off-pump coronary artery bypass graft surgery for preparing a porcine model of chronic myocardial ischemia

Adjunctive Exosome Therapy: Enhancing Myocardial Recovery Post-CABG Surgery

Globally, severe CAD affects over a hundred million patients, and although the mortality rate has decreased, it remains one of the leading causes of death1,2. 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 CABG6,7. 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-1&#945;12, 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.

Author Spotlight: Enhancing Coronary Artery Revascularization

Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft

The overall goal of this surgical procedure is to create a surgical model of coronary artery revascularization using LIMA to LAD bypass, and the use of exosome-laden collagen patch as an adjunct to demonstrate physiological improvement in cardiac function. Our in-vivo model is a single-vessel model that does not include any comorbidities, which is not representative of the typical clinical situation for patients undergoing treatment for multi-vessel disease. However, despite this limitation, our model has consistently produced reliable results and has maintained low mortality rates.We developed a clinically relevant adjuvant therapy to be used during CABG and aim to understand the cellular mechanisms driving the myocardial functional improvement in chronic myocardial ischemia. Complete recovery of ischemic tissue appears to hinge on restoration of mitochondrial capabilities and reduced inflammation;representing a novel area of study.

Off-Pump Coronary Artery Bypass Graft Surgery for Preparing a Porcine Model of Chronic Myocardial Ischemia

To begin, position the anesthetized and intubated animal on its back and cover it with sterile towels. Use monopolar electrocautery to make a 20 centimeter incision, extending from the sternal notch proximally down to the xiphoid process distally, and to incise layers of muscles, subcutaneous fat and connective tissue down to the sternum. Then carry out a medium sternotomy, employing an oscillating saw.For efficient visualization of the mediastinum, use a specialized chest retractor. Dissect adhesions using monopolar electrocautery or Metzenbaum scissors. Then expose LIMA by dissecting the parasternal muscle and fat.Next, start the dissection of the LIMA by elevating the left sternal border to ensure optimal visualization. Apply gentle traction to the adventitia and expose the arterial and venous branches of the LIMA. Employ blunt dissection using electrocautery tip to carefully separate it from the chest wall.Use hemo clips on the branches and cauterize the chest wall side of these branches. Then clip the distal end of the LIMA just before the point of bifurcation and divide the conduit. Secure the distal end with a freely tied 2-O silk suture.After preparing the proximal end for grafting, evaluate the flow quality manually by allowing the graft to bleed briefly. Now clamp the distal end of the LIMA conduit with an atraumatic bulldog clamp to prevent bleeding. Stabilize the left anterior descending, or LAD artery, using silicone retraction tape and a tissue stabilizer that's secured to the sternal retractor.Perform an arteriotomy in the LAD artery distal to the stenosis, using an 11 blade, and extend it with iris scissors. Position an appropriately sized coronary shunt in the LAD artery. Conduct the LIMA to LAD anastomosis using a 7-O running, non-absorbable suture in an off-pump bypass technique.Take a three millimeter exosome suspension and use a five milliliter syringe equipped with an 18 gauge needle to mix it. Then gradually pipette 1.5 milliliters of this suspension onto two absorbable collagen sponges, placed in a medium sized Petri dish. To target the hibernating region of the heart, place the exosome sponge upside down on the epicardium of the anterior septal region in the distribution of the LAD artery.Carefully arrange two sponges to fully cover the hibernating region. Use a polyglactin mesh on each collagen sponge to ensure proper coverage and sew the mesh onto the epicardium with fine 7-O interrupted sutures. Make a separate stab incision near the inferior aspect of the sternotomy incision and place the chest tube carefully on the anterior aspect of the heart.Approximate the sternum using non-absorbable sutures, employing a figure eight pattern. Close layers of muscle and skin using 2-O and 3-O absorbable sutures, respectively, following the standard procedure. After evacuating the chest cavity with a chest tube, seal the wound using a purse string suture.Once the wound is closed, remove the chest tube. At rest, the peak filling rate over end diastolic volume is compared among four animal groups. In low dose dobutamine infusion, the group with hibernating myocardium had a decrease in peak filling rate.The coronary artery bypass graft surgery group showed improvement, while the coronary artery bypass graft, plus mesenchymal stem cell group, saw a significant increase. MRI analysis revealed that coronary artery bypass graft, plus mesenchymal stem cell group does not alter regional systolic function at rest. However, under stress this group showed significant improvement in regional systolic function compared to coronary artery bypass graft.

off-pump-coronary-artery-bypass-graft-surgery-for-preparing-porcine

Research

JoVE Journal

Medicine

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.

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.

Chronic myocardial ischemia resulting from progressive coronary artery stenosis leads to hibernating myocardium (HIB), defined as myocardium that adapts ...