Critical warm ischemia time point for cardiac donation after circulatory death.

Donation after circulatory death (DCD) represents a promising opportunity to overcome the relative shortage of donors for heart transplantation. However, the necessary period of warm ischemia is a concern. This study aims to determine the critical warm ischemia time based on in vivo biochemical changes. Sixteen DCD non-cardiac donors, without cardiovascular disease, underwent serial endomyocardial biopsies immediately before withdrawal of life-sustaining therapy (WLST), at circulatory arrest (CA) and every 2 min thereafter. Samples were processed into representative pools to assess calcium homeostasis, mitochondrial function and cellular viability. Compared to baseline, no significant deterioration was observed in any studied parameter at the time of CA (median: 9 min; IQR: 7-13 min; range: 4-19 min). Ten min after CA, phosphorylation of cAMP-dependent protein kinase-A on Thr197 and SERCA2 decreased markedly; and parallelly, mitochondrial complex II and IV activities decreased, and caspase 3/7 activity raised significantly. These results did not differ when donors with higher WLST to CA times (≥9 min) were analyzed separately. In human cardiomyocytes, the period from WLST to CA and the first 10 min after CA were not associated with a significant compromise in cellular function or viability. These findings may help to incorporate DCD into heart transplant programs.Fundación Mutua Madrileña.S

Characteristics of the nicorandil-mediated protection against doxorubicin-induced ROS.

<p>Rates of ROS production were measured at 37°C in Tyrode medium following the DCF accumulation when the process was started by adding 5 μM doxorubicin. (A) Dependence on nicorandil concentration when added in preincubation for 30 min before measurements (●) or when the preincubation with nicorandil included 100 μM <i>L</i>-NAME (○) or 1 μM KT5823 (□). (B) Dependence on 5-HD when added in preincubation with 100 μM nicorandil for 30 min before measurements (●). (C) Fluorescence confocal images from live cells once loaded with 10 μM H₂-DCFDA and exposed to 5 μM doxorubicin for 30 min. Control images of DCF production (green) induced by doxorubicin (red) (<i>upper row</i>). Effect of 100 μM nicorandil (Nic) when added in preincubation for 30 min before exposure to doxorubicin (<i>middle row</i>). Effect of 100 μM 5-HD when added in preincubation with 100 μM nicorandil for 30 min before the 30 min treatment with doxorubicin (<i>lower row</i>). Superimposed images from the corresponding left and middle panels are shown on the right. (D) Red fluorescence was measured by confocal microscopy when plated cells were exposed to 5 μM doxorubicin for 30 min before or after 30 min preincubation with different nicorandil concentrations. Plotted data are average of mean fluorescence ± SEM in 20 μm² cytoplasmic regions (<i>n</i> = 20) from randomly selected cell fields after background subtraction.</p

Protection of cytosolic Ca²⁺ transient and lipid peroxidation by nicorandil preincubation.

<p>(A) Plated cells were loaded with Fluo-3/AM and maintained in Tyrode medium at 37°C during the experiments. Fluorescence trace of cytosolic free Ca²⁺ before and after application of 10 mM caffeine (arrow) for the irreversible discharge of the sarcoplasmic reticulum Ca²⁺ store (<i>trace a</i>). Ca²⁺ transient induced by 10 mM caffeine after exposing plated cells to 5 μM doxorubicin for 1 h (<i>trace b</i>). Cells were preincubated with 100 μM nicorandil for 30 min and then exposed to 5 μM doxorubicin for 1 h before caffeine addition (<i>trace c</i>). Cells preincubated with 100 μM 5-HD plus 100 μM nicorandil for 30 min were then exposed to 5 μM doxorubicin for 1 h before caffeine addition (<i>trace d</i>). Traces are average of repeated experiments (<i>n</i> = 5) collected from confocal images of representative cell fields. (B) MDA content as index of lipid peroxidation when plated cells were treated or not with 5 μM doxorubicin (Dox) for 1 h, preincubated for 30 min with 100 μM nicorandil in the absence or presence of 100 μM 5-HD before doxorubicin treatment, or exposed to nicorandil or 5-HD alone. Significant difference <i>vs</i>. control (*). Significant difference between absence and presence of nicorandil (#).</p

Role of ΔΨ_m in the mechanism of protection by nicorandil.

<p>(A) Time-course of ΔΨ_m when cells were left untreated (●), supplemented with 30 (○), 50 (Δ), 70 (▲) or 100 μM nicorandil (□) or supplemented with 100 μM 5-HD before the 100 μM nicorandil addition (▽). (B) Time-course of ROS accumulation in the absence (○) or presence of 5 μM doxorubicin (●) and effect of 5 μM CCCP (■) or 1 mM ZnCl₂ (▲) when added in preincubation before doxorubicin. (C) Evolution of ΔΨ_m when cells were left untreated (○) or exposed to 5 μM doxorubicin (●), 5 μM CCCP (■) or 1 mM ZnCl₂ (▲). (D) Initial rates of mitoNOX activity were measured in samples from the mitochondrial fraction before or after preincubation with a given nicorandil concentration.</p

Sensitivity of doxorubicin-induced ROS to other agents.

<p>(A) Dependence of the rate of ROS production on pinacidil concentration when added in preincubation for 30 min before the addition of 5 μM doxorubicin. (B) Dependence on glibenclamide concentration when added in preincubation with 50 μM pinacidil for 30 min before doxorubicin.</p

Effect of nicorandil on NO production.

<p>(A) Time-course of the intracellular NO appearance when plated cells at 37°C were preincubated for 90 min in the absence (●) or presence of 30 (○), 50 (Δ) or 100 μM nicorandil (□) before the reaction was started. (B) Dependence of the NO production rate on nicorandil concentration when added in preincubation for 90 min. Data are expressed with respect to the rate measured in the absence of nicorandil (100%). (C) NO production rate before and after preincubation with 100 μM <i>L</i>-NAME for 30 min and effect when the preincubation with <i>L</i>-NAME was prolonged for 90 min in the presence of nicorandil before measurements.</p