**3. Results**

#### *3.1. Role of Oxidation of Fatty Acids in Regulation of Oxidative Phosphorylation and Mitochondrial Swelling*

Oxidation of fatty acids, the major myocardial respiratory substrates (palmitoyl-l-carnitine, palmitoyl-CoA + l-carnitine and octanoyl-l-carnitine) caused the drastic decrease of the apparent KmADP specific for pyruvate+malate oxidation [9], but the mechanisms of this effect have not been elucidated yet. In this study, we investigated which factors are responsible for the low apparent KmADP observed during fatty acid oxidation. Firstly, we evaluated if the fatty acid related decrease of the apparent KmADP depend on the different fatty acid chain length and degree of saturation. The principal scheme of fatty acid and pyruvate and malate oxidation in mitochondria is presented in Figure 1.

**Figure 1.** The principal scheme of the fatty acid and pyruvate and malate oxidation in mitochondria. LCFA—long chain fatty acids, MCFA—medium chain fatty acids, LACS—long chain acyl-CoA synthetase, CPT—carnitine palmitoyl-transferase, CACT—carnitine acylcarnitine translocase, MACS—medium chain acyl-CoA synthetase, ME—malic enzyme, MCT—monocarboxylate transporter, PDH—pyruvate dehydrogenase, TCA—tricarboxylic acid cycle, NAD—nicotinamide adenine dinucleotide, FAD—flavin adenine dinucleotide, I-V—mitochondrial electron transport chain complexes, Q—coenzyme Q, Cyt c—cytochrome c, ANT—ADP/ATP carrier, PiC—inorganic phosphate carrier, UCP—uncoupling protein.

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In the case of pyruvate+malate oxidation the apparent K m ADP of mitochondria in saponin-permeabilized rat cardiac fibers (K m ADP = 217.8 ± 8 μM) is by 3–10 folds higher if compared with the apparent K m ADP for oleoyl-CoA + l-carnitine+malate (55.7 ± 5.1 μM ADP), decanoic acid + pyruvate + malate (75.8 ± 7.6 μM ADP) or isolated rat heart mitochondria (K m ADP = 23 μM, Liobikas et al., 2001) (Figure 2). It is evident that the oxidation of both decanoic acid and oleoyl-CoA in situ induces a marked increase in the a ffinity of mitochondrial oxidative phosphorylation system for ADP.

**Figure 2.** Influence of di fferent respiratory substrates: pyruvate + malate (6 mM + 6 mM), oleoyl-CoA + l-carnitine+malate (6 μM + 2.5mM + 0.24mM) and decanoic acid + pyruvate + malate (0.3 mM + 6 mM + 6 mM) on the apparent K m ADP (**a**) and Vmax (**b**) of saponin-permeabilized rat cardiac fibers. *n* = 5; 37 ◦C; \* *p* < 0.05 vs. control (pyruvate + malate). The results were analyzed with one-way analysis of variance (ANOVA) followed by the Dunnett post hoc test.

In the next series of experiments on di fferent skinned cardiac fiber samples we tested the concentration dependence of the e ffect of fatty acids on the apparent K m ADP (Figure 3). The high apparent K m ADP with pyruvate + malate decreased to 30% and 33% of control values, when pyruvate + malate was supplemented with 2.2 μM or 9 μM palmitoyl-l-carnitine.

**Figure 3.** Influence of di fferent respiratory substrates: pyruvate + malate (P + M, 6 mM + 6 mM), pyruvate + malate + palmitoyl-l-carnitine (6 mM + 6 mM + 2.2 μM) and pyruvate + malate + palmitoyl-l-carnitine (6 mM + 6 mM + 9 μM) on the apparent K m ADP of saponin-permeabilized rat cardiac fibers. Here, *n* = 5; 37 ◦C; \* *p* < 0.05 vs. control (pyruvate + malate). The results were analyzed with one-way analysis of variance (ANOVA) followed by the Dunnett post hoc test.

Thus, four times lower concentration of palmitoyl-l-carnitine (2.2 μM) also e ffectively decreased the apparent K m ADP. Noteworthy, at 2.2 μM palmitoyl–l-carnitine (alone) the State 3 respiration rate was equal to 30–40% of that estimated at 9 μM. In the separate set of experiments, we revealed also that even 0.5 μM of palmitoyl-l-carnitine (used together with pyruvate+malate) decreased the

apparent KmADP (*p* < 0.05) to similar level as 9 μM of palmitoyl-l-carnitine (94.6 ± 13 μM and 77.9 ± 11 μM, respectively, compared with pyruvate+malate alone, 253.3 ± 23 μM; data of 4–6 paired experiments; 37 ◦C). Essentially similar results were obtained in the separate group of experiments when octanoyl-l-carnitine at two different concentrations (0.36 and 0.1 mM) was used as respiratory substrate in the presence of pyruvate + malate (Figure 4).

**Figure 4.** Influence of different respiratory substrates: pyruvate + malate (P + M, 6 mM + 6 mM), pyruvate + malate + octanoyl-l-carnitine (6 mM + 6 mM + 0.36 mM) and pyruvate + malate + octanoyl-l-carnitine (6 mM + 6 mM + 0.1 mM) on the apparent KmADP of saponin-permeabilized rat cardiac fibers. In this case, *n* = 4; 37 ◦C; \* *p* < 0.05 vs. control (pyruvate + malate). The results were analyzed with one-way analysis of variance (ANOVA) followed by the Dunnett post hoc test.

In this case, the apparent KmADP decreased, respectively, to 34% and 26% of control values estimated with pyruvate+malate (*p* < 0.05, *n* = 4, 37 ◦C). Thus, our results showed that the decrease in the apparent KmADP value did not depend upon the concentration of fatty acids.

Interestingly, during palmitoyl-l-carnitine oxidation, the apparent KmADP decreased by about 2.6-fold with elevation of temperature from 25 ◦C to 37 ◦C, i.e., from 123 ± 22 μM ADP to 48 ± 5 μM ADP, respectively (Figure 5).

**Figure 5.** Influence of temperature on the apparent KmADP of saponin-permeabilized rat cardiac fibers oxidizing palmitoyl-l-carnitine (9 μM) in the presence of malate (0.24 mM). The results were analyzed with paired t test; *n* = 7; \* *p* < 0.05.

When pyruvate+malate were added immediately after the complete oxidation of a limited quantity (1.6 nmol) of palmitoyl-l-carnitine (Figure 6), the apparent KmADP of mitochondria in cardiac fibers remained at a high level (211 ± 40 μM ADP), similar as in the case of pyruvate+malate oxidation alone (223 ± 56 μM ADP) (data of five paired experiments, 37 ◦C). Thus, the effect of fatty acids on the apparent KmADP of mitochondria in permeabilized rat cardiac fibers is reversible.

**Figure 6.** Representative respiration curve after the complete oxidation of a limited quantity of palmitoyl-l-carnitine. The black curve—oxygen consumption, the blue curve—oxygen consumption derivative. Additions: O—skinned rat cardiac fibers (registration of respiration on endogenous substrates), then A1—1.6 nmol of palmitoyl-l-carnitine, A2—pyruvate + malate (6 mM + 6 mM, respiration rate: 24.8 nmol/O/min/mg dry weight), A3—10 μM ADP (respiration rate: 32 nmol/O/min/mg dry weight), A4—1.2 mM ADP (respiration rate: 57.7 nmol/O/min/mg dry weight).

To exclude that the decrease in the apparent KmADP during fatty acid oxidation is related to the detergent activity of palmitoyl-l-carnitine (reviewed in Goni FM et al. 1966) and its damage to mitochondrial membranes, we measured the swelling of isolated rat heart mitochondria oxidizing palmitoyl-l-carnitine (9, 27 and 80 μM) and malate (0.24 mM). Our data (Figure 7) showed that palmitoyl-l-carnitine at a concentration of 9 μM did not induce mitochondrial swelling.

**Figure 7.** Swelling of isolated rat heart mitochondria respiring on palmitoyl-l-carnitine (9–80 μM) and malate (0.24 mM). *n* = 3, typical absorption traces are shown.

However, the higher concentrations (starting from 27 μM) of a palmitoyl-l-carnitine-induced concentration–dependent increase in mitochondrial swelling. Importantly, palmitoyl-l-carnitine did not change the mitochondrial outer membrane integrity (external cytochrome c did not stimulate the State 3 respiration; its effect varied between 0.98 and 1.02 at distinct palmitoyl-l-carnitine concentrations; *n* = 8).

#### *3.2. Regulation of Mitochondrial Respiration by Endogenous versus Exogenous ADP: Influence of Palmitoyl-*l*-Carnitine*

The endogenous ADP is produced from exogenous ATP by ATPases in the myofibrils and in the sarcoplasmic reticulum, and is delivered directly to the mitochondria [8]. In the next series of experiments, the respiration of fibers was supported by palmitoyl-l-carnitine or pyruvate + malate (control), and titrated by ADP and ATP (Figure 8).

**Figure 8.** Influence of the adenosine diphosphate (ADP) source (endogenous (from exogenous adenosine triphosphate (ATP)) or exogenous) on the apparent KmADP of saponin-permeabilized rat cardiac fibers oxidizing palmitoyl-l-carnitine (9 μM) in the presence of malate (0.24 mM) or pyruvate + malate (control, 6 mM + 6 mM). Experiments were performed at 37 ◦C. \* *p* < 0.05 vs. control. *n* = 5 (paired experiments). The results were analyzed with one-way analysis of variance (ANOVA) followed by Dunnett post hoc test.

The apparent KmADP and KmATP were very low with palmitoyl-l-carnitine as the substrate (55 ± 9 and 59 ± 26 μM, respectively compared to pyruvate+malate (KmADP, 312 ± 28 μM). Thus, in our study the decrease in the apparent KmADP during fatty acid oxidation did not depend on the exogenous or endogenous supply of ADP.

#### *3.3. Influence of Palmitoyl-*l*-Carnitine on the Permeability of Mitochondrial Outer Membrane for ADP*

Using an exogenous ADP-trapping system consisting of pyruvate kinase + phosphoenolpyruvate (PK + PEP), which effectively competes with mitochondria for extramitochondrial ADP [8], we investigated the interactions of functional complexes of mitochondria with Ca, Mg ATPases of myofibrils and sarcoplasmic reticulum under different conditions (25 and 37 ◦C), and mitochondrial outer membrane permeability for ADP. The results of these experiments are presented in Figure 9. By the addition of 2 mM ATP we stimulated the basal respiration of mitochondria (similarly with both substrates, glutamate + malate and palmitoyl-l-carnitine), due to endogenously generated ADP. Consecutive addition of PK (40 U/mL, in the presence of PEP in the medium) suppressed respiration by about 30%; a similar effect (23%) was observed with palmitoyl-l-carnitine at 25 ◦C. The inhibitory effect of PK + PEP at 37 ◦C was equal with both respiratory substrates, glutamate + malate (33%) and palmitoyl-l-carnitine+malate (31%).

**Figure 9.** Influence of pyruvate kinase + phosphoenolpyruvate (PK + PEP) ADP-consuming system and creatine on the respiration of rat cardiac fibers using glutamate+malate and palmitoyl-l-carnitine + malate as respiratory substrates at (**a**) 25 ◦C and (**b**) 37 ◦C. The effect of pyruvate kinase (PK) (40 U/mL) on VATP and the effect of creatine on VPK (40 U/mL) are shown. \* *p* < 0.05 versus 37 ◦C with palmitoyl-l-carnitine, *n* = 5 (paired experiments). The results were analyzed with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.

It is important to note that the exogenous cytochrome c test showed the intactness of the mitochondrial outer membrane (no stimulation of respiration in State 3 by cytochrome c was observed; its effect on respiration varied from 0.90±0.02 to 1.04±0.01; *<sup>n</sup>*=5, paired experiments) and inaccessibility of PK to ADP localized in the intermembrane space of mitochondria. Thus, neither the increase in temperature (from 25 to 37 ◦C) nor fatty acid oxidation, which both largely increased the affinity of mitochondrial oxidative phosphorylation for exogenous and endogenous ADP (i.e., decrease in the apparent KmADP and KmATP), affected the inhibitory effects of exogenous ADP-trapping system, and possibly, the concentration of ADP in the medium. In case palmitoyl-l-carnitine could increase the mitochondrial outer membrane permeability to ADP or ATP, the inhibitory effect of PK + PEP would have been different during palmitoyl-l-carnitine oxidation compared to the oxidation of pyruvate + malate. Our data show that the effect was similar during the oxidation of either substrate. Thus, the data presented above mean that the oxidation of palmitoyl-l-carnitine does not increase the mitochondrial outer membrane permeability to ADP or ATP.

#### *3.4. Influence of Creatine Kinase on Regulation of Mitochondrial Respiration Supported by Fatty Acids*

In the next series of experiments, we investigated the effect of fatty acid oxidation on the functional coupling between mitochondrial creatine kinase (mi-CK) and the ADP/ATP carrier.

The addition of 20 mM creatine in the presence of 2 mM ATP and exogenous PK + PEP system give notable and similar (1.35–1.56-fold) increase in the respiration rate in all conditions investigated (with both substrates, glutamate and palmitoyl-l-carnitine, at two different temperatures 25 and 37 ◦C, Figure 9).

Furthermore, the exogenous creatine, in the medium devoid of PK and PEP, stimulated the respiration of cardiac fibers oxidizing pyruvate + malate or palmitoyl-l-carnitine in presence of low 60 μM ADP concentration, accordingly by 1.37 ± 0.05 times and 1.42 ± 0.04 times (data of six unpaired experiments, 20 ◦C) with a similar efficacy as in the medium containing PK + PEP (Figure 9). It should be noted that in these experiments, exogenous cytochrome c had no significant stimulating effect on the mitochondrial respiration in State 3 with both substrates, indicating the intactness of the mitochondrial outer membrane.

In other series of experiments, fibers respiration supported by palmitoyl-l-carnitine was stimulated by exogenous ATP. In these conditions, endogenous ADP is produced from exogenous ATP by ATPases in myofibrils and the sarcoplasmic reticulum, and it is delivered directly to the mitochondria [13]. This approach should be regarded as more physiological. In this case the stimulating effect of creatine

on mitochondrial respiration appeared to be very close to that observed in the above described experiments with the exogenous ADP; i.e., 1.38 ± 0.06 and 1.47 ± 0.05 times, respectively, with 23 and 37 μM of ATP (data of five unpaired experiments, 20 ◦C).

The maximal effect of creatine on mitochondrial respiration with octanoyl-dl-carnitine, similarly to palmitoyl-l-carnitine, was observed at 60 μM concentration of exogenous ADP (*n* = 3–5; Figure 10). Similar results (ADP concentration) were obtained also in the case of pyruvate + malate oxidation. When octanoyl-dl-carnitine was used as a respiratory substrate, the estimated apparent KmADP values were significantly different in the presence and absence of creatine, accordingly 98 ± 11 μM ADP and 235 ± 16 μM ADP (Figure 10).

**Figure 10.** The dependence of the respiration rates of rat cardiac fibers on the external ADP concentration: the effect of creatine. The substrate: octanoyl-dl-carnitine 0.36 mM + malate 0.24 mM. The data of five separate paired experiments are presented. Vmax values (in nmol O/min/mg dry weight) were found to be similar: 29.9 ± 1.8 (the medium without creatine) and 31.1 ± 1.6 (the medium with 20 mM creatine).

Thus, the creatine-induced decrease in the apparent KmADP reflects the maintenance of functional coupling in the intermembrane space between ADP/ATP carrier and mi-CK in the mitochondria respiring on fatty acids.

#### *3.5. E*ff*ects of Oncotic Pressure on Mitochondrial Fatty Acid Oxidation*

In our further study, 5% of dextran T-70 was added to the respiration medium to mimic the oncotic pressure of the cellular cytoplasm and to test its effect on the respiration of saponin-permeabilized rat cardiac fibers oxidizing pyruvate + malate or palmitoyl-l-carnitine. 5% dextran T-70 similarly (by 25%) decreased the maximal respiration rate (with ADP) of mitochondria in situ, oxidizing both substrates palmitoyl-l-carnitine and pyruvate + malate (Figure 11). Noteworthy, dextran did not affect the high apparent KmADP of mitochondria in cardiac fibers oxidizing pyruvate + malate, but significantly (by 50–60%) increased the low apparent KmADP of mitochondria oxidizing palmitoyl-l-carnitine. The apparent KmADP values during the oxidation of palmitoyl-l-carnitine in the medium containing 5% dextran (110.6 ± 20.7 μM) were much lower than the high apparent KmADP values (255.7 ± 42 μM) characteristic for the oxidation of pyruvate + malate.

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**Figure 11.** Influence of 5% dextran T70 on the apparent KmADP (**a**) and Vmax (**b**) of saponin-permeabilized rat cardiac fibers using different respiratory substrates. *n* = 5 (paired), 37 ◦C, \* *p* < 0.05 versus control. The results were analyzed with one-way analysis of variance (ANOVA) followed by a Dunnett post hoc test.

#### *3.6. Fatty Acid-Oxidation and Oncotic Pressure-induced Changes of Mitochondrial Morphology*

Electron microscopy was used to evaluate morphological changes in mitochondria in situ caused by fatty acids oxidation in presence and absence of 5% dextran T-70 (Figure 12).

**Figure 12.** Morphology of mitochondria in skinned rat cardiac fibers. 5 min incubations of saponin-skinned cardiac fibers were performed in physiological salt solution at 37 ◦C in the presence of: (**a**) pyruvate + malate (6 mM + 6 mM); (**b**) palmitoyl-l-carnitine (9 μM); (**c**) palmitoyl-l-carnitine (9 μM) + 5% dextran T-70; (**d**) pyruvate + malate (6 mM + 6 mM) and ADP/ATP carrier inhibitor carboxyatractyloside (1.3 μM); (**e**) pyruvate + malate (6 mM + 6 mM) and ADP/ATP carrier inhibitor bongkrekic acid (17.6 μM).

The results revealed that mitochondrial morphology in saponin-permeabilized cardiac fibers significantly alters during 5 min oxidation of palmitoyl-l-carnitine in the absence of dextran (Figure 12b); compared with pyruvate and malate oxidation (Figure 12a): increased mitochondrial intermembrane space and condensed mitochondrial matrix were observed.

In fibers treated with dextran (in the presence of palmitoyl-l-carnitine), the whole population of mitochondria was dark (Figure 12c) indicating that they all have intact outer membranes. This finding is in accordance with the functional evaluation of intactness of mitochondrial outer membrane by cytochrome c test. Dextran prevented the palmitoyl-l-carnitine-induced morphological changes of mitochondria, where condensed mitochondria were observed; i.e., the appearance of mitochondria became similar to that in fibers incubated with pyruvate and malate, or similar as with in vivo conditions.

It is noteworthy that the incubation of fibers with carboxyatractyloside (1.3 μM; at this concentration the state 3 respiration with pyruvate and malate was completely inhibited)-induced ultrastructural changes of mitochondria similar to those induced by palmitoyl-l-carnitine, corresponding to the orthodox state of mitochondria; that is, enlarged cristae volume, contracted matrix space (Figure 12d). Bongkrekic acid (17.6 μM) induced completely di fferent changes of mitochondrial ultrastructure; mitochondria were dark, cristae compressed, matrix space enlarged, little vacuoles were noticed, corresponding to the condensed state of mitochondria (Figure 12e). Thus, our results sugges<sup>t</sup> that fatty acid oxidation may cause the morphological changes of mitochondrial ultrastructure, with an orthodox conformation when the ADP/ATP carrier is in the cytosol-oriented state.
