**1. Introduction**

The major part of energy supply in cells comes from the fatty acid oxidation in mitochondria. Fatty acids as the main respiratory substrates are important, not only for cardiac function [1,2], but also they recently have been shown to provide energy for the proliferation and survival of tumors [3]. An increased fatty acid consumption reduces cardiac efficiency, and among the mechanisms involved, a modulation of the ADP/ATP carrier has been suggested [4–6].

The studies of saponin-permeabilized heart and skeletal muscle fibers demonstrated that, in contrast to isolated mitochondria, the mitochondrial outer membrane in situ possesses a low permeability for exogenous ADP (high apparent KmADP of oxidative phosphorylation for external

ADP), and therefore, is crucial in the mechanism of regulation of mitochondrial respiration in vivo [7,8]. KmADP is drastically (up to 10-fold) decreased in the case of the oxidation of saturated fatty acids (CoAor carnitine-esters of palmitate and octanoate; alone or in combination with pyruvate), but not during the transport of fatty acids into mitochondria [9]; however, the mechanism of this phenomenon has not been elucidated yet.

Mitochondria and intracellular ATPases in cardiomyocytes are in close proximity, and are arranged into tightly coupled structural and functional complexes known as intracellular, energetic units [8,10,11]. Strict interpositions of mitochondrion optimizes the energy fluxes and interactions of mitochondria with surrounding organelles; however, at high workload the direct ATP transfer does not fulfil the energy need in heart cells [12,13]. In these conditions, the creatine kinase–phosphocreatine system is useful: creatine (the substrate of mitochondrial creatine kinase) significantly stimulates the production of ADP (when the concentration of ADP is suboptimal, i.e., about 50–10 μM) by mitochondrial creatine kinase functionally coupled with the ADP/ATP carrier. This significantly enhances the respiration of heart mitochondria and ATP production. The stimulating effect of creatine on respiration decreases when the permeability of the mitochondrial outer membrane for ADP increases (the apparent KmADP decreases) due to the treatment of cardiac cells with proteolytic enzymes (trypsin or collagenase) [14,15], after the isolation of mitochondria [16,17], or due to some clustering of mitochondria possessing an intact mitochondrial outer membrane and the alteration of their position within the cells of the non-ischemic zone of the low-flow-perfused rat heart [18].

Fatty acids have been demonstrated to change the conformation of uncoupling protein [19]. Furthermore, other studies revealed structural, and to some extent functional, homology between the uncoupling protein and the ADP/ATP carrier [20,21]. There is also an indirect evidence that low apparent KmADP in mitochondria from cancerous cells [22,23] and fetal or neonatal mitochondria [24] could be related to mitochondrial ultrastructural changes, namely increased, diffuse the mitochondrial matrix volume corresponding to orthodox mitochondrial conformation [25,26].

Mitochondria are significantly more abundant in hearts compared to skeletal muscle, brain, kidney and liver [27]. Furthermore, cardiac mitochondria primarily use fatty acids as respiratory substrates, whereas most other organs use glucose as the major energy substrate [28], and therefore, we have chosen skinned cardiac fibers as the experimental object in our study. We investigated the mechanism of fatty acid–oxidation-induced changes and their relations with mitochondrial morphology and ADP/ATP carrier conformation on the kinetics of the regulation of mitochondrial respiration (the apparent KmADP) in situ; i.e., in the mitochondria of saponin-permeabilized rat heart muscle fibers, where mitochondria and the ATPases in myofibrils and in the sarcoplasmic reticulum remain intact, corresponding to the physiological conditions in the cell [8,10].

#### **2. Materials and Methods**

All chemicals used in this work were from Sigma-Aldrich (St. Louis, MO, USA).

The male Wistar rats ~3 months old and weighing 250–300 g were obtained from the Vivarium of the Lithuanian University of Health Sciences, where they were housed at 23 ± 2 ◦C with a 12-h light/dark cycle and free access to food and water. The experimental procedures used in the present study were performed according to the permission of the Lithuanian Committee of Good Laboratory Animal Use Practice (number 0228/2012). Rats were killed by cervical dislocation. Rat hearts were excised and rinsed in ice-cold 0.9% KCl solution. The bundles of cardiac fibers, approximately 0.3–0.4 mm in diameter, were prepared by using sharp-ended needles from the muscle strips cut out from the left ventricular endocardium in an ice-cold preparation solution containing 20 mM imidazole, 20 mM taurine, 0.5 mM dithiothreitol, 7.1 mM MgCl2, 50 mM 2-(NMorpholino)ethanesulfonic acid (MES), 5 mM adenosine triphosphate (ATP), 15 mM phosphocreatine, 2.62 mM CaK2EGTA and 7.38 mM K2EGTA (ionic strength of the solution 160 mM, free Ca2+ 0.1 μM, free Mg<sup>2</sup>+ 3 mM; pH 7.0, adjusted with KOH), then permeabilized by saponin (50 μg/mL, 30 min), washed for 10 min in a physiological salt solution containing 20 mM imidazole, 20 mM taurine, 0.5 mM dithiothreitol, 1.61 mM MgCl2, 100 mM MES, 3 mM KH2PO4, 2.95 mM CaK2EGTA and 7.05 mM K2EGTA (ionic strength of the solution 160 mM, free Ca2+ 0.1 μM, free Mg<sup>2</sup>+ 1 mM; pH 7.1, adjusted with KOH) [29]. All procedures were carried out under intensive shaking (120 times/min). The washed bundles of fibers were rinsed once in the physiological salt solution, transferred into the tubes with the same solution and then kept on ice.

Respiration rates of skinned cardiac fibers were determined in the closed respiration chamber in physiological salt solution at 37 ◦C or 25 ◦C by the means of the Clark-type oxygen electrode. Pyruvate + malate (6 mM + 6 mM), glutamate 6 mM + malate (6 mM + 6 mM), palmitoyl-l-carnitine+malate (9 μM + 0.24 mM), oleoyl-CoA + l-carnitine + malate (6 μM + 2.5 mM + 0.24 mM) or decanoic acid + pyruvate + malate (0.3 mM + 6 mM + 6 mM) were used as respiratory substrates as indicated in the Results section or in the Figure legends. Respiration rates were expressed as nmol O/min/mg fibers' dry weight. Dry weight = wet weight before respiration measurement/factor '*W*'. The factor '*W*' was calculated to be 4.85 for heart muscle fibers [14]. The solubility of oxygen was taken to be 422 nmol O/mL at 37 ◦C and 452 nmol O/mL at 25 ◦C [30]. The adenosine diphosphate (ADP) regenerative system, consisting of 1.2 IU/mL lyophilized yeas<sup>t</sup> hexokinase (Type V; EC 2.7.1.1) and 24 mM glucose, was added to the chamber before the addition of heart muscle fibers. Titration was made by different ADP concentrations in each separate probe. ΔV was expressed as a difference between respiration rates in the presence and in the absence of added ADP. The apparent KmADP and Vmax were estimated from the least-squares fit to the Michaelis–Menten equation (ΔV vs. ADP concentration).

Mitochondria were isolated by a differential centrifugation procedure. Hearts were excised and rinsed in ice-cold isolation medium, containing 160 mM KCl, 10 mM NaCl, 20 mM Tris, 5 mM EGTA (pH 7.7, adjusted with KOH at 2 ◦C). Mitochondria were isolated in the same medium supplemented with 2 mg/mL bovine serum albumin (BSA). Homogenate was centrifugated for 5 min at 750× *g*, then the supernatant was centrifugated for 10 min at 6740× *g* and the pellet was washed once in the medium containing 180 mM KCl, 20 mM Tris, 3 mM EGTA (pH 7.3 adjusted with KOH at 2 ◦C), suspended in it and kept on ice. The mitochondrial protein concentration was determined by the biuret method (Piotrowski's test) [31]. The final mitochondrial protein concentration was 0.5 mg/mL. Mitochondrial swelling was recorded as the decrease of light scattering at 540 nm with the Heλios α spectrophotometer in physiological salt solution supplemented with 24 mM glucose and 1.2 IU/mL hexokinase, 0.24 mM malate and palmitoyl-l-carnitine (9–80 μM).

An exogenous ADP-trapping system consisting of pyruvate kinase + phosphoenolpyruvate (PK + PEP), which effectively competes with mitochondria for the extramitochondrial ADP, and therefore, decreases the respiration rate in the State 3, was used to investigate the interactions of the functional complexes of mitochondria with Ca, the MgATPases of myofibrils and the sarcoplasmic reticulum under the different conditions (25 ◦C and 37 ◦C; mitochondria oxidizing different substrates: glutamate 6 mM + malate 6 mM or palmitoyl-l-carnitine 9 μM + malate 0.24 mM). The sequence of additions to the respiration chamber: 8 mM PEP, ~3 mg of cardiac fibers, 2 mM ATP, 20 + 20 U/mL (or 40 U/mL) PK, 20 mM creatine, 35 μM cytochrome c, 125 μM atractyloside. After each addition, the respiration rate was estimated. The cytochrome c test was used to evaluate the intactness of the mitochondrial outer membrane.

The coupling of mitochondrial creatine kinase (mi-CK) and the ADP/ATP carrier was estimated using two approaches. (1) The apparent KmADP and Vmax values were estimated from ΔV vs. ADP concentration relationships in the presence or in the absence of 20 mM creatine; the results were compared with corresponding kinetic parameters without creatine; (2) 60 μM ADP was added into the respiration chamber followed by the addition of 20 mM of creatine. The stimulation of respiration by creatine, i.e., the creatine effect, was expressed as the ratio of the respiration rates with creatine and with 60 μM of ADP without creatine.

For the electron microscopy studies, the saponin-permeabilized rat cardiac fibers were incubated aerobically in the physiological salt solution containing pyruvate and malate, 6 mM both (without, as control, or with carboxyatractyloside 1.3 μM or bongkrekic acid 17.6 μM), or palmitoyl-l-carnitine 9 μM, or palmitoyl-l-carnitine 9 μM plus 5% dextran T-70, for 5 min at 37 ◦C under intensive stirring. Subsequently, the fibers were placed into 2.5% glutaraldehyde solution in 0.1 M cacodylate buffer (pH 7.4) and kept in it for 5 min at room temperature under gentle shaking. Afterwards, the fibers were left in the same fixative overnight at 4 ◦C. Later on, the fibers were washed several times in cacodylate buffer and post-fixed for 1 h at 4 ◦C with 1% osmium tetraoxide solution in the same buffer. After that, they were dehydrated through a graded ethanol series and embedded in a mixture of resins Epon 812 and Araldite. Ultrathin sections were cut with a with ultra-microtome, stained with uranyl acetate and lead citrate, and examined with a PHILIPS EM300 electron microscope, using AGFA electron image films.

The results are presented as means ± S.E. The data were analyzed with one-way analysis of variance (ANOVA) by Prism v. 5.04 (GraphPad Software Inc., La Jolla, CA, USA). Then *p* <0.05 was taken as the level of significance.
