The ESP and dp/dtmax decreased significantly in DCD and DCD-DNC compared to Control (Fig. 2). In DCD-HTK-N, ESP and dp/dtmax were significantly higher than in DCD and DCD-DNC. The EDP was significantly increased in DCD-DNC compared to Control (Fig. 2). In DCD-HTK-N, EDP and dp/dtmin were significantly lower than DCD-DNC. In DCD-DNC, dp/dtmin was significantly increased compared to Control. During PCM (Fig. 3), at low perfusion pressures, ESP was significantly higher in DCD-HTK-N and DCD–DNC compared to DCD. Nevertheless, ESP increased in all groups, except DCD-DNC, by increasing perfusion pressures. In DCD-HTK-N, dp/dtmax was significantly higher than in DCD and DCD-DNC, especially at high perfusion pressures. Dp/dtmin showed a comparable course in DCD-HTK-N and Control and was significantly higher in DCD-HTK-N compared to DCD-DNC.

Left-ventricular contractility. After (A) 30 min, (B) 60 min and (C) 120 min of reperfusion. DCD: Donation after circulatory death. DNC: Del Nido cardioplegia. HTK-N: Histidine-tryptophane-ketoglutarate-N. *p < 0.05 and **p < 0.001 vs. Control. #p < 0.05 and ##p < 0.001 vs. DCD. $p < 0.05 and $$p < 0.05 vs. DNC

Pressure-Contractility-Matching during final reperfusion with blood after EVMP with HTK-N or DNC. CF: Coronary flow. DCD: Donation after circulatory death. DNC: Del Nido cardioplegia. EVMP: Ex-vivo machine perfusion. HTK-N: Histidine-tryptophane-ketoglutarate-N. LDP: Laser-Doppler-Perfusion. *p < 0.05 and **p < 0.001 vs. Control. #p < 0.05 and ##p < 0.001 vs. DCD. $p < 0.05 vs. DNC

The total coronary flow was comparable between DCD-HTK-N and –DNC (Fig. 4). Nevertheless, the relative LDP was increased by higher perfusion pressures in DCD-HTK-N compared to DCD-DNC. The coronary arterial compliance C was higher in DCD-HTK-N compared to DCD-DNC.

Myocardial microcirculation during final reperfusion with blood after EVMP with HTK-N or DNC. In DCD-HTK-N, LDP was measured only in N = 7 hearts due to detachment of the probe in one case. CF: Coronary flow. DCD: Donation after circulatory death. DNC: Del Nido cardioplegia. EVMP: Ex-vivo machine perfusion. HTK-N: Histidine-tryptophane-ketoglutarate-N. LDP: Laser-Doppler-Perfusion

In general, in vivo ischemia (DCD) induced a predominant downregulation of transcripts compared to the controls. Subsequently, cardioplegic EVMP reinforces this effect in the myocardium but reverses it in the LAD to different extents. In the myocardium, 14 genes were down- and 4 genes were upregulated in DCD-DNC vs. DCD-HTK-N (Fig. 5). In the LAD, a total of 1355 transcripts were significantly regulated. 34 genes were down- and 1321 genes were upregulated in DCD-DNC compared to DCD-HTK-N. The individual heart samples of each group clustered well together.

Heat maps and volcano plots. (A) Number of regulated genes. (B) Gene expression in the myocardium. (C) Gene expression in the LAD. DCD: Donation after circulatory death. DNC: Del Nido cardioplegia. HTK-N: Histidine-tryptophane-ketoglutarate-N. LAD: Left anterior descending coronary artery

EVMP can treat the heart during transportation. Thus, we investigated how both perfusion solutions affected the gene expression compared to the DCD group. In the myocardium of DCD-DNC, pathways associated with cytokine signaling, cytokine receptor CXCR3, and cytokines such as INFα/β, TNF, IL-6, IL-17, IL-18, IL-23, IL-27 were downregulated compared to DCD. Also, chemokine binding and signaling, the JAK/STAT pathway, and p53 were downregulated. Only CYSLTR1 was significantly upregulated in the myocardium in the DCD-DNC compared to the DCD group.

In the myocardium of DCD-HTK-N hearts, pathways associated with cytokine signaling involving cytokines such as INFα/β, TNF, IL-4, IL-6, IL-17, IL-18, cytokine receptor activation, and inflammatory response pathways in general were also downregulated compared to DCD hearts. Next to the chemokine binding and signaling, the JAK/STAT pathway and p53, pathways associated with ferroptosis, the complement system, and eicosanoid metabolism were additionally downregulated. The miRNA regulation of DNA damage response was upregulated.

In the LAD, pathways involved in the electron transport chain, oxidative phosphorylation, citric acid cycle, and cellular response to starvation or stress were downregulated in the DCD-DNC compared to the DCD group. Upregulated pathways included inflammatory response, cytokines such as IL-2 to IL-7, IL-9, IL-11, IL-12, IL-17, IL-27, TNF, TGFβ, IFNα and INFγ, pathways for cytokine production, such as JAK/STAT, and elements of the immune or cytokine-mediated stress reaction such as the t-cell receptor, AP-1, MAPK signaling, P53. Other upregulated pathways were associated with vascular development, such as VEGF-VEGRFR2 signaling, angiogenesis, extracellular matrix, vascular function, such as cGMP-PKG or development in general, such as Wnt signaling, C-MYC, RAC1, focal adhesion, selenocysteine synthesis, and seleno amino acid metabolism, PI3K-Akt-mTOR, PIP3 activates Akt, FGF signaling and respective receptors FGFR, FGFR2. Additionally, pathways associated with post-translational-protein modification, senescence, and cell death, such as necroptosis and apoptosis, AGE-RAGE, and EPO, were upregulated in the LAD of DCD-DNC compared to the DCD group.

In the LAD of DCD-HTK-N hearts compared to DCD hearts, respiratory electron transport, oxidative phosphorylation, citric acid cycle, cellular response to chemical stress, and selenocysteine synthesis were downregulated. Upregulated pathways were associated with inflammatory response, MAPK signaling, and cytokines such as IL-2, IL-4, IL-6, IL-17, IL-18, TGFβ and its receptor, and NFkB. Additionally, pathways responsible for vascular development, such as VEGFA/VEGFR2, extracellular matrix, focal adhesion PI3K-Akt-mTOR signaling pathway, and Akt signaling, were upregulated. Pathways that counteract development, such as repression of WNT target genes and EGFR tyrosine kinase inhibitor, were also upregulated. Other upregulated pathways were associated with cellular response to stress, HSF-1-dependent transactivation, and HIF-1-alpha transcriptional network.

In light of the different compositions, a manual analysis of the array data was also performed to outline potential component-dependent regulations and highlight possibilities to improve preservation solutions. The prostaglandin-receptor PTGER2 (EP2) transcript was downregulated in the myocardium in HTK-N vs. DNC. PTGFR showed the same tendency. In the LAD, the receptor transcription was not altered. However, PTGIS, a prostaglandin-synthase, adenylyl cyclase 6, and beta-catenin 1 (CTNNB1) transcripts were downregulated as are cAMP-specific and dependent enzymes (PKIA, PRKAG2, PRKAR1A, PRKAR2A, PDE4D, PDE5A). Furthermore, several potassium channels had deregulated transcripts in DNC compared to Control (downregulated: HCN3, KCNA3, KCNA4, KCTD11, KCNK3), compared to DCD (downregulated: KCNK7), compared to Control and DCD (upregulated: SLC12A6, KCTD12, KCNMA1, KCNMB1, KCMF1) and compared to HTK-N (upregulated: KCTD20) which were not significantly regulated in HTK-N vs. Control or DCD. A variety of transcripts for sodium channels were deregulated similarly, with a pronounced upregulation of SCN7A in DNC vs. HTK-N. Furthermore, CLCC1, a chloride channel, was also upregulated in DNC vs. HTK-N, as was its binding partner, calreticulin. Several immune-cell-related transcripts were differently upregulated in HTK-N vs. DNC in the myocardium: CCL5, CCL8, CCL3L1, CD209, and TNF[11]. In contrast, transcripts of selectin P (CD62) and selectin e were downregulated in HTK-N compared to DCD alone and in the case of CD62 also vs. DNC in LAD and myocardium. DNC perfusion led to a more pronounced activated gene response in LAD than HTK-N (76% to 68% vs. Control; 88% to 67% vs. DCD upregulated transcripts). Within this data, biological processes of ubiquitin-mediated protein degradation and vesicular transport were differentially enriched (DNC vs. HTK-N). Of note, 52 transcripts containing"ubiquitin"in the description were regulated in DNC vs. HTK-N in LAD; the only downregulated transcript was ubiquitin B. MYSM1, a deubiquitinase of H2A, was strongly upregulated by DNC in the LAD as compared to HTK-N, but downregulated by HTK-N in the myocardium, if compared to Control. In contrast, the YOD1 deubiquitinase transcript was only regulated in the myocardium, displaying a compensatory upregulation by HTK-N. On the other hand, DNC downregulated the counteracting ubiquitin-ligase TRIM31 and showed a tendency for compensatory upregulation in LAD (HTK-N vs. DNC). A similar pattern could be found for transcripts of kinases, of which 72 were up- and 2 downregulated in DNC vs. HTK-N in LAD and phosphatases, of which 27 were up- and 0 were downregulated.

Figure 6 shows the top 3, or less (if less were identified), transcripts of the LAD and the myocardium that significantly correlated with the leftventricular functional parameters dp/dtmax and dp/dtmin in DCD-HTK-N. CEP128, SFRP1, and BVES in the LAD, and CCL4 and TNF significantly correlated with dp/dtmax. RSP16, SELP, and TALDO1 in the LAD and NFκBID in the myocardium significantly correlated with dp/dtmin.

Top 3 genes that correlate with left-ventricular function after HTK-N perfusion