a-heterotopic-rat-heart-transplantation-model-using-circulatory-death

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There ...

Virginia Commonwealth University

The control of p53 protein stability is critical to its tumor suppressor functions. The CREB binding protein (CBP) transcriptional co-activator co-operates with MDM2 to maintain normally low physiological p53 levels in cells via exclusively cytoplasmic E4 polyubiquitination activity. Using mass spectrometry to identify nuclear and cytoplasmic CBP-interacting proteins that regulate compartmentalized CBP E4 activity, we identified deleted in breast cancer 1 (DBC1) as a stoichiometric CBP-interacting protein that negatively regulates CBP-dependent p53 polyubiquitination, stabilizes p53, and augments p53-dependent apoptosis. TCGA analysis demonstrated that solid tumors often retain wild-type p53 alleles in conjunction with DBC1 loss, supporting the hypothesis that DBC1 is selected for disruption during carcinogenesis as a surrogate for p53 functional loss. Because DBC1 maintains p53 stability in the nucleus, where p53 exerts its tumor-suppressive transcriptional function, replacement of DBC1 functionality in DBC1-deleted tumors might enhance p53 function and chemosensitivity for therapeutic benefit.

DBC1 Regulates p53 Stability via Inhibition of CBP-Dependent p53 Polyubiquitination.

Heart transplantation is the ultimate treatment option for patients with advanced heart failure. Since hearts from donation after brain death (DBD) donors are limited, donation after circulatory death (DCD) donor hearts could be another source for heart transplantation. DCD process involves ischemia-reperfusion (IR) injury. Mitochondrial dysfunction contributes to IR and is well established in the  (buffer perfused) ischemia animal model. However, DCD hearts undergo  ischemia with a variable "ischemic period." In addition, the DCD hearts are exposed to an intense catecholamine surge that is not seen with  perfused hearts. Thus, the severity of mitochondrial damage in  ischemia hearts could differ from the  ischemia hearts even following the same period of ischemia. The aim of our current study is to identify the mitochondrial dysfunction in DCD hearts and propose strategies to protect mitochondria. Adult Sprague Dawley rat hearts underwent  or  ischemia for 25 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from hearts following ischemia. We found that both  and  ischemia led to decreased oxidative phosphorylation in SSM and IFM compared to time control or DBD hearts. The proportion of damage to SSM and IFM, including proton leak through the inner membrane, was higher with  ischemia compare to  ischemia. Time control hearts showed a decrease in SSM and IFM function compared to DBD hearts. The calcium retention capacity (CRC) was also decreased in SSM and IFM with  and  ischemia, indicating that ischemic damage to mitochondria sensitizes mitochondrial permeability transition pores (MPTP). Our study found differential mitochondrial damage between the  ischemia and the  ischemia setup. Therefore, consideration should be given to the mode of ischemia while evaluating and testing myocardial protective interventions targeting mitochondria to reduce IR injury in hearts.

The Commonalities and Differences in Mitochondrial Dysfunction Between  and  Myocardial Global Ischemia Rat Heart Models: Implications for Donation After Circulatory Death Research.

The ultimate treatment for patients with end-stage heart failure is heart transplantation. The number of donor hearts which are primarily procured from donation after brain death (DBD) donors is limited, but donation after circulatory death (DCD) donor hearts can increase the heart donor pool. However, ischemia and reperfusion injuries associated with the DCD process causes myocardial damage, limiting the use of DCD hearts in transplantation. Addressing this problem is critical in the exploration of DCD hearts as suitable donor hearts for transplantation. In this study, rat hearts were procured following the control beating-heart donor (CBD) or DCD donation process. Changes in mitochondria and cardiac function from DCD hearts subjected to 25 or 35 minutes of ischemia followed by 60 minutes of reperfusion were compared to CBD hearts. Following ischemia, rates of oxidative phosphorylation and calcium retention capacity were progressively impaired in DCD hearts compared to CBD hearts. Reperfusion caused additional mitochondrial dysfunction in DCD hearts. Developed pressure, inotropy and lusitropy, were significantly reduced in DCD hearts compared to CBD hearts. We, therefore, suggest that interventional strategies targeted before the onset of ischemia and at reperfusion could protect mitochondria, thus potentially making DCD hearts suitable for heart transplantation.

Ischemia and reperfusion injury to mitochondria and cardiac function in donation after circulatory death hearts- an experimental study.

Heart transplantation is a lifesaving procedure, which is limited by the availability of donor hearts. Using hearts from donors after circulatory death, which have sustained global ischemia, requires thorough studies on reliable and reproducible models that developing researchers may not have mastered. By combining the most recent literature and our recommendations based on observations and trials and errors, the methods here detail a sound in vivo heterotopic heart transplantation model for rats in which protective interventions on the ischemic heart can be studied, and thus allowing the scientific community to advance organ preservation research. Knowledge gathered from reproducible animal models allow for successful translation to clinical studies.

Refining murine heterotopic heart transplantation: A model to study ischemia and reperfusion injury in donation after circulatory death hearts.

Donation after circulatory death (DCD) hearts requires machine perfusion preservation, the conditions of which are not well defined.

Article	Year
DBC1 Regulates p53 Stability via Inhibition of CBP-Dependent p53 Polyubiquitination. Cell reports\| PubMed ID: 30893604	2019
The Commonalities and Differences in Mitochondrial Dysfunction Between and Myocardial Global Ischemia Rat Heart Models: Implications for Donation After Circulatory Death Research. Frontiers in physiology\| PubMed ID: 32714203	2020
Ischemia and reperfusion injury to mitochondria and cardiac function in donation after circulatory death hearts- an experimental study. PloS one\| PubMed ID: 33370296	2020
Refining murine heterotopic heart transplantation: A model to study ischemia and reperfusion injury in donation after circulatory death hearts. Animal models and experimental medicine\| PubMed ID: 34557655	2021
Assessment of machine perfusion conditions for the donation after circulatory death heart preservation. Artificial organs\| PubMed ID: 35167122	2022

Oluwatoyin Akande

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