Supplementary Material for: Hemodynamic Assessment of a Murine Heterotopic Biventricularly Loaded Cardiac Transplant in vivo Model

<i>Background:</i> Heterotopic heart transplantation (HHT) in rodent animal models represents an important technique enabling studies on organ transplantation immunology and pharmaceutical development. Recent investigations used nonworking HHT designs, with the left ventricle (LV) bypassed in the anastomosis system. In spite of their principal success, the lack of orthogonal ventricular filling leads to myocardial atrophy. However, when focusing on the cellular and molecular mechanisms involved in the in vivo remodeling of the myocardium or cell-based cardiovascular implants, a nonworking model is suboptimal as it lacks the native-analogous hemodynamic and metabolic situation. Here we present the hemodynamic and electrical assessment of a biventricularly loaded murine HHT method without the need for a combined heart-lung transplantation approach. <i>Methods:</i> Heterotopic transplantations (n = 13) were performed on C57BL/6J-(H-2b) inbred mice (n = 13 donors, n = 13 recipients) by creating end-to-side anastomoses between the donors' cranial vena cava (CrVC) and the recipients' abdominal caudal vena cava (CVC), between the donors' ascending aorta and the recipients' abdominal aorta (aAo), and between the grafts' pulmonary trunk and the left atrium. After transplantation, a hemodynamic assessment using echocardiography (including 2D speckle tracking analysis) and electrocardiography was performed. <i>Results:</i> The loaded HHT procedure in the mice was performed with an overall success rate of 61%. In 3 of the remaining 5 cases, only atrial function was restored. The median duration of the entire surgical procedure for the recipient animal was 190 (IQR 180-250) min. The mean heart rate in the loaded HHT group was 355 ± 6 bpm in comparison to the control group with an in situ heart rate of 418 ± 61 bpm. A native-like closing and opening pattern of the aortic and mitral valves (visible on both 2D and M-mode images) was observed, confirming a native-analogous loading of the LV. Pulsed-wave Doppler provided visualization of the flow across the region of anastomoses between the pulmonary trunk and the left atrium, reaching a mean maximum velocity of 382 ± 12 mm/s. Exemplary 2D speckle tracking analysis of the LV free wall and interventricular septum revealed some differences in vector directions in one animal when compared to the orthotopic native heart, indicating an asynchronous movement of the LV. <i>Conclusions:</i> These results demonstrate the technical (micro)surgical feasibility of a fully loaded HHT procedure in the murine model without using a combined heart-lung transplantation approach. The acute hemodynamic performance of the HHT grafts approximated the native orthotopic situation. This model may open up new options for the investigation of cellular and molecular questions in the murine cardiovascular in vivo system in the near future.