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Ann Thorac Surg 1995;60:338-344
© 1995 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery and Department of Pathology, New England Deaconess Hospital and Harvard Medical School, Boston, Massachusetts
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. To investigate the relationship between increased mitochondrial and nuclear calcium and DNA fragmentation, mature and aged rabbit hearts were subjected to normothermic global ischemia with and without the addition of ruthenium red to block mitochondrial calcium influx. Cytosolic calcium accumulation was measured in a parallel experiment using fura-2.
Results. Ruthenium red ameliorated mitochondrial calcium accumulation and was associated with both decreased DNA fragmentation and decreased nuclear calcium accumulation.
Conclusions. Nuclear calcium accumulation was correlated with increased mitochondrial calcium accumulation but not increased cytosolic calcium accumulation in the aged heart. Modulation of mitochondrion ``futile calcium cycling'' may be of significance in the modulation of ischemic myocardial injury.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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The mechanism(s) leading to myocardial cellular dysfunction have yet to be elucidated; however, mitochondrial calcium ([Ca2+]mt) accumulation has been suggested as one possible mechanism contributing to reduced functional recovery after normothermic GI in the aged as compared with the mature heart. Increased [Ca2+]i accumulation has been shown to increase [Ca2+]mt accumulation [11]. In addition, increased [Ca2+]mt accumulation has been shown to parallel the severity of contracture and the lack of diastolic relaxation during reperfusion [12].
Previous reports have suggested that the reduction of ischemic tolerance in the aged myocardium may be related to the observed alterations in calcium homeostasis in the aging mitochondria [3, 13]. Under homeostatic conditions the mitochondrial inner membrane (cristae), which contains the electron transport chain, expels protons to the cytosol, creating a charge gradient that provides the passive energy for Ca2+ influx by the Ca2+ uniporter. Increased [Ca2+]mt accumulation destabilizes the inner mitochondrial membrane and causes the inner membrane pore to open and permit further cation movement (``futile calcium cycling'') [3]. It has been speculated that this futile calcium cycling in the mitochondrion, an energy-dependent process requiring adenosine triphosphate (ATP) to transport calcium against the electrochemical gradient out of the mitochondrion, utilizes needed ATP required for the maintenance of cell viability [14]. With subsequent depletion of ATP during ischemia, mitochondrial function may play a central role in the molecular events leading to tissue injury, especially in the aged myocardium.
Recently we have shown that in the aged myocardium reduced functional recovery was correlated with increased nuclear calcium ([Ca2+]n) accumulation and increased DNA fragmentation during normothermic global ischemia [9]. In the mature heart these changes were not evident [9]. These events have led us to speculate that the modulation of intracellular organellar calcium may be important in the overall recovery of the aged myocardium. These findings further suggest that myocardial susceptibility to ischemic injury in the aged as compared with the mature heart may involve an underlying mechanism(s) that is altered or at least made vulnerable by the aging process such that the functional recovery of the myocardium is compromised.
Benzi and Lerch [15] have reported that postischemic perfusion with ruthenium red, a hexavalent dye that inhibits [Ca2+]mt uptake, significantly decreased oxygen consumption and enhanced contractile function in the rat isolated perfused heart. These authors suggested that the modulation of intracellular calcium accumulation may be involved in the mechanism(s) underlying decreased myocardial functional recovery [15]. It is unclear, however, if age-related alterations in [Ca2+]mt accumulation during global ischemia affect [Ca2+]n accumulation and DNA fragmentation. In this article, we have used ruthenium red to modulate [Ca2+]mt accumulation during GI. Our results show that decreased [Ca2+]mt accumulation during GI is associated with decreased [Ca2+]n accumulation and DNA fragmentation in the aged heart. These findings may have important implications in understanding the mechanism of ischemia-induced reduction of myocardial function and may allow for the reduction of morbidity and mortality in the aged patient.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Langendorff Perfusion
All rabbits were anesthetized with sodium pentobarbital (100 mg/kg intravenously), and heparin (200 units/kg intravenously). The heart was excised and placed in a 4°C bath of Krebs-Ringer solution containing (in mmol/L) NaCl, 100; KCl, 4.7; CaCl2, 1.7; MgSO4, 1.2; NaHCO3, 25; KH2PO4, 1.1; glucose, 11.5; sodium pyruvate, 4.9; and sodium fumarate, 5.4; equilibrated with 95% O2 and 5% CO2 (pH 7.4 at 37°C). Spontaneous beating ceased within a few seconds. Polyethylene cannulas were advanced into the main pulmonary artery, the right superior vena cava, and the left atrium via the pulmonary vein and held in place by sutures. The inferior and left superior venae cavae were closed near their insertion into the right atrium, and the left atrium was opened. A latex balloon containing a catheter-tip transducer (Millar Instruments, Inc, Houston, TX) was inserted into the left ventricle and held in place by a pursestring suture. The volume of the water-filled balloon was maintained at a constant physiologic end-diastolic pressure in a range of 5 to 10 mm Hg using a calibrated microsyringe. The aorta was cannulated with a metal cannula, and the heart was subjected to Langendorff retrograde perfusion at a constant pressure of 75 cm H2O at 37°C. Left ventricular pressure was recorded, and an electrocardiogram was obtained with electrodes placed on the epicardial surface of the right ventricle. The heart was placed into the water-jacketed chamber, and myocardial temperature was maintained at 37°C.
Preparation of Ruthenium Red
Crude ruthenium red was purified by the method according to Luft [16]. The purified ruthenium red powder was dissolved in Krebs-Ringer solution, filtered through a 0.2-µm filter, and diluted in Krebs-Ringer solution to a final 6 µmol/L concentration. Spectrophotometry was employed to confirm concentration. The modified Langendorff apparatus was preequilibrated with the ruthenium red perfusate for 2 hours before rabbit heart perfusion to eliminate loss of ruthenium red secondary to adsorbance onto the apparatus surfaces.
Langendorff Perfusion With Ruthenium Red
Langendorff perfusion was performed as described above but with the following inclusions. Isolated hearts (mature, n = 18; aged, n = 18) were perfused retrograde for 30 minutes with Krebs-Ringers solution (pH 7.4, 37°C) to allow equilibration to stable pulse rate. The heart then was perfused with 6 µmol/L ruthenium red in Krebs-Ringer solution for 40 minutes. After the ruthenium red perfusion, the cannula was clamped to provide for 30 minutes of normothermic ischemia. Control hearts were perfused at a constant pressure of 75 cm H2O at 37°C for 160 minutes (30 minutes of equilibrium, 40 minutes of sham perfusion, and 30 minutes of sham global ischemia). Global ischemia hearts were perfused at a constant pressure of 75 cm H2O at 37°C for 70 minutes (30 minutes of equilibrium, 40 minutes of sham perfusion) then subjected to 30 minutes of normothermic GI. All hearts were freeze clamped at the end of the experimental protocol and stored in liquid nitrogen before subsequent determinations.
Comparison of Wet and Dry Weights
Frozen samples from all experimental groups were weighed (wet weight) and then dried at 80°C for 24 hours for reweighing (dry weight) and determination of wet/dry weight ratios using previously described methods [9].
Measurement of Cytosolic Calcium Accumulation
The fluorescent calcium indicator fura-2 was used to measure [Ca2+]i accumulation quantitatively. After 30 minutes of Langendorff retrograde circulation for equilibrium, background fluorescence and hemodynamic data of left ventricular pressure were recorded as control data. The hearts (mature, n = 18; aged, n = 18) then were loaded with 2.5 µmol/L fura-2 in Krebs-Ringer solution and recirculated for 15 minutes. After the loading process, the heart was perfused for 30 minutes with Krebs-Ringer solution to wash out unincorporated fura-2 acetoxymethyl ester. Fura-2 epifluorescence (510 nm) from the epicardial surface of the left ventricle was measured using an in-house spectrofluorescence system, which supplied rapidly alternating excitation wavelengths (340 nm, 380 nm) to the isolated perfused heart and allowed for the quantitative determination of [Ca2+]i from the ratio of emission induced by the two excitation wavelengths [9, 10]. The fura-2 fluorescence ratio was calculated as described previously [9, 10].
Isolation of Nuclei and Mitochondria
Mitochondria were isolated by differential centrifugation according to the method of Welter and associates [17]. Myocardial nuclei were isolated by the nonenzymatic method of Liew and co-workers [18]. All procedures were performed at 4°C unless otherwise stated.
Determination of Nuclear and Mitochondrial Calcium
Mitochondrial and nuclear pellets were suspended in 1% hydrochloric acid and sonicated using an Ultrasonic Homogenizer 36260 Series (Cole-Parmer Instruments, Chicago, IL). Quantitative, colorimetric determinations were determined by the o-cresolphthalein complexone reaction in the presence of 8-hydroxyquinoline [9]. Nuclear and mitochondrial DNA (mtDNA) concentration was determined as described previously [9].
Nuclear DNA Isolation and Purification
Nuclear DNA was purified using previously described methods [9].
Determination of Nuclear DNA Fragmentation
Nuclear DNA fragmentation was determined by agarose gel electrophoresis and scanning laser densitometry [9]. Semiquantitative determination of DNA fragmentation was performed by scanning laser densitometry using an LKB Ultrascan XL laser densitometer (Pharmacia LKB, Cambridge, England). The integral for each blot was calculated using the LKB GelScan XL software program for one-dimensional analysis. 
-DNA (HindIII digest) was used as a molecular size standard. Intact DNA was determined arbitrarily to be DNA resolving at greater than or equal to 23 kB. The percent of DNA fragmentation was determined based on the amount of DNA found to resolve at less than 23 kB [9].
Statistical Analysis
Statistical analysis was performed using the Stat View II software package. The mean and standard deviation for all data was calculated for all variables and significance determined within and between groups using a one-way analysis of variance followed by Tukey's range test. Statistical significance was claimed only at p less than 0.05.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Mitochondrial DNA Concentration
To allow for standardization of results and to account for possible differences in mitochondrial populations in the mature and aged heart, mtDNA concentration per gram of wet weight was measured. No significant difference in mtDNA concentrations per gram of wet weight was found between mature and aged hearts (results not shown). These results allowed for the use of mtDNA to provide a basis for the comparison of intraorganelle cation concentration.
Effect of Global Ischemia on Mitochondrial Calcium Accumulation in Mature and Aged Myocardium: Effect of Ruthenium Red
The effect of GI on [Ca2+]mt accumulation, with and without the addition of ruthenium red, in the mature and aged heart is shown in Figure 1
. In the mature heart, [Ca2+]mt in control was 220 ± 14 nmol/L Ca2+/µg mtDNA (p < 0.05 versus aged control). After 30 minutes of normothermic GI [Ca2+]mt in the mature heart was increased to 321 ± 25 nmol/L Ca2+/µg mtDNA (p < 0.05 versus mature control). In the aged heart, [Ca2+]mt in control was 136 ± 26 nmol/L Ca2+/µg mtDNA, a concentration approximately 62% less than that found in mature hearts. After 30 minutes of normothermic GI [Ca2+]mt in the aged heart was significantly increased to 227 ± 22 nmol/L Ca2+/µg mtDNA, and was not significantly different from the value in mature heart. When [Ca2+]mt data were normalized, with control being expressed as 100%, the percent increase in [Ca2+]mt (nmol/L Ca2+/µg mtDNA) in the mature hearts after 30 minutes of normothermic GI was increased 121% ± 13% (p < 0.05 versus mature control), whereas in the aged heart [Ca2+]mt was increased 166% ± 16% (p < 0.05 versus aged control). Mitochondrial calcium level was increased to a significantly greater level in the aged heart during normothermic GI than in mature heart (p < 0.05 versus mature GI).
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Effect of Global Ischemia on Cytosolic Calcium Accumulation in Mature and Aged Myocardium: Effect of Ruthenium Red
To determine if the addition of ruthenium red (6 µmol/L) to the perfusate altered [Ca2+]i accumulation in the mature and aged heart after 30 minutes of normothermic GI, a parallel experiment was performed using the Ca2+-sensitive fluorescent dye fura 2 to measure [Ca2+]i accumulation. Results shown in Figure 2 indicate that [Ca2+]i accumulation was not significantly different in control between mature (200 ± 11 nmol/L) and aged (207.0 ± 28.7 nmol/L) hearts during the preischemic perfusion period. In mature hearts after 30 minutes of normothermic GI, [Ca2+]i was significantly increased to 410 ± 21.0 nmol/L (p < 0.05 versus control) and 501.0 ± 50.5 nmol/L in aged hearts (p < 0.05 versus control and mature GI).
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Effect of Global Ischemia on Nuclear Calcium Accumulation in Mature and Aged Myocardium: Effect of Ruthenium Red
To investigate the effect of 30 minutes of normothermic GI on [Ca2+]n accumulation, myocardial nuclei were isolated and [Ca2+]n was determined (Fig 3). Myocardial nuclei were examined for integrity and purity by light microscopy and Trypan blue exclusion. No differences were observed between nuclei from mature and aged hearts. All results were standardized to nuclear DNA content and are expressed as nmol [Ca2+]n/µg DNA. Results shown in Figure 3 indicate that control [Ca2+]n was not significantly different between mature (7.3 ± 1.4 nmol/µg DNA) and aged (7.3 ± 1.2 nmol/µg DNA) hearts. In mature hearts subjected to 30 minutes of normothermic GI, [Ca2+]n was 9.3 ± 1.3 nmol/µg DNA. In aged hearts, [Ca2+]n was significantly increased to 18.2 ± 2.5 nmol/µg DNA (p < 0.05 versus control and mature GI).
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Effect of Global Ischemia on Nuclear DNA Fragmentation in Mature and Aged Myocardium: Effect of Ruthenium Red
To investigate the effect of 30 minutes of normothermic GI on nuclear DNA fragmentation, nuclear DNA was isolated separately from both mature and aged hearts, the DNA was fractionated by gel electrophoresis, and the percent DNA fragmentation was semiquantified using scanning laser densitometry (Fig 4). No significant difference in the percent DNA fragmentation was found between mature and aged hearts in control. In mature hearts, percent nuclear DNA fragmentation remained stable in GI and GI plus ruthenium red. In aged hearts 30 minutes of normothermic GI significantly increased DNA fragmentation from 8.12 ± 1.25 nmol/µg DNA (control) to 19.64 ± 0.6 nmol/µg DNA (GI) (p < 0.05). The addition of ruthenium red (6 µmol/L) to the perfusate significantly decreased the percent DNA fragmentation in aged heart to 8.66 ± 1.4 nmol/µg DNA (p < 0.05 versus GI). No significant difference in the percent DNA fragmentation was found between control and aged GI when perfused with ruthenium red, nor was any difference found in the percent DNA fragmentation between mature and aged hearts with GI and ruthenium red.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Several lines of evidence indicate that mitochondrial efficiency decreases with age and ischemia. Investigation of myocardial energy metabolism with aging in intact hearts and isolated mitochondria has shown that maximal myocardial substrate oxidation rates decline approximately 20% from the mature heart to the aged heart in the rat [19]. In addition, studies in isolated mitochondria have shown that aging is associated with reduced oxidation rates [20]. Adenosine triphosphate synthase activity in isolated mitochondria has been shown to decrease with age and also has been shown to decrease more rapidly in the heart as compared with the liver [21]. In the isolated perfused rat heart, anoxia followed by reoxygenation in isolated mitochondria has been shown to induce mitochondrial enzyme release, and the magnitude of the release was dependent on the duration of the anoxic period and the concentration of cytosolic ATP [22]. Nohl and associates [23] have shown that in isolated heart mitochondria treated with ischemia and reperfusion, there was incomplete collapse of the transmembrane proton gradient and impairment of respiration-linked ATP generation.
Benzi and Lerch [15] have shown that postischemic perfusion with ruthenium red, a hexavalent dye that inhibits mitochondrial calcium uptake, significantly decreased oxygen consumption in the ischemic heart and enhanced contractile function. Benzi and Lerch [15] provided no mechanism of action but speculated that the protective mechanism of ruthenium red could involve the maintenance of essential processes required for cell viability or ruthenium red could act by the direct inhibition of calcium entry into the cell. Our results show that in hearts subjected to 30 minutes of normothermic GI with the addition of ruthenium red to the perfusate, [Ca2+]mt (nmol/L Ca2+/µg mtDNA) was decreased significantly in both the mature and aged heart. Under similar conditions, however, ruthenium red was found to be more efficient in decreasing [Ca2+]mt accumulation in the mature heart as compared with the aged heart.
It was possible that the observed differences in [Ca2+]mt accumulation seen in the mature and aged heart were the result of differences in the accumulation of [Ca2+]i resulting from the action of ruthenium red as a general calcium inhibitor [24]. To investigate this possibility a parallel experiment was performed using the Ca2+-sensitive fluorescent dye fura 2 to measure [Ca2+]i accumulation. Our results indicate that [Ca2+]i accumulation after 30 minutes of normothermic GI with the addition of ruthenium red in the perfusate was significantly increased in the mature heart as compared with control and with mature hearts subjected to the same protocol without ruthenium red. In aged hearts [Ca2+]i accumulation was increased significantly after 30 minutes of normothermic GI but no statistical difference between those hearts treated with and without ruthenium red was observed.
The increased [Ca2+]i accumulation in mature hearts after 30 minutes of normothermic GI with the addition of ruthenium red is most likely the result of the differences in the inhibitory action of ruthenium red on the mature as compared with the aged heart mitochondrion. At least part of the difference in [Ca2+]i accumulation in the mature as compared with the aged heart can be accounted for by the differences observed in the action of ruthenium red on [Ca2+]mt accumulation in the mature heart as compared with the aged heart. These results suggest that the decrease in [Ca2+]mt accumulation in the aged and the mature heart is not the result of differences in [Ca2+]i accumulation and further suggest that in the aged heart mitochondrial regulatory function is compromised. This would agree with Pieri and associates [25], who have shown a lower respiratory activity per unit of mitochondrial mass in old mitochondria than in young mitochondria.
Previously, we have shown that 30 minutes of normothermic GI results in increased [Ca2+]i accumulation in both mature and aged hearts. These effects were ameliorated using magnesium-supplemented potassium cardioplegia [9, 10]. We also have shown that under similar conditions [Ca2+]n accumulation was increased after 30 minutes of normothermic GI in aged hearts but not in mature hearts. These changes in [Ca2+]i and [Ca2+]n were associated with increased nuclear DNA fragmentation in aged hearts but not mature hearts, and again were ameliorated by magnesium-supplemented potassium cardioplegia [9].
The accumulation of [Ca2+]i has been reported to involve the activation of endogenous catabolic enzymatic mechanisms that compromise cellular integrity, eventually resulting in cellular injury or necrosis [26]. Much attention has been focused on the association between the accumulation of [Ca2+]i and the activation of endogenous proteases and phospholipases [6]. The modulation of [Ca2+]n has been proposed to be regulated by an ATP-dependent and a calmodulin-dependent mechanism, which allows for the selective accumulation of calcium in the nucleus [27]. Recently, Brini and colleagues [28] have reported that [Ca2+]n concentration closely follows [Ca2+]i concentration, suggesting that the nuclear membrane does not represent a major barrier to the diffusion of calcium ions [28]. An increase in [Ca2+]n has been proposed as a possible mechanism involved in the regulation of key nuclear processes, such as gene expression, degradation of the nuclear envelope, and apoptosis [29]. Our results would indicate that [Ca2+]n accumulation is significantly increased after 30 minutes of normothermic GI in the aged heart but not in the mature heart. The addition of ruthenium red ameliorated [Ca2+]mt accumulation and was associated with both decreased DNA fragmentation and decreased [Ca2+]n accumulation. These results indicate that [Ca2+]n accumulation does not appear to be correlated with increased [Ca2+]i accumulation in either the mature or the aged heart and suggest that modulation of the mitochondrion, and in particular futile calcium cycling, may be of significance in the modulation of ischemic myocardial injury.
To date ruthenium red and magnesium are the only two reported agents to block calcium influx via the mitochondrial uniporter [30]. The mechanism of action of magnesium or magnesium-supplemented potassium cardioplegia remains to be clarified but may involve the maintenance of essential processes required for cell viability or act through synergistic intracellular homeostasis. The development of cardioplegic strategies that would allow for the exploitation of these age-related mechanisms may allow for the amelioration of morbidity and mortality in the aged patient.
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Acknowledgments |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Address reprint requests to Dr Levitsky, Division of Cardiothoracic Surgery, New England Deaconess Hospital, 110 Francis St, Suite 2C, Boston, MA 02215.
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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