ObjectiveThe adoption of hearts from donation after circulatory death (DCD) is a promising approach for the shortage of suitable organs in heart transplantation. However, DCD hearts suffer from serious ischemia/reperfusion injury (IRI). Recent studies demonstrate that nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome-mediated pyroptosis is a novel target to ameliorate myocardial IRI. Melatonin is shown to inhibit NLRP3 inflammasome-mediated pyroptosis. Therefore, this study is designed to verify the hypothesis that melatonin can protect the heart graft preserved with ex vivo heart perfusion (EVHP) against myocardial IRI via inhibiting NLRP3 inflammasome-mediated pyroptosis in a rat model of DCD.MethodsDonor-heart rats were randomly divided into three groups: (1) Control group: non-DCD hearts were harvested from heart-beating rats and immediately preserved with allogenic blood-based perfusate at constant flow for 105 min in the normothermic EVHP system; (2) DCD-vehicle group; and (3) DCD-melatonin group: rats were subjected to the DCD procedure with 25 min of warm ischemia injury and preserved by the normothermic EVHP system for 105 min. Melatonin (200 μmol/L) or vehicle was perfused in the cardioplegia and throughout the whole EVHP period. Cardiac functional assessment was performed every 30 min during EVHP. The level of oxidative stress, inflammatory response, apoptosis, and NLRP3 inflammasome-mediated pyroptosis of heart grafts submitted to EVHP were evaluated.ResultsTwenty five-minute warm ischemia injury resulted in a significant decrease in the developed pressure (DP), dP/dtmax, and dP/dtmin of left ventricular of the DCD hearts, while the treatment with melatonin significantly increased the DP, dP/dtmax of the left ventricular of DCD hearts compared with DCD-vehicle group. Furthermore, warm ischemia injury led to a significant increase in the level of oxidative stress, inflammatory response, apoptosis, and NLRP3 inflammasome-mediated pyroptosis in the hearts preserved with EVHP. However, melatonin added in the cardioplegia and throughout the EVHP period significantly attenuated the level of oxidative stress, inflammatory response, apoptosis, and NLRP3 inflammasome-mediated pyroptosis compared with DCD-vehicle group.ConclusionEVHP combined with melatonin post-conditioning attenuates myocardial IRI in DCD hearts by inhibiting NLRP3 inflammasome-mediated pyroptosis, which might expand the donor pool by the adoption of transplantable DCD hearts.

Objective: Atrial fibroblasts are the main component of atrial fibrosis. Data in previous studies proved the implication of miRNAs in AF progression and the association of miR-205 with cancer associated-fibroblasts, while no evidence supported the implication of miR-205 in atrial fibrosis. Therefore, this study aims to explore the effect and mechanism of miR-205/P4HA3 axis on atrial fibrosis.Methods: Angiotensin II (Ang II) was used to induce atrial fibrosis model in rats, which was verified by H&E staining and Masson staining. qRT-PCR and Western blot were applied to measure the expressions of miR-205, P4HA3, collagen I, and α-SMA. The rat atrial fibroblasts were isolated and then subjected to Ang II treatment or cell transfection for determination of cell biological functions using CCK-8, BrdU assay, TUNEL staining, and cell scratch assay. qRT-PCR and Western blot was applied to analyze the expressions of miR-205, P4HA3, collagen I, α-SMA, JNK, and p-JNK in atrial fibroblasts. Dual-luciferase reporter gene assay and RNA immune-precipitation experiment was employed to verify the binding relationship between miR-205 and P4HA3.Results: Ang II induced rats had disordered arrangement of atrial muscles with uneven nuclear sizes and necrotic atrial myocytes, and increased collagen deposition, in which elevated expressions of P4HA3, collagen I, and α-SMA as well as suppressed expression level of miR-205 were found. In vitro, Ang II treatment in atrial fibroblasts with overexpression of P4HA3 facilitated cellular migration and proliferation, with the induction of JNK signaling pathway. However, these trends were reversed after transfection with miR-205 mimic. P4HA3 is a target gene of miR-205.Conclusion: The miR-205/P4HA3 axis is implicated in atrial fibrosis by inhibition of rat fibroblast proliferation and migration and the inactivation of JNK signaling pathway.

Background: In heart transplantation, the adoption of hearts from donation after circulatory death (DCD) is considered to be a promising approach to expanding the donor pool. Normothermic ex situ heart perfusion (ESHP) is emerging as a novel preservation strategy for DCD hearts. Therefore, pre-clinical animal models of ESHP are essential to address some key issues before efficient clinical translation. We aim to develop a novel, reproducible, and economical rat model of DCD protocol combined with normothermic ESHP.Methods: Circulatory death of the anesthetized rats in the DCD group was declared when systolic blood pressure below 30 mmHg or asystole was observed after asphyxiation. Additional 15 min of standoff period was allowed to elapse. After perfusion of cold cardioplegia, the DCD hearts were excised and perfused with allogenic blood-based perfusate at constant flow for 90 min in the normothermic ESHP system. Functional assessment and blood gas analysis were performed every 30 min during ESHP. The alteration of DCD hearts submitted to different durations of ESHP (30, 60, and 90 min) in oxidative stress, apoptosis, tissue energy state, inflammatory response, histopathology, cell swelling, and myocardial infarction during ESHP was evaluated. Rats in the non-DCD group were treated similarly but not exposed to warm ischemia and preserved by the normothermic ESHP system for 90 min.Results: The DCD hearts showed compromised function at the beginning of ESHP and recovered over time, while non-DCD hearts presented better cardiac function during ESHP. The alteration of DCD hearts in oxidative stress, apoptosis, tissue energy state, histopathological changes, cell swelling, and inflammatory response didn't differ among different durations of ESHP. At the end of 90-min ESHP, DCD, and non-DCD hearts presented similarly in apoptosis, oxidative stress, inflammatory response, myocardial infarction, and histopathological changes. Moreover, the DCD hearts had lower energy storage and more evident cell swelling compared to the non-DCD hearts.Conclusion: We established a reproducible, clinically relevant, and economical rat model of DCD protocol combined with normothermic ESHP, where the DCD hearts can maintain a stable state during 90-min ESHP.