pack list	Femoral and carotid cutdowns and sternotomy
Surgical Spring Clip	Applied Medical	A1801	Clamp end of LIMA after takedown
Syringe pump	Harvard		Delivers IV Dobutamine infusion
SYTO RNASelect Green Fluorescent cell Stain – 5 mM Solution in DMSO	Millipore Sigma	S32703
Telazol 100 mg/mL	Fort Dodge	01L60030	Pre operative Sedative
Telpha pad	Covidien	2132	Sterile wound dressing
Timer			Time collection of blood samples
Total Exosome Isolation Reagent (from cell culture media)	ThermoFisher Scientific	4478359
TPP Tissue Culture Flask, T75, Filter Cap w/ 0.22uM PTFE	ThermoFisher Scientific	TP90076
Triple Antibiotic Ointment	Johnson & Johnson	23734	Topical over wound
Vicryl mesh	Ethicon	VKML	Patch for epicardial cell application
Vortex			Mix microspheres
Xylazine 100 mg/mL	Vedco	468RX	Pre operative Sedative/ analgesic