Keywords: heart transplantation, ischemia-reperfusion, mitochondria, mitochondrial permeability transition pore
Abstract
Donation after circulatory death (DCD) donor hearts are not routinely used for heart transplantation (HTx) because of ischemic damage, which is inherent to the DCD process. HTx outcomes are suboptimal in males who received female donor hearts. The exact mechanism for suboptimal outcomes from female donor hearts has not been defined. Differential susceptibility to ischemia tolerance, which would play a significant role in DCD donation, could be a reason but has not been studied. We studied the influence of sex on global myocardial ischemia tolerance and mitochondrial function. Sprague-Dawley rats of both sexes were assigned to DCD (n = 32) or control beating-heart donor (CBD, n = 28) groups. DCD hearts underwent 25 min of in vivo global myocardial ischemia and 90 min of ex vivo Krebs-Henseleit buffer perfusion at 37°C. CBD hearts were procured without ischemia. Infarct size was determined in hearts following 90 min of reperfusion, and in another set of hearts, mitochondrial function (oxidative-phosphorylation) was studied following 60 min of reperfusion. Infarct size was increased 3.3-fold in male and 3.1-fold in female DCD hearts compared with CBD hearts. However, infarct size (%) was comparable in female and male DCD hearts (male: 25.4 ± 3.7 vs. female 19.0 ± 3.3, P = NS). Oxidative phosphorylation was similarly decreased in male and female DCD hearts’ mitochondria compared with CBD hearts’ mitochondria. Thus, neither infarct size nor mitochondrial dysfunction was higher in female DCD hearts. These results suggest that the susceptibility to ischemia is not the reason for suboptimal HTx outcomes with female donor hearts.
NEW & NOTEWORTHY The current study shows cardiac injury is not increased in female DCD hearts following global ischemia-reperfusion compared with male DCD hearts. In addition, mitochondrial dysfunction with DCD ischemia-reperfusion is comparable in both sexes. Sex-specific immune responses and hormone receptor modulation may contribute to suboptimal outcomes in male HTx recipients with female donor hearts.
INTRODUCTION
Heart transplantation (HTx) is a lifesaving procedure for a patient with advanced heart failure (1). The median life expectancy after HTx is 15 years; however, in a subset of patients, such as female recipients and males who received hearts from female donors, the long-term outcomes are suboptimal (2). In a recent study, male HTx recipients of female heart donors had inferior survival compared with recipients of male donors (2). There is no clear explanation for the observed suboptimal outcomes of heart transplantation in males who received female donor hearts. Possible reasons mentioned in the literature are relatively smaller female heart size, hormonal influences, enhanced immune reactivity in female recipients, and varying endothelial response to the stressors, particularly ischemia (2–5).
To date, the HTx outcomes literature based on the sex differences is primarily from donation after brain death (DBD) donors only (2). To expand the donor heart pool, donation after circulatory death (DCD) donor hearts are being used for clinical transplantation (1, 6). Limited data show that overall survival and rejection episodes of DCD heart recipients are comparable to the outcomes of DBD donors. However, DCD heart recipients required more mechanical circulatory support for delayed graft function (2). Overall, these are encouraging results and should expand the use of DCD hearts for clinical HTx in the future.
Global myocardial ischemia of varying duration is innate to DCD organ donation. Our previous study shows that a longer period of global ischemia increases cardiac injury in male DCD hearts (7). In addition, we noticed that global ischemia as seen with DCD process leads to mitochondrial dysfunction in male DCD hearts (7–9). Although mitochondria are a key source of energy production to support cardiac function (10, 11), the damaged mitochondria also are the primary source of cardiac injury during ischemia-reperfusion (12). Cardiac mitochondria include subsarcolemmal mitochondria (SSM) located underneath the plasma membrane and interfibrillar mitochondria (IFM) located among the myofibrils (13, 14). We noted more severe damage in SSM than in IFM in DCD hearts (8). Therapeutic interventions, including blockade of electron transport chain using amobarbital or inhibition of mitochondrial permeability transition pore (MPTP) opening using cyclosporine A, lead to decreased cardiac injury in male DCD hearts (7, 15), indicating that mitochondrial injury from ischemia-reperfusion can be modulated. Very limited data are available to draw information on the susceptibility of mitochondria to ischemia by sex. Increased susceptibility to ischemic injury may be the reason for suboptimal outcomes in male recipients of female donor hearts. As ischemia and reperfusion are innate to the DCD organ donation, data on sex-related ischemia tolerance will have a direct impact on DCD organ donation.
We sought to study the influence of sex on global in vivo myocardial ischemia tolerance and mitochondrial function derangements, which are potentially amenable to modulation. Our findings may guide donor heart selection, especially in the setting of DCD HTx.
METHODS
In Vivo and Ex Vivo Experimental Animal Models
The experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of Virginia Commonwealth University (VCU) and the McGuire Department of Veterans Affairs Medical Center.
Adult male and female Sprague-Dawley rats (10–15 wk old) were randomly assigned to a CBD (continuous beating-heart donor without in vivo ischemia) or a DCD (hearts subjected to 25 min of global in vivo ischemia) group (9). The rats were first anesthetized with sodium pentobarbital (100 mg/kg) administered intraperitoneally. The rats were then placed on a heating pad to maintain a body temperature of 37°C, followed by endotracheal intubation with a 14-gauge tube for respiratory support. Heart activity was monitored by continuous EKG recording. Heparin (300 U) was administered intraperitoneally and allowed to circulate for 1 min, followed by intramuscular administration of a paralytic agent (vecuronium bromide, 40 mg/kg) and allowed to circulate for 5 min (9). Rats in the CBD group underwent a thoracotomy, hearts were procured and stored (<2 min) in cold Krebs-Henseleit (K-H) buffer for ex vivo perfusion (details below). In the DCD group, the ventilator support was terminated, leading to immediate respiratory arrest followed by cardiac asystole monitored by the EKG. Twenty-five minutes following termination of the ventilator, thoracotomy was performed, and hearts were procured and also stored (<2 min) in cold K-H buffer for ex vivo perfusion.
The procured hearts were mounted on the Langendorff setup and perfused at a constant pressure of 72 mmHg with a modified K-H buffer, consisting of (in mM) 115 NaCl, 4.0 KCl, 2.5 CaCl2, 26 NaHCO3, 1.1 MgSO4, 0.9 KH2PO4, and 5.5 glucose and 5 IU insulin/L at 37°C, oxygenated with 95% O2-5% CO2 to maintain a pH of 7.4. Cardiac function was monitored with a balloon catheter inserted into the left ventricle and analyzed with ADInstruments (Colorado Springs, CO) (16). In the infarct size study, rat hearts subjected to 25 or 35 min of ischemia were procured after 90 min of K-H buffer perfusion. In the mitochondrial functional study, rat hearts were procured after 60 min of K-H buffer perfusion.
Isolation of Mitochondria from Rat Hearts
SSM and IFM were isolated using the previously published protocol (17, 18). Briefly, the rat hearts were washed with isolation buffer A, consisting of (in mM) 100 KCl, 50 3-(N-morpholino)propanesulfonic acid (MOPS), 1 EGTA, 5 MgSO4·7H2O, and 1 ATP (pH 7.4) at 4°C. Heart tissue was then minced and resuspended in buffer B, containing buffer A + 0.2% bovine serum albumin, and homogenized with a polytron tissue processor (Brinkman Instruments, Westbury, NY) for 2.5 s at the setting of 10,000 rpm. The polytron homogenate was first centrifuged at 500 g for 10 min to separate supernatant and pellet. The supernatant was collected and centrifuged at 3,000 g for 10 min to sediment SSM. The pellet from the polytron homogenate was used to isolate IFM. The pellet was resuspended in mitochondrial isolation buffer A and homogenized again and incubated with trypsin (5 mg/g wet wt) for 10 min at 4°C with constant stirring. The homogenate was centrifuged at 500 g for 10 min, and the supernatant was collected and further centrifuged at 3,000 g for 10 min to sediment IFM. SSM and IFM were washed and suspended in KME, consisting of (in mM) 100 KCl, 50 MOPS, and 0.5 EGTA, for functional study (19).
Measurement of Oxidative Phosphorylation in Isolated SSM and IFM
The rate of oxidative phosphorylation (OXPHOS) was measured using a Clark-type oxygen electrode at 30°C as previously described (19). SSM or IFM were incubated in OXPHOS buffer, consisting of (in mM) 80 KCl, 50 MOPS, 1 EGTA, and 5 KH2PO4 and 1 mg of defatted, dialyzed bovine serum albumin/mL at pH 7.4. Glutamate (20 mM, complex I substrate), succinate (20 mM, complex II substrate), and TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine, 1 mM)-ascorbate (10 mM, complex IV substrate) were used. Rotenone (7.5 µM) was used to block potential reverse electron flow when succinate and TMPD were used as substrates (19). ADP (0.2 mM) was used to determine the rate of state 3 (ADP-stimulated respiration) and rate of state 4 (ADP-limited respiration) OXPHOS in the isolated mitochondria (19). ADP (2 mM) was used to measure the maximal rate of ADP-stimulated OXPHOS in the isolated mitochondria (19). Dinitrophenol (DNP, 0.3 mM) was used as an uncoupler to assess respiration independent of complex V in the isolated mitochondria (19).
Measurement of Calcium Retention Capacity in Isolated Mitochondria
The calcium retention capacity (CRC) is used to reflect the sensitivity of calcium-induced MPTP opening in isolated mitochondria (20). SSM or IFM (250 μg/mL) was incubated in buffer containing (in mM) 150 sucrose, 50 KCl, 2 KH2PO4, 5 succinate in 20 Tris·HCl at pH 7.4. Calcium (5 nmol/pulse) was sequentially added into the medium to induce MPTP opening. Succinate was used as the substrate for CRC measurement in that the CRC value is higher in mitochondria oxidizing a complex II substrate compared with a complex I substrate (21, 22). 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 (20).
Measurement of H2O2 Generation in SSM and IFM
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 peroxidase (23). H2O2 generation was initiated in mitochondria resuspended in a buffer including (in mM) 150 KCl, 5 KH2PO4, and 1 EGTA using 10 glutamate as complex I substrate or 5 succinate as complex II substrate. Rotenone (2.4 µM) was added to the incubation medium to inhibit complex I when succinate was used as a complex II substrate. Fluorescence was recorded with 530 nm excitation and 590 nm emission wavelengths.
Infarct Size Measurement
The 2,3,5-triphenyl-tetrazolium chloride (TTC) staining method was used to measure infarct size (7, 24). Hearts perfused for 90 min were transversely sectioned and incubated in 1% of 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) to determine the total infarct size in relation to the heart weight.
Statistical Analysis
Statistical analysis was performed using SigmaStat 3.5 (Systat, Richmond, CA). Data were expressed as means ± SE. Differences in cardiac function between four groups were compared by one-way ANOVA after passing a normality and equal variance test (25). If the overall ANOVA was significant, pairwise group comparisons were further conducted using the Student-Newman-Keuls analysis. Alternatively, if the parametric one-way ANOVA model assumption was violated including RPP function at 60 and 90 min of reperfusion, the nonparametric Kruskal-Wallis one-way ANOVA and Dunn’s test were used for comparison. Two-way ANOVA was used for infarct size and mitochondria functional analysis. If the overall ANOVA was significant, pairwise group comparisons were further conducted using the Holm–Sidak test. An unpaired Student’s t test was used for infarct size analysis after DCD hearts following 35 min of ischemia and 90 min of reperfusion. Statistical significance was defined as a value of P < 0.05.
RESULTS
Infarct Size Was Not Increased in Female DCD Hearts Compared with Male Hearts
Consistent with our previous study, there was a small infarct (5%–7%) in male and female CBD hearts following 90 min of ex vivo reperfusion. The infarct size in male and female CBD hearts was comparable (Fig. 1A). The infarct size in male and female DCD hearts was significantly larger compared with their corresponding CBD hearts [means ± SE, infarct size: male, 7.7 ± 1.2 (CBD) vs. 25.4 ± 3.7% (DCD), P < 0.05; female, 6.2 ± 1.8 (CBD) vs. 19.0 ± 3.3% (DCD), P < 0.05] (Fig. 1A). Interestingly, infarct size in female DCD hearts was not larger than male DCD hearts (Fig. 1A). To further explore the effect of sex on cardiac injury with a longer duration of ischemia, we measured the infarct size in both male and female DCD hearts subjected to 35 min of global ischemia. We found that infarct size in female DCD hearts was relatively smaller compared with male DCD hearts (means ± SE, female vs. male, 25.8 ± 2.2 vs. 40.9 ± 1.4%, n = 8, P < 0.05). These results suggest that cardiac injury is not higher in female DCD hearts compared with male DCD hearts, even with a longer ischemia duration.
Figure 1.
Alteration of cardiac function and infarct size in male and female DCD hearts. Both male and female DCD hearts underwent 25 min of in vivo global ischemia and 90 min of ex vivo buffer perfusion. CBD hearts underwent 90 min of ex vivo buffer perfusion without in vivo ischemia. A: there was small infarct size in both male and female CBD hearts following 90 min of buffer perfusion. Infarct size was significantly increased in both male and female DCD hearts compared with their CBD hearts. However, there was no significant difference in infarct size between male and female DCD hearts. B: heart rate (HR) was decreased in both male and female DCD hearts compared with CBD hearts at 15 min of reperfusion. HR in female DCD hearts was also decreased compared with CBD hearts at 60 min of reperfusion. C: LVDP was decreased in both male and female DCD hearts compared with CBD hearts during reperfusion. The degree of decreased LVDP in female DCD hearts was less compared with male DCD hearts. D: RPP (HRXLVDP) was also decreased in both male and female DCD hearts compared with their CBD hearts. The decreased degree of RPP in female DCD hearts was less compared with male DCD hearts. Data were expressed as means ± SE. Two-way ANOVA was used for infarct size analysis. One-way ANOVA and Student-Newman-Keuls analysis were used for cardiac function analysis. The nonparametric Kruskal–Wallis one-way ANOVA and Dunn’s test were used for comparison in RPP function at 60- and 90-min reperfusion in that data did not pass normality test. *P < 0.05 vs. CBD; †P < 0.05 vs. female DCD hearts. The n values represent independent animals. Male CBD, n = 8; female CBD, n = 8; male DCD, n = 9; female DCD, n = 8. CBD, control beating-heart donor; DCD, donation after circulatory death; LVDP, left ventricular developed pressure; RPP, rate pressure product.
Cardiac Function on Langendorff Setup in Male and Female DCD Hearts
Cardiac function was monitored during heart perfusion. Heart rate (HR) and left ventricle developed pressure (LVDP) were measured at 15-min intervals. RPP (rate pressure product) was also calculated (HR × LVDP) to account for cardiac contractile function attributable to variable heart rate. HR was decreased in both male and female DCD hearts during initial 15 min of perfusion compared with corresponding CBD hearts [means ± SE, HR (beats/min): male, 300 ± 18 (CBD) vs. 200 ± 21 (DCD), P < 0.05; female, 271 ± 17 (CBD) vs. 226 ± 20 (DCD), P < 0.05] (Fig. 1B). There were no differences in HR between male and female CBD and DCD hearts during the remaining reperfusion duration (Fig. 1B). There were no differences in LVDP between male and female CBD hearts (Fig. 1C). The LVDP was decreased in male and female DCD hearts compared with their corresponding CBD hearts [LVDP (mmHg) at 90 min of reperfusion: means ± SE, male, 86 ± 14 (CBD) vs. 30 ± 3 (DCD), P < 0.05; female, 88 ± 6 (CBD) vs. 53 ± 9 (DCD), P < 0.05] (Fig. 1C). The LVDP in female DCD hearts was higher than that in male DCD hearts (Fig. 1C). There were no differences in RPP between male and female CBD hearts (Fig. 1D). The RPP was decreased in male and female DCD hearts compared with their corresponding CBD hearts [means ± SE, RPP (beats/min × mmHg) at 90 min of reperfusion: male, 22,681 ± 4,144 (CBD) vs. 6,911 ± 743 (DCD), P < 0.05; female, 22,464 ± 1,858 (CBD) vs. 12,792 ± 2,022 (DCD), P < 0.05] (Fig. 1D). Similar to the LVDP, the RPP in female DCD hearts was higher than in male DCD hearts (Fig. 1D). These results indicate that cardiac function was better preserved in female DCD hearts during reperfusion compared with male DCD hearts.
OXPHOS Function in SSM and IFM from CBD and DCD Hearts
Functional data of SSM are shown in Table 1. The protein yield of SSM in both male and female DCD hearts was slightly decreased compared with corresponding CBD hearts. There were no differences in state 3 respiration (stimulated by 0.2 mM ADP) in SSM between male CBD and female CBD hearts using glutamate as a complex I substrate. The state 3 rate of SSM in both male and female DCD hearts was decreased compared with their corresponding CBD hearts using complex I substrates. The state 3 respiration was better preserved in female DCD hearts compared with male DCD hearts with complex I substrates. There were no differences in state 4 respiration (ADP-limited respiration) between the four groups oxidizing complex I substrates. The RCR (respiratory control ratio) was only decreased in male DCD hearts in the presence of complex I substrate. Maximal respiration rate stimulated by 2 mM ADP and uncoupled rate (stimulated by dinitrophenol) was also decreased in both male and female DCD SSM compared with corresponding CBD SSM in the presence of complex I substrate. There were no significant differences in maximal respiration or uncoupled rate between male and female DCD SSM. The state 3 respiratory rate, maximal respiration, and uncoupled rate of respiration in SSM of both male and female DCD hearts was decreased compared with their corresponding CBD hearts when succinate was used as a complex II substrate. These rates were also better preserved in female DCD hearts compared with male DCD hearts with succinate as a complex II substrate. The state 4 respiration was slightly decreased in male DCD hearts compared with its corresponding CBD hearts oxidizing complex II substrates. The maximal respiration of SSM in male and female DCD hearts decreased compared with their corresponding CBD hearts when TMPD-ascorbate was used as a complex IV substrate.
Table 1.
Oxidative phosphorylation in SSM from male and female CBD and DCD hearts
| Male |
Female |
|||
|---|---|---|---|---|
| CBD | DCD | CBD | DCD | |
| n | 8 | 8 | 6 | 7 |
| Protein yield, mg/g | 13.1 ± 1.1 | 10.2 ± 0.4* | 14.7 ± 1.1 | 11.5 ± 0.6* |
| Complex I substrates (glutamate + malate) | ||||
| State 3, nAO/min/mg | 234 ± 22 | 119 ± 8* | 245 ± 19* | 174 ± 16*† |
| State 4, nAO/min/mg | 36.1 ± 4.8 | 31.6 ± 2.3 | 35.7 ± 5.2 | 35.1 ± 6.6 |
| RCR | 6.8 ± 0.4 | 3.9 ± 0.5* | 7.2 ± 0.5 | 7.1 ± 2.3 |
| ADP (2 mM), nAO/min/mg | 266 ± 26 | 128 ± 11* | 244 ± 26 | 166 ± 18* |
| DNP (0.3 mM), nAO/min/mg | 264 ± 24 | 126 ± 12* | 259 ± 24 | 171 ± 24* |
| Complex II substrates (succinate + rotenone) | ||||
| State 3, nAO/min/mg | 257 ± 25 | 141 ± 8* | 266 ± 32 | 212 ± 16*† |
| State 4, nAO/min/mg | 100 ± 11 | 65 ± 4* | 99 ± 15 | 80 ± 9 |
| RCR | 2.6 ± 0.2 | 2.2 ± 0.2 | 2.8 ± 0.3 | 2.8 ± 0.4 |
| ADP (2 mM), nAO/min/mg | 254 ± 30 | 135 ± 9* | 285 ± 31 | 197 ± 14*† |
| DNP (0.3 mM), nAO/min/mg | 239 ± 33 | 119 ± 9* | 270 ± 36 | 178 ± 12*† |
| Complex IV substrates (TMPD-ascorbate + rotenone) | ||||
| ADP (2 mM), nAO/min/mg | 751 ± 90 | 405 ± 23* | 792 ± 87 | 640 ± 31† |
Values are means ± SE. ADP, adenosine diphosphate; ADP/O, ratio of ADP over atom oxygen; CBD, control beating-heart donor; DNP, dinitrophenol; IFM, interfibrillar mitochondria; RCR, respiratory control ratio (state 3/state 4); SSM, subsarcolemmal mitochondria; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine. *P < 0.05 vs. corresponding vehicle treatment. †P < 0.05 vs. male-donation after circulatory death (DCD) subsarcolemmal mitochondria.
Functional data of IFM are shown in Table 2. There were no differences in protein yield of IFM between the four groups. There were no differences in state 3 respiration in IFM between male CBD and female CBD hearts using complex I substrate. The state 3 rate of IFM in male DCD hearts was decreased compared with male CBD hearts using complex I substrate. However, state 3 respiration of female DCD IFM was not decreased compared with female CBD IFM. In addition, the state 3 respiration of female DCD IFM was better preserved than that of male DCD IFM oxidizing complex I substrate. There were no differences in state 4 respiration of IFM between the four groups oxidizing complex I substrates. The RCR was decreased in male DCD IFM in the presence of complex I substrate. Maximal respiration rate stimulated by 2 mM ADP and uncoupled rate (stimulated by dinitrophenol) was decreased in male DCD IFM but not in female DCD IFM compared with corresponding CBD IFM using complex I substrate. The rate of state 3, maximal respiration, and uncoupled respiration was decreased in male DCD IFM but not in female DCD IFM compared with their corresponding CBD hearts when succinate was used as a complex II substrate. These rates were also better preserved in the female DCD hearts compared with male DCD hearts using complex II substrates. The maximal respiration of SSM in male DCD IFM was decreased compared with its CBD IFM when TMPD-ascorbate was used as a complex IV substrate. The rate of TMPD oxidation in female DCD IFM was higher than that in male DCD IFM.
Table 2.
Oxidative phosphorylation in IFM from male and female CBD and DCD hearts
| Male |
Female |
|||
|---|---|---|---|---|
| CBD | DCD | CBD | DCD | |
| n | 8 | 8 | 6 | 7 |
| Protein yield, mg/g | 10.2 ± 0.7 | 10.9 ± 0.5 | 10.9 ± 1.0 | 10.8 ± 0.5 |
| Complex I substrates (glutamate + malate) | ||||
| State 3, nAO/min/mg | 264 ± 25 | 176 ± 14* | 256 ± 19 | 243 ± 17† |
| State 4, nAO/min/mg | 44.8 ± 4.0 | 44.7 ± 3.7 | 32.6 ± 6.6 | 47.2 ± 8.4 |
| RCR | 6.2 ± 0.6 | 4.2 ± 0.5* | 10.6 ± 2.7 | 8.4 ± 3.9 |
| ADP (2 mM), nAO/min/mg | 302 ± 33 | 190 ± 20* | 264 ± 23 | 235 ± 21 |
| DNP (0.3 mM), nAO/min/mg | 308 ± 36 | 187 ± 24* | 279 ± 26 | 241 ± 25 |
| Complex II substrates (succinate + rotenone) | ||||
| State 3, nAO/min/mg | 312 ± 28 | 199 ± 16* | 293 ± 24 | 291 ± 18† |
| State 4, nAO/min/mg | 132 ± 9 | 96 ± 2* | 91 ± 11‡ | 109 ± 7 |
| RCR | 2.4 ± 0.2 | 2.1 ± 0.2 | 3.4 ± 0.4‡ | 2.7 ± 0.1† |
| ADP (2 mM), nAO/min/mg | 320 ± 38 | 197 ± 14* | 282 ± 26 | 294 ± 29† |
| DNP (0.3 mM), nAO/min/mg | 290 ± 38 | 178 ± 13* | 256 ± 24 | 269 ± 34† |
| Complex IV substrates (TMPD-ascorbate + rotenone) | ||||
| ADP (2 mM), nAO/min/mg | 821 ± 121 | 479 ± 26* | 1,061 ± 95 | 899 ± 70† |
Values are means ± SE. ADP, adenosine diphosphate; ADP/O, ratio of ADP over atom oxygen; CBD, control beating-heart donor; DNP, dinitrophenol; IFM, interfibrillar mitochondria; RCR, respiratory control ratio (state 3/state 4); TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine. *P < 0.05 vs. corresponding vehicle treatment. †P < 0.05 vs. male-donation after circulatory death (DCD) interfibrillar mitochondria. ‡P < 0.05 vs. male CBD interfibrillar mitochondria.
CRC in SSM and IFM from Male and Female Hearts
There were no differences in the CRC of SSM between male and female CBD hearts. The CRC of SSM from male and female DCD hearts was decreased compared with the corresponding SSM from CBD hearts (Fig. 2A). The CRC was better preserved in SSM from female DCD hearts compared with male DCD hearts [means ± SE, male, 275 ± 17 vs. female, 350 ± 17 nmol Ca2+/mg, P < 0.05] (Fig. 2A). The CRC in IFM from female CBD hearts was decreased slightly compared with IFM from male CBD hearts (Fig. 2B). The CRC in IFM from both male and female DCD hearts was reduced compared with corresponding IFM from CBD hearts (Fig. 2B). There were no differences in the CRC of IFM between male and female DCD hearts (Fig. 2B).
Figure 2.
Alteration of calcium retention capacity (CRC) in male and female heart mitochondria. A: CRC change in subsarcolemmal mitochondria (SSM). There was no difference in the CRC between male and female CBD SSM. The CRC was decreased in both male and female DCD SSM compared with CBD SSM. Interestingly, the CRC in female DCD SSM was less impaired compared with male DCD SSM. B: CRC change in interfibrillar mitochondria (IFM). The CRC in female CBD IFM was slightly decreased compared with male CBD IFM. The CRC was decreased in both male and female DCD IFM compared with CBD IFM. Data were expressed as means ± SE. Two-way ANOVA was used for statistical analysis. *P < 0.05 vs. male or female CBD; †P < 0.05 vs. male DCD; ‡P < 0.05 vs. male CBD. The n values represent independent animals. Male CBD, n = 8; female CBD, n = 6; male DCD, n = 8; female DCD, n = 7. CBD, control beating-heart donor; DCD, donation after circulatory death
H2O2 Generation in SSM and IFM from Male and Female Hearts
A net release of H2O2 was higher in SSM from female CBD hearts compared with male CBD hearts using glutamate as a complex I substrate [CBD, 37 ± 2 vs. DCD, 62 ± 3 pmol/min/mg, P < 0.05] (Fig. 3A). A net release of H2O2 (pmol/min/mg) was higher in SSM from male DCD hearts compared with SSM from female CBD hearts oxidizing glutamate [male DCD, 62 ± 3 vs. female CBD, 47 ± 4 pmol/min/mg, P < 0.05] (Fig. 3A). A net release of H2O2 was not higher in SSM from female DCD hearts compared with SSM from female CBD hearts oxidizing glutamate (Fig. 3A). A net release of H2O2 was less in SSM from female CBD hearts compared with SSM from male CBD hearts using succinate as a complex II substrate in the presence of rotenone to block potential reverse electron flow (Fig. 3B). H2O2 generation was also less in SSM from female DCD hearts compared with SSM from male DCD hearts oxidizing succinate in the presence of rotenone [male DCD, 320 ± 28 vs. female DCD, 157 ± 9 pmol/min/mg, P < 0.05] (Fig. 3B).
Figure 3.
H2O2 generation in male and female heart mitochondria. H2O2 generation was measured in 0.2-mg protein of freshly isolated SSM and IFM using glutamate or succinate + rotenone as complex I or complex II substrates, respectively. A: H2O2 generation in SSM using glutamate as complex I substrate. H2O2 generation was slightly increased in female CBD SSM compared with male CBD SSM. H2O2 generation was increased in male DCD SSM compared with male CBD. There was no difference in H2O2 generation between male and female DCD SSM. B: H2O2 generation in SSM using succinate as complex II substrate in the presence of rotenone to block reverse electron flow. H2O2 generation was decreased in female CBD SSM compared with male CBD SSM. H2O2 generation was also decreased in female DCD IFM compared with male DCD IFM. C: H2O2 generation in IFM using glutamate as complex I substrate. H2O2 generation was slightly increased in female CBD IFM compared with male CBD IFM. H2O2 generation was also increased in female DCD IFM compared with male DCD IFM. There was no difference in H2O2 generation between male and female DCD IFM. D: H2O2 generation in IFM using succinate as complex II substrate in the presence of rotenone to block reverse electron flow. H2O2 generation was decreased in female CBD IFM compared with male CBD IFM. H2O2 generation was also decreased in female DCD IFM compared with male DCD IFM. Data were expressed as means ± SE. Two-way ANOVA was used for statistical analysis. *P < 0.05 vs. male or female CBD; †P < 0.05 vs. male DCD, ‡P < 0.05 vs. male CBD. The n values represent independent animals. Male CBD, n = 8; female CBD, n = 6; male DCD, n = 8; female DCD, n = 7. CBD, control beating-heart donor; DCD, donation after circulatory death; IFM, interfibrillar mitochondria; SSM, subsarcolemmal mitochondria.
A net release of H2O2 (pmol/min/mg) was slightly higher in IFM from female CBD hearts compared with IFM from male CBD hearts using glutamate as a complex I substrate [male CBD, 39 ± 4 vs. female CBD, 55 ± 3, P < 0.05] (Fig. 3C). H2O2 generation was not higher in male and female IFM from DCD hearts compared with their corresponding IFM from CBD hearts oxidizing glutamate (Fig. 3C). H2O2 generation was higher in IFM from female DCD hearts compared with IFM from male DCD hearts oxidizing glutamate [male DCD, 42 ± 4 vs. female DCD, 57 ± 3, P < 0.05] (Fig. 3C). A net release of H2O2 was less in IFM from female CBD hearts compared with IFM from male CBD hearts using succinate as a complex II substrate in the presence of rotenone [male CBD, 365 ± 25 vs. female CBD, 173 ± 35, P < 0.05] (Fig. 3C). A net release of H2O2 was less in IFM from female DCD hearts compared with IFM from male DCD hearts oxidizing succinate in the presence of rotenone [male DCD, 420 ± 45 vs. female DCD, 196 ± 13, P < 0.05] (Fig. 3D).
DISCUSSION
Factors attributed to suboptimal HTx outcomes with female donor hearts, especially in male recipients, are not well defined. Susceptibility to damage resulting from storage and transportation of heart (ischemia) is one of the factors that is linked to suboptimal HTx outcomes from female donor hearts. The extent of damage from ischemia is one of the important factors that will guide the future DCD HTx practice. Our study quantified the size of infarct in male and female hearts with 25 min of global in vivo ischemia and noticed no increase in infarct size in female hearts. In addition, consistent with our previous publications (8), mitochondrial function was impaired in male and female DCD hearts (SSM > IFM) compared with CBD hearts. However, the extent of mitochondrial dysfunction was more severe in male DCD hearts compared with female DCD hearts. Similarly, susceptibility of MPTP opening was increased in male and female DCD heart mitochondria compared with CBD hearts. The increased susceptibility of MPTP opening in DCD hearts was comparable for both sexes. In addition, infarct size was smaller in female DCD hearts with 35 min ischemia compared with male DCD hearts. Thus, our results suggest that suboptimal outcomes with female donor hearts are unlikely due to increased susceptibility to ischemic injury or greater mitochondrial damage. Other factors such as donor comorbid conditions, hormonal influences, endothelial response to stress, and immunological differences may contribute to suboptimal HTx outcomes in recipients of female donor hearts.
Influence of Sex on Cardiac Injury in HTx
HTx is an established therapy for patients with end-stage heart failure (3). Although previous studies show that female HTx recipients have slightly decreased survival compared with male recipients (26), a recent study found no difference in posttransplant survival between women and men after matching for recipient and donor characteristics (3). However, male recipients using hearts from female donors had inferior survival compared with male recipients of male donor hearts (2). Although women only represent ∼20% of HTx recipients (3), their contribution to the heart donor pool is 31% (2). At present, with the brain death donors, there is a reluctance to accept a female donor heart for a male recipient due to potential suboptimal outcomes. It is likely that this reluctance may extend to the DCD HTx practice in the future. Therefore, we studied the cardiac injury in female DCD hearts to quantify the differences in ischemia tolerance. Our results indicated that female DCD hearts tolerate ischemia-reperfusion injury similarly and in some aspects better than male DCD hearts. The results are consistent with the notion that female hearts are more resistant to ischemia/reperfusion injury (27–30).
Our previous studies led us to select 25 min of in vivo warm ischemia as the limit to study DCD heart function without any modulating factors (8). The infarct size was not larger in female DCD hearts after 25 min of ischemia than in male DCD hearts. Interestingly, with a longer duration of ischemia, 35 min, the infarct size was relatively smaller in female DCD hearts compared with male DCD hearts. We do not have a clear explanation for better ischemia tolerance in female DCD hearts, but it could be secondary to better mitochondrial function preservation. Our results provide objective evidence that cardiac injury, as measured with infarct size and mitochondrial function, is not increased in female DCD hearts compared with male DCD hearts. Hence, the suboptimal outcomes in male recipients receiving female donor hearts are less likely due to increased cardiac injury. As ischemic injury is inherent to DCD organ donation, it is important to keep our study findings in perspective while evaluating a female DCD donor heart.
Mitochondrial Dysfunction in DCD Hearts
Our previous studies show that 25 min of global ischemia leads to a decreased OXPHOS in SSM and IFM, with more damage in SSM than IFM from DCD hearts (8). In the current study, we found that 25 min of ischemia decreased OXPHOS in SSM from female DCD hearts without significant alteration in IFM. The decreased OXPHOS can be due to a damaged electron transport chain (ETC), including complex I, II, III, and IV, or damaged phosphorylation apparatus (complex V). To localize the damage in the ETC, DNP was used in the current study. DNP is an uncoupler that brings protons across the inner mitochondrial membrane back to the mitochondrial matrix bypassing the phosphorylation apparatus (19). Thus, DNP enhances uncoupled respiration, which indicates intactness of the ETC, and reduction of uncoupled respiration in the presence of DNP implies impairment of electron transfer between the complexes. The decreased uncoupled respiration with DNP, as seen in our study of mitochondria from DCD hearts, indicates that the ETC in DCD heart mitochondria was damaged (19).
Although state 3 respiration was slightly better preserved in SSM from female DCD hearts compared with SSM from male DCD hearts, maximal ADP- and DNP-stimulated respiration in SSM from female DCD hearts was comparable to SSM from male DCD hearts. These results suggest that the degree of damage in SSM from male and female DCD hearts was comparable. However, the degree of damage in IFM from female DCD hearts was less compared with IFM from male DCD hearts. As IFM are the primary source of energy supply to the contractile apparatus (12, 31), a relatively less damaged IFM may provide more energy supply in female DCD hearts during reperfusion. This may explain better functional recovery in female DCD hearts during reperfusion, as shown with improved RPP (male 6,911 ± 743 vs. female 12,792 ± 2,022, P < 0.05).
MPTP Opening in Mitochondria from DCD Hearts
The opening of MPTP is a final step preceding cell death due to ischemia-reperfusion injury (31, 32). MPTP is a nonselective pore in the inner mitochondrial membrane functioning in communication between the mitochondria matrix and cytoplasm (33–35). MPTP opening leads to loss of mitochondrial membrane integrity, decreased energy production, and ultimately, cell death (33–35). Susceptibility to MPTP opening was increased during reperfusion in mitochondria from male DCD hearts (8, 9). MPTP opening contributes to cardiac injury in DCD hearts. Inhibition of MPTP opening with cyclophilin D or amobarbital treatment at reperfusion in DCD hearts led to decreased cardiac injury (7, 15). In the present study, we found that the CRC was decreased in male and female DCD hearts compared with corresponding CBD hearts. However, CRC is better preserved in female DCD hearts than in male DCD hearts. These results indicate that susceptibility of MPTP opening is affected less in female DCD hearts compared with male DCD hearts.
ROS generation is one of the activators for increased MPTP opening during reperfusion (36, 37). Mitochondrial ETC is a key site for ROS generation (23, 38). Complex I and complex III are the two principal sites for ROS generation, with complex III playing a dominant role in ROS generation in cardiac mitochondria (23, 38). Ischemia-induced ETC damage increases ROS generation in cardiac mitochondria (39). ROS generation was increased in male DCD heart mitochondria (7, 8). In the current study, ROS generation was slightly increased in SSM and IFM from female CBD hearts compared with male CBD hearts in the presence of complex I substrate. However, the amount of ROS generation using complex I substrate was much less than that using complex II substrates. The ROS generation in female SSM and IFM was decreased compared with male DCD hearts in the presence of complex II substrates. These results suggest that less ROS production in female DCD hearts may contribute to better CRC and reduced cardiac injury following 35 min of ischemia.
Potential Other Factors That Affect Donor Heart Function
Many factors affect posttransplantation outcomes, including the age and morbidities in heart donor, ischemia duration, organ preservation strategy, and recipient factors (3). Although ischemic cardiac injury is minimal during CBD heart donation, it is unavoidable in DCD heart donation. As ischemia is inherent to DCD organ donation, we elected to examine the effect of sex on heart damage with 25 min of ischemia that was shown to induce significant injury in male hearts (8). Our study shows that ischemic injury as measured by IS and mitochondrial function is not increased in female DCD hearts. Future studies focused on studying the role of inflammation (40, 41) and sex-specific immune responses (2) may contribute to further understanding of the suboptimal outcomes in men receiving female donor hearts.
Limitations
Our study shows that cardiac injury is not increased in female DCD hearts following in vivo ischemia and ex vivo reperfusion. The major limitation is the lack of in vivo reperfusion in our study. As discussed earlier, sex-specific immune responses and hormone receptor modulators may play a key role in posttransplant survival; this information needs to be separately addressed using in vivo ischemia-reperfusion models. A model of rat heart transplantation using donor hearts from both male and female DCD donors will address factors associated with in vivo reperfusion of DCD donor hearts.
Conclusion
Ischemia-reperfusion injury, as measured by the infarct size and mitochondrial function in DCD rat hearts with 25 min of ischemia, was not influenced by sex.
DATA AVAILABILITY
Data sharing will be made available upon request from the authors.
GRANTS
This work was supported by Veterans Affairs Merit Review Grants 2IO1 BX003859 (to M. Quader) and 2IO1 BX001355 (to E. J. Lesnefsky) and funds from the Pauley Heart Center (to M. Quader and Q. Chen).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Q.C., E.J.L., and M.Q. conceived and designed research; Q.C. and O.A. performed experiments; Q.C., O.A., and M.Q. analyzed data; Q.C., O.A., E.J.L., and M.Q. interpreted results of experiments; Q.C. prepared figures; Q.C. drafted manuscript; E.J.L. and M.Q. edited and revised manuscript; O.A., E.J.L., and M.Q. approved final version of manuscript.
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Associated Data
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Data Availability Statement
Data sharing will be made available upon request from the authors.
