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Ann Thorac Surg 2007;83:1102-1109
© 2007 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Harvard Medical School, Boston, Massachusetts
b Harvard School of Public Health, Boston, Massachusetts
Accepted for publication October 23, 2005.
* Address correspondence to Dr McCully, Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Rm 144, Boston, MA 02115 (Email: james_mccully{at}hms.harvard.edu).
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Abstract |
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Methods: To test this hypothesis, 104 male and female rabbit hearts, mature (15 to 20 weeks) and aged (>32 months), were subjected to Langendorff perfusion. Control hearts were perfused for 75 minutes. Global ischemia hearts were underwent 30 minutes of equilibrium, 30 minutes of global ischemia, and 15 minutes of reperfusion. Cardioplegia (potassium/magnesium) ± diazoxide was infused 5 minutes before global ischemia. Mitochondria were isolated from left ventricular tissue and used for the measurement of oxygen consumption and [Ca2+]Mito.
Results: Mitochondrial oxygen consumption was significantly increased in the mature and aged female hearts in all treatment groups (p < 0.001 versus male). Cardioplegia ± diazoxide modulated mitochondrial oxygen consumption, but these effects were significantly decreased in the aged heart and in the female heart (p < 0.001 each versus male). Cardioplegia (potassium/magnesium) significantly decreased [Ca2+]Mito (p < 0.001 versus global ischemia) in aged but not mature hearts. The addition of diazoxide to potassium/magnesium significantly decreased [Ca2+]Mito in mature and aged males (p < 0.001 versus potassium/magnesium) but not in females.
Conclusions: These results demonstrate that mitochondrial oxygen consumption and [Ca2+]Mito are modulated by age and gender and play an important role in the differences observed between mature and aged male and female response to global ischemia and the cardioprotection afforded by cardioplegia ± diazoxide.
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Introduction |
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We also demonstrated that the effects of global ischemia were partially ameliorated by cardioplegia ± diazoxide, but this cardioprotection was significantly decreased in the aged compared with the mature heart and significantly decreased in the aged female compared with the aged male rabbit heart [2]. These animal studies are consistent with human studies indicating that women have a significantly greater risk potential compared with men and worse outcomes after cardiac surgery [3, 4].
The Society of Thoracic Surgeons National Cardiac Surgery Database provides evidence that in patients undergoing coronary artery bypass grafting (CABG), women have a significantly higher operative mortality than men, and long-term survival is generally less favorable in women than in men [5]. Multivariate analysis also shows that women have higher mortality rates than equally matched men in low-risk and medium-risk groups [5]. It is only among very high-risk patients that sex is not found to be an independent predictor of adverse outcomes [5].
Our model was chosen because previous studies in animal models have shown that age-related ischemic intolerance develops well before senescence, being primarily evident by middle age [2, 6, 7]. It has been demonstrated in animal models that there is an apparent nadir in ischemic tolerance in middle-aged to aged hearts, with a subsequent modest improvement with senescence [6, 7]. This pattern is consistent with empiric clinical observations and with prior studies showing an improvement in the senescent compared with the aged heart that may be related to selection of a subpopulation resistant to myocardial injury [6, 7]. Support for this hypothesis comes from the study of Vaccarino and colleagues [8], who have reported that in CABG patients aged between 50 and 60 years, women experienced an 86% higher risk of in-hospital death than men; however, sex differences in in-hospital mortality were less marked in the older-age subgroup.
The mechanism(s) modulating age-associated and gender-associated differences in cardioprotection remain to be fully elucidated; however, we and others have suggested that alterations in mitochondrial function and mitochondrial free matrix calcium accumulation ([Ca2+]Mito) occurring after global ischemia and apparent during early reperfusion may play a significant role [9–15]. Results from our recent studies led us to hypothesize that age and gender modulated mitochondrial function and [Ca2+]Mito and that these changes are associated with the age-specific and gender-specific differential cardioprotection afforded by cardioplegia ± diazoxide. To test this hypothesis, age-matched mature (sexually mature) and aged (retired breeders, not senescent) male and female hearts were subjected to either no ischemia (control) or ischemia–reperfusion with and without cardioplegia ± diazoxide. Mitochondria were isolated from postischemic hearts to determine the effects of ischemia on mitochondrial function and calcium accumulation.
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Material and Methods |
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Langendorff Perfusion
All rabbits were anesthetized with pentobarbital sodium (Nembutal, 100 mg/kg; Abbott Laboratories, North Chicago, IL), and heparin (200 U/kg) was given intravenously through the marginal ear vein [1, 2, 10]. Langendorff perfusion was performed as previously described [1, 2, 10]. Hearts were paced via the right atrium so that heart rate was maintained at 180 ± 3 beats/min throughout the experiment using a Medtronic Model 5330 stimulator (Minneapolis, MN). Hemodynamic variables were acquired using the PO-NE-MAH digital data acquisition system (Gould, Valley View, OH), with an Acquire Plus processor board (Gould, Valley View, OH), and left ventricular pressure analysis software [1, 2].
Experimental Protocol
The experimental protocol is shown in Figure 1. To determine the effects of ischemia–reperfusion on mitochondrial function and mitochondrial free calcium accumulation during early reperfusion, hearts were perfused for 30 minutes to establish equilibrium hemodynamics. Control hearts were perfused without global ischemia at 37°C for 75 minutes. Global ischemia (GI) hearts underwent 30 minutes of perfusion for equilibrium, 30 minutes of global ischemia, and 15 minutes of reperfusion. The K/Mg hearts and K/Mg + DZX hearts were perfused for 30 minutes of equilibrium, received either K/Mg or K/Mg + DZX at the start of global ischemia, and then underwent 15 minutes of reperfusion. The reperfusion time of 15 minutes was used because previous investigations have shown that a 15-minute reperfusion allows for verification of hemodynamic recovery of samples and for comparison with previous results using 120 minutes of reperfusion as an end point [2].
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A separate group of hearts consisting of mature male hearts (n = 6 each group), mature female hearts (n = 6 each group), aged male hearts (n = 6 each group), and aged female hearts (n = 6 each group) were immediately freeze-clamped at the end of reperfusion and stored in liquid nitrogen. They were then used for mitochondrial free calcium determination and tissue ATP content.
Mitochondrial Oxygen Consumption
Mitochondria were isolated from postischemic left ventricular tissue and suspended in 3 to 4 mL of respiration medium as previously described [10]. Mitochondrial oxygen consumption was determined using a Clark-type electrode (Yellow Springs Instruments Co, Yellow Springs, OH) as previously described [10]. In brief, oxygen consumption in isolated mitochondria was determined in the presence of the substrate malate/glutamate (complex I) or succinate (complex II). The addition of adenosine diphosphate (ADP) causes a sudden burst of oxygen uptake as the ADP is converted into ATP. Before the assay, the instrument was calibrated to 100% saturation in air-saturated respiration media at 30°C.
Oxygen consumption at complex I, the first of the complexes of the electron transport chain, was determined using glutamate and malate (5 mM of each) added to the respiration media. Complex II mitochondrial oxygen consumption, the second of the complexes of the electron transport chain, was determined using succinate (8 mM) and rotenone (4 µM), an inhibitor of complex I, added to the respiration media in the place of glutamate and malate. State 3, (active) mitochondrial oxygen consumption defined as ADP-stimulated respiration, was initiated by the addition of 1 mmol/L ADP to the respiration media and allowed to proceed for 2 minutes, as we have previously described [10]. Oxygen consumption was calculated based on an initial air-saturated [O2] = 195 nM/mL [10].
Mitochondrial Free Matrix Calcium
[Ca2+]Mito was determined using an in-house spectrofluorescence system as previously described [9]. Two observations per heart were determined for [Ca2+]Mito, and the mean was used as n = 1 observation. Calibration was performed as previously described [10].
Tissue Adenosine 5'-Triphosphate Content
Tissue ATP content was determined in freeze-clamped tissue samples (approximately 200 mg) according to the method as described by Lowry and Passonneao [17] using a LS 55 luminescence spectrometer (Perkin Elmer, Wellesley, MA). Tissue ATP content was expressed as ATP content per gram of dry weight, based on an ATP standard curve (ATP standard, FL-AAS, Sigma-Aldrich, St. Louis, MO). Two observations per heart were determined for ATP content and the mean used as n = 1 observation. Wet weight/dry weight ratios were determined as previously described [18].
Statistical Analysis
Statistical analysis was performed with SAS 8.02 software (SAS Institute, Cary, NC). The mean ± standard error of the mean is shown for all results. Standard t tests were used for comparing GI results with the control group. The same approach was used for comparisons between male and female hearts within an age and treatment group. Post hoc pair-wise testing was performed using a Bonferroni correction. Values for p for the between-group comparisons were reported adjusted for the six comparisons within each analysis of variance. A two by two analysis of variance with factors for age, gender, and age by gender interaction was performed. Results presented always include the age by gender interaction. Statistical significance was set at p < 0.05.
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Results |
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Tissue adt content after 15 minutes of reperfusion
No significant difference in tissue ATP content was observed between mature and aged male and female control hearts (p = 1.000, Table 1). Tissue ATP content after 15 minutes of reperfusion was significantly decreased compared with control in GI, K/Mg, and K/Mg + DZX in both mature and aged male and female hearts (p < 0.001 for each). K/Mg and K/Mg + DZX significantly increased tissue ATP content compared with GI in both mature and aged male and female hearts (p < 0.001 for each). Tissue ATP content was significantly decreased in aged males and females compared with mature male and female hearts in GI, K/Mg, and K/Mg + DZX hearts (Table 1). Of significance, tissue ATP content was significantly decreased in aged female K/Mg + DZX hearts compared with aged male K/Mg + DZX hearts (Table 1).
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K/Mg + DZX cardioplegia significantly decreased [Ca2+]Mito in mature male (p < 0.001 versus GI), in aged male (p < 0.001 versus GI), and in mature female (p = 0.019 versus GI), but not in aged female hearts (p = 1.000 versus GI). [Ca2+]Mito was significantly increased in mature female hearts compared with mature male K/Mg + DZX hearts and in the aged female hearts compared with the aged male K/Mg + DZX hearts (Table 2).
State 3 (active) mitochondrial oxygen consumption
Malate and succinate state 3 (active) mitochondrial oxygen consumption was significantly increased in both mature and aged female hearts compared with male hearts (Fig 3;
Table 3). Age significantly decreased both malate and succinate state 3 mitochondrial oxygen consumption.
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K/Mg + DZX significantly decreased succinate state 3 mitochondrial oxygen consumption in mature male and female hearts compared with K/Mg but had no affect in aged male or aged female hearts (Fig 3; Table 3). K/Mg + DZX significantly increased succinate state 3 mitochondrial oxygen consumption only in mature female hearts; in all others, no significant difference compared with GI was observed (Fig 3).
A significant difference between K/Mg and K/Mg + DZX malate state 3 mitochondrial oxygen consumption was observed in mature male hearts (p < 0.001). No significant difference was observed in any other experimental treatments (p = 1.000; Table 3).
Both malate and succinate state 3 mitochondrial oxygen consumption were affected by age, significantly decreased in aged compared with mature and gender, significantly increased in female hearts compared with male and by age by gender, significantly increased in mature female hearts compared with male, and significantly increased in aged female hearts compared with aged male, except for GI (Table 3). Significant differences between mature male and female hearts and aged male and female hearts were observed in all experimental groups (Table 3).
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Comment |
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Our data show that after unprotected global ischemia, there is a significant decrease in tissue ATP content, a significant decrease in both complex I and complex II mitochondrial oxygen consumption, and a significant increase in [Ca2+]Mito. In global ischemia, increased [Ca2+]Mito accumulation has been shown to destabilize the inner mitochondrial membrane and cause the inner membrane pore to open and permit further cation movement [11, 12, 14, 22]. This is an energy-dependent process requiring ATP to transport calcium against the electrochemical gradient out of the mitochondrion, thus reducing tissue ATP content, a process called futile calcium cycling [10].
In the aged heart, [Ca2+]Mito is significantly increased and would result in increased futile calcium cycling and decreased tissue ATP content, which in turn would limit postischemic functional recovery and cell viability. Support for this mechanism is seen in our earlier observations demonstrating that global ischemia significantly decreases postischemic functional recovery and significantly increases infarct size in the GI heart compared with the control heart and that these effects are exacerbated in the aged compared with the mature heart [2].
Our data further indicate that the cardioprotection afforded by cardioplegia ± diazoxide modulates tissue ATP content, mitochondrial oxygen consumption, and [Ca2+]Mito. The proportionate role of each of these mechanisms is affected by age and gender, however. In the mature male and female heart, K/Mg cardioplegia had significantly increased tissue ATP content and significantly increased both complex I and complex II mitochondrial oxygen consumption but had no effect on [Ca2+]Mito compared with GI. The increase in both complex I and complex II mitochondrial oxygen consumption would allow for increased high-energy phosphate resynthesis, which would allow for proper mitochondrial function in the presence of increased [Ca2+]Mito concentration.
In the aged male and female heart, K/Mg cardioplegia did not enhance either complex I or complex II mitochondrial oxygen consumption but significantly decreased [Ca2+]Mito levels. This action would decrease futile calcium cycling, thus maintaining high-energy phosphate stores for cell survival and function.
The addition of diazoxide to K/Mg cardioplegia further decreased [Ca2+]Mito compared with K/Mg alone in both the mature and aged male heart and allowed for decreased complex I and complex II mitochondrial oxygen consumption. In the mature and aged female heart, however, the addition of diazoxide to K/Mg cardioplegia had no effect on [Ca2+]Mito compared with K/Mg alone, and both complex I and complex II mitochondrial oxygen consumption were significantly increased compared with mature and aged males, respectively. This increase in [Ca2+]Mito levels, which are significantly increased in the aged compared with the mature heart, would be expected to increase futile calcium cycling and significantly decrease tissue ATP content, as evidenced in our results. These events would further impact negatively on postischemic functional recovery and infarct size as we demonstrated previously [2].
It has been proposed that increased [Ca2+]Mito destabilizes the mitochondrial inner membrane, causing the inner membrane pores to open and thus permitting further movement of cations across the mitochondrial membrane [22]. The opening of the inner membrane pores renders the mitochondrion incapable of synthesizing ATP and has been suggested to be a key event in the process leading to myocardial cell death.
We have suggested that the early opening of the mitoKATP channels would allow for K+ entry into the mitochondria and result in the depolarization of the mitochondrion, which would reduce Ca2+ entry through the mitochondrial calcium uniporter and thus decrease [Ca2+]Mito during global ischemia. Subsequently, these events would result in ATP preservation and reduce the requirement for high-energy resynthesis during reperfusion and reduce energy flux through the electron transport with resultant and reactive oxygen species generation. These events would putatively allow for enhanced postischemic functional recovery and the limitation of infarct size. The inability of diazoxide to decrease [Ca2+]Mito in the mature and aged female heart would increase futile calcium cycling and decrease the high-energy phosphates required for cell maintenance and cell survival. These results are in agreement with our previous studies demonstrating that the addition of diazoxide to cardioplegia is less effective in females compared with males and significantly less effective in the aged female compared with the aged male [2].
The mechanism(s) which differentiate female from male heart response to cardioplegia ± diazoxide remain to be fully elucidated. Our data demonstrate that mitochondrial oxygen consumption is significantly increased in the mature and aged female compared with the male. The reason for these differences is at present unknown; however, we speculate that estradiol may play an important role.
Previous studies have shown that the mechanism of action of 17ß-estradiol is modulated by the mitoKATP channels and that the infarct-size-limiting effects of 17ß-estradiol are abolished by 5-hydroxydecanoate, a mitoKATP channel-blocker [23–25]. Whether these changes are related to hormonal changes or other factors remains at the level of speculation and more involved studies are required.
It is important to note that our results have been obtained in an isolated perfused heart and require further studies using an in situ blood perfused model. Our data do demonstrate, however, that mitochondrial oxygen consumption and [Ca2+]Mito are modulated by age and gender and are associated with observed age-related and gender-related differences in the response to global ischemia and the cardioprotection afforded by cardioplegia ± diazoxide.
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