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

Abstract

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.

Fig 1. Experimental design.

Horizontal black arrows represent the reperfusion timeline for donation after brain death (CBD) or donation after circulatory death (DCD) hearts. Black arrows pointing downwards represent time intervals where the cardiac function was measured. Red arrows pointing upwards represent time points where rat hearts were collected for mitochondria isolation to study oxidative phosphorylation and calcium retention capacity.

Fig 2. Oxidative phosphorylation in mitochondria from DCD and CBD hearts.

Oxygen consumption using glutamate, succinate, or TMPD-ascorbate as complex I, II and IV substrates respectively, were measured in SSM and IFM. (A.) ADP-stimulated glutamate oxidation (complex I) from 0.25mg of SSM and IFM from CBD and DCD hearts. (B.) ADP-stimulated succinate oxidation (complex II) from 0.25mg of SSM and IFM from CBD and DCD hearts. (C.) ADP-stimulated TMPD oxidation (complex IV) from 0.125mg of SSM and IFM from CBD and DCD hearts. n = 5 in each group. Data are expressed as means ±SEM. *p<0.05 vs CBD, using one-way ANOVA.

Fig 3. Effect of reperfusion on oxidative phosphorylation in mitochondria from CBD hearts.

Oxidative phosphorylation was measured in subsarcolemmal (SSM) or interfibrillar mitochondria (IFM) from CBD hearts with no reperfusion (n = 5) or with 60 minutes of reperfusion (RPF), n = 8. Oxygen consumption using glutamate, succinate, or TMPD-ascorbate as complex I, II and IV substrates respectively, were measured in SSM and IFM (A-C). Data are expressed as mean ±SEM.

Fig 4. Effect of reperfusion on oxidative phosphorylation in mitochondria from DCD hearts.

Oxidative phosphorylation was measured in subsarcolemmal (SSM) or interfibrillar mitochondria (IFM) from DCD hearts with no reperfusion (n = 5), with 10 minutes of reperfusion (n = 8) or with 60 minutes of reperfusion (n = 8). Oxygen consumption using glutamate, succinate, or TMPD-ascorbate as complexes I, II and IV substrates respectively, were measured in SSM and IFM (A-C). Data are expressed as mean ± SEM. *p<0.05 vs DCD hearts with no reperfusion group; ^ǂp<0.05 vs DCD hearts with 10 min reperfusion group, using one-way ANOVA.

Fig 5. Calcium retention capacity in mitochondria from CBD and DCD hearts with and without reperfusion.

(A). Subsarcolemmal (SSM) or interfibrillar mitochondria (IFM) were isolated from CBD hearts (n = 5) or from DCD hearts with 25 or 35 minutes of ischemia (n = 5, each). Pulses of calcium were added at 1minute intervals to 0.4mg protein of SSM or IFM incubated with calcium retention capacity (CRC) buffer and calcium green. *p<0.05 vs CBD group, using one-way ANOVA. (B.) SSM and IFM were isolated from CBD hearts with no reperfusion or with 60 minutes of reperfusion (n = 8). CRC was measured as described above. *p<0.05 vs CBD group with no reperfusion, using two tailed non paired t-test. (C.) SSM and IFM were isolated from DCD hearts with no reperfusion (n = 5), with10 or 60 minutes of reperfusion (n = 8, each). CRC was measured as described above. Data are expressed as mean ±SEM. * p<0.05 vs DCD with no reperfusion group; ^ǂp<0.05 vs DCD with 10 minutes of reperfusion group, using one -way ANOVA.

Fig 6. Cardiac function and lactate dehydrogenase release in CBD and DCD hearts.

Heart function (left ventricle developed pressure- LVDP) or (B.) rate pressure product (RPP) was measured with a balloon tip catheter placed in the left ventricle of DCD and CBD hearts at 15, 30, 45 and 60 minutes. (C.) Timed coronary sinus samples were collected from CBD and DCD hearts (n = 8, each) to measure lactate dehydrogenase release. Data are expressed as mean ±SEM. *p<0.05 vs CBD group, using two tailed non-paired t-test.

Fig 7. Reactive oxygen species production from mitochondria and cytosol cytochrome c levels in reperfused CBD and DCD hearts.

(A.) H₂O₂ production in CBD and DCD hearts subjected to 60 minutes of reperfusion (RPF), n = 8, each. *p<0.05 vs CBD group, using two tailed non-paired t-test. (B.) Upper panel shows representative blot for immunoblotting of cytosolic cytochrome c with anti-cytochrome c antibody. Lower panel graph represents ratio of cytochrome c to GAPDH, the loading control. Data are expressed as mean ±SEM. *p<0.05 vs. CBD group; ^ǂp<0.05 vs. DCD group; ^Ŧp<0.05 vs. CBD with reperfusion group, using one-way ANOVA. n = 4 in each group. (C). Total infarct size as measured by triphenyl tetrazolium chloride (TTC) staining, in CBD and DCD hearts subjected to reperfusion (RPF), n = 8, each. *p<0.05 vs CBD group, using two tailed non-paired t-test. (D.) Coronary flow from CBD and DCD hearts following 60 minutes of reperfusion. *p<0.05 vs CBD group, using two tailed non-paired t-test.