Heterotopic heart transplantation and postoperative assessment

At the end of the perfusion period, the heart was rearrested and removed from the device. The heart was not washed out after the perfusion period. The heart was then prepared for transplantation in a standard fashion and transplanted into the recipient pig in an intra-abdominal position through a laparotomy and an aorto–aortic anastomosis and pulmonary artery–IVC anastomosis between the heart and the recipient²⁰ (Fig. 1). Postoperatively, the pigs received maintenance immunosuppression with methylprednisolone 8 mg/kg daily, cyclosporine 10–20 mg/kg daily, and mycophenolate mofetil 500 mg twice daily. Pigs underwent weekly sedation procedures to obtain complete blood counts (CBC), comprehensive metabolic panels (CMP), and echocardiographic assessment of the allograft. Cyclosporine levels were assessed on a weekly basis to ascertain therapeutic blood levels (100–300 ng/mL). The animals were examined daily by palpation of the graft.

AAV-mediated genetic modification of a donor heart during cardiac transplantation. Gene delivery to a porcine donor heart is achieved during normothermic EVMP by (A) infusing rAAVs into the perfusion circuit. (B) The rAAVs circulate in the perfusate and are trafficked to cardiac cells. (C) The rAAVs are then able to bind to their receptors on the cellular target where they are internalized and transduction ensues leading up to active protein expression of the transgene. The composition of the perfusate can enhance this part of the gene delivery process. (D) After 2-h of EVMP, the transduced heart is then heterotopically transplanted into the abdomen of the recipient pig. AAV, adeno-associated viral vector; EVMP, ex vivo machine perfusion; rAAV, recombinant AAV.

The pigs were survived for 30–35 days post-transplantation. The allograft and native heart were both dissected and arrested simultaneously with del Nido cardioplegia (mixed at Duke Compounding Facility, Durham, NC) solution infusion. Following arrest, both hearts were excised and biopsies of each region (atria, ventricles, septa) subdivided by level (base, mid, apex) were sectioned and preserved by flash freezing, Tissue-Tek optimal cutting temperature (OCT) compound embedding (Sakura, Torrance, CA), or formalin fixation. Biopsies of the left anterior descending artery, lungs, liver, spleen, and psoas muscle were also collected and preserved similarly.