a-high-fidelity-porcine-model-orthotopic-heart-transplantation

A High-Fidelity Porcine Model of Orthotopic Heart Transplantation Following Donation after Circulatory Death

The number of advanced heart failure patients who can receive a heart transplant is limited by a shortage of suitable organ donors. In efforts to expand ...

Duke University Medical Center

The porcine intra-abdominal heterotopic heart transplantation model allows for the assessment of immunologic effects on cardiac transplantation without relying on the allograft to maintain hemodynamic support for the animal. Historically, allograft function and histology is monitored by physical exam, echocardiogram evaluation, percutaneous core biopsy, and open biopsy. We performed transvenous endomyocardial biopsies in three pigs that had undergone heterotopic heart implantation. We describe the procedure to be feasible and reproducible, and that histologic results from these biopsies correlated with those from corresponding tissue collected by surgical dissection at the time of allograft explantation. The ability to perform endomyocardial biopsies in the heterotopic heart transplantation model allows for serial non-invasive monitoring of allograft histology.

Transvenous Endomyocardial Biopsy Technique for Intra-abdominal Heterotopic Cardiac Grafts.

Transplantation, the gold standard intervention for organ failure, is a clinical field that is ripe for applications of gene therapy. One of the major challenges in applying gene therapy to this field is the need for a method that achieves consistent and robust gene delivery to allografts. Normothermic  perfusion is a growing organ preservation method and a device for cardiac preservation was recently approved by the Food and Drug Administration (FDA) (Organ Care System, OCS™; TransMedics, Inc., Andover, MA); this device maintains donor hearts in a near physiologic state while they are transported from the donor to the recipient. This study describes the administration of recombinant adeno-associated viral vectors (rAAVs) during  normothermic perfusion for the delivery of transgenes to porcine cardiac allografts. We utilized a myocardial-enhanced AAV3b variant, SASTG, assessing its transduction efficiency in the OCS perfusate relative to other AAV serotypes. We describe the use of normothermic  perfusion to deliver SASTG carrying the Firefly Luciferase transgene to porcine donor hearts in four heterotopic transplant procedures. Durable and dose-dependent transgene expression was achieved in the allografts in 30 days, with no evidence of off-target transgene expression. This study demonstrates the feasibility and efficiency of delivering genes to a large animal allograft utilizing AAV vectors during  perfusion. These findings support the idea of gene therapy interventions to enhance transplantation outcomes.

 Gene Delivery to Porcine Cardiac Allografts Using a Myocardial-Enhanced Adeno-Associated Viral Vector.

Reliable biomarkers for assessing the viability of the donor hearts undergoing  perfusion remain elusive. A unique feature of normothermic  perfusion on the TransMedics® Organ Care System (OCS™) is that the donor heart is maintained in a beating state throughout the preservation period. We applied a video algorithm for an  assessment of cardiac kinematics, video kinematic evaluation (Vi.Ki.E.), to the donor hearts undergoing  perfusion on the OCS™ to assess the feasibility of applying this algorithm in this setting.

Video analysis of  beating hearts during preservation on the TransMedics® organ care system.

The serine/threonine kinase, PINK1, and the E3 ubiquitin ligase, Parkin, are known to facilitate LC3-dependent autophagosomal encasement and lysosomal clearance of dysfunctional mitochondria, and defects in this process contribute to a variety of cardiometabolic and neurological diseases. Although recent evidence indicates that dynamic actin remodeling plays an important role in PINK1/Parkin-mediated mitochondrial autophagy (mitophagy), the underlying signaling mechanisms remain unknown. Here, we identify the RhoGAP GRAF1 (Arhgap26) as a PINK1 substrate that regulates mitophagy. GRAF1 promotes the release of damaged mitochondria from F-actin anchors, regulates mitochondrial-associated Arp2/3-mediated actin remodeling and facilitates Parkin-LC3 interactions to enhance mitochondria capture by autophagosomes. Graf1 phosphorylation on PINK1-dependent sites is dysregulated in human heart failure, and cardiomyocyte-restricted Graf1 depletion in mice blunts mitochondrial clearance and attenuates compensatory metabolic adaptations to stress. Overall, we identify GRAF1 as an enzyme that coordinates cytoskeletal and metabolic remodeling to promote cardioprotection.

GRAF1 integrates PINK1-Parkin signaling and actin dynamics to mediate cardiac mitochondrial homeostasis.

Cardiomyocytes rely on proper mitochondrial homeostasis to maintain contractility and achieve optimal cardiac performance. Mitochondrial homeostasis is controlled by mitochondrial fission, fusion, and mitochondrial autophagy (mitophagy). Mitophagy plays a particularly important role in promoting the degradation of dysfunctional mitochondria in terminally differentiated cells. However, the precise mechanisms by which this is achieved in cardiomyocytes remain opaque. Our study identifies GRAF1 as an important mediator in PINK1-Parkin pathway-dependent mitophagy. Depletion of GRAF1 () in cardiomyocytes results in actin remodeling defects, suboptimal mitochondria clustering, and clearance. Mechanistically, GRAF1 promotes Parkin-LC3 complex formation and directs autophagosomes to damaged mitochondria. Herein, we found that these functions are regulated, at least in part, by the direct binding of GRAF1 to phosphoinositides (PI(3)P, PI(4)P, and PI(5)P) on autophagosomes. In addition, PINK1-dependent phosphorylation of Parkin promotes Parkin-GRAF1-LC3 complex formation, and PINK1-dependent phosphorylation of GRAF1 (on S668 and S671) facilitates the clustering and clearance of mitochondria. Herein, we developed new phosphor-specific antibodies to these sites and showed that these post-translational modifications are differentially modified in human hypertrophic cardiomyopathy and dilated cardiomyopathy. Furthermore, our metabolic studies using serum collected from isoproterenol-treated WT and GRAF1 mice revealed defects in mitophagy-dependent cardiomyocyte fuel flexibility that have widespread impacts on systemic metabolism. In summary, our study reveals that GRAF1 co-regulates actin and membrane dynamics to promote cardiomyocyte mitophagy and that dysregulation of GRAF1 post-translational modifications may underlie cardiac disease pathogenesis.

Article	Year
Transvenous Endomyocardial Biopsy Technique for Intra-abdominal Heterotopic Cardiac Grafts. Journal of cardiovascular translational research\| PubMed ID: 36344902	2023
Gene Delivery to Porcine Cardiac Allografts Using a Myocardial-Enhanced Adeno-Associated Viral Vector. Human gene therapy\| PubMed ID: 36927038	2023
Video analysis of beating hearts during preservation on the TransMedics® organ care system. Frontiers in cardiovascular medicine\| PubMed ID: 37408655	2023
GRAF1 integrates PINK1-Parkin signaling and actin dynamics to mediate cardiac mitochondrial homeostasis. Nature communications\| PubMed ID: 38081847	2023
GRAF1 Acts as a Downstream Mediator of Parkin to Regulate Mitophagy in Cardiomyocytes. Cells\| PubMed ID: 38474413	2024

Ryan T. Gross

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