Marys Medicine

Bioscience Reports, Vol. 25, Nos. 5/6, October/December 2005 (Ó 2005)DOI: 10.1007/s10540-005-2897-2 Cytochrome c Effect on Respiration of Heart Mitochondria:Influence of Various Factors Adolfas Toleikis,1,2 Sonata Trumbeckaite,1 and Daiva Majiene1 The effect of exogenous cytochrome c on respiration rate of the rat and human heartmitochondria was assessed in situ, using permeabilized fibers. It was (i) much more pro-nounced in State 2 and 4 than in State 3 with all the respiratory substrates (pyru-vate+malate, succinate, palmitoyl-CoA+carnitine and octanoyl-L-carnitine), (ii) differentwith different substrates, (iii) much higher after ischemia in both metabolic states, partic-ularly in the case of succinate oxidation compared to pyruvate+malate, (iv) the highest inState 4 with succinate as a substrate. Similar results were obtained with the isolated rat andrabbit heart mitochondria. The differences in the degree of stimulation of mitochondrialrespiration by cytochrome c and, thus, sensitivity of cytochrome c test in evaluation of theintactness/injury of outer mitochondrial membrane are probably determined by the differ-ences in the cytochrome c role in the control of mitochondrial respiration in the above-described conditions.
KEY WORDS: Saponin-permeabilized heart muscle fibers; isolated mitochondria; outermitochondrial membrane permeability; cytochrome c test; oxidative phosphorylation;ischemia.
ABBREVIATIONS: OMM, outer mitochondrial membrane; IMM, inner mitochondrialmembrane; ATR, atractyloside; CCCP, carbonyl cyanide-m-chlorophenyl hydrazone.
It is well known that mitochondria undergo significant structural and functionalchanges during their isolation. It was demonstrated (Wojtczak et al., 1972; see forrev. Wojtczak, 1974) that routinely prepared control heart mitochondria contained10–44% particles with damaged outer membrane. These alterations can be enhancedby pathological processes. For example, ischemia-damaged mitochondria are morefragile than normal (Jennings and Ganote, 1976) and thus more susceptible to injuryduring their isolation.
To overcome the difficulties mentioned above, saponin-permeabilized cardiac fibers have been applied for studies of mitochondrial respiration in situ. One of the 1Institute for Biomedical Research, Kaunas University of Medicine, Eiveniu St.4, LT-50009, Kaunas - 7, 2To whom correspondence should be addressed.
0144-8463/05/1200-0387/0 Ó 2005 Springer Science+Business Media, Inc.
Toleikis, Trumbeckaite, and Majiene advantages of the saponin-skinned fiber technique (Saks et al., 1998) is that only fewmilligrams of tissue are needed for respiration measurements, which allows one toinvestigate small human muscle biopsies.
The outer mitochondrial membrane (OMM) is a barrier for macromolecules (proteins, enzymes, etc.) and for smaller molecules of adenine nucleotides, creatinephosphate, creatine, etc. Therefore, OMM is relevant in the compartmentalization ofvarious metabolites and enzymes, and in the regulation of oxidative phosphorylationand energy transport in the cell (Gellerich et al., 2000; Saks et al., 1998, 2001;Toleikis et al., 2001). These processes may be disturbed by various factors (isolationof the mitochondria, ischemia, hypoosmotic conditions, fatty acids, etc.) leading todisruption of OMM and loss of cytochrome c from the mitochondria. It inhibits therespiration of mitochondria and decreases the extent of coupling between respirationand phosphorylation (Shur-Perek and Avi-Dor, 1971). Cytochrome c release fromthe intermembrane space of mitochondria to the cytoplasm is a critical signalingevent during many forms of apoptotic cell death (see for rev. Skulachev, 1998;Murphy, 1999; Borutaite and Brown, 2003).
Thus, the evaluation of OMM intactness (permeability to exogenous cyto- chrome c) is relevant for the assessment of quality of mitochondrial preparations andfor interpretation of experimental results. Electron microscopy is tedious andqualitative rather than quantitative. For this purpose, the semi-quantitative assay -spectrophotometric and oxygraphic—was applied many years ago (Wojtczak et al.,1972). The assay consists in measuring the rate of oxidation of external reducedcytochrome c (30–100 lM) by isolated mitochondria and comparing it with the totalcytochrome oxidase activity of completely disrupted (by Lubrol, Tween-60, TritonX-100, etc.) mitochondria taken as 100%.
In studies of saponin-skinned cardiac fibers, the oxygraphic method is based on the measurement of the degree of stimulation of mitochondrial respiration, mostly inState 3, by the exogenous cytochrome c (8–30 lM) (Kay et al., 1997a, b; Gellerichet al., 2000). In addition, various but mostly NAD-dependent respiratory substratesare used. Usually, no stimulation of fiber respiration by external cytochrome c isobserved, in contrast to studies with isolated mitochondria (see for rev. Wojtczak,1974). Accordingly, it was concluded that mitochondria in the control saponin-skinned heart fibers are completely intact (Saks et al., 1998).
However, the role of the particular effector or component of the oxidative phosphorylation system in the regulation of respiration appeared to be cruciallydependent on the metabolic state of mitochondria and on the respiratory substrate(Mildaziene et al., 1993; Ainscow and Brand, 1995).
Thus, the aim of this work was to assess the influence of these two factors on the exogenous cytochrome c induced stimulation of mitochondrial respiration and, thus,sensitivity of cytochrome c test in evaluation of the intactness of OMM in controland ischemia-damaged mitochondria.
MATERIALS AND METHODS Hearts of male Wistar rats weighing 250–300 g were excised and rinsed in ice- cold 0.9% KCl solution. Total ischemia was induced in vitro by autolysis (37°C, The Intactness of Outer Mitochondrial Membrane 60 min). The human heart atrial appendage tissue (about 10–20 mg biopsy speci-mens) was obtained from the patients with ischemic heart disease undergoing openheart surgery.
Bundles of the heart muscle fibers, approximately 0.2–0.3 mm in diameter, were prepared as described in Ref (Toleikis et al., 1996, 1997, 2001). Bundles of fiberswere transferred to cooled solution A containing 20 mM imidazole, 20 mM taurine,0.5 mM dithiothreitol, 7.1 mM MgCl2, 50 mM 2-[N-Morpholino]ethanesulfonicacid (MES), 5 mM ATP, 15 mM phosphocreatine, 2.6 mM CaK2EGTA and7.4 mM K2EGTA (free Ca2+ concentration 0.1 lM) (pH 7.0 adjusted with KOH at2°C), supplemented with 50 lg/ml saponin (from Gypsophila; sapogenin content17%; Sigma) alone or in combination with 3 mg/ml collagenase (Type IV, Sigma)and incubated for 30 min. Then the bundles were washed for 10 min in solution B,containing 20 mM imidazole, 20 mM taurine, 0.5 mM dithiothreitol, 1.6 mM MgCl2, 100 mM MES, 3 mM KH2PO4, 3.0 mM CaK2EGTA and 7.1 mM K2EGTA(free Ca2+ concentration 0.1 lM) (pH 7.1 adjusted with KOH at 37°C).
Rat heart mitochondria were isolated in the medium, containing 220 mM mannitol, 70 mM sucrose, 5 mM N-tris[Hydroxymethyl]methyl-2aminoethanesulf-onic acid and 0.5 mM EGTA (pH 7.4, adjusted with Trizma base; 2°C) and 2 mg/mlbovine serum albumin (BSA; fraction V, A4503, Sigma) (Kopustinskiene et al.,2003). Rabbit heart mitochondria were isolated and investigated as indicated in theTable 1 (Scholte, 1973). Some other conditions are indicated in tables and figures.
Table 1. The effect of cytochrome c on respiration of isolated heart mitochondria (fold increase in res- piration rate due to addition of cytochrome c): dependence on the respiratory substrate and metabolic state of the mitochondria Effect of cytochrome c Respiratory substrate 3-hydroxybutyrate (10 mM) 3-hydroxybutyrate (10 mM), 2 hr ischemia Pyruvate + malate (5 mM+5 mM) Succinate (20 mM) Succinate (20 mM), 2 hr ischemia Succinate (20 mM)a Succinate (20 mM)b Pyruvate + malate (5 mM+5 mM) Succinate (10 mM) Succinate (10 mM), control Succinate (10 mM), 0.5 hr ischemia Experiment 1 and 2—incubation medium contains: 0.15 M KCl, 5 mM KH2PO4, pH 7.0. Experiment3—amitochondria were isolated in medium containing 0.3 M sucrose, 10 mM EDTA (pH 7.5); bmito-chondria were isolated with trypsin (Scholte, 1973); incubation medium: 0.15 M KCl, 5 mM KH2PO4,1 mM MgCl2, pH 7.4. Experiment 4—for experimental conditions see ‘‘Materials and Methods''.
Experiment 5—incubation medium: 110 mM KCl, 1 mM free Mg2+, 10 mM TrisHCl, 5 mM KH2PO4, 4U/ml creatine kinase, 50 mM creatine, 10 mM dithiothreitol, 1 mM ATP, pH 7.2. Measurements wereperformed in the presence of 0.12 mM atractylosidec; or 1 lg oligomycind per 1 mg mitochondrial protein.
Concentration of cytochrome c was 10 lM (exp. 1 and 2), 30 lM (exp. 3 and 4) and 75 lM (exp. 5).
RCI—respiratory control index. The numbers in parentheses indicate number of independent experiments.
Toleikis, Trumbeckaite, and Majiene Oxygen uptake rates of skinned fibers were measured at 37°C with Clark-type electrode in solution B, supplemented with 2 mg/ml of BSA (Fraction V; A4503,Sigma). Respiration measurements were started immediately after preparation ofcardiac fibers and isolated mitochondria. For this purpose, permeabilized heartfibers, ADP, ATR, cytochrome c and, in some experiments, CCCP were addedconsequtively into incubation medium containing respiratory substrate(s) (seeFig. 4). In order to assess the OMM intactness, cytochrome c was added aftermeasurement of ATR-uninhibitable respiration rate (State 4) or after addition ofADP. We chose this protocol instead of that proposed by Wojtczak et al. (1972)mainly due to following reasons: (i) the main purpose of studies was to assess NAD-and FAD- dependent respiration and the OMM integrity in one probe due to verylimited amount of human tissue biopsies; and (ii) due to low oxygen concentration atthe end of the measurement the estimation of the cytochrome c oxidase activitywithout and with detergent as proposed by Wojtczak et al. (1972) is hardly possible.
Solubility of oxygen was taken to be 422 ngatoms/ml. Fibers respiration rates were expressed as ngatomsOmin)1mg fibers dry weight )1 (dry weight = wetweight before respiration measurement/4.85) (Toleikis et al., 1996). Mitochondrialrespiration rates were expressed as ngatomsOmin)1mg protein)1. The mito-chondrial protein concentration was determined by the biuret method (Gornallet al., 1949). The final mitochondrial protein concentration in all experiments was0.5 mg/ml.
Data are presented as means ± S.E.M. Statistical analysis was performed using Student's t test, and p<0.05 was taken as the level of significance.
RESULTS AND DISCUSSION As can be seen in Fig. 1(a, b), the stimulating effect of the exogenous cyto- chrome c on respiration of the control rat heart (ventricles) mitochondria in State 3,measured in situ using saponin- and saponin+collagenase-permeabilized cardiacfibers, is slightly higher in the case of succinate as a respiratory substrate than in thecase of pyruvate+malate. It was negligible or absent with the latter substrate. Theeffects of cytochrome c and their difference between substrates largely increased inthe case of ischemia (1 hr), which is known to produce the injury to OMM (Kayet al., 1997a, b; Toleikis et al., 1997).
It is noteworthy, that the degree of exogenous cytochrome c-induced stimula- tion of respiration of the control and ischemic rat cardiac fibers with both therespiratory substrates was much higher in State 4 than in State 3, and in the case ofsuccinate used as a substrate (Fig. 1(c)). In these experiments, cytochrome c wasadded to the incubation medium after atractyloside—an inhibitor of adeninenucleotide translocase—which decreased the maximum State 3 respiration rate of themitochondria to the level characteristic of State 4.
Much higher effects of cytochrome c on State 4 respiration with succinate than with pyruvate + malate were also observed in other two separate groups ofexperiments with 1 hr ischemia-injured saponin+collagenase-treated rat cardiacfibers (2.56 and 2.73-fold stimulation of succinate oxidation, n=5, data not shown;compare with Fig. 1(c) for pyruvate + malate oxidation).
The Intactness of Outer Mitochondrial Membrane ControlIschemia 60min Fig. 1. The effect of cytochrome c on the State 3 (a and b) and State 4(c) respiration rate of saponin—(a, c) and saponin+collagenase-per-meabilized (b) rat cardiac fibers—the influence of ischemia. The standardincubation medium (see ‘‘Materials and Methods'') was supplementedwith pyruvate + malate (6 mM + 6 mM, n=5), or succinate + rote-none (10 mM + 5 lM, n=5). Additions: 1 mM ADP, 30 lM cyto-chrome c, 0.12 mM atractyloside. VADP+C/VADP—the effect ofcytochrome c on State 3 respiration rate; VATR+C/VATR—the effect ofcytochrome c on State 4 respiration rate. *p<0.05 vs. control, **p<0.05vs. pyruvate+malate as substrates.
saponin + collagenase-treated appendage fibers respiring on succinate (Fig. 2) also demonstrated that after addi-tion of cytochrome c to the incubation medium in State 4, respiration rate increasesmuch more (about 1.4-fold) than in State 3 (when no increase was observed).
Metabolic state dependent differences in the cytochrome c effect on fibers res- piration were confirmed also with other substrates, palmitoyl-CoA (+L-carnitine)and octanoyl-L-carnitine (Fig. 3). The dependence of sensitivity of cytochrome c test(applied in State 4) in evaluation of the intactness of OMM on the respiratorysubstrate is also obvious. Essentially the same results as with cardiac fibers wereobtained in experiments with isolated rabbit and rat heart mitochondria (Table 1).
Data presented in Table 2, show that in absolute values (ngatomsOmin)1mg fibers dry weight)1 ) exogenous cytochrome c-induced increase in State 4 respirationrate is also higher than in State 3. It is in agreement with the above described relativeeffects (%) of cytochrome c on respiration rate.
It should be noted that the higher cytochrome c effect on succinate oxidation compared to other substrates cannot be ascribed to succinate itself, i.e. its deterio-rating action on the integrity of OMM, because almost 4-fold increase in the suc-cinate concentration (up to 37 mM) did not increase the cytochrome c effect on State Toleikis, Trumbeckaite, and Majiene Effect of cytochrome c 1.2
Fig. 2. The effect of cytochrome c on State 3 andState 4 respiration rate of the human atrial appendagefibers permeabilized with saponin plus collagenase.
The standard incubation medium (see ‘‘Materials andMethods'') was supplemented with succinate +rotenone (10 mM +5 lM, n=4). Additions: 1 mMADP, 0.12 mM atractyloside, 30 lM cytochrome c.
1—the effect of cytochrome c on State 3 respirationrate; VADP+C/VADP; 2—the effect of cytochrome c onatractyloside-insensitive respiration rate, VATR+C/VATR. *p<0.05 vs. VADP+C/VADP.
3 respiration rate (1.17±0.01, compared to 1.13±0.03 at 10 mM succinate, n=3).
This is also true for State 4 respiration of the fibers (not shown).
In some of our experiments, in order to assess the OMM intactness cytochrome c was added after addition of ATR and measurement of State 4, i.e.
ATR-uninhibitable respiration rate as it is shown on Fig. 4. It is well known, thatATR acts as inducer of mitochondrial permeability transition (MPT) (for review seeZoratti and Szabo, 1995), and, thus may cause cytochrome c release from mito-chondria and related increase in the stimulating effect of exogenous cytochrome c onrespiration. It is also shown (Machida et al., 2002) that ATR (0.5–2 mM) can inducecytochrome c release from isolated mouse liver mitochondria by a permeabilitytransition-independent mechanism.
However, in the presence of high concentration of ADP, Mg2+, dithiothreitol, BSA and, in some of our experiments, carnitine and acylcarnitines, mitochondrialmembrane permeabilizing action of ATR seemed to be impossible. This assumptionwas confirmed in further experiments with fibers and isolated heart mitochondria. Itdemonstrated that: (i) the cytochrome c equally stimulates pyruvate + malate andsuccinate oxidation in State 2 and in State 4 (Table 3), (ii) cyclosporin A, MPTinhibitor, is not effective in these experiments, i.e. does not change the cytochrome ceffect on respiration (Tables 3 and 4; this finding was also confirmed with the dogheart ventricular fibers respiring on succinate; not shown), (iii) cytochrome c isequally effective when ATR is substituted for oligomycin, an inhibitor of MPT The Intactness of Outer Mitochondrial Membrane Effectof cytochrome c 1.2 Fig. 3. The effect of cytochrome c on State 3 andState 4 respiration rate of the saponin-permeabilizedrat cardiac ventricular fibers. 1—6 mM pyruvate +6 mM malate (n=4); 2—12.2 lM palmitoyl-CoA +2.41 mM L-carnitine + 0.24 mM malate (n=7);3—0.36 mM octanoyl-L-carnitine + 0.24 mM malate(n=5); 4—10 mM succinate + 5 lM rotenone(n=4). Cytochrome c concentration was 33 lM, *p<0.05 VADP+C/VADP, **p< 0.05 vs. pyr- uvate+malate as substrates. #p<0.05 vs. octanoyl-L-carnitine+malate as substrates.
(Tables 1 and 4) and (iv) CCCP, when added after ADP, ATR and cytochrome c,substantially increases respiration rate of isolated mitochondria (Fig. 4) and sapo-nin-skinned heart fibers (Fig. 5). Noteworthy, CCCP effect was observable also inhuman atrial appendage fibers after post-ischemic (cardioplegic) reperfusion of thehearts (not shown) and, even after severe 1 hr total ischemia of the rat heart (Fig. 5).
In the latter case it was small because of significant uncoupling of oxidative phos-phorylation induced by ischemia. Thus, these data unequivocally contradict thepossibility of MPT pore opening due to the presence of ATR in the medium andrelated increase of cytochrome c effect on the ATR-uninhibitable respiration rate(State 4 respiration). Moreover, it is worth mentioning that, in agreement with Sakset al. (1998), in our experiments, low saponin concentration (50 lg/ml) used forfibers preparation did not affect the OMM permeability: rat heart fibers preparedwith or without saponin showed the identical cytochrome c-induced increase in State3 respiration rate with succinate (1.08±0.07 and 1.06±0.02 times, respectively;n=3).
The mitochondrial respiratory parameters of skinned fibers in different exper- imental groups are shown in Table 5. It can be seen that (i) the State 3 and State 2respiration rates are different with different substrates and (ii) the State 3 fibersrespiration rate with pyruvate + malate and succinate is largely decreased by 1 hrischemia showing a significant injury of mitochondria.
Toleikis, Trumbeckaite, and Majiene Table 2. The Cytochrome c-induced Increase in Rat Heart Fibers Respiration Rate (in nga- tomsOmin)1mg Fibers Dry Weight)1) with Different Substrates: Dependence on the Metabolic State Pyruvate + malate (4) Octanoyl-carnitine (4) For experimental conditions, see ‘‘Materials and Methods'' and legend for Fig. 3.
The numbers in parentheses indicate number of paired experiments.
*p<0.05 vs. State 3.
Table 3. The Effect of Cytochrome c on Mitochondrial Respiration in Rat Heart Fibers (Fold Increase): Dependence on the Metabolic State and Presence of Cyclosporine A Pyruvate + malate Pyruvate + malate Succinate + (rotenone) For experimental conditions, see ‘‘Materials and Methods'' and legend for Fig. 3.
CsA—cyclosporin A (2 lM). Notes: (1) in experiment 2, measurements were performed using fibersprepared from the same heart; (2) State 3 respiration rate was not affected by exogenous cytochrome c(33 lM).
The numbers in parentheses indicate number of experiments.
Table 4. The Effect of Cytochrome c on Respiration of Isolated Rat Heart Mitochondria with Succinate Fold increase of respiration rate by cytochrome c ADP, 1 mM + atractyloside, 0.12 mM ADP, 1 mM + oligomycin, 1 lg/mg protein ADP, 1 mM + atractyloside, 0.12 mM, + cyclosporin A, 2 lM For composition of incubation medium see ‘‘Materials and methods.'' Respiratory control index (respi-ration rate in State 3 divided by rate in State 2, before addition of ADP) were 1.61±0.09 (Exp. 1),1.78±0.05 (Exp. 2), 1.74±0.01 (Exp. 3) and 1.63±0.03 (Exp. 4). *p<0.05 vs. Exp. 1; no statisticallysignificant differences were observed between experiments 2, 3 and 4.
It is known that the role of a particular component of the multienzyme system in the control of the flow through the system depends on the respiration rate/metabolic state of the mitochondria (Mildaziene et al., 1993; Ainscow and Brand,1995) and respiratory substrate (Mildaziene et al., 1993). Therefore, the higherstimulating effects of cytochrome c on State 4 than on State 3 respiration rate (withall substrates) and on respiration with succinate than with pyruvate + malate (inboth metabolic states) revealed in this work may be explained by the higher role ofcytochrome c in the control of mitochondrial respiration in these conditions. Onthe other hand, estimation of the intactness of OMM by the cytochrome c test, in

The Intactness of Outer Mitochondrial Membrane Fig. 4. Respiration of isolated rat heart mitochondria with succinate. The uppertrace indicates the oxygen concentration in the oxygraph, the lower trace repre-sents the first derivative of these signals indicating the respiratory rate. Additions:O—isolated A2—Atractyloside (0.12 mM) A3—cytochrome c (30 lM), A4—CCCP ( 0.5 lM).
Effect of cytochrome c was 2.12 times.
Total ischemia 1 h Fig. 5. Permeabilized rat heart fibers respiration in different metabolicstates of mitochondria: effect of total ischemia (succinate as a substrate).
For experimental conditions see Fig. 2. p<0.05 vs. control.
the ischemia-damaged mitochondria respiring on NAD-dependent substrates aspyruvate + malate, etc., is complicated (OMM injury underestimated) becausesevere ischemia causes the damage to OMM and IMM, the loss of cytochrome c,NAD(H) as well as the other components of the mitochondrial matrix (enzymes,adenine nucleotides, etc.) and complex I activity. These alterations significantlydecrease the State 3 respiration rate of mitochondria when the NAD-dependentsubstrates are used, whereas the succinate oxidation is affected to a lesser degree. Inaddition, the succinate oxidation can be nearly completely restored by simple Toleikis, Trumbeckaite, and Majiene Table 5. Main Respiratory Parameters of Rat Cardiac Fibers Treated with Saponin alone (1) or in Combination with Collagenase (2 and 3): Effect of 1 hr Ischemia Control pyruvate+malate Control succinate Control pyruvate+malate ischemia pyruvate+malate Control succinate ischemia succinate For experimental conditions, see ‘‘Materials and Methods'' and legend for Fig. 3.
* denotes statisticaly significant effect of ischemia p<0.05.
addition of cytochrome c to the incubation medium which is in contrast to pyru-vate+malate oxidation (Borutaite et al., 1996; Balasevicius et al., 1985). It means,that succinate oxidase activity decrease is mostly, if not completely, dependent onthe deficiency of cytochrome c. Therefore, in ischemia or other cases causing severedamage to the both mitochondrial membranes, outer and inner, succinate can besuggested as a preferable oxidizable substrate for accurate assessment of OMMintegrity.
In conclusion, it is possible to affirm that in most cases the degree of injury of OMM is most sensitively estimated by cytochrome c test when the mitochondriaoxidize succinate in State 4. Probably, in these conditions, the role of cytochrome cin the control of mitochondrial respiration is mostly expressed compared to othermetabolic states and respiratory systems.
The authors wish to thank Vilmante Borutaite PhD for critical reading of the manuscript and valuable suggestions. This work was in part supported by theLithuanian State Science and Studies Foundation.
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Coumarin: The Real Story (Updated Jan. 2008). Copyright © Tony Burfield 2006-2008 What is it? Coumarin (2H-1-benzopyran-2-one) CAS No 91-64-5, is a crystalline white solid when seen pure, with a hay-like, sweet aromatic creamy odour with certain nutty shadings, much used in synthetic form as a fragrance chemical for perfumes and for fragranced soaps and detergents. Coumarin has a widespread occurrence in natural products too (see separate section below), and is a representative of the lactones (where a lactone is an ester group integrated into a carbon ring system).