This study was approved by the Duke University Institutional Animal Care and Use Committee. Female Yucatan pigs (Sinclair Bio Resources, Auxvasse, MO, United States) aged 7–9 months were utilized for this study. Baseline cardiac troponin I values and cardiac magnetic resonance imaging (cMRI) were obtained prior to surgery. In preparation for surgery, the animals were anesthetized and intubated for mechanical ventilation. The donor hearts (n = 6) were procured in a standard fashion through a sternotomy. The hearts were then prepared on a back table and subsequently mounted on an OCS™ and underwent 2 h of NEVP at 34°C–35°C as described by Mendiola Pla et al. (11). During this time, the perfusion parameters of aortic flow, aortic pressure, heart rate, perfusion temperature, venous oxygen saturation (SvO₂), perfusate lactate, and perfusate hematocrit (Hct) were obtained.

Video recordings were obtained using either a Nikon D5600 equipped with a Nikon 18–55 mm f/3.5–5.6G VR lens (Nikon Inc., Melville, NY, United States) or a Canon EOS Rebel T8i equipped with a Canon EFS 18–55 mm lens (Canon, Inc., Melville, NY, United States). Once perfusion of the donor heart was established on the OCS™ and the heart was beating, the camera was positioned approximately 30–40 cm perpendicularly in front of the heart (Figure 1). The distance between the camera and the heart, the focus, lighting, and orientation of the heart remained unchanged during and between recordings once these parameters were established. Serial high-resolution videos were recorded every 15 min during the perfusion period at a recording frequency of 30 frames per second (fps). All hearts were in normal sinus rhythm during the recordings.

Following the ex vivo perfusion and video acquisition, the hearts were cooled to 14°C–16°C, then arrested and removed from the OCS™. The heart was then prepared for transplantation in a standard fashion and transplanted into the recipient pig in an intra-abdominal position with the graft aorta anastomosed to the recipient aorta and the graft pulmonary artery anastomosed to the recipient inferior vena cava (11). Echocardiographic assessments of each transplanted heart were obtained between 2–6 post-operative days.

Cardiac performance on the OCS™ was evaluated using Vi.Ki.E. by extraction of the kinematic parameters every 15 min during spontaneous beating, while monitoring the heart for 2 h (Figure 1). As shown in Supplementary Video S1, a virtual marker was placed on top of the beating heart and followed using a video spot tracker (VST), an open-software (https://cismm.web.unc.edu/resources/software-manuals/video-spot-tracker-manual/) capable of returning the XY coordinates of the marker movement for every cardiac beat. The selected VST kernel followed the heart movement and created the trajectory of contraction and relaxation (Figure 2) in the XY plane.

The coordinates are then analyzed using the Vi.Ki.E. system, which is written in MATLAB programming language (MathWorks, Inc., Natick, MA, United States) and returns the kinematic parameters such as contractility (maximal contraction velocity), cardiac force, energy expenditure, and trajectory perimeter (tissue compliance), as previously described by Rozzi et al. (10).

To investigate whether the kinematic parameters such as contractility (expressed as maximal contraction velocity), contraction force (indicating cardiac fatigue), energy (expenditure of energy during contraction/relaxation), and trajectory perimeter (indication of cardiac compliance) were modified over time, data acquisition was divided into four temporal windows (0–30, 31–60, 61–90, and 91–120 min) (Figure 3).

Data are expressed as mean ± SD. Normality was assessed by the Kolmogorov–Smirnov test. Comparisons were performed using one-way ANOVA with Bonferroni post-hoc test for multiple comparisons. Statistical analyses were performed using GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, CA, United States). The details of the specific test used for each experiment are reported in the figure legends. P-values <0.05 were considered statistically significant.

The baseline cardiac MRI demonstrated no evidence of compromised function in any of the donor hearts with the ejection fraction (EF) measuring >50%. The representative MRI data are shown in Supplementary Figure S1 and Supplementary Video S2. The baseline troponin levels for each pig are shown in Supplementary Table S1. The median cardiac troponin I value was 22 ng/L with an interquartile range from 19 to 31 ng/L. Cardiac troponin I was elevated in only one of the pigs (865 ng/L) for unknown reasons. However, on gross inspection of the heart at the time of sternotomy, there was no evidence of cardiac injury or compromised activity.

The composition of OCS™ perfusate is shown in Supplementary Table S2. It is donor blood-based and supplemented with several additives to maintain near physiologic function of the donor heart throughout the perfusion period. Supplementary Figure S2 demonstrates the OCS™ perfusion parameters measured throughout the perfusion period. The parameters were largely consistent between each of the donor hearts: average aortic flow, 0.61–0.72 L/min; average aortic pressure, 56.2–65.6 mmHg; average heart rate, 56–105 bpm; temperature, 33.9°C; SvO₂, 88.4%–96.0%; and Hct, 18.7%–28.4%. The average total perfusion time was 140 min with a standard deviation of 18 min. Supplementary Figure S3 shows the plotted lactate trends of each heart during perfusion on the OCS, with minimal differences noted between each heart and each remaining within normal limits (<1.5 mmol/L).

While monitoring the heart for 2 h, we did not observe changes in spontaneous beating frequency (Figure 2). This may be attributed to the accommodation of the heart to the new environment. Despite the wide distribution of the data, likely due to differences between each of the hearts, we did not observe significant changes in both contractility and energy parameters over time in any of the hearts. This kinematic parameter ranged from 918.0 ± 430 px/s at the start of perfusion to 1,535 ± 728.5 px/s at the end of perfusion (Figure 3A). Force measurements ranged from 3,776 ± 1,357 (px/s²)/beat at the start of perfusion to 3,350 ± 897.4 (px/s²)/beat at the end of perfusion (Figure 3B). Energy measurements ranged from 6,274 ± 3,240 (px/s)²/beat at the start of perfusion to 16,948 ± 11,262 (px/s)²/beat at the end of perfusion (Figure 3C). Finally, the trajectory perimeter measurements ranged from 199.3 ± 72.52 px/beat at the start of perfusion to 241.7 ± 83.59 px/beat at the end of perfusion (Figure 3D).

This was further assessed and confirmed by linear regression analysis (Figure 4). Contractility and energy showed a slightly increasing slope in the regression lines y = 6.260 × x + 969.8 and y = 91.49 × x + 8,296, respectively. On the other hand, force and trajectory perimeter exhibited a nearly flat regression over time in the regression lines y = 2.937 × x + 3,844 and y = 0.4509 × x + 198.3, suggesting that cardiac fatigue and tissue compliance remained constant over the period of the experiment.

Each heart demonstrated robust biventricular contractility on post-operative echocardiography, which were all consistently performed by MMP. A representative recording is shown in Supplementary Video S3.