Determinación del tiempo de isquemia caliente óptimo en el trasplante cardíaco en donación en asistolia a través del análisis del proceso de lesión del cardiomiocito durante la parada cardiaca en donantes tipo III de Maastricht

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Introduction: Heart transplantation is the gold standard for treating end-stage heart failure. In recent years, our country has developed record donation and transplantation activity, based primarily on the donation of deceased patients in a situation of brain death (BD). However, despite a transplantation activity in 2022 of 46.3 donors and 113.4 procedures per million population (pmp), this activity is not enough to cover the transplantation requirements of our population. Donation after circulatory death (DCD) represents a promising opportunity to overcome the relative shortage of donors for heart transplantation. Nowadays Spain is the only country in the world that transplants all types of organs from DCD donors. In 2022, DCD grew by 38% and already represents 42% of the total number of donors in our country. Including DCD donors could increase the number of heart transplants by 15-30%. However, unlike conventional donation after BD, DCD organs undergo a period of warm ischemia during the withdrawal of life-sustaining therapy (WLST) and between circulatory arrest (CA) and heart procurement. In this context, there is concern about the functionality and quality of the graft. Objectives: The main objective of the study was to analyse the process of cell degradation and cell death in CA from a biochemical point of view and to relate it temporally in order to identify the moment at which irreversible cardiomyocyte injury occurs. The secondary objective was to determine the critical warm ischemia time based on in vivo biochemical changes during DCD procedure of abdominal organs and lungs. Methodology: This is a prospective single-center study conducted in Hospital Clínico Universitario Virgen de la Arrixaca. All consecutive DCD Maastricht type III donors were screened from June 2017 to June 2019. Participants who met all the following inclusion criteria were included: age 18 to 75 years, no structural heart disease and signed informed consent provided by the legal representatives according to donor regulations. DCD non-cardiac donors underwent serial endomyocardial biopsies immediately before WLST, at CA and every 2 min thereafter. Samples were processed into representative pools to assess calcium homeostasis, mitochondrial function and cellular viability. SDS-Polyacrylamide gel electrophoresis (PAGE) and western blots were used to evaluate the phosphorylation of cAMP-dependent protein kinase A (PKA) and phospholamban (PLN), two proteins related to intracellular calcium transients. Myocardial cells at the onset of apoptosis activation were quantified by Caspase 3/7 activation levels, measured using luminescent assays. Mitochondrial function, as a measure of the energy-generating capacity of the isolated myocardium, was assessed by measuring the enzyme activities of complexes I and IV from the mitochondrial electron transport chain. Results: The final sample population of our study comprised 16 DCD Maastricht type III donors (from a total of 56 DCD donors who were handled during the study period: 26 subjects did not meet the inclusion criteria and of the 30 DCD donors who met the inclusion criteria, 14 subjects were excluded for social or logistical reasons). For most DCD donors, the cause of WLST was intracranial hemorrhage. The median time from WLST to CA was 9 min (25th-75th percentile: 7–13 min; range: 4–19 min). Hypoperfusion (systolic blood pressure <60 mmHg) occurred at a median of 5.5 min before CA. It was observed that the levels of the phosphorylated form of the enzyme PKA on its activating residue Thr197 significantly decreased 10 min after CA (p < 0.001). Similarly, the phosphorylation state of PLN on the Ser16 site decreased markedly 10 min after CA compared to the control assay (phosphorylation state assessed at baseline). When we evaluated the state of PLN in its monomeric form (total non-phosphorylated form) the level of accumulation was significantly increased 10 min after CA. Both the combined and isolated activities of complex II and complex IV in the mitochondrial electron transport chain decreased in a time-dependent way 10 min after CA. Caspase 3/7 activity remained unchanged until 10 min after CA, but increased significantly and time-dependently from 11 min after CA. We also performed a sensitivity analysis by analyzing the samples from donors whose times from WLST to CA were above and below the median time (9 min) separately. In this analysis, we found no differences between subjects with shorter (4–8 min) and longer (9–19 min) times in terms of cardiac contractility, mitochondrial dysfunction or apoptotic cell death. Conclusions: The results of this study suggest that myocardial contractility and cellular viability are significantly compromised 10 min after CA. The WLST period before CA did not affect the processes examined here. These findings suggest that, in human cardiomyocytes, the period from WLST to CA and the first 10 min after CA are not associated with a significant and irreversible compromise in cellular function or viability.