Modulation of mitochondrial respiration during early reperfusion reduces cardiac injury in donation after circulatory death hearts

Abstract

Donation after circulatory death (DCD) donors are a potential source for heart transplantation. The DCD process has unavoidable ischemia and reperfusion (I/R) injury, primarily mediated through mitochondria, which limits routine utilization of hearts for transplantation. Amobarbital (AMO), a transient inhibitor of the electron transport chain, is known to decrease cardiac injury following ex-vivo I/R. We studied whether AMO treatment during reperfusion can decrease injury in DCD hearts. Sprague Dawley rat hearts subjected to 25 minutes of in-vivo ischemia (DCD hearts), or control beating donor (CBD) hearts, were treated with AMO or vehicle for the first 5 minutes of reperfusion, followed by Krebs-Henseleit buffer reperfusion for 55 minutes (for mitochondrial isolation) or 85 minutes (for infarct size determination). Compared to vehicle, AMO treatment led to decreased infarct size (25.2 ± 1.5% vs. 31.5 ± 1.5%; p≤0.05), and troponin I release (4.5 ± 0.05 ng/ml vs. 9.3 ± 0.24 ng/ml, p≤0.05). AMO treatment decreased H₂O₂ generation with glutamate as complex I substrate in both subsarcolemmal mitochondria (SSM) (37± 3.7 pmol/mg/min vs. 56.9 ± 4.1 pmol/mg/min; p≤0.05), and interfibrillar mitochondria (IFM) (31.8 ± 2.8 pmol/mg/min vs. 46±4.8 pmol/mg/min; p≤0.05), and improved calcium retention capacity (CRC) in SSM (360 ±17.2 nmol/mg vs. 277 ± 13nmol/mg; p≤0.05), and IFM (483 ± 20nmol/mg vs. 377± 19 nmol/mg; p≤0.05) compared to vehicle treatment. SSM and IFM retained more cytochrome c with AMO treatment compared to vehicle. In conclusion, brief inhibition of mitochondrial respiration during reperfusion using amobarbital is a promising approach to decrease injury in DCD hearts.

Introduction

Heart transplantation (HTx) is a life-saving procedure for patients with advanced heart failure.¹ Currently, most of the hearts used for transplantation come from donation after brain death (DBD) donors.² The availability of DBD donor hearts is limited; therefore, other potential donor sources such as donation after circulatory death (DCD) donor hearts are being evaluated for HTx.^{3, 4} The critical limitation of the DCD process is the inherent ischemia-reperfusion (I/R) injury due to cardiac arrest and reperfusion following transplantation. I/R injury limits the routine utilization of DCD hearts for transplantation.^{5, 6} Cardioprotective interventions are thus necessary for the DCD hearts to be routinely considered for HTx.

Mitochondria are critical to maintaining heart function by continuous energy production.^{7, 8} In addition, mitochondria play a key role in the regulation of cell death.^9–11 I/R leads to dysfunctional mitochondria, which increases cardiac injury by reducing energy production, increasing reactive oxygen species (ROS) generation, increasing sensitization to mitochondrial permeability transition pore (MPTP) opening, and triggering apoptosis.^12–17 Thus, modulation of mitochondrial respiration using amobarbital (AMO), a reversible inhibitor of complex I, is a potential intervention to decrease I/R injury.^18–22

The mitochondrial electron transport chain (ETC) is a source of ROS generation.²³ The damaged ETC especially complex I increase ROS generation during I/R.²⁴ We recently showed that the mitochondrial ETC, predominantly complex I, is impaired in DCD hearts undergoing ischemia alone or ischemia and reperfusion.^{15, 17} Studies have indicated that administration of AMO at the onset of reperfusion transiently blocked ETC and mitigated cardiac injury in aged hearts.^{25, 26} Therefore, in this study, we sought to examine if AMO-induced transient blockage of the ETC at the onset of reperfusion can decrease cardiac injury in DCD hearts.

Materials and Methods

Ethical aspects

All experimental animals were cared in accordance with institutional guidelines and the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 86–23, revised 2011)²⁷. The study protocol was approved by the McGuire Veterans Administration Hospital and Virginia Commonwealth Institutional Animal Care and Use Committees.

Experimental animal model

Adult male Sprague Dawley rats (10–15 weeks old with bodyweight < 400 grams) were randomly assigned to control beating-heart donor (CBD) group, DCD group, or AMO-treated CBD or DCD groups. The rats were anesthetized using sodium pentobarbital (100 mg/Kg) administered intraperitoneally, followed by endotracheal intubation for respiratory support (Figure 1). Heart rate was monitored by continuous EKG recording. Heparin (1000 U/kg, intraperitoneally) for anticoagulation, and vecuronium (40mg/kg, intramuscularly) to paralyze skeletal muscles, were administered and allowed to circulate for 5 minutes. CBD rat hearts were procured without terminating the ventilation. In the DCD group, rat hearts underwent a 25 min of in-vivo ischemia (the total time from the withdrawal of mechanical ventilation to initiation of coronary perfusion) followed by procurement and perfusion with a Langendorff model for 60 or 90 minutes with modified Krebs-Henseleit (K-H) buffer (115 mM NaCl, 4.0 mM KCl, 2.5 mM CaCl₂, 26 mM NaHCO₃, 1.1 mM MgSO₄, 0.9 mM KH₂PO₄, 5.5 mM glucose, and 5 IU of insulin/liter), oxygenated with 95% O₂ - 5% CO₂. In AMO treated CBD and DCD groups, the isolated rat hearts were initially perfused with 2 mM AMO (dissolved in K-H buffer) for 5 min followed by an additional 55 minutes or 85 minutes of perfusion with K-H buffer. After 10 min of reperfusion, a latex balloon tip catheter was inserted into the left ventricle to monitor left ventricle function. Left ventricle developed pressure (LVDP), Left ventricle diastolic pressure (LVEDP), myocardial contractility (+dP/dt) and myocardial relaxation (-dP/dt) were measured and calculated using Labchart software (ADInstruments Inc., Colorado Springs, CO). Rate pressure product (RPP = heart rate X LVDP) was used to account for cardiac function variability with heart rate (HR). At the end of perfusion, hearts were collected for mitochondrial isolation or infarct size measurement (Figure 1).

Figure 1: Study design with four groups of rats.

The following groups of rats were studied: Control beating-heart donor (CBD)+ vehicle group, CBD + AMO group, donation after circulatory death (DCD) + vehicle group, and DCD + AMO group. AMO (2mM) was administered to CBD+ AMO and DCD + AMO groups for 5 minutes at the start of 60 minutes or 90 minutes reperfusion duration, using Krebs-Henseleit (K-H) buffer. Vehicle groups were treated with K-H buffer. AMO=amobarbital

Isolation of rat heart mitochondria

Mitochondria were isolated using our previously published protocol²⁵. Briefly, the procured hearts were minced in cold (4°C) buffer containing 100 mM KCl, 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS), 1 mM EGTA, 5 mM MgSO_4ˑ7H₂O, 0.2% bovine serum albumin, and 1 mM ATP, pH 7.4. The minced cardiac tissue was homogenized with a polytron tissue processor (Brinkman Instruments, Westbury, NY) for 2.5 seconds at 10,000 rpm. The polytron homogenate was subjected to sequential centrifugation steps to sediment subsarcolemmal mitochondria (SSM). The pellet was re-suspended in buffer without bovine serum albumin, homogenized and incubated with trypsin (5 mg/g wet weight) for 10 min at 4°C followed by sequential centrifugation of the homogenate to sediment interfibrillar mitochondria (IFM). SSM and IFM were washed twice and then suspended in 100 mM KCl, 50 mM MOPS, and 0.5 mM EGTA. Mitochondrial protein content was measured by the Lowry method²², using bovine serum album as a protein standard.

Mitochondrial oxidative phosphorylation

Oxygen consumption in mitochondria was assessed using a Clark-type oxygen electrode at 30°C as previously described.²⁸ Briefly, mitochondria were incubated in 80 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM KH₂PO₄, and 1 mg/ml defatted, dialyzed bovine serum albumin at pH 7.4. 20mM Glutamate, 20mM succinate plus 7.5 μM rotenone, or 10mM TMPD (N,N,N’,N’ tetramethyl p-phenylenediamine, 1 mM)-ascorbate plus 7.5 μM rotenone were used as substrates for complex I, II or IV, respectively²¹.

Calcium retention capacity in isolated mitochondria

Calcium retention capacity (CRC) assay was used to assess calcium-induced mitochondrial permeability transition pore opening in isolated mitochondria. Mitochondria (40 μg/ml) were incubated in buffer containing 150 mM sucrose, 50 mM KCl, 2 mM KH₂PO₄, 5 mM succinate in 20 mM Tris/HCl, pH 7.4. Sequential pulses of calcium (20 nmol) were added to induce MPTP opening in isolated SSM and IFM. Extra-mitochondrial Ca²⁺ concentration was recorded with 0.5 μM Calcium Green-5N and fluorescence was monitored with excitation and emission wavelengths set at 500 and 530 nm, respectively, using LS-5 fluorimeter (Perkin Elmer, Waltman, MA).

H₂O₂ generation in cardiac mitochondria

A net release of H₂O₂ generation from SSM and IFM was examined using the oxidation of the fluorogenic indicator amplex red in the presence of horseradish peroxidise.²⁹ H₂O₂ generation was initiated in mitochondria using glutamate (10mM) or succinate (5mM). Rotenone (2.4μM) was added to the incubation medium to inhibit complex I. Fluorescence was recorded with 530nm excitation and 590nm emission wavelengths.

Infarct size measurement

The 2,3,5- triphenyl tetrazolium chloride (TTC) staining method was employed to measure infarct size.³⁰ Hearts perfused for 90 minutes were transverse sectioned and incubated in TTC solution for 20 min at 37°C followed by storing the heart slices in 10% formaldehyde overnight. Once stained, the heart slices were individually weighed and scanned. The scanned images were processed using image tool software (an automated computerized planimetry package, version-3.0 Slimware Utilities Holdings, Inc.) to determine the total infarct size in relation to the heart weight.

Measurement of cytochrome content

Mitochondrial cytochrome contents (c, b, c1, and aa3) were determined as previously described.²⁸ Briefly, SSM or IFM (0.4 mg) were first solubilized in 5% sodium deoxycholate (DOC) followed by resuspension in 10 mM sodium phosphate, pH 7.0. Cytochrome contents were calculated by the difference between oxidized and reduced spectra. Sodium dithionite was used to generate the reduced spectra²⁸.

Measurement of Cardiac Troponin

The coronary eluate was collected from the coronary sinus at 15 minutes intervals during reperfusion. Cardiac troponin I (cTnI) was measured in the eluate using a commercially available ELISA kit (Fisher Scientific, Waltham, MA) and expressed as ng/mL.

Statistical Analysis

Values are expressed as the mean ± standard error of the mean (SEM). Data were not analyzed blindly. Data were tested for normality and equal variance before performing statistical comparisons between 4 groups (CBD, CBD+AMO, DCD, and DCD+AMO) based on parametric one-way analysis of variance (ANOVA). If the parametric one-way ANOVA model assumption was violated, the nonparametric Kruskal-Wallis one-way ANOVA was considered. A p-value less than 0.05 was considered significant. If the overall ANOVA was significant, pairwise group comparisons were further conducted using the Student-Newman-Keuls analysis, or Dunn’s test if appropriate, with necessary multiplicity adjustments. Specifically, comparisons among the four groups for LVDP, LVEDP, RPP, cTnI, RCR in SSM with glutamate as complex I substrate, SSM TMPD-ascorbate oxidation, H₂O₂ generation in both SSM and IFM with succinate as substrate, CRC in both SSM and IFM, failed the test of normality and were compared using the nonparametric Kruskal-Wallis one-way ANOVA followed by Dunn’s test for multiple groups. SigmaStat software (Version 3.5, Systat Software, Inc., San Jose, CA) were used for the statistical analysis.

Results

AMO reduces myocardial injury in DCD hearts

To measure the effects of AMO on the size of the infarct, CBD or DCD hearts received AMO or vehicle for 5 minutes at the onset of ex-vivo reperfusion followed by K-H reperfusion for 85 minutes. Infarct size was significantly larger in vehicle-treated DCD hearts compared to vehicle-treated CBD hearts (31 ± 1.5% vs. 8 ± 1.2%, p ≤0.05) (Figure 2A). In the DCD groups, AMO treatment resulted in a 20% reduction in infarct size (Figure 2A). AMO treatment alone did not change the infarct size in CBD hearts (Figure 2A). Similarly, cardiac troponin I (cTnI) release was higher in vehicle-treated DCD hearts compared to vehicle-treated CBD hearts treated (Figure 2B), and AMO treatment during reperfusion significantly decreased cTnI content in DCD hearts compared to vehicle-treated DCD hearts (4.5 ±0.05 ng/ml vs. 9.3± 0.2ng/ml, p ≤0.05) (Figure 2B). We also measured cardiac function in response to AMO treatment. Left ventricular developed pressure (LVDP), rate pressure product (RPP), ±dP/dt, and coronary effluent were decreased in vehicle-treated DCD hearts compared to vehicle-treated CBD hearts at 45 minutes and 60 minutes of reperfusion (Table 1). LVEDP was decreased in AMO-treated CBD hearts compared to vehicle-treated CBD hearts at 45 and 60 minutes of reperfusion (Table 1). LVEDP was elevated in vehicle-treated DCD hearts compared to AMO-treated CBD hearts at 60 minutes of reperfusion (Table 1). However, AMO treatment did not improve these physiological parameters in DCD hearts compared to vehicle-treated DCD hearts (Table 1). These data suggest that AMO treatment decreases acute myocardial injury in DCD hearts, as shown by reduced infarct size and cTnI release.

Figure 2: Amobarbital (AMO) decreases infarct size and a release of cardiac troponin I in coronary effluent in DCD hearts.

(A) Total infarct size as measured by triphenyl tetrazolium chloride (TTC) staining in CBD+ vehicle group (n=9), CBD + AMO group (n=8), DCD + vehicle group (n=8), and DCD + AMO group (n=8), or (B) cardiac troponin I, were assessed in CBD + vehicle (n=9), CBD + AMO (n=8), DCD + vehicle (n=8) and DCD + AMO hearts (n=8). AMO (2 mM) or vehicle (K-H buffer) was administered for 5 minutes at the onset of a 90 minute reperfusion duration. The n values represent independent animals. Not every coronary effluent sample was used for cardiac troponin I analysis. * p<0.05 vs CBD + vehicle; Ŧ p<0.05 vs. CBD + AMO; ‡ p<0.05 vs. DCD + vehicle. Infarct size data were analyzed with one-way ANOVA with the Student-Newman-Keuls multiple comparison post-hoc test. Cardiac troponin I data was analyzed using the nonparametric Kruskal-Wallis one-way ANOVA followed by Dunn’s test for multiple groups analysis.

Table 1.

Physiologic heart function in CBD and DCD hearts with and without amobarbital treatment.

	CBD + Vehicle (n=10)	CBD + AMO (n=8)	DCD + Vehicle (n=9)	DCD + AMO (n=9)
Physiologic parameters at 45 min
Heart Rate-bpm	275 ± 15	223 ± 21	226 ± 30	272 ± 21
Coronary perfusion- ml/min	21 ± 1.3	17 ± 1.8	12 ± 1.7*	14 ± 1.6*
LVDP-mmHg	112 ± 17	97 ± 10	28 ± 5* Ŧ	36 ± 5* Ŧ
LVEDP-mmHg	19.2 ± 1.1	10.3 ± 2.7*	20.1 ± 2.9	17.2 ± 3.8
RPP-mmHg × bpm	30657 ± 4939	22397 ± 4213	6444 ± 1548* Ŧ	9412 ± 1145 * Ŧ
+dP/dt -mmHg/s	4985± 631	2806 ± 455	2273 ± 323*	2331 ± 253*
−dP/dt- mmHg/s	−4072 ± 411	−2245 ± 384	−2242 ± 410*	−2096 ± 410*
Physiologic parameters at 60 min
Heart Rate-bpm	276 ± 15	226 ± 20*	274 ± 10 Ŧ	250 ± 14
Coronary perfusion- ml/min	20 ± 1.5	16 ± 1.7	12 ± 1.7*	13 ± 1.6*
LVDP-mmHg	97 ± 9	87 ± 12	32 ± 6* Ŧ	36 ± 5* Ŧ
LVEDP-mmHg	19.2 ± 1.0	9.8 ± 2.5*	26.9 ± 4.8 Ŧ	17.2 ± 3.6
RPP-mmHg × bpm	26648 ± 2786	20739 ± 4259	8962 ± 1930* Ŧ	8942 ± 1194* Ŧ
+dP/dt -mmHg/s	4550 ± 427	2700 ± 469*	3750 ± 974	2242 ± 148*
−dP/dt- mmHg/s	−3768 ± 321	−2091 ± 385*	−3063 ± 475	−2012 ± 154*

The hemodynamic data were collected in hearts used for infarction study. CBD = Control beating-heart donor, DCD = Donation after Circulatory Death, AMO = amobarbital (2mM). LVDP = left ventricle developed pressure, LVEDP = left ventricle diastolic pressure, RPP = rate pressure product (heart rate times LVDP), +dP/dt = rate of positive LVDP/second, -dP/dt = rate of negative LVDP/second. The n values represent independent animals. Mean ± SEM.

^*p <0.05 vs. CBD + vehicle group,

^Ŧp <0.05 vs. CBD + AMO group, using one-way ANOVA with the Student-Newman-Keuls multiple comparison post-hoc test.

Data including LVDP, LVEDP, and RPP were analyzed using the Kruskal-Wallis one-way ANOVA followed by Dunn’s test for multiple groups.

Oxidative phosphorylation (OXPHOS) was not affected by AMO treatment in DCD hearts.

OXPHOS was assessed in the two mitochondrial populations, SSM and IFM, from CBD, or DCD hearts. Compared to vehicle-treated CBD hearts, the rate of ADP-stimulated OXPHOS was decreased in SSM and IFM from vehicle-treated DCD hearts when glutamate, succinate or TMPD+ascorbate were used as complex I, II or IV substrates, respectively (Tables 2a and 2b). AMO-treated DCD hearts exhibited similar mitochondrial respiration with complexes I, II IV substrates, compared to vehicle-treated DCD hearts (Tables 2a and 2b). In the CBD groups, AMO improved ADP-stimulated OXPHOS with glutamate, succinate or TMPD+ascorbate as substrates, in SSM and IFM compared with vehicle-treated CBD hearts (Tables 2a and 2b).

Table 2a.

Oxidative phosphorylation and calcium retention capacity in SSM from CBD and DCD hearts with and without amobarbital treatment.

SSM	CBD + Vehicle (n=8)	CBD + AMO (n=8)	DCD + Vehicle (n=12)	DCD + AMO (n=10)
OXPHOS- With Glutamate
State 3 (nAO/mg/min)	223 ± 22	274 ± 14*	135 ± 10* Ŧ	136± 112* Ŧ
State 4 (nAO/mg/min)	32 ± 3	34 ± 4	44± 5	44 ± 5
RCR	7.0 ± 0.3	8.6 ± 0.8	3.4 ± 0.4* Ŧ	3.8 ± 1.0* Ŧ
2mM ADP (nAO/mg/min)	259 ± 28	306 ± 21	141 ± 11* Ŧ	138 ± 14* Ŧ
DNP	256 ± 26	291 ±25	135 ± 13* Ŧ	134 ± 14* Ŧ
OXPHOS- With Succinate
State 3 (nAO/mg/min)	220 ± 20	336 ± 21*	156± 11* Ŧ	154 ± 12* Ŧ
State 4 (nAO/mg/min)	76 ± 6	110 ± 6*	78 ± 5 Ŧ	84 ± 4 Ŧ
RCR	3.0 ± 0.3	3.1 ± 0.1	2.0 ± 0.1* Ŧ	1.9 ± 0.2* Ŧ
2 mM ADP (nAO/mg/min)	216 ± 22	347 ± 20*	150 ± 12* Ŧ	156 ± 17* Ŧ
DNP	201 ± 21	314 ± 17*	137 ± 12* Ŧ	149 ± 12* Ŧ
OXPHOS-With TMPD-ascorbate
2 mM ADP (nAO/mg/min)	644 ± 64	998 ± 83*	499 ± 39 Ŧ	505± 41 Ŧ

SSM = subsarcolemmal mitochondria, CBD = Control beating-heart donor, DCD = Donation after Circulatory Death, AMO = amobarbital (2mM), Veh = vehicle, OXPHOS = oxidative phosphorylation, nAO = nanomole of atom oxygen, ADP = adenosine diphosphate, DNP = 2,4-dinitrophenol, RCR = respiratory control ratio, TMPD = N,N,N,N′-Tetramethyl-p-phenylenediamine. The n values represent independent animals. Mean ± SEM.

^*p<0.05 vs. CBD + Vehicle;

^Ŧp<0.05 vs. CBD + AMO, using one-way ANOVA with the Student-Newman-Keuls multiple comparison post-hoc test.

Data including RCR with glutamate as substrate and rate of TMPD oxidation were analyzed using the Kruskal-Wallis one-way ANOVA followed by Dunn’s test for multiple groups.

Table 2b.

Oxidative phosphorylation and calcium retention capacity in IFM from CBD and DCD hearts with and without amobarbital treatment.

IFM	CBD + Vehicle (n=8)	CBD + AMO (n=8)	DCD + Vehicle (n=12)	DCD + AMO (n=10)
OXPHOS- With Glutamate
State 3 (nAO/mg/min)	255 ± 24	341 ± 19*	174 ± 10* Ŧ	176 ± 13* Ŧ
State 4 (nAO/mg/min)	41 ± 3	39 ± 4	56 ± 6* Ŧ	57 ± 4* Ŧ
RCR	6.6 ± 0.7	9.3 ± 0.8*	3.5 ± 0.4* Ŧ	3.3 ± 0.4* Ŧ
2mM ADP (nAO/mg/min)	300 ± 29	393 ± 26*	177 ± 12* Ŧ	172 ± 14* Ŧ
DNP	316 ± 38	390 ± 36	167 ± 17* Ŧ	169 ± 17* Ŧ
OXPHOS- With Succinate
State 3 (nAO/mg/min)	301 ± 26	435 ± 28*	204 ± 14* Ŧ	203 ± 15* Ŧ
State 4 (nAO/mg/min)	116 ± 6	135 ± 12	101 ± 4 Ŧ	112 ± 8
RCR	3.0 ± 0.2	3.3 ± 0.1*	2.0 ± 0.1* Ŧ	1.9 ± 0.2* Ŧ
2 mM ADP (nAO/mg/min)	292 ± 31	453 ± 32*	201 ± 14* Ŧ	206 ± 12* Ŧ
DNP	266 ± 30	406 ± 28*	181 ± 15* Ŧ	195 ± 12 * Ŧ
OXPHOS-With TMPD-ascorbate
2 mM ADP (nAO/mg/min)	730 ± 91	1304 ± 98*	531 ± 29 Ŧ	526 ± 52 Ŧ

IFM = Interfibrillar mitochondria, CBD = Control beating-heart donor, DCD = Donation after Circulatory Death, AMO = amobarbital (2mM), Veh = vehicle, OXPHOS = oxidative phosphorylation, nAO = nanomole of atom oxygen, ADP = adenosine diphosphate, DNP = 2,4-dinitrophenol, RCR = respiratory control ration, TMPD = N,N,N,N′-Tetramethyl-p-phenylenediamine. The n values represent independent animals. Mean ± SEM.

^*p<0.05 vs. CBD + vehicle;

^Ŧp<0.05 vs. CBD + AMO, using one-way ANOVA with the Student-Newman-Keuls multiple comparison post-hoc test.

AMO decreases sensitivity to MPTP opening in DCD hearts

We examined the effect of AMO on calcium retention capacity (CRC) that reflects the susceptibility to MPTP opening in isolated SSM and IFM. AMO treatment caused a 30% and 28% increase in CRC in SSM and IFM respectively, from DCD hearts, compared to SSM and IFM from vehicle-treated DCD hearts (Figures 3A and 3B). AMO treatment did not alter the CRC in both SSM and IFM from CBD hearts. These results indicate that brief AMO treatment at the start of reperfusion decreases MPTP opening in DCD hearts.

Figure 3: Calcium Retention Capacity (CRC) is improved with amobarbital (AMO) treatment in DCD hearts.

(A) Subsarcolemmal (SSM) or (B) Interfibrillar mitochondria (IFM), were isolated from CBD + vehicle (n=8), CBD+ AMO (n=8), DCD + vehicle (n=12), and DCD + AMO hearts (n=10). The n values represent independent animals. Pulses of calcium were added at 1 minute intervals to 0.4mg protein of SSM or IFM incubated with calcium retention capacity (CRC) buffer and calcium green. * p<0.05 vs CBD + vehicle; Ŧ p<0.05 vs. CBD + AMO; ‡ p<0.05 vs. DCD + vehicle. Data were analyzed using the nonparametric Kruskal-Wallis one-way ANOVA followed by Dunn’s test for multiple groups analysis.

AMO mitigates H₂O₂ generation in DCD hearts

Compared to vehicle-treated CBD hearts, DCD hearts with vehicle treatment markedly increased H₂O₂ generation from both SSM and IFM, when glutamate was used as complex I substrate (Figures 4A and 4B). H₂O₂ generation was decreased in AMO-treated SSM (decreased by 34%) and IFM (decreased by 30%) from DCD hearts compared to vehicle-treated DCD hearts (Figures 4A and 4B). AMO did not improve glutamate-induced H₂O₂ generation in CBD hearts (Figures 4A and 4B). H₂O₂ generation in SSM and IFM oxidizing succinate in the presence of rotenone, from vehicle-treated DCD hearts was not altered compared to CBD hearts with vehicle treatment (Figures 4C and 4D). However, AMO treatment decreased H₂O₂ generation in both SSM and IFM when succinate + rotenone was used as complex II substrate (53% or 51%, respectively) compared to vehicle-treated DCD hearts (Figure 4C and 4D). AMO treatment also decreased H₂O₂ generation in the CBD groups using succinate as a complex II substrate. These data indicate that AMO treatment can decrease H₂O₂ generation in both CBD and DCD hearts.

Figure 4: H₂O₂ generation decreases with amobarbital (AMO) treatment in DCD hearts.

Subsarcolemmal (SSM) or Interfibrillar mitochondria (IFM), were isolated from CBD + vehicle (n=8), CBD + AMO (n=8), DCD + vehicle (n=12), and DCD + AMO (n=10) hearts. The n values represent independent animals. (A and B) H₂O₂ generation was measured in 0.2mg protein of SSM and IFM using glutamate or in (C and D) using succinate + rotenone. * p<0.05 vs CBD + vehicle; Ŧ p<0.05 vs. CBD + AMO; ‡ p<0.05 vs. DCD + vehicle, using one-way with the Student-Newman-Keuls multiple comparison post-hoc test. Data including H₂O₂ generation in both SSM and IFM with succinate as substrate were analyzed using the nonparametric Kruskal-Wallis one-way ANOVA followed by Dunn’s test for multiple groups.

AMO decreased the release of cytochrome c and cytochrome aa3 from mitochondria of DCD hearts

The contents of cytochrome c, and cytochrome aa3 (a component of complex IV) in both SSM and IFM of vehicle-treated DCD hearts were decreased compared to SSM and IFM from vehicle-treated CBD hearts (Table 3). In the DCD group, AMO treatment improved cytochrome c content in SSM and IFM (64% and 41%), respectively, and preserved cytochrome aa3 content in SSM and IFM (52% and 37%), respectively (Table 3). There were no significant differences in the content of cytochrome b and c1 (a component of complex III) between AMO-treated DCD hearts and vehicle-treated DCD hearts (Table 3). In the AMO-treated CBD group, cytochrome c content was higher in SSM but not IFM, compared to SSM and IFM of vehicle-treated CBD group, whereas cytochrome aa3 content was not altered by AMO treatment (Table 3).

Table 3:

Cytochrome content in SSM and IFM from CBD and DCD hearts with and without amobarbital treatment.

	CBD + Vehicle (n=8)	CBD + AMO (n=8)	DCD + Vehicle (n=12)	DCD + AMO (n=10)
SSM
c (nmol/mg)	0.24 ± 0.01	0.29 ± 0.04*	0.13 ± 0.02* Ŧ	0.18 ± 0.01 Ŧ ‡
c1 (nmol/mg)	0.14 ± 0.02	0.21 ± 0.01*	0.15 ± 0.01 Ŧ	0.15 ± 0.00
b (nmol/mg)	0.22 ± 0.02	0.24 ± 0.03	0.13 ± 0.03* Ŧ	0.19 ± 0.01
aa3 (nmol/mg)	0.60 ± 0.04	0.70 ± 0.07	0.29 ± 0.07* Ŧ	0.41 ± 0.02 *Ŧ ‡
IFM
c (nmol/mg)	0.35 ± 0.02	0.39 ± 0.05	0.18 ± 0.03* Ŧ	0.24± 0.02* Ŧ ‡
c1 (nmol/mg)	0.18 ± 0.02	0.20 ± 0.01	0.13 ± 0.02	0.15 ± 0.02
b (nmol/mg)	0.24 ± 0.01	0.31 ± 0.04*	0.19 ± 0.03 Ŧ	0.21 ± 0.004 Ŧ
aa3 (nmol/mg)	0.67 ± 0.03	0.74 ± 0.13	0.30 ± 0.08* Ŧ	0.52 ± 0.01 Ŧ ‡

SSM = subsarcolemmal mitochondria, IFM = Interfibrillar mitochondria, CBD = Control beating-heart donor, DCD = Donation after Circulatory Death, AMO = amobarbital (2mM). The n values represent independent animals. Mean ± SEM,

^*p <0.05 vs. CBD + vehicle group,

^Ŧp <0.05 vs. CBD + AMO group,

^‡p <0.05 vs. DCD + vehicle group, using one-way ANOVA with the Student-Newman-Keuls multiple comparison post-hoc test.

Discussion

The current study found that AMO treatment at the onset of reperfusion led to decreased cardiac injury in DCD hearts, as shown by reduced infarct size and cTnI release. AMO treatment also led to decreased MPTP opening and decreased cytochrome c loss during reperfusion in DCD hearts, likely by reducing H₂O₂ generation. These results indicate that AMO treatment during early reperfusion decreases cardiac injury in DCD hearts by inhibiting MPTP opening through reduction of ROS generation.

AMO is a short-acting barbiturate that reversibly inhibits complex I of ETC.^{31, 32} AMO treatment before ischemia led to decreased cardiac injury during I/R in several animal models including, rabbits, guinea pigs, rats, and mice.^19–22 Since pre-mortem interventions are not allowed in the DCD donation process, interventions that would diminish cardiac damage in DCD hearts can only be used during reperfusion. AMO administered at the onset of reperfusion decreased cardiac injury in isolated rat hearts.^{25, 26} The current study found that AMO treatment applied during early reperfusion also decreased cardiac injury in DCD hearts. Reperfusion injury contributes up to half of the final myocardial damage,³³ therefore, interventions targeted during reperfusion are critical for DCD heart protection. In our study, AMO treatment at early reperfusion decreased infarct size in DCD hearts (Figure 2A). This finding is further corroborated with a decrease in cTnI release in AMO-treated DCD hearts (Figure 2B).

Cytosolic calcium loading, which occurs during ischemia, correlates with the degree of post-ischemic cardiac dysfunction, also known as myocardial stunning.³⁴ Prevention of the rise in cytosolic calcium during ischemia attenuated myocardial stunning during reperfusion.^{34, 35} It is, therefore, possible that AMO given during reperfusion did not prevent cytosolic calcium loading, which already occurred in the ischemic DCD hearts, such that mitigation of myocardial stunning during reperfusion was not achieved during the experimental duration of 60 minutes. This may explain why AMO treatment did not improve cardiac function including contraction and relaxation despite reducing the infarct size and cTnI level in DCD hearts (Table 1). The decrease in infarct size and cTnI demonstrate that AMO treatment at reperfusion protects DCD hearts from reperfusion injury. The functional benefits of this injury protection may be studied in more extended perfusion duration experiments.

The opening of MPTP is central to I/R injury and cardiomyocyte death. MPTP is a large conductance non-selective pore in the inner mitochondrial membrane, which, when formed, allows communication between the mitochondria matrix and cytoplasm.^{13, 14, 16} MPTP opening results in mitochondrial swelling, loss of inner membrane potential, inhibition of oxidative phosphorylation, rupture of the outer mitochondrial membrane, culminating in cell death.^{13, 14, 16} Several studies have indicated that MPTP opening is favored by conditions such as the depletion of ATP, elevated mitochondrial Ca²⁺, loss of mitochondrial inner membrane potential, ROS generation, and physiological pH; conditions seen with I/R of DCD process ultimately contribute to cardiomyocyte death.^{13, 14, 16, 36, 37} Our previous work revealed that MPTP opening triggered during ischemia, worsens during reperfusion in DCD hearts.^{15, 17} Although the exact molecular component of MPTP is uncertain, there is evidence of the involvement of certain protein components: the voltage-dependent anion channel in the outer mitochondrial membrane, the adenine nucleotide transporter in the outer mitochondrial membrane, and cyclophilin D (Cyp D), and subunits of mitochondrial ATP synthase. Inhibition of MPTP opening with cyclosporine A (CyA), (a potent inhibitor of MPTP that prevents interaction of Cyp D with other mitochondrial membrane components to initiate pore opening), led to a decrease in cardiac injury in the in-vivo model of myocardial I/R, and in DCD hearts following reperfusion,^38–40 suggesting that MPTP inhibition is cardioprotective. In this study, we found that AMO treatment at the onset of reperfusion improved CRC (an index of sensitivity to MPTP opening) in DCD hearts (Figure 3). The degree of CRC improvement was higher in SSM than IFM (Figure 3), probably because of the greater susceptibility of SSM to ischemic injury.²⁸ One explanation for the AMO-induced MPTP opening inhibition in DCD hearts could be due to decreased ROS generation at reperfusion.

During early reperfusion, the burst of ROS generation is a major activator of MPTP opening, which in turn plays a role in cyclical ROS-induced ROS release⁴¹. ROS generation begins during ischemia.²⁴ We previously demonstrated mitochondrial ETC defects in rat heart ex-vivo and DCD in-vivo ischemia models and proposed complex I as the predominant source of ROS production upon reperfusion.^{15, 17} Our previous work and studies from others support the notion that ischemia-induced defects of the ETC which results in a burst of ROS generation during reperfusion, contribute to myocardial injury.^{15, 41, 42} The present study revealed that H₂O₂ generation with glutamate (complex I substrate) was decreased with AMO treatment during reperfusion in DCD hearts (Figures 4A and B), suggesting that inhibition of complex I by AMO leads to a decrease in ROS generation from complex I, and a decrease in electron flow to complex III. The decrease in H₂O₂ generation with succinate + rotenone (complex II substrate) in AMO-treated DCD hearts (Figures 4C and D), confirms inhibition of electron flow from complex I without any backflow of electrons from complex II to complex I. AMO treatment at reperfusion mitigates cardiac injury in DCD hearts by decreasing ROS generation during reperfusion.

Permeabilization of the mitochondrial outer membrane, which facilitates the release of cytochrome c into the cytosol, is a consequence of MPTP opening.^{13, 14, 16} The release of cytochrome c from the intermembrane space of mitochondria to cytosol is a critical signal for initiating the downstream apoptotic pathway leading to cardiomyocyte death.^{13, 14, 16} We demonstrated in this study that AMO treatment improved mitochondrial cytochrome c content in DCD hearts (Table 3), suggesting less cytochrome c release into the cytosol. This result provides additional evidence that AMO treatment decreases MPTP opening in DCD hearts during reperfusion. Further, AMO treatment preserved mitochondrial cytochrome aa3 content in DCD hearts (Table 3). Cytochrome aa3 is a component of cytochrome c oxidase (complex IV), the terminal enzyme in the ETC that catalyzes the reduction of molecular oxygen to water, coupled with proton translocation across the inner mitochondrial membrane for ATP production.^{43, 44} Impaired cytochrome c oxidase activity contributed to I/R injury in rats.^{43, 44} These results further confirm that AMO treatment at early reperfusion can protect against cardiac injury in DCD hearts by attenuating of cytochrome c release.

We previously demonstrated that an ischemia duration of 25 minutes impaired mitochondrial OXPHOS function prior to reperfusion in DCD hearts,^{15, 17} supporting the concept that mitochondrial damage mainly occurs during ischemia.¹² In the current study, AMO treatment at the onset of reperfusion did not improve OXPHOS function in DCD hearts (Tables 2a and 2b). This observation implies that other factors apart from mitochondrial respiration contribute to cardiac injury during reperfusion. It is also possible that the impaired mitochondrial OXPHOS, which already occurred during ischemia, was at a climax, and AMO treatment could not improve it. CBD hearts, however, exhibited improved OXPHOS function (Tables 2a and 2b). CBD hearts were procured without termination of ventilation and exposed to approximately 5 minutes of ischemia between heart procurement and the start of ex-vivo reperfusion (Figure 1). The improved OXPHOS observed in CBD hearts suggests that AMO treatment can benefit hearts perhaps with brief periods of ischemia.

Taken together, our findings show that AMO reduces cardiac injury in DCD hearts by decreasing the burst of ROS generation at reperfusion, consequently reducing susceptibility to MPTP opening (Figure 5). Our future studies will evaluate the transitional benefit of decreasing cardiac injury in DCD hearts by performing heterotopic heart transplantation in rats.

Figure 5: Graphical Summary.

Reperfusion of DCD hearts increases ROS generation and MPTP opening, and decreases mitochondrial cytochrome c content, resulting in increased myocardial damage. AMO treatment at early reperfusion protects DCD hearts by decreasing ROS generation and MPTP opening, and increasing mitochondrial cytochrome c. Red arrow = increase, Blue arrow = decrease, cTnI = cardiac troponin I.

Limitations:

Due to the complexity of blood-based perfusates, we used the K-H buffer for reperfusion in this study. Although K-H buffer is not a physiologic solution, we maintained constant perfusion conditions by not recirculating the K-H buffer, which is not practical with blood-based perfusates. Infarct size determination could be limited due to 90 minutes of reperfusion times. A longer duration of perfusion could have resulted in improved heart function; we plan to pursue this objective with a heterotopic heart transplantation model in rats where graft function can be monitored for weeks.^{45, 46}

Conclusion:

The reversible blockade of electron transport with AMO, early during reperfusion, attenuates cardiac injury in DCD hearts. Thus, cardiac injury can be decreased in DCD hearts at reperfusion despite the unavoidable ischemic injury. Timely intervention with AMO applied at the onset of reperfusion is a promising approach to decrease cardiac injury in DCD hearts.

Abbreviations:

AMO

Amobarbital

ADP

Adenosine diphosphate

ATP

Adenosine triphosphate

CBD

Control beating-heart donor

CRC

Calcium retention capacity

cTnI

Cardiac troponin I

DBD

Donation after brain death

DCD

Donation after circulatory death

DNP

Dinitrophenol

DP

Developed pressure

ETC

Electron transport chain

HF

Heart failure

HTx

Heart transplantation

IFM

Interfibrillar mitochondria

I/R

Ischemia-reperfusion

KH

Krebs-Henseleit

LV

Left ventricular

LVDP

Left ventricular developed pressure

MPTP

Mitochondrial permeability transition pore

OXPHOS

Oxidative phosphorylation

ROS

Reactive oxygen species

SSM

Subsarcolemmal mitochondria

TMPD

N,N,N’,N’ tetramethyl p-phenylenediamine