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Validity of measurements of respiration in isolated mitochondria

Validity of measurements of respiration in isolated mitochondria


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I've recently read a couple of papers on exercise and mitochondria, in which state 4 and state 3 respiration rates and ROS production are assessed in vitro after exercise has been performed (i.e., rat heart mitochondria are isolated).


Question:

Given that it takes 1 hour for the mitochondria to be removed from the animal and isolated, that additional (saturating) substrate is added before respiration parameters are measured, and that the medium is saturated to the ambient oxygen concentration (much higher than in vivo), are this type of experiment valid to assess mitochondrial function in vivo in active animals? Opinions and/or pointers to resources that clarify how useful these measurements are would be helpful.


Evaluation of Respiration with Clark-Type Electrode in Isolated Mitochondria and Permeabilized Animal Cells

In many studies, the evaluation of mitochondrial function is critical to understand how disease conditions or xenobiotics alter mitochondrial function. One of the classic end points that can be assessed is oxygen consumption, which can be performed under controlled yet artificial conditions. Oxygen is the terminal acceptor in the mitochondrial respiratory chain, namely, at an enzyme called cytochrome oxidase, which produces water in the process and pumps protons from the matrix to the intermembrane space. Several techniques are available to measure oxygen consumption, including polarography with oxygen electrodes or fluorescent/luminescent probes. The present chapter will deal with the determination of mitochondrial oxygen consumption by means of the Clark-type electrode, which has been widely used in the literature and still remains to be a reliable technique. We focus our technical description in the measurement of oxygen consumption by isolated mitochondrial fractions and by permeabilized cells.

Keywords: ADP/O ratio Cellular respiration Clark-type electrode Mitochondria Mitochondrial respiratory chain Oxygen consumption rate Permeabilized cells Respiratory control ratio Respirosome.


Abstract

Evaluating the activity of cardiac mitochondria is probably the best way to estimate early cellular damage in chronic pathology. Early diagnosis allows rapid therapeutic intervention thus increasing patient survival rate in a number of diseases. However, data on human cardiac mitochondria are scarce in the international literature. Here, we describe a method to extract and study functional mitochondria from the small-sized right atrial aliquots (minimum of 400 mg) obtained during extracorporeal circulation and usually considered as surgical waste products. The mitochondria were purified through several mechanical processes (fine myocardial cutting, tissue grinding and potter Elvehjem homogenising), an enzymatic proteolytic action (subtilisin) and differential centrifugations. In chronic pathologies, including obesity, early disturbances of mitochondrial function can occur. The effects of obesity on the rate of mitochondrial oxygen consumption and H2O2 release were thus determined with three different substrates (glutamate/malate, succinate/rotenone and palmitoylcarnitine/malate). The human atrial mitochondria were of high quality from a functional viewpoint, compared to rat ventricle organelles, but the extraction yield of the human mitochondria was twice lower than that of rat mitochondria. Tests showed that glutamate/malate-related ADP-stimulated respiration was strongly increased in obese subjects, although the oxidation of the other two substrates was unaffected. Reactive oxygen species (ROS) production by the isolated mitochondria was low in comparison with that of the lean subjects. These results confirm those found in one of our previous studies in the ventricles of rats fed a high-fat diet. In conclusion, the described method is simple, reliable and sensitive. It allows for the description of the impact of obesity on the function of atrial mitochondria while using only a small patient sampling (n = 5 in both the lean and the obese groups).


RESULTS AND DISCUSSION

The protocol described here successfully determined the mitochondrial respiration rate of permeabilized gill tissue from fish (Table 1, Fig. 2A). The respiration profile obtained using this technique demonstrated increases in O2 consumption rate at each step when the substrates pyruvate, malate, glutamate and succinate were sequentially added to stimulate electron transport chain components, and responded in an expected manner to the known mitochondrial complex inhibitors oligomycin and antimycin A (Fig. 2A). The addition of cytochrome c led to only a small percentage increase in respiration rate (Table 1), confirming that the preparations were of high quality. In comparison to semi-isolated, Dounce-homogenized, gill tissues (Braz-Mota et al., 2018), our preparation shows greater RCR values (7.4 versus 2.6–5.5), further confirming the high quality of mitochondrial preparations using this technique (Table 1). It is interesting to note that our respiration values are lower than those reported by Braz-Mota et al. (2018), although this is expected and has been shown in studies comparing different mitochondrial preparations in other taxa (Saks et al., 1998 Picard et al., 2011 Mahalingam et al., 2017). Mitochondrial respiration rates have classically been normalized using markers of mitochondrial volume, including CS and COX activity (Barrientos, 2002 Larsen et al., 2012 Dawson et al., 2018), and our mitochondrial respiration rates showed an excellent linear relationship (P=0.011, R 2 =0.689 Fig. 2B) with CS activity, suggesting that our measurements were the result of mitochondrion-based oxygen consumption. This is in line with previous experiments showing a strong correlation between mitochondrial content and CS activity (Larsen et al., 2012). While Larsen et al. (2012) also found a linear relationship between mitochondrial content and COX activity, this relationship was marginally non-significant in our study (P=0.079, R 2 =0.426 Fig. 2C), but a small sample size (n=8) meant that statistical power was low. Given the greater ease of measurement and stronger relationship with respiration rate, we suggest that CS is a more appropriate and more accessible method of normalizing gill mitochondrial respiration rates.

Mitochondrial parameters from duplicate measurements of permeabilized gills in brown trout


Abstract

Hibernating mammals may suppress their basal metabolic rate during torpor by up to 95% to reduce energy expenditure during winter, but the underlying mechanisms remain poorly understood. Here we show that hydrogen sulfide (H2S), a ubiquitous signaling molecule, is a powerful inhibitor of respiration of liver mitochondria isolated from torpid 13-lined ground squirrels, but has a weak effect on mitochondria isolated during summer and hibernation arousals, where metabolic rate is normal. Consistent with these in vitro effects, we find strong seasonal variations of in vivo levels of H2S in plasma and increases of H2S levels in the liver of squirrels during torpor compared to levels during arousal and summer. The in vivo changes of liver H2S levels correspond with low activity of the mitochondrial H2S oxidizing enzyme sulfide:quinone oxidoreductase (SQR) during torpor. Taken together, these results suggest that during torpor, H2S accumulates in the liver due to a low SQR activity and contributes to inhibition of mitochondrial respiration, while during arousals and summer these effects are reversed, H2S is degraded by active SQR and mitochondrial respiration rates increase. This study provides novel insights into mechanisms underlying mammalian hibernation, pointing to SQR as a key enzyme involved in the control of mitochondrial function.


4. Discussion

Proper selection of stains for use in flow cytometry panels measuring mitochondrial function is crucial in everyone's efforts to provide meaningful and insightful data. Our data confirms that MTG and NAO localize to mitochondria independent of ΔΨm. However, MTG does not provide accurate measurements of mitochondrial mass under all conditions. In our whole cell studies we observed a correlation with MTG MFI and our markers for cytosolic and mitochondrial oxidative stress. Potentially MTG might be acting as a substrate for the reduction of oxidized mitochondrial proteins, like peroxiredoxin, thioredoxin, or glutaredoxin, and the increase in MTG fluorescence we are detecting could be due to one of two mechanisms: (1) oxidation by cysteines in mitochondrial proteins or (2) ROS generated by the electron transport chain (ETC). These mechanisms could be possible explanations for why others have reported increased MTG signal after altering mitochondrial function, independent of whether the compound enhanced or dissipated the ΔΨm [5]. Many of these treatments also alter the redox environment and may impact MTG fluorescence. Our data showed that the only treatment that did not result in a significant change in mitochondrial mass by MTG was the reducing agent NAC. NAC treatment caused minimal changes to ROS and RNS levels in mitochondria after 2 h ( Figure 1a ). Staining mitochondria in whole cells with NAO, which binds to cardiolipin, showed a decrease in fluorescence in samples treated with NO. While no significance was noted by our correlation analysis both DAF-FM and DHR (markers for NO and RNS) did show a negative trend. This is possibly due to the oxidation of cardiolipin by peroxynitrite [8] resulting in decreased binding sites for NAO.

When we isolated mitochondria after treatment we saw no change to mitochondrial mass by FSC, despite the large changes in mass from MTG staining of whole cells. The lack of change in mass by FSC but increased MTG staining could be a result of enhanced biogenesis of mitochondria coupled with fission of the mitochondria. However, the lack of measurable change in mitochondrial protein levels by western blotting suggests that this is not the case. When we further compare our mitochondrial mass measurements in whole cells to our western blot results we observe NAO mass measurements more closely resemble the changes seen in protein levels by western blotting, providing evidence that NAO is a more accurate mass marker than MTG.

While analysis of whole cells showed dramatic changes in MTG fluorescence, isolated mitochondria had greater changes in NAO signal. This suggests that NAO, while cell permeable, does not react the same way in whole cells as it does in isolated mitochondria, or that cardiolipin may become more exposed as a result of our isolation protocol. These discrepancies should be taken into account when selecting a mitochondrial mass dye for studies.

Our flow cytometric analysis of isolated mitochondria identified two subpopulations that strongly stained positive with both MTG and NAO but had minimal TMRM signal. These two populations were consistent in their location within the forward versus side scatter plots between each treatment group ( Figure 2 ). These two populations of mitochondria might be from different subsets of cells within the isolated PBL. With different cell types having different work load requirements the mitochondrial structure in each subset would be different and might explain why we see these distinct groups. The use of isolated cell populations from PBL might help to elucidate where these populations derive from.

A significant decrease in NDUFS3 protein levels was measured by western blot after 15 min treatment with 3 mM NAC. This decrease was not present in our 2 h treated samples. With no difference seen in the level of either outer mitochondrial membrane protein VDAC, or inner mitochondrial membrane protein ANT after treatment with NAC, we propose that there may be a specific interaction between NAC and complex I. Given that we have previously described complex I respiration is directly inhibited by NAC [9], this decrease could be a result of NAC's inhibition of complex I. One possible mechanism this might occur through is NAC's ability to disrupt disulfide bonds [10], leading to loss of some complex I protein. Further work is needed to explore this hypothesis.

In this study, we have provided evidence that MTG does not accurately measure mitochondrial mass under all conditions and that this is not due to changes in ΔΨm, but rather is the result of altered oxidative and nitrosative stress. While both MTG and NAO may be good markers for mitochondrial mass under physiological conditions, we believe that it is critical to understand the ROS and RNS state of the mitochondria before using either of these dyes for determining mitochondrial mass by flow cytometry.


Results

Validation of the spectrophotometric assay procedure

No detectable ferric protein is formed in the presence of superoxide dismutase and catalase. Control experiments gave identical results whether or not the cuvette contents were stirred completely filled cuvettes with no gas phase were used in these control experiments. Accordingly, the contents of the Thunberg cuvette were not stirred in the experiments reported here. Material exchange between the gas phase and the aqueous solution in the lower part of the cuvette, where the light beam passes, is very slow. Control experiments indicate that it may be neglected in these brief experiments. The mitochondria settle slowly, so that the number entering and leaving the light path is the same, and mitochondrial density in the light path does not change during the course of these relatively brief experiments.

The conditions of the spectrophotometric assay of mitochondrial oxygen consumption differ radically from those of the familiar polarographic assay. Mitochondrial density is approximately 100-fold greater ADP concentration ranges from 4- to 50-fold greater during the course of the determinationoxygen pressure is markedly less than that used in the polarographic assay. An experiment, presented in Fig. 1, was designed to show that the parameters of mitochondrial function are not much changed. The sharp breaks in the rate of oxygen uptake,where ADP is calculated to be exhausted, evidence tight coupling of oxygen uptake to ADP usage. Initial specific activities are ∼200 mol O2 (mol cytochrome aa3) –1 min –1 . P/O ratios are: 2.3, 2.4 and 2.4 at initial ADP concentrations of 500, 1000 and 2000 μmol l –1 , respectively, with respiratory control indices of 3.1–3.4. These are comparable to those found in the polarographic assay: P/O ratio 2.4, with RCI>6 (N=24). This shows that mitochondrial oxygen uptake, measured in this assay, is tightly coupled to ADP usage and proceeds at rates commensurate with state III oxygen uptake measured polarographically.

Oxygen consumption reported by myoglobin deoxygenation monitored at 625 nm is determined in mitochondrial suspensions containing myoglobin and limiting amounts of ADP. ADP is exhausted at the arrows. Myoglobin, 500 μmol l –1 . A, 500 μmol l –1 ADP B, 1000 μmol l –1 ADP C, 2000 μmol l –1 ADP. Initial rates are ∼200 mol O2 (mol cytochrome aa3) –1 min –1 . P/O ratios are: 2.3, 2.4 and 2.4, respectively, with respiratory control indices of 3.1–3.4. The P/O ratio of this preparation of mitochondria,determined polarographically, was 2.4 with RCI>6. This shows that mitochondrial oxygen uptake is tightly coupled to phosphorylation when the mitochondria are consuming myoglobin-bound oxygen. AU, absorbance units.

Oxygen consumption reported by myoglobin deoxygenation monitored at 625 nm is determined in mitochondrial suspensions containing myoglobin and limiting amounts of ADP. ADP is exhausted at the arrows. Myoglobin, 500 μmol l –1 . A, 500 μmol l –1 ADP B, 1000 μmol l –1 ADP C, 2000 μmol l –1 ADP. Initial rates are ∼200 mol O2 (mol cytochrome aa3) –1 min –1 . P/O ratios are: 2.3, 2.4 and 2.4, respectively, with respiratory control indices of 3.1–3.4. The P/O ratio of this preparation of mitochondria,determined polarographically, was 2.4 with RCI>6. This shows that mitochondrial oxygen uptake is tightly coupled to phosphorylation when the mitochondria are consuming myoglobin-bound oxygen. AU, absorbance units.

Myoglobin does not interact with the mitochondrial surface

The polarographically measured rate of oxygen uptake by a suspension of pigeon heart mitochondria is changed only slightly by the addition of 500μmol l –1 oxymyoglobin, a concentration greater than that in the pigeon ventricle, approximately 200 μmol kg –1 wet mass tissue (Schuder and Wittenberg, 1979), Fig. 2. The P/O ratio,likewise, was not affected by the presence of 500 μmol l –1 myoglobin. As discussed below, each of the six radically different hemoglobins used in these experiments supported mitochondrial oxygen uptake to approximately the same extent, Table 1 and Table 2.

Oxygen affinity and equilibrium constants of monomeric heme protein used

. P1/2 . . Combination constant (mol l -1 s -1 ×10 -6 ) . Dissociation constant (s -1 ) .
Heme protein . (kPa) . (mmHg) . . .
Busycon Mb a 0.11 0.81 48 71
Horse Mb b 0.056 0.43 14 11
Lucina Hb I c 0.044 0.34 100 61
Lucina Hb II c 0.021 0.16 0.39 0.11
Soybean Lb c1 d 0.003 0.021 124 4.9
Gasterophilus Hb e 0.009 0.067 10 1.2
. P1/2 . . Combination constant (mol l -1 s -1 ×10 -6 ) . Dissociation constant (s -1 ) .
Heme protein . (kPa) . (mmHg) . . .
Busycon Mb a 0.11 0.81 48 71
Horse Mb b 0.056 0.43 14 11
Lucina Hb I c 0.044 0.34 100 61
Lucina Hb II c 0.021 0.16 0.39 0.11
Soybean Lb c1 d 0.003 0.021 124 4.9
Gasterophilus Hb e 0.009 0.067 10 1.2

Hb, hemoglobin Mb, myoglobin Lb, leghemoglobin. Kinetic constants from: a Busycon Mb (Schreiber and Parkhurst, 1984) b Horse Mb(Schenkman et al., 1997) c Lucina Hb I and Hb II (Kraus and Wittenberg, 1990) d Leghemoglobin c(Martin et al., 1990) e Gasterophilus Hb (Phelps et al., 1972).

Oxygen pressure and fractional saturation of heme proteins at half-maximal oxygen uptake of state III isolated cardiac mitochondria

. Fractional saturation . Oxygen pressure . .
Heme protein . (%) . (kPa) . (mmHg) .
Busycon Mb 3.9 0.0047 0.035
Horse Mb 4.7 0.0094 0.071
Lucina Hb I 9.1 0.0024 0.018
Lucina Hb II 24 0.0067 0.050
Soybean Lb c41 0.0067 0.050
Gasterophilus Hb 31 0.0015 0.011
Mean 0.0052 0.040
. Fractional saturation . Oxygen pressure . .
Heme protein . (%) . (kPa) . (mmHg) .
Busycon Mb 3.9 0.0047 0.035
Horse Mb 4.7 0.0094 0.071
Lucina Hb I 9.1 0.0024 0.018
Lucina Hb II 24 0.0067 0.050
Soybean Lb c41 0.0067 0.050
Gasterophilus Hb 31 0.0015 0.011
Mean 0.0052 0.040

Hb, hemoglobin Mb, myoglobin Lb, leghemoglobin.

Progress of hemoglobin deoxygenation during the experiment

These experiments explore low oxygen pressures comparable to sarcoplasmic oxygen pressure, where oxygen uptake would be limited by oxygen availability in the absence of hemoglobin or myoglobin. The progress of experiments in which deoxygenation of myoglobin (P50=0.09 kPa ∼0.7 mmHg) or leghemoglobin c (P50=0.009 kPa ∼0.07 mmHg) reports mitochondrial oxygen uptake is presented in Fig. 3. The ordinate reports hemoglobin oxygenation. Initially, oxygen is present in small excess the hemoglobin is largely or fully oxygenated, and the traces curve downward as mitochondrial respiration draws on reserves of both dissolved and hemoglobin-bound oxygen. Subsequently, the store of dissolved oxygen becomes small relative to the store of hemoglobin-bound oxygen, equilibrium between oxyhemoglobin and free oxygen buffers the rate of change of oxygen pressure,and the traces approach linearity. The rate of hemoglobin deoxygenation now reports a near-steady-state rate of hemoglobin-supported mitochondrial oxygen consumption. The oxygen pressure at half-maximal rate of oxygen uptake is reported by the points at which the rate of change of hemoglobin deoxygenation is half that during the earlier quasi-linear portion of the progress curve,indicated by the arrows in Fig. 3. The two progress curves differ. Half-maximal respiration in the presence of the high-affinity leghemoglobin occurs when the protein is 57%,oxygenated that in the presence of the lower affinity myoglobin occurs when the protein is 94%, deoxygenated. Accordingly, mitochondrial function is independent of the nature of the supporting hemoglobin and its fractional saturation with oxygen.

Oxygen pressure in a suspension of mitochondria reported polarographically as a function of time (1 torr=0.133 kPa). ADP (500 nmol) is added at the arrows. A, myoglobin absent B, 500 μmol l –1 oxymyoglobin. Average oxygen uptake in the presence of adequate ADP is 193 and 157 mol O2 (mol cytochrome aa3) –1 min –1 in the absence and presence of myoglobin, respectively. P/O ratios are 2.21 and 2.20. The concentration of myoglobin used here exceeds the volume-average concentration in pigeon heart, 209 μmol l –1 (Schuder et al.,1979). These results show that myoglobin in the solution does not affect the respiratory parameters of isolated mitochondria.

Oxygen pressure in a suspension of mitochondria reported polarographically as a function of time (1 torr=0.133 kPa). ADP (500 nmol) is added at the arrows. A, myoglobin absent B, 500 μmol l –1 oxymyoglobin. Average oxygen uptake in the presence of adequate ADP is 193 and 157 mol O2 (mol cytochrome aa3) –1 min –1 in the absence and presence of myoglobin, respectively. P/O ratios are 2.21 and 2.20. The concentration of myoglobin used here exceeds the volume-average concentration in pigeon heart, 209 μmol l –1 (Schuder et al.,1979). These results show that myoglobin in the solution does not affect the respiratory parameters of isolated mitochondria.

Oxygen pressure at half-maximal rate of mitochondrial oxygen uptake

The oxygen pressures obtained at half-maximal mitochondrial oxygen uptake,supported by six hemoglobins with very different kinetic and equilibrium constants in their reactions with oxygen(Table 1), are presented in Table 2. It is apparent that nearly the same oxygen pressure (range 0.0015–0.0095 kPa0.011–0.071 mmHg. Average PO2= 0.005 kPa0.040 mmHg) prevails at half-maximal oxygen delivery by each of the hemoglobins. There is no correlation of half-maximal rate with oxygen affinity of the proteins nor with the combination or dissociation rate constants. The values presented in Table 2 are roughly comparable to the PO2 for half-maximal respiration of mitochondria isolated from a variety of sources, the so-called Km for oxygen (reviewed by Wilson et al., 1988). Even the highest value (≈0.01 kPa ∼0.10 mmHg), encountered in mitochondria where specific activity has been roughly doubled, falls within the envelope of values reported for Km.

Mitochondrial oxygen uptake as a function of hemoglobin concentration

Mitochondrial oxygen uptake is enhanced in the presence of myoglobin. We examined the relation between myoglobin concentration and this enhancement. The rate of mitochondrial oxygen uptake, measured by the slope of the near-linear portion of the progress curve(Fig. 3), increases monotonically with hemoglobin concentration to attain a plateau where oxygen uptake is independent of hemoglobin concentration(Fig. 4). The functions presented in Fig. 4 are the same for leghemoglobin, myoglobin and Busycon myoglobin (data not shown),proteins that differ 10-fold in the kinetics and equilibrium of their reactions with oxygen (Table 1). Oxygen uptake is also independent of the degree of hemoglobin oxygen saturation within the linear range of experiments presented in Fig. 3. These findings confirm the conclusion that hemoglobin-dependent oxygen uptake does not involve reaction of the hemoglobin with the mitochondrial surface. The maximum rates of uptake of hemoglobin-delivered oxygen do not differ significantly from the rates of state III oxygen uptake determined polarographically in the absence of hemoglobins. We conclude that added oxyhemoglobins do not change mitochondrial-specific activity. Instead they relieve a limitation to mitochondrial oxygen uptake imposed by limited availability of dissolved oxygen at low PO2.

Myoglobin and leghemoglobin deoxygenation as functions of time in hemoglobin-containing suspensions of mitochondria. Initially the hemoglobins are fully oxygenated. Deoxygenation is down. (A) Myoglobin, 50 μmol l –1 mitochondria (as cytochrome aa3) 170 nmol l –1 , 10 mm light path. (A, inset) Myoglobin 500 μmol l –1 mitochondria (as cytochrome aa3) 700 nmol l –1 , 2 mm light path. (B) Leghemoglobin c, 50μmol l –1 mitochondria (as cytochrome aa3) 65 nmol l –1 , 10 mm light path. The broken lines are drawn at a tangent to the nearly linear portions of the curves. The arrows indicate the points at which the rates of mitochondrial oxygen uptakes are half that during the near-linear portion of the progress curves. The quasi-steady state oxygen uptakes calculated from the slopes of the broken lines are 204 and 358 mol O2 (mol cytochrome aa3) –1 min –1 for myoglobin and leghemoglobin, respectively. The oxygen pressure at half-maximal oxygen uptake is found to be the same for two proteins for which affinities differ 10-fold (see Table 2). This shows that the PO2 for half-maximal mitochondrial oxygen uptake is not related to the oxygen affinity of the hemeprotein supplying oxygen. AU, absorbance units.

Myoglobin and leghemoglobin deoxygenation as functions of time in hemoglobin-containing suspensions of mitochondria. Initially the hemoglobins are fully oxygenated. Deoxygenation is down. (A) Myoglobin, 50 μmol l –1 mitochondria (as cytochrome aa3) 170 nmol l –1 , 10 mm light path. (A, inset) Myoglobin 500 μmol l –1 mitochondria (as cytochrome aa3) 700 nmol l –1 , 2 mm light path. (B) Leghemoglobin c, 50μmol l –1 mitochondria (as cytochrome aa3) 65 nmol l –1 , 10 mm light path. The broken lines are drawn at a tangent to the nearly linear portions of the curves. The arrows indicate the points at which the rates of mitochondrial oxygen uptakes are half that during the near-linear portion of the progress curves. The quasi-steady state oxygen uptakes calculated from the slopes of the broken lines are 204 and 358 mol O2 (mol cytochrome aa3) –1 min –1 for myoglobin and leghemoglobin, respectively. The oxygen pressure at half-maximal oxygen uptake is found to be the same for two proteins for which affinities differ 10-fold (see Table 2). This shows that the PO2 for half-maximal mitochondrial oxygen uptake is not related to the oxygen affinity of the hemeprotein supplying oxygen. AU, absorbance units.

Effects of increased mitochondrial-specific activity

Mitochondria of heart and red skeletal muscle adapt their rate of oxidative phosphorylation to meet very large, say 10- or more-fold, changes in steady-state work output of the muscle. Accordingly, it is of interest to investigate the response of myoglobin-dependent oxygen delivery to change in mitochondrial-specific activity. Approximately doubling mitochondrial-specific activity roughly doubles the maximum rate of oxygen uptake at the plateau and more than doubles the oxygen pressure required to achieve half-maximal rate to a value of ∼0.01 kPa (∼0.10 mmHg)(Fig. 5). The myoglobin concentration (approximately 300 μmol l –1 ) required to sustain the full respiratory rate is approximately fivefold greater than that required at lesser rates, and is comparable to the volume-average myoglobin concentration in pigeon ventricle, 209 μmol kg –1 wet mass(Schuder et al., 1979).

Mitochondrial oxygen uptake as functions of myoglobin or leghemoglobin concentration. Closed symbols, myoglobin open symbols, leghemoglobin c. Different symbols represent different experiments. With the exception of the two lowest points, oxygen uptake rates are those reported spectrophotometrically by the near-steady-state rates of deoxygenation of myoglobin or leghemoglobin, as in Fig. 3. Each point is the average of measurements on seven (myoglobin)or two (leghemoglobin) preparations of isolated mitochondria. The two lowest points were determined using trace concentrations of myoglobin or leghemoglobin as a reporter of oxygen pressure. Oxygen uptakes are normalized to a plateau value determined by the polarographically determined specific activity. The ratios of the rates of oxygen uptake at the plateau of this figure to those determined polarographically were 0.91, 0.77 and 1.30 in experiments using myoglobin and 0.76, 0.96 and 0.92 in experiments using leghemoglobin.

Mitochondrial oxygen uptake as functions of myoglobin or leghemoglobin concentration. Closed symbols, myoglobin open symbols, leghemoglobin c. Different symbols represent different experiments. With the exception of the two lowest points, oxygen uptake rates are those reported spectrophotometrically by the near-steady-state rates of deoxygenation of myoglobin or leghemoglobin, as in Fig. 3. Each point is the average of measurements on seven (myoglobin)or two (leghemoglobin) preparations of isolated mitochondria. The two lowest points were determined using trace concentrations of myoglobin or leghemoglobin as a reporter of oxygen pressure. Oxygen uptakes are normalized to a plateau value determined by the polarographically determined specific activity. The ratios of the rates of oxygen uptake at the plateau of this figure to those determined polarographically were 0.91, 0.77 and 1.30 in experiments using myoglobin and 0.76, 0.96 and 0.92 in experiments using leghemoglobin.


Results

Confocal microscopic imaging of the cellular systems studied

Figs 1, 2, 3 show the detailed characteristics of the permeabilized cardiomyocytes and skinned myocardial fibers in solution B with pCa=7.0, which corresponds to the resting state of the cell. Fig. 1 shows the confocal microscopic imaging, both by autofluorescence of flavoproteins(Fig. 1A) and intramitochondrial calcium (Fig. 1B), of mitochondria in isolated and permeabilized rat cardiomyocytes in solution B. The cardiomyocytes are relaxed and of rod-like shape, and, inside the cells, mitochondria are regularly arranged between myofibrils. Fig. 2 shows that the permeabilization process is complete and allows excellent immunofluorescence staining of the microtubular network in the cells. The network is intact, showing that the cytoskeleton of the cells is well preserved during the permeabilization procedure. An alternative, and more rapid and simple, technique for isolation of cardiomyocytes is to use the permeabilized (`skinned') muscle fibers(Fig. 3). This technique requires careful separation of muscle bundles into fibers, where the cardiac cells maintain their contacts with each other at intercalated discs(Veksler et al., 1987 Saks et al., 1998a Fig. 3) but the fiber diameter is the same as that of cardiomyocytes (10–20 μm), and the diffusion distances (correspondingly, 5–10 μm) and diffusion kinetics for substrates and ADP are always the same as those of cardiomyocytes(Table 1). While isolation of cardiomyocytes requires the use of whole rat hearts and is rather time-consuming, the permeabilized fiber technique needs only a few milligrams of muscle tissue, which is crucial, for example, in human and clinical studies(Walsh et al., 2001 Kuznetsov et al., 1998). The two methods, however, are equally good for studying the whole population of mitochondria inside the cell in their natural surroundings.

Measured apparent Km for exogenous ADP in different permeabilized cell preparations, and diffusion distances from the medium into the center of cells (Rdif)

Preparation . Measured diameter (μm) . Rdif (μm) . Apparent Km (ADP) (μmol 1 -1 ) . Reference .
Cardiomyocytes 10-25 5-12.5 - Opie, 1998
- - 329±50 * Present study
16 8 250±38 Saks et al., 1991
20 10 200-250 Kay et al., 1997
`Ghost' cardiomyocytes 20 10 200-250 Kay et al., 1997
Skinned cardiac fibers - - 297±35 Saks et al., 1993
20 10 300±23 Saks et al., 2001
- - 300-400 Seppet et al., 2001
- - 370±70 Boudina et al., 2002
- - 234±24 Liobikas et al., 2001
22 11 - Menin et al., 2001
- - 334±48 * Present study
`Ghost' cardiac fibers 20 10 349±24 Saks et al., 1993
- - 349±34 * Present study
Skinned cardiac fibers + creatine - - 79±8 Saks et al., 1991
- - 85±5 Kuznetsov et al., 1996
Rat heart mitochondria in vitro∼1 0 17.6±1 Saks et al., 1991
Preparation . Measured diameter (μm) . Rdif (μm) . Apparent Km (ADP) (μmol 1 -1 ) . Reference .
Cardiomyocytes 10-25 5-12.5 - Opie, 1998
- - 329±50 * Present study
16 8 250±38 Saks et al., 1991
20 10 200-250 Kay et al., 1997
`Ghost' cardiomyocytes 20 10 200-250 Kay et al., 1997
Skinned cardiac fibers - - 297±35 Saks et al., 1993
20 10 300±23 Saks et al., 2001
- - 300-400 Seppet et al., 2001
- - 370±70 Boudina et al., 2002
- - 234±24 Liobikas et al., 2001
22 11 - Menin et al., 2001
- - 334±48 * Present study
`Ghost' cardiac fibers 20 10 349±24 Saks et al., 1993
- - 349±34 * Present study
Skinned cardiac fibers + creatine - - 79±8 Saks et al., 1991
- - 85±5 Kuznetsov et al., 1996
Rat heart mitochondria in vitro∼1 0 17.6±1 Saks et al., 1991

Measured in this work for preparations similar to those described in Fig. 1. The diffusion distances are given to evaluate the rate of Brownian movement of ADP in water, if necessary (Saks et al.,2001).

Respiratory characteristics of permeabilized cell systems

The rate of oxidative phosphorylation (mitochondrial ATP production) is regulated by ADP due to the respiratory control phenomenon(Chance and Williams, 1956). The affinity of oxidative phosphorylation for ADP is quantitatively characterized by an apparent Km for ADP. For isolated mitochondria in the homogenous suspension, the value of this constant for ADP in the medium (exogenous ADP) is very low, 10–20 μmoll -1 ,due to the high permeability of the outer mitochondrial membrane(Klingenberg, 1970) and high affinity of adenine nucleotide translocator for this substrate(Vignais, 1976). However, when mitochondria are studied in the permeabilized cells in situ, the results are very different (Saks et al.,1998a).

Table 1 summarises the measured affinities of mitochondria for exogenous ADP in different preparations before (intact permeabilized cells) and after (ghost cells or fibers) extraction of myosin. In spite of the very small diffusion distances(mean=10 μm) from the medium into the core of the cells (Figs 1, 2, 3), in all cases the affinities are very low compared with the very high values of apparent Km for exogenous ADP (300–400μmoll -1 ). An important observation shown in Table 1 is that activation of the mitochondrial creatine kinase (miCK) reaction decreases the value of apparent Km for exogenous ADP. This is due to functional coupling of the miCK reaction to the oxidative phosphorylation viathe adenine nucleotide translocator(Barbour et al., 1984 Joubert et al., 2002 Saks et al., 1975, 1994, 1995 Wallimann et al., 1992 Wyss and Kaddurah-Daouk,2000), which leads to increased local turnover of adenine nucleotides in mitochondria, effective aerobic phosphocreatine production and metabolic stability of the heart (Garlid,2001 Kay et al.,2000 Saks et al.,1995). This emphasizes the role of miCK in regulation of mitochondrial respiration in muscle cells(Joubert et al., 2002 Kay et al., 2000 Saks et al., 2001 Walsh et al., 2001).

Further evidence for this kind of local control of respiration by miCK is provided in Fig. 4, which shows the oxygraph recordings of mitochondrial respiration in skinned cardiac fibers when ADP was produced endogenously in the cellular MgATPase reactions in the presence of 2mmoll -1 MgATP. Endogenous ADP production activates respiration several times. Subsequent addition of a system competing with mitochondria for ADP (Gellerich and Saks,1982), consisting of pyruvate kinase (PK) in high concentrations and phosphoenolpyruvate (PEP), reduced the respiration rate, but not by more than 40%. Addition of creatine increased the respiration rate to its maximal value observed in State 3. This is again due to activation of local production of ADP by miCK in the mitochondrial intermembrane space, and this locally produced ADP is totally inaccessible for exogenous PK but is channeled to the adenine nucleotide translocator and transported into the mitochondrial matrix(see above).

Recordings of the respiration rate in permeabilized myocardial fibers activated by endogenous ADP production in MgATPase reactions. Traces show the rate of change of oxygen concentration in time in an oxygraph cell. Respiration rates were measured in the presence of 5mmoll -1 glutamate plus 2mmoll -1 malate, as described in Materials and methods. Addition of 2mmoll -1 ATP, and various final concentrations of pyruvate kinase (PK) in the presence of 2mmoll -1 phosphoenolpyruvate (PEP) in the medium are indicated. At the end of experiments, 20mmoll -1 creatine was added. Arrows show the time of addition. The results show some inhibitory effect of the competitive pyruvate kinase (PK)–PEP system for endogenous ADP on the respiration rate and a stimulatory effect of creatine, due to coupled creatine kinase reaction, in the presence of PK. Only minor inhibition of respiration by very high PK activity (20i.u.ml -1 ) demonstrates compartmentation and direct channeling of endogenous ADP. These effects of direct channeling are increased after activation of mitochondrial creatine kinase.

Recordings of the respiration rate in permeabilized myocardial fibers activated by endogenous ADP production in MgATPase reactions. Traces show the rate of change of oxygen concentration in time in an oxygraph cell. Respiration rates were measured in the presence of 5mmoll -1 glutamate plus 2mmoll -1 malate, as described in Materials and methods. Addition of 2mmoll -1 ATP, and various final concentrations of pyruvate kinase (PK) in the presence of 2mmoll -1 phosphoenolpyruvate (PEP) in the medium are indicated. At the end of experiments, 20mmoll -1 creatine was added. Arrows show the time of addition. The results show some inhibitory effect of the competitive pyruvate kinase (PK)–PEP system for endogenous ADP on the respiration rate and a stimulatory effect of creatine, due to coupled creatine kinase reaction, in the presence of PK. Only minor inhibition of respiration by very high PK activity (20i.u.ml -1 ) demonstrates compartmentation and direct channeling of endogenous ADP. These effects of direct channeling are increased after activation of mitochondrial creatine kinase.

Explanation of these experimental data and of the low efficiency of inhibition of respiration by exogenous PK can be found by using the mathematical model of ADP diffusion and energy transfer inside the cells (see Materials and methods). Fig. 5shows the results of calculations of the respiration rate for two different situations. First, the diffusion coefficient, D, for ADP and ATP was taken to be equal to that in the cellular bulk water phase, D0=145 μm -2 s -1 (Aliev and Saks, 1997). In this case, because of the small diffusion distance and the high rate of diffusion(high value of D), ATP and ADP are rapidly exchanged between extra-and intracellular spaces, and ADP produced endogenously in the cellular MgATPase reactions is very rapidly consumed by PK (solid line in Fig. 5). The result is that respiration is effectively suppressed already in the presence of PK at activities below 5i.u.ml -1 in the medium (solid line in Fig. 5). These data are in agreement with our previous conclusions that the Brownian movement of ADP in water phase across 10 μm is much faster than its metabolic turnover in heart mitochondria (Saks et al.,2001).

The computer modeling of data shown in Fig. 4 on the reduction of respiration rate of mitochondria in situ in permeabilized cardiac cells dependent on endogenous ADP (in the presence of 2mmoll -1 MgATP) by increasing pyruvate kinase (PK) activity for two different systems. V., respiration rate under given conditionsV̇ respiration rate before addition of ATPV̇ATP, respiration rate in the presence of 2mmoll -1 ATP. Solid line: diffusion constant of ADP was taken as D0 (characteristic for Brownian movement in water phase). Broken line: apparent diffusion coefficient was changed to fit the experimental data (separate points show the mean values ± S.D. of respiration rate for given pyruvate kinase activity). Good correlation between simulations and the measurements was obtained for DF=10 -1.8 .

The computer modeling of data shown in Fig. 4 on the reduction of respiration rate of mitochondria in situ in permeabilized cardiac cells dependent on endogenous ADP (in the presence of 2mmoll -1 MgATP) by increasing pyruvate kinase (PK) activity for two different systems. V., respiration rate under given conditionsV̇ respiration rate before addition of ATPV̇ATP, respiration rate in the presence of 2mmoll -1 ATP. Solid line: diffusion constant of ADP was taken as D0 (characteristic for Brownian movement in water phase). Broken line: apparent diffusion coefficient was changed to fit the experimental data (separate points show the mean values ± S.D. of respiration rate for given pyruvate kinase activity). Good correlation between simulations and the measurements was obtained for DF=10 -1.8 .

However, the curve for D0 is much lower than the experimental dependence (Fig. 5, experimental points). To fit the experimental results described in Figs 4 and 5, we decreased the mean,apparent diffusion coefficient(Dapp=DF×D0), inside the cells,assuming that the high degree of intracellular structural organization (see Figs 1, 2, 3) may restrict the diffusion of adenine nucleotides (Saks et al.,2001). A good fit between the results of the modeling and the experimental data was observed when the DF approached the value of 10 -2 (Fig. 5). This means that the intracellular diffusion of ADP (and ATP) is likely to be very heterogeneous and strongly restricted in some areas inside the cells. Probably, this occurs both at or near the outer mitochondrial membrane and between the ICEUs (Aliev and Saks,1997 Saks et al., 1994, 2001).

It is also known that the high values of apparent Kmfor exogenous ADP are significantly decreased from 300–350μmoll -1 to 40–70 μmoll -1 by selective proteolysis (Kuznetsov et al.,1996 Saks et al.,2001). Treatment of permeabilized cardiomyocytes for a short time with 1 μmoll -1 trypsin also results in rapid disorganisation of the regular arrangement of mitochondria in cardiomyocytes and a collapse in the microtubular network (Appaix et al.,2003). Thus, evidently under these conditions, the specific structure of ICEUs is lost and the local intracellular restrictions for ADP diffusion are eliminated. This may well explain the decrease in apparent Km for exogenous ADP. In addition, we have shown previously that, after similar proteolytic treatment, the endogenous ADP becomes more accessible for the exogenous PK reaction(Saks et al., 2001).

The hypothesis of the heterogeneity of intracellular diffusion of ADP related to the structure of ICEUs is consistent with the new surprising findings described below.

The apparent link between sarcomere length and kinetic parameters of respiration regulation

The results described in Figs 6, 7, 8 show a new interesting phenomenon – an apparent link between sarcomere length and the affinity of mitochondria for exogenous ADP, measured as an apparent Km for this substrate in regulation of mitochondrial respiration in the permeabilized cells in situ. This phenomenon was observed when the kinetics of regulation of respiration by ADP were studied at different free calcium concentrations in two systems: permeabilized cardiac muscle fibers and permeabilized `ghost' fibers after extraction of myosin. The free calcium concentration was increased from 0.1 μmoll -1 to 3μmoll -1 , which corresponds to the physiological range of concentrations (Bers, 2001). Fig. 6 shows that, in the presence of ATP (or respiratory substrates and ADP), an increase of free Ca 2+ concentration to 3 μmoll -1 results in strong contraction of sarcomeres and shortening of fiber length in intact permeabilized cardiac fibers. If the fibers are not fixed, intermyofibrillar mitochondria seem to fuse as a result of being pressed together if the fibers are fixed in flexiperm and contract isometrically, one observes the appearance of the empty areas and of a rather long distance between mitochondria. In both cases, the structure of the cell and the structure of ICEUs are deformed.

Imaging of mitochondria in permeabilized myocardial fibers by the membrane-potential-sensitive probe teramethylrhodamine ethyl ether (TMRE). (A)Fibers in the presence of 2mmoll -1 ATP, 2mmoll -1 malate and 5mmoll -1 glutamate (concentration of free Ca 2+ in Ca-EGTA buffer: 0.1 μmoll -1 ). (B) The same fibers after addition of calcium chloride (final concentration of free Ca 2+ in Ca-EGTA buffer: 1.0 μmoll -1 ). The left fiber in a flexiperm chamber was not fixed, while the right (longer) fiber was fixed by its ends. In A, both fibers are relaxed. In B, the left fiber is contracted, while the right fiber,which is contracting almost isometrically, shows significant structural changes due to sarcomere contraction. Note the empty spaces between mitochondria.

Imaging of mitochondria in permeabilized myocardial fibers by the membrane-potential-sensitive probe teramethylrhodamine ethyl ether (TMRE). (A)Fibers in the presence of 2mmoll -1 ATP, 2mmoll -1 malate and 5mmoll -1 glutamate (concentration of free Ca 2+ in Ca-EGTA buffer: 0.1 μmoll -1 ). (B) The same fibers after addition of calcium chloride (final concentration of free Ca 2+ in Ca-EGTA buffer: 1.0 μmoll -1 ). The left fiber in a flexiperm chamber was not fixed, while the right (longer) fiber was fixed by its ends. In A, both fibers are relaxed. In B, the left fiber is contracted, while the right fiber,which is contracting almost isometrically, shows significant structural changes due to sarcomere contraction. Note the empty spaces between mitochondria.

Imaging of mitochondria in permeabilized myocardial fibers after extraction of myosin (ghost fibers) by membrane-potential-sensitive probe teramethylrhodamine ethyl ether (TMRE). (A) Ghost fibers in the presence of 2mmoll -1 ATP, 2mmoll -1 malate and 5mmoll -1 glutamate (concentration of free Ca 2+ in Ca-EGTA buffer: 0.1μmoll -1 ). (B) The same ghost fibers after addition of calcium chloride (final concentration of free Ca 2+ in Ca-EGTA buffer: 1.0μmoll -1 ). No structural changes were seen. The same result was obtained for a free Ca 2+ concentration of 3μmoll -1 .

Imaging of mitochondria in permeabilized myocardial fibers after extraction of myosin (ghost fibers) by membrane-potential-sensitive probe teramethylrhodamine ethyl ether (TMRE). (A) Ghost fibers in the presence of 2mmoll -1 ATP, 2mmoll -1 malate and 5mmoll -1 glutamate (concentration of free Ca 2+ in Ca-EGTA buffer: 0.1μmoll -1 ). (B) The same ghost fibers after addition of calcium chloride (final concentration of free Ca 2+ in Ca-EGTA buffer: 1.0μmoll -1 ). No structural changes were seen. The same result was obtained for a free Ca 2+ concentration of 3μmoll -1 .

Effect of different free Ca 2+ concentrations on parameters of ADP kinetics of mitochondrial respiration in permeabilized myocardial fibers and in myosin-extracted (ghost) fibers. (A) Effect of Ca 2+ on apparent Km for ADP. A dramatic decline in the apparent Km for ADP was observed in control fibers. By contrast, no changes in apparent Km for ADP can be seen in ghost fibers. (B) Effect of different free Ca 2+ concentrations on Vmax.

Effect of different free Ca 2+ concentrations on parameters of ADP kinetics of mitochondrial respiration in permeabilized myocardial fibers and in myosin-extracted (ghost) fibers. (A) Effect of Ca 2+ on apparent Km for ADP. A dramatic decline in the apparent Km for ADP was observed in control fibers. By contrast, no changes in apparent Km for ADP can be seen in ghost fibers. (B) Effect of different free Ca 2+ concentrations on Vmax.

Extraction of myosin prevents these Ca 2+ -induced structural changes (Fig. 7). The removal of a significant proportion of myosin decreased the total MgATPase activity of fibers (measured in the presence of 3mmoll -1 MgATP) from approximately 4.5–5.0nmol/minmg -1 wetmass (initial mass) to 0.9–1.0nmol/minmg -1 wetmass. In ghost fibers, a very regular arrangement of mitochondria with a precise, parallel fixation in the yz plane of cells was observed (direction of fiber orientation perpendicular to the x-axis), giving the impression of a striated pattern for the intracellular distribution of mitochondria. In these ghost fibers, the regular distance between mitochondria, corresponding to sarcomere length, is not changed with alteration of calcium concentration(Fig. 7). Thus, there is no deformation of the internal, modified structure of the ICEUs in the cell.

Fig. 8 shows that,surprisingly, the apparent Km for exogenous ADP in regulation of mitochondrial respiration in intact permeabilized fibers decreases from 350 μmoll -1 to 30 μmoll -1 with elevation of the free calcium concentration to 3 μmoll -1 and deformation of the cell structure (Fig. 8A). A decrease in the Vmax of respiration was also observed (Fig. 8B). None of these changes are observed in ghost fibers. In spite of removal of myosin and a 5-fold decrease in the overall MgATPase activity, the apparent Km for exogenous ADP (349±34 μmoll -1 )is initially (at 0.1 μmoll -1 free calcium concentration) equal to that of intact permeabilized fibers and always stays above 250μmoll -1 , even when free Ca 2+ concentration is increased to 3 μmoll -1 (Fig. 8A). Vmax does not change either with alteration of the free Ca 2+ concentration(Fig. 8B in comparison with intact permeabilized fibers, Vmax is elevated in ghost fibers due to extraction of a large proportion of the protein, i.e. myosin). Stability of all mitochondrial functions in ghost fibers shows that changes in the free Ca 2+ concentration in the range used does not alter the mitochondria, which might result from a mechanism of the permeability transition pore (PTP) opening (Lemasters et al., 1998).

An important conclusion from these data is that their seems to be a direct structural and functional link between sarcomere structure and mitochondrial function, which is in agreement with the concept of ICEUs.


Results

Mass spectrometry and in silico analysis of the mitochondrial proteome do not verify the existence of a mitochondrial RAS

First, in order to obtain unbiased evidence for the presence of RAS in mitochondria, we used purified mitochondrial fractions for proteomic analysis. The Crude mitochondrial fraction (CM), along with nuclei, microsomes, lysosomes and cytoplasm were purified by differential centrifugation. From the CM fraction, pure mitochondria (PM) were separated from mitochondria associated membranes (MAM), using isopicnic ultracentrifugation on a self-forming Percoll density gradient from rat livers 24 . The MAM fraction represents the interface of mitochondria with other cellular organelles, in particular the endoplasmic reticulum (ER), where signalling and metabolic interactions take place. It thus contains components of the OMM and other loosely associated cellular membranes. In contrast, pure mitochondria are devoid of other organelles, and highly enriched in matrix, IMM, OMM and intermembrane space components (for recent reviews see 25 ,26 ). Proteins from the CM, PM and MAM fractions were separated by SDS-PAGE and subjected to mass spectrometry analysis (Supplementary Fig. S1 and Supplementary Dataset S1). We compiled a list of RAS-related genes using the AmiGO gene ontology (GO) database, and sought the presence of their transcription products amongst those identified by mass spectrometry analysis. Importantly, from the three RAS related components found in the CM and MAM and PM fractions, none is involved directly in angiotensin generation and binding (see Supplementary Dataset S1).

The validity of these findings is supported by interrogation of unbiased catalogues of the mitochondrial proteome. The MitoMiner database aggregates findings from 47 proteomic surveys across several species 27 , while MitoCarta combines proteomics, imaging and sequence analysis to score the probability of mitochondrial localization of individual proteins in humans and mouse, further increasing the sensitivity to identify mitochondrial proteins 28 . In addition to our proteomic analysis in rat liver, the use of these databases allowed us to test the presence of RAS components and all related genes from a series of mammalian species and tissues, including rodent, bovine and human gene sets (Supplementary Dataset S2). Again, we generated gene sets from those belonging to all RAS related GO terms in the AmiGO database across all species. We then searched against the predicted mitochondrial genes in the MitoMiner and MitoCarta databases. This revealed mitochondrial localization of gene products involved in aldosterone synthesis, as targets of RAS, but no intrinsic RAS components have been experimentally proven or were bioinformatically predicted to localize to mitochondria (Supplementary Dataset S2).

While these approaches together rendered the presence of RAS in the mitochondria unlikely, they cannot formally exclude the possibility of the presence of components at low abundance. Thus, we proceeded to analyse the purified mitochondrial sub-fractions for the presence of main RAS components using immunoprecipitation and immunoblotting.

Rat liver mitochondria do not contain detectable ACE

If a functional, self-sufficient RAS exists in mitochondria, it should contain enzymes generating AngII, a role mostly fulfilled by ACE, the most evolutionarily conserved enzyme, which is directly responsible for the generation of AngII from AngI throughout the body. We thus sought to identify ACE in purified mitochondria from rat liver tissue. As shown in Fig. 1A , an antibody directed to the C-terminus of the protein recognised a high molecular weight band (MW ≈ 150 kDa, predicted MW of ACE) in the homogenate, nuclear, lysosomal and microsomal fractions, but not in mitochondria. Conversely, a low MW (� kDa) band was enriched in the crude mitochondrial fraction. This band was also present in the pure mitochondrial fractions from three independent preparations albeit at lower and variable intensity. Since a shorter natural human and rat ACE isoform exists (ACE-T, Uniprot <"type":"entrez-protein","attrs":<"text":"P47820","term_id":"1351843","term_text":"P47820">> P47820), we sought to confirm the identity of the lower MW mitochondrial band. We thus purified it by immunoprecipitation ( Fig. 1B ) and analysed by mass spectrometry. However, the results of the analysis did not return any ACE related sequences, indicating that the band represents non-specific binding by the antibody. Altogether, these results confirmed the proteomic analysis excluding the presence of ACE in mitochondria.

(A). Western blot using anti ACE and anti-grp75 antibodies from rat liver subcellular fractions. H-homogenate, N-nuclear fraction, C-cytosol, L-lysosomes, Mc-microsomes. Rat liver mitochondria were purified by differential centrifugation to obtain crude mitochondria (CM) and further separated to mitochondria associated membranes (MAM) and pure mitochondrial (PM) fractions by isopicnic Percoll centrifugation (for details see Methods). Mitochondrial fractions contain only a non-canonical 50 kDa immunoreactive band. Images of immunoblots were cropped to delineate the regions of interest. For the two immunoblots different aliquots of the same samples were loaded and separated under identical conditions. See full images on Supplementary Figure S3. (B). Immunoprecipitation of ACE using the CM fraction as input using the Pierce Crosslink IP approach as described in Methods. 10 μg ACE C-20 antibody was crosslinked to the protein A/G agarose resin and 1 mg protein was used as input. The immunoprecipitation was performed either in Tris-Buffered Saline, (TBS, 0.025 M Tris, 0.15 M NaCl pH 7.2) or TBS supplemented with 0.5 mM EDTA, 0.5% NP-40, 2.5% glycerol to promote ACE binding (D). An unrelated goat antibody (UR) and the resin without antibody crosslinked (R) were used as controls in both TBS and D solutions. Upper panel shows immunodetection using the same ACE antibody, lower panel shows silver stained SDS-PA gels. The 50 kDa band in the ACE-D fraction has been analysed by mass spectrometry, and identified as a non-ACE or RAS related protein. Images were cropped to show all visible bands.

AT1R is present in the MAM, but no AT1R, AT2R or AngII binding is detectable in the PM fraction

Whilst the previous results excluded the possibility of intra-mitochondrial generation of AngII by ACE, mitochondria can still be the target of AngII generated at other intracellular sites or imported from extracellular sources. We therefore sought the presence of functional AngII receptors in rat liver subcellular and submitochondrial fractions, using two independent strategies. First, we measured specific binding of [ 125 I]-AngII in the PM and MAM fractions. As shown in Fig. 2A , we detected a single high affinity binding site in the MAM fraction as indicated by Hill slopes close to unity, with a subnanomolar equilibrium dissociation constant (KD) and a maximal receptor density (Bmax) comparable to the range found in the plasma membrane 29 , indicating that functional AngII binding sites are present in mitochondria associated membranes. However, the same high affinity binding sites showed ≈ 50 times less density in purified mitochondria, most likely indicating contamination from the MAM fraction, and unlikely to be consistent with efficient AngII mediated signalling. Binding of AngII to the MAM-localized binding sites was inhibited by the specific AT1R antagonist Losartan, but not affected by the AT2R antagonist PD123319, indicating that binding was AT1R-related ( Fig. 2B ).

(A). Affinity (KD), density (BMAX) and cooperativity (Hill coefficient, nH) of specific AngII binding sites in the PM and MAM fractions. (B). Specific binding of [ 125 I]-AngII in the MAM fraction in the presence of AT1R (losartan) or AT2R (PD123319) blockers. The concentration of losartan was calculated from inhibition constants 29 to block 㺕% of the AT1 receptor but ς% of AT2R. [PD123319] was calculated to block 99% of AT2R but ς% of AT1R. (C). Western blot detection of AT1R in subcellular fractions. H-homogenate, C-cytosol, L-lysosomes, Mc-microsomes. Rat liver mitochondria were purified by differential centrifugation (CM) and further separated to mitochondria associated membranes (MAM) and pure mitochondrial (PM) fractions by isopicnic Percoll centrifugation (for details see Methods). Cytochrome oxidase subunit IV (CoxIV) was used as mitochondrial inner membrane marker. Images of immunoblots were cropped to delineate the regions of interest. The same membrane was used for both immunoblots. See full images on Supplementary Figure S4.

In order to further analyse the presence and localization of AT1R and AT2R subtypes, we next performed Western blot analysis of the rat liver subcellular and sub-mitochondrial fractions. In agreement with the binding studies, we were able to detect AT1R in the CM and MAM fraction, but not in the PM ( Fig. 2C ). In contrast, antibodies against AT2R gave a series of immunoreactive bands ( Fig. 3A ), even in the expected molecular weight range of the receptor (�� kD Fig. 3B ). Thus we followed the strategy previously applied to ACE, using the antibody to immunoprecipitate its binding partners and identified the resulting proteins by mass spectrometry. Immunoprecipitation with the anti-AT2R antibody pulled down three bands in the range of 30� kD in the MAM and partly in the PM fraction ( Fig. 3C ). However, mass spectrometry identified these bands as unrelated proteins.

(A). Immunoreactive bands detected by the sc9040, rabbit AT2R antibody in subcellular fractions: H-homogenate, C-cytosol, L-lysosomes, Mc-microsomes. Rat liver mitochondria were purified by differential centrifugation (CM) and further separated to mitochondria associated membranes (MAM) and pure mitochondrial (PM) fractions by isopicnic Percoll centrifugation (for details see Methods). For immunoblotting the same membrane was used as in Fig. 2C . Images were cropped to show all visible bands. The bands appearing

15 kD represent CoxIV staining. (B). The method reveals several bands also in around the predicted molecular weight of AT2R. The AT2R panel was cropped from (A). Cytochrome oxidase subunit IV (CoxIV) was used as mitochondrial inner membrane marker, immunoblotted from the same membrane as used in (A) and Fig. 2C . The full images are shown in Supplementary Fig. S4. (C). AT2R immunoprecipitations from all subcellular fractions and IgG controls separated by SDS PAGE and stained with Coomassie-blue. To maintain integrity of IgG at 130 kDa, we used a non-denaturating 4× loading buffer without dithiothreitol and β-mercaptoethanol (200 nM Tris HCl pH 6.8, 8% SDS, 40% glycerol, 0.1% bromophenol blue). Three immunoprecipitated bands from the MAM fraction (red circles) have been identified by mass spectrometry as non-RAS related proteins. Images were cropped to show all bands.

Together, these results confirm the lack of functional AngII receptors in rat liver mitochondria, but raised the possibility that intracellular AngII might alter mitochondrial function through agonist action at receptors located on the MAM. We thus performed further experiments using purified crude mitochondria (which contain pure mitochondria and their associated membranes) and evaluated the effect of AngII on oxidative phosphorylation.

AngII exerts marginal inhibition on respiration of isolated mitochondria at supra-physiological concentrations

Physiological concentrations of AngII in the plasma are in the picomolar range, in accordance with the sub-nanomolar affinity of angiotensin receptors on the cell surface. We have found AT1Rs with similarly high affinity in the MAM fraction. Tissue AngII levels vary from picomolar to the low nanomolar range depending on tissue type and conditions 30 ,31 , while no intracellular concentrations of AngII have been reported so far. Thus, we analysed the effect of AngII on isolated mitochondria (crude mitochondrial fraction, containing both the PM and MAM sub-fractions) in a broad concentration range. Supra-physiological (1 μM) saturating AngII had no significant effect on basal endogenous mitochondrial respiration rate (5 to 25 min in the absence of exogenous substrates and ADP) as compared to controls ( Figure 4A and Supplementary Figure S2). Similarly, acute addition of AngII, using concentrations in the physiological range (10� nM) had no effect on the activity of complexes I and II in the presence of substrates and ADP (state 3 respiration, Fig. 4B𠄽 ). Finally, we applied AngII in the range of 1 nM-1 μM range for 20 min and measured the state 3 activities of complexes I, II and IV ( Fig. 4E–G ). Again, AngII exerted no significant effect on respiration when applied in the physiological range (1� nM), while 1 μM AngII induced a minor but significant reduction in the maximal ADP-stimulated complex I-, II, and IV-dependent state 3 respiration. These results suggest that AngII does not exert a specific effect on oxidative phosphorylation in mitochondria isolated from rat liver, but might target non-specific binding sites at supra-physiological concentrations.

(A). Basal oxygen consumption of isolated rat liver mitochondrial 5 min after transfer to the respiration chamber, and at 5, 10, 15, 20 and 25 minutes after addition of AngII (1 μM) (n = 18). (B𠄽). ADP-induced oxygen consumption of isolated rat liver mitochondria for complexes I and II, with and without (control) acute addition of AngII at the indicated concentrations. Data are summarized on panel (B). (C) and (D) show individual representative traces from 9 experiments from 3 independent preparations. (E–G). ADP-induced oxygen consumption of isolated rat liver mitochondria for complexes I, II and IV after 20 minutes of incubation with and without (control) AngII for 20 min (n = 9 (1� nM) n = 16 (1 μM)). Data represent mean ±SEM. Statistical analysis: for basal respiration rates, differences along time between samples were assessed by ANOVA for repeated measures (time-group interaction: p > 0.05). For ADP-stimulated respiration rates differences between samples were assessed using paired sample t-test (* p < 0.05 AngII vs controls for comparisons of complexes I, II and IV).


Researchers solve decades old mitochondrial mystery that could lead to new disease treatments

Credit: CC0 Public Domain

Penn Medicine researchers have solved a decades old mystery around a key molecule fueling the power plant of cells that could be exploited to find new ways to treat diseases, from neurodegenerative disorders to cancer.

Reporting in a new study published today in Nature, researchers from the Department of Physiology in the Perelman School of Medicine at the University of Pennsylvania and other institutions found that the SLC25A51 gene dictates the transport of nicotinamide adenine dinucleotide (NAD+), a fundamental coenzyme in cellular metabolism, to the mitochondria, where energy from nutrients is converted into chemical energy for the cell. A low level of NAD+ is a hallmark of aging and has been associated with diseases including muscular dystrophy and heart failure.

"We have long known that NAD+ plays a critical role in the mitochondria, but the question of how it gets there had been left unanswered," said co-senior author Joseph A. Baur, Ph.D., an associate professor of Physiology and member of Penn's Institute for Diabetes, Obesity, and Metabolism. "This discovery opens up a whole new area of research where we can actually manipulate—selectively deplete or add—NAD+ at a subcellular level, now that we know how it's transported."

Xiaolu Ang Cambronne, Ph.D., an assistant professor in the department of Molecular Biosciences in The University of Texas at Austin, served as co-senior author.

The finding closes out a longstanding unknown around how NAD+ finds its way into the mitochondrial matrix. Several hypotheses had been circulating, including the idea that mammalian mitochondria were incapable of NAD+ transport, instead relying entirely on synthesis of NAD+ within the organelle, but in 2018, Baur's lab put that idea to rest when it reported in an eLife study that a transporter was in fact responsible.

From there, the team began its search for the genetic identity of the mammalian mitochondrial NAD+ transporter, homing in on several genes, including SLC25A51, that were predicted to be transporters, but for which the function remained unknown. SLC25A family members encode mitochondrially-localized proteins that carry materials across mitochondrial membranes.

"In our approach, we focused in on genes that were determined to be essential for cellular viability. NAD+ is a fundamental molecule required for maintaining the mitochondrial-mediated energy production. We predicted that loss-of mitochondrial NAD+ transport would disrupt oxidative phosphorylation and possibly reduce cell survival," said lead author Timothy S. Luongo, Ph.D., a postdoctoral fellow in the Baur lab.

In laboratory experiments, the researchers isolated the mitochondria from human cells and measured the levels of NAD+ after knocking out SLC25A51 or overexpressing it. Using mitochondrially-targeted NAD+ "biosensors," they showed that a change in the gene expression level controls mitochondria NAD+ levels specifically.

"We observed that loss of SLC25A51 expression dramatically altered the mitochondria's ability to consume oxygen and generate ATP as well as transport NAD+ into the matrix. Also, in collaboration with the Cambronne lab, we were able to demonstrate that expression of SLC25A51 in yeast lacking their endogenous mitochondrial NAD+ transporters restored NAD+ mitochondrial transport," said Luongo.

NAD+ levels can be targeted in various disease treatments however, it has been more of a catch-all approach, where levels are increased or reduced in all parts of the cell, which runs the risk of unintended alterations of gene expression or other types of metabolism. This study is the first published case where researchers identified a specific target and reduced the levels solely in the mitochondria and no other parts of the cell.

Controlling the levels of NAD+ and thus metabolic processes in the mitochondria could have major implications for the study and development of new treatments for diseases. Activating the transport mechanism could potentially make cells favor a state of respiration to make energy, instead of glycolysis. Different cancer types, for example, rely heavily on glycolysis, so creating an unfavorable environment without that metabolism could be one strategy. Or, conversely, it might be possible to deny highly respiratory cancer cells mitochondrial NAD+, so they're forced to rely on glycolysis. The heart requires abundant quantities of mitochondrial-produced energy to continually supply blood to peripheral tissue. A major contributor to heart failure is mitochondrial dysfunction, so targeting the mitochondria's capacity to transport NAD+ may improve cardiac function of the failing heart. With respect to exercise, shifting towards a more oxidative metabolism could boost endurance.

The work is in its early days, but a door has been opened for new investigations centered on mitochondrial NAD+ and this gene. Next, the researchers will study the physiological function of NAD+ transport and how this mechanism is regulated, as well as ways to turn the transport on and off outside of reducing or increasing gene expression.

"An approach to specifically alter the mitochondrial NAD+ pool is something many researchers have been looking for, so I would expect that we will see this gene targeted in a multitude of systems," Baur said. "I think this is going to be a really valuable tool to help us better understand the function of mitochondrial NAD+ and its therapeutic potential."


RESULTS

No differences between incubators were detected for all the parameters measured, consequently data from incubators A and B were pooled.

Mitochondrial respiration

State 3m

For both complexes, state 3m exhibited the same pattern between the mitotypes for 12°C, 18°C and 24°C (Fig. 2A,B) as well as among the temperatures for the two mitotypes. There were significant differences between the two mitotypes at 18°C (P=0.0168 for pyruvate + malate + proline and P<0.0001 for sn glycerol-3-phopshate), siII being higher than siIII.

siII showed a slight increase from 12°C to 24°C, with significant differences between 12°C and 18°C (P-values ≤0.0201 for both complexes), and between 18°C and 24°C (P<0.0001 for both complexes). We detected a significant decrease when comparing 28°C with 24°C (P<0.0001 for both mitotypes). The state 3m of complex III markedly increased from 18°C to 24°C (P<0.0001) while staying similar at 24°C and 28°C (siII) or slightly increasing for siIII (P=0.0426).

State 4o

siII had a significantly higher state 4o than siIII at 18°C (P≤0.0021 for both complexes) and 24°C (P-values ≤0.0123 for both complexes), while at 12°C and 28°C, it was significantly higher for siIII (P-values ≤0.0497) but only when substrates are provided to complex I (Fig. 2C,D).

We also observed significant differences between 12°C and 18°C (complex I, P<0.0001), 12°C and 24°C (P<0.0001 for both complexes), and between 18°C and 24°C (P-values ≤0.0011 for both complexes) in siII. At 28°C, a significant decrease was observed comparatively with 24°C for complex I in siII (P=0.0002).

In siIII state 4o of complex I significantly increased from 12°C to 28°C while in complex III it decreased between 12°C and 18°C (P=0.0106) and increased between 18°C, 24°C and 28°C (Fig. 2C,D).

ADP/O ratios

When using substrates for complex I (Fig. 2E), there was a statistical difference between the two lines at 12°C, with a higher ADP/O ratio for the siII mitotype (P=0.0003). Among the temperatures, siII and siIII exhibited significant differences, with a higher ADP/O ratio at 24°C compared with 12°C, 18°C and 28°C (all P-values ≤0.0002).

For complex III (Fig. 2F), the only significant difference between mitotypes occurred at 28°C, with siIII being lower (P=0.0093). Among the temperatures, no significant differences were detected for siII while siIII exhibited significant increases at 18°C and 24°C compared with 12°C (P-values ≤0.0054) and with 28°C (P-values ≤0.0026).

RCRs and UCRs

Results of RCRs are presented in Fig. 3. Comparisons between mitotypes showed that there were significantly higher RCRs for siII at 12°C at the complex I level (P=0.0231), as well as at 28°C at the complex III level (P=0.0143). Surprisingly, the RCR of siII was significantly lower than siIII at 24°C (P=0.0292) when the substrates were provided to complex I.

In both mitotypes, RCRs of complex I were not different between 18°C and 28°C but both were significantly higher at 12°C (P-values ≤0.0309 for both mitotypes) and 24°C (P-values ≤0.0139 for both mitotypes). Moreover, RCR at 12°C was higher than at 24°C for siII (P=0.0238), while we observed the opposite trend for siIII (P=0.0043). We observed slight but significant increases from 12°C to 28°C at the complex III level. Results of UCRs are shown in Table 2. No significant differences were detected in UCRs between strains or between temperatures.

Enzymatic measurements

No significant differences were detected between mitotypes (Fig. 4A) in ACO activity at any temperature, suggesting no differences in the oxidative stress supported by each of these lines, at least in mitochondria. This is reflected by absences of significant differences between both mitotypes in MDA levels (data not shown).

Mitochondrial functions measured at four different temperatures in isolated mitochondria from the two mitotypes of Drosophila simulans siII and siIII. State 3 respiration with (A) complex I substrates (pyruvate + malate + proline) and (B) sn glycerol-3-phosphate State 4 respiration with inhibitor olygomycin at level of (C) complex I and (D) complex III ADP/O ratios calculated from (E) complex I and (F) complex III. Results are means ± s.d. for 10 mitochondrial preparations. Significance was set as P<0.05 * denotes differences between mitotypes letters denote differences between temperatures with a statistically different from b and c, b statistically different from c.

Mitochondrial functions measured at four different temperatures in isolated mitochondria from the two mitotypes of Drosophila simulans siII and siIII. State 3 respiration with (A) complex I substrates (pyruvate + malate + proline) and (B) sn glycerol-3-phosphate State 4 respiration with inhibitor olygomycin at level of (C) complex I and (D) complex III ADP/O ratios calculated from (E) complex I and (F) complex III. Results are means ± s.d. for 10 mitochondrial preparations. Significance was set as P<0.05 * denotes differences between mitotypes letters denote differences between temperatures with a statistically different from b and c, b statistically different from c.

COX activity was significantly higher for siIII only at 12°C (P<0.0001 Fig. 4B). Moreover, we observed a slight increase from 12°C to 28°C for both mitotypes with significant differences between 12°C, 18°C, 24°C and 28°C.

At 18°C CAT activity was higher in siII compared wih siIII (P=0.0116) whereas at 28°C it was lower (P=0.0005) (Fig. 4C).

COX excess capacity and flux metabolic control

Complex IV activity and mitochondrial respiration with pyruvate, malate, l -proline and sn glycerol-3-phosphate were inhibited by sodium azide. We examined the apparent excess capacity of COX at high flux through the ETS using a combination of substrates that maximally reduce complexes I and III. Azide titration resulted in hyperbolic inhibition of COX. The threshold plots display pathway flux as a function of COX activity the threshold for inhibition of COX is defined as the intercept of the initial slope with the linear fit of the final slope (Fig. 5). The apparent excess capacity of COX is the intercept of the extrapolation of the linear regression for the final slope with the axis at zero COX inhibition. We detected a threshold, and consequently, a COX excess capacity at 12°C with no distinctions between the two mitotypes (604%, R 2 =0.9139 for siII and 613%, R 2 =0.9301 for siIII).

Surprisingly, with increasing temperatures the COX excess capacity vanished and comparisons were made between the different Ki and Ci. We observed increasing Ki with increasing temperatures but no differences between mitotypes were detected (Table 1).


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