Survival after partial heart transplantation in a piglet model

Partial heart transplantation (PHT) is a novel surgical approach that involves transplantation of only the part of the heart containing a valve. The rationale for this approach is to deliver growing heart valve implants that reduce the need for future re-operations in children. However, prior to clinical application of this approach, it was important to assess it in a preclinical model. To investigate PHT short-term outcomes and safety, we performed PHT in a piglet model. Yorkshire piglets (n = 14) were used for PHT of the pulmonary valve. Donor and recipient pairs were matched based on blood types. The piglets underwent PHT at an average age of 44 days (range 34–53). Post-operatively, the piglets were monitored for a period of two months. Of the 7 recipient piglets, one mortality occurred secondary to anesthesia complications while undergoing a routine echocardiogram on post-operative day 19. All piglets had appropriate weight gain and laboratory findings throughout the post-operative period indicating a general state of good health and rehabilitation after undergoing PHT. We conclude that PHT has good short-term survival in the swine model. PHT appears to be safe for clinical application.


Recipient operation
The recipients were pre-medicated with amiodarone, antibiotics and induction immunotherapy.The recipient piglet underwent the same preoperative preparation and anesthesia induction as the donor.Analgesia was provided with fentanyl.A midline incision was performed from the thyroid cartilage to the xyphoid process.The carotid artery and internal jugular vein were exposed and cannulated for intraoperative hemodynamic monitoring.A median sternotomy was then performed and a pericardial well created for optimal exposure.The recipient was then systemically heparinized and CPB initiated via aortic and right atrial cannulas.The main pulmonary artery was circumferentially dissected, and the native pulmonary valve resected.The donor graft was then positioned anatomically for partial heart transplantation.The distal anastomosis was created, and the graft was filled retrograde to test the valve for competency.Once this was ensured, the proximal anastomosis was completed, and the recipient was weaned off bypass.Hemostasis was obtained, the sternotomy was closed, and multi-modality analgesia was provided followed by extubated.

Postoperative management
Immediately after the surgical procedure, piglets were taken to their recovery cages where they received pain management.Postoperative monitoring involved routine postoperative checks every 2 h for the first 24 h, which were then extended to every 6 h on post-operative day 2 and every 12 h for the remainder of the experiment.The study animals were immunosuppressed using mycophenolate mofetil and tacrolimus, along with methylprednisolone.Serum immunosuppressive agent levels were measured at least twice weekly.Postoperative labs included weekly hematocrit and creatinine.Antibiotics and peptic ulcer prophylaxis were provided with ceftiofur and omeprazole, respectively.A routine echocardiogram was performed postoperatively.Piglets were followed for approximately 2 months until they had doubled in weight after surgery to ensure adequate assessment time (Fig. 1).

Statistical analysis
Categorical variables were presented as percentage (number) and continuous variables were presented as mean (range).Survival analysis was performed using Kaplan-Meier methodology.Statistical analysis was performed using GraphPad Prism (version 10.1.1 for Mac OS, GraphPad Software).

Baseline characteristics
Our cohort consisted of 7 donor piglets and 7 recipient piglets.Piglets were taken to the operating room when they were approximately 10 kg, which is comparable in size to human children undergoing pulmonary valve replacement.Recipient animals comprised of four females and three males.The mean age and weight at time of PHT was 44 days (range 34-53 days) and 11.3 kg (range 9.1-14.0kg), respectively (Table 1).

Morbidity
On average, animals voided and had a bowel movement within 2-6 h of surgery.Normal activity levels and food intake were achieved by post-operative day two.Given that surgical blood loss can be a significant stressor and affect recovery after surgery, hematocrit levels were monitored post-operatively (Fig. 2A).After undergoing PHT, routine labs demonstrated appropriate recovery and maintenance of hematocrit after surgery.Similarly, hemoglobin levels recovered and remained stable throughout the study period (Fig. 2B).Creatinine, a surrogate parameter for renal failure 10 , was trended throughout the study (Fig. 2C).Lastly, white blood cell and platelet counts were monitored closely in the early post-operative period (Fig. 2D,E).Study animals doubled their weight in approximately two months, which is in line with previously described weight trajectories of healthy piglets (Fig. 3) 11 .

Importance of animal model
While there are other large animal models available to examine the safety and feasibility of PHT, we have chosen the swine model for the experiments presented in this study.The swine model is one of the more popular large animal models for experimental congenital cardiac surgery 12,13 .The anatomic structure of the swine heart and its physiology are akin to that of humans, making them an ideal translational model 14 .With these similarities, piglets are well suited for the use of standard pediatric CPB, enabling researchers to perform the same operative procedures that would be done in human neonates.In addition, the piglet's immune system mimics that of humans, allowing for the use of the same immunosuppressive agents across species 15 .Lastly, piglets grow quickly and are able to transition from infancy to adulthood in a matter of months, making them a particularly useful model in congenital heart surgery 8 .While sheep are another model commonly used in translational cardiac research, their anatomic variations from pigs pose technical challenges for cardiac surgery: Firstly, the sternum  in sheep is narrow, making sternotomies more difficult compared to their swine counterparts 16 .Additionally, sheep have a short ascending aorta 16 , making cannulation onto cardiopulmonary bypass complex, often resulting in exposure of the descending aorta for arterial access.Lastly, a fast-growing model is particularly important to study the transition from infancy to adulthood.Sheep tend to have slower rates of growth, requiring studies to have longer follow-up periods in CHD research 17 .

Valve transplantation choice
While PHT can occur using any combination of aortic and pulmonic valves in either position, the pulmonary valve transplanted into its anatomic position was particularly well-suited for the swine model.The more anterior positioning of the pulmonary valve allows for less extensive dissection and minimal cardiac manipulation compared to the aortic valve 18 .Importantly, swine hearts are particularly arrhythmogenic when extensively manipulated 14 .Additionally, by transplanting solely the pulmonary valve, cardioplegic arrest of the heart can be avoided, thus allowing for a quicker recovery.Finally, transplantation of the pulmonary valve is less technically demanding given that reimplantation of coronary buttons is avoided 19 , therefore minimizing the risk for ischemia.For these reasons, we have decided to focus on the pulmonary valve to demonstrate the safety and feasibility of PHT.

Mortality assessment
Out of the 7 recipient piglets that underwent partial heart transplantation, the only mortality occurred during sedation for a routine echocardiogram.Echocardiograms were obtained via a transthoracic approach, which necessitated sedation of the piglets.While under sedation for the echocardiogram, the piglet was no longer able to protect its airway, aspirated, and died shortly afterwards.This incident occurred early in the study protocol and as such, the amount of sedation provided to piglets during echocardiograms was still being optimized.

Risks and expected complications
While PHT is overall safe, there are some risks that can be expected.Although arrhythmias can lead to intraoperative complications, we have been able to mitigate the risk for severe arrhythmias by premedicating with amiodarone.During PHT, the mediastinum must be entered.While care is taken to avoid entering the pleural space, cases in which the pleural space was violated, a chest drain must be placed to prevent pneumothoraxes.Prior to waking the piglets from general anesthesia, care must be taken to ensure the sternum and soft-tissue incision are closed properly.Given that piglets are quadrupeds, the sternum becomes weight-bearing once the piglets begin walking after surgery.If the chest is not closed properly, sternal dehiscence may occur.Similarly, the soft tissue above the sternum must be closed and topped with a layer of skin glue.Animal housing does not allow for a pristine post-operative environment and the incision often comes in contact with fecal matter when the animal lays down.To prevent wound infection, skin glue is used to provide a physical barrier.As piglets are waking from anesthesia, they must be monitored closely to prevent aspiration.In cases of suspected aspiration, animals must be observed for signs and symptoms of pneumonia, which can be treated with antibiotics and antipyretics.Lastly, it is important to diurese the piglets post-operatively since piglets do not tolerate pulmonary edema 20 .

Clinical translation
Our study demonstrates the overall safety and feasibility of PHT in a large animal model.This pre-clinical work led to our center performing the world's first human PHT 21 .As this procedure gains traction in the field of pediatric cardiac surgery, just allocation of donor grafts will become an issue because pediatric donor hearts are a scarce resource.Children in need of a conventional heart transplant have no alternative treatment options as ventricular assist devices suitable for destination therapy are only available at adult size.In contrast, children in need of a PHT who decompensate waiting for a PHT can reasonably be treated with a non-growing homograft.Therefore, children awaiting partial heart transplantation should not compete with patients urgently awaiting a conventional hear transplant.Importantly, evidence suggests that every year, there are pediatric hearts which are not utilized for conventional heart transplant as they are deemed low likelihood for transplant success 22,23 .We estimate that approximately one third of pediatric donor hearts are unsuitable for full organ transplantation and could be used for PHT 24 .Thus, PHT may represent an avenue for improved organ stewardship, by allowing for use of donor hearts that may otherwise be discarded.

Limitations
Our study examined PHT of the pulmonary valve in a piglet model.The pulmonary valve was chosen because it is a technically easier operation.However, the design chosen for this study does not allow for examination of hemodynamic perturbations that can be expected with PHT of the aortic valve.Finally, although the immune systems of pigs are similar to that of humans, there are species-specific differences.As such, the efficacy of the immunosuppressive medication regimen used in human neonates and potential immunomodulatory effects remain not fully understood.

Conclusions
Through the use of a swine large animal model, partial heart transplantation of the pulmonary valve has been shown be a safe and effective alternative to mechanical or biologic valve replacement.Animals undergoing pulmonary valve PHT have demonstrated good short-term survival and outcomes.These results not only demonstrate the feasibility of PHT but also indicate that it can be safely performed in humans.

Figure 1 .
Figure 1.Graphic of experimental design: (A) Harvesting of the donor heart (B) dissection of the pulmonary valve (C) partial heart transplant into recipient (D) growing piglet after transplant.

Figure 3 .
Figure 3. Trend in piglet weight gain across duration of study.

Figure 4 .
Figure 4. (A) Pie chart of mode of death (B) Kaplan-Meier survival analysis of piglets undergoing partial heart transplantation.

Table 1 .
Baseline characteristics of recipient piglets undergoing partial heart transplantation.