The growth of xenotransplanted hearts can be reduced with growth hormone receptor knockout pig donors

Abstract

Objective: Genetically engineered pigs are thought to be an alternative organ source for patients in end-stage heart failure unable to receive a timely allograft. However, cardiac xenografts exhibit growth and diastolic heart failure within 1 month after transplantation. Grafts function for up to 6 months, but only after administration of temsirolimus and afterload-reducing agents to reduce this growth. In this study we investigated the growth and hemodynamics of growth hormone receptor (GHR) knockout xenografts, without the use of adjuncts to prevent intrinsic graft growth after transplantation.

Methods: Genetically engineered pig hearts were transplanted orthotopically into weight-matched baboons between 15 and 30 kg, using continuous perfusion preservation before implantation (n = 5). Xenografts included knockout of carbohydrate antigens and knockin of human transgenes for thromboregulation, complement regulation, and inflammation reduction (grafts with intact growth hormone, n = 2). Three grafts contained the additional knockout of GHR (GHR knockout grafts; n = 3). Transthoracic echocardiograms were obtained twice monthly and comprehensively analyzed by a blinded cardiologist. Hemodynamics were measured longitudinally after transplantation.

Results: All xenografts demonstrated life-supporting function after transplantation. There was no difference in intrinsic growth, measured using septal and posterior wall thickness and left ventricular mass, on transthoracic echocardiogram out to 1 month in either GHR knockout or GHR intact grafts. However, hypertrophy of the septal and posterior wall was markedly elevated by 2 months post transplantation. There was minimal hypertrophy out to 6 months in GHR knockout grafts. Physiologic mismatch was present in all grafts after transplantation, which is largely independent of growth.

Conclusions: Xenografts with GHR knockout show reduced post-transplantation xenograft growth using echocardiography >6 months after transplantation, without the need for other adjuncts.

Keywords: allotransplantation; cardiac xenotransplantation; diastolic heart failure; echocardiography; growth hormone; heart failure; heart transplantation; hypertrophy; organ growth; physiologic mismatch.

Figure 1-. Study Overview.

Life supporting pig-to-baboon cardiac xenotransplantations were performed from genetically engineered (GE) pig donors after procurement of xenografts using non-ischemic cardiac preservation. After transplantation, echocardiographic examination was performed and invasive implanted telemetric data was obtained. Anti-CD40 monoclonal antibody (mAb)-based immunosuppression was utilized. Human transgenes in GE pigs are categorized by thromboregulatory, complement regulation and anti-inflammatory proteins. GGTA1= α1,3-galactosyltransferase, β4GalNT2=β1,4-N-acetylgalactosyltransferase, CMAH= CMP-N-acetylneuraminic acid hydroxylase, TBM=thrombomodulin, EPCR=endothelial protein C receptor, DAF=decay accelerating factor, HO1=hemeoxygenase.

Figure 2-. Overview of xenograft assessment by TTE.

Septal thickness, posterior wall thickness and left ventricular (LV) dimension in end-diastole were measured longitudinally over time after pig-to-baboon orthotopic cardiac xenotransplantation. LV mass was calculated using interventricular septum (IVS), left ventricular internal diameter (LVID) and posterior wall thickness (PWT). IVS and LVID were derived from septal thickness and LV dimension in end-diastole, respectively. Depicted in short axis here, in actuality measurements conducted in parasternal long-axis views.

Figure 3-. Xenograft measurements post-transplantation by TTE.

Septal thickness, LV dimension in end-diastole, relative wall thickness (rWT), posterior wall thickness, LV Mass and Ejection Fraction were measured longitudinally after transplantation. LV Mass percent change was calculated by the average LV Mass in the 30 days after transplantation and 30 days prior to euthanasia or 6 months post-transplantation, whichever came first. POD=post-operative day, LV=left ventricle, TTE=transthoracic echocardiography. Blue=growth hormone receptor knockout, red=growth hormone receptor intact xenografts.

Figure 4-. Histology between Growth Hormone Receptor Intact and Knockout Grafts.

a) B32988, septum 20X- significant fibrosis and myocyte degeneration b) B33121, left ventricle 20X- congestion, interstitial hemorrhage, myodegeneration c) B32863, left ventricle 40x- normal histology after euthanasia and explanation at 182 days after transplantation d) B32651 left ventricle 25x- focal areas of early ischemia as demonstrated by myofiber degeneration and infiltration of macrophages.

Figure 5-. Hemodynamics in xenografts grafts after xenotransplantation.

Heart rate (HR) and blood pressure were measured from an implanted telemetry device in the subxiphoid space. Average naïve baboon and piglet HR and blood pressures are noted by dashed lines, which was referenced from a prior study in anesthetized baboons (HR, baboon: 93±13, piglet: 108±22; systolic pressure, baboon: 124±24, piglet: 83±11; diastolic pressure, baboon: 79±20, piglet: 47±12; mean arterial pressure, baboon: 99±22, piglet: 62±11). B32988 had a malfunctioning aortic pressure sensor and thus systolic, diastolic and mean arterial pressures were derived from a non-invasive cuff upon scheduled routine sedation and examination.

Figure 6-. Average hemodynamics in xenografts grafts after xenotransplantation.

a-d) No statistical differences existed between growth hormone receptor knockout and intact xenografts after xenotransplantation. e-f) There is significant physiologic mismatch between transplanted xenografts before and after implantation from pig donors to baboon recipients as depicted by increase in HR and MAP (a measure of afterload experienced by the xenograft). Before=the HR and MAP just after induction of anesthesia for procurement of the cardiac xenograft from the pig donor. After=Average HR and MAP over the survival of the recipient. Mean SBP, DBP, MAP and HRs were calculated and then compared by Student’s t-test. SBP=systolic blood pressure, DBP=diastolic blood pressure, MAP=mean arterial pressure, ns=not significant at a p-value of 0.05, *= p-value<0.05, **= p-value<0.01. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median.

Figure 7-. Graphical Abstract.

Post-transplantation xenograft growth is a life-limiting phenomenon seen in recipients after cardiac xenotransplantation. In this study, genetically engineered hearts expressing multiple human transgenes, with or without intact growth hormone receptor, were transplanted orthotopically into weight-matched baboons after continuous perfusion preservation. Post-transplantation xenograft growth was investigated by echocardiography and physiologic mismatch was characterized. Results indicate markedly elevated heart rates and mean arterial pressures experienced by all transplanted xenografts. Growth hormone intact grafts demonstrated marked post-transplantation xenograft growth compared to those with growth hormone receptor knocked out. This study demonstrates reduced xenograft growth after transplantation by using xenografts with multiple human transgenes and growth hormone receptor knockout. Implications include the possible elimination for the need of adjuncts to reduce post-transplantation cardiac xenograft growth.

Central Picture:

Modulation of growth after xenotransplantation from pig donors with genetic engineering

Central Message:

Cardiac growth after xenotransplantation can be reduced by genetically engineering pig donors with growth hormone receptor knockout as measured by transthoracic echocardiography.