Báo cáo khoa học: Mitochondrial affinity for ADP is twofold lower in creatine kinase knock-out muscles Possible role in rescuing cellular energy homeostasis pptx

Mitochondrial affinity for ADP is twofold lower in creatine kinase knock-out muscles Possible role in rescuing cellular energy homeostasis Frank ter Veld 1 , Jeroen A. L. Jeneson 2,3 and Klaas Nicolay 3 1 Department of Experimental In Vivo NMR, Image Sciences Institute, University Medical Center, Utrecht, the Netherlands 2 Department of Physiology, School of Veterinary Medicine, Utrecht University, the Netherlands 3 Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, the Netherlands Excitable mammalian cells contain high activities of creatine kinase (CK, EC 2.7.3.2), which catalyses the reversible exchange of a phosphoryl group between phosphocreatine (PCr) and ATP. The tissue-specific CK enzymes are subcellularly compartmentalized and consist of three cytosolic dimers: BB-CK (brain- and smooth muscle-specific), MM-CK (muscle-specific) and MB-CK heterodimers. Furthermore, there is mitochondrial CK (Mi-CK) which is located in the intermembrane space of the mitochondrion and con- sists mainly of octamers in vivo [1]. Mi-CK and M-CK have been hypothesized to jointly form an energy transport network in which creatine (Cr) and PCr function as diffusible intermediates between sites of ATP synthesis and utilization, thereby buffering fluctuations in the ATP free energy potential, i.e. the ATP ⁄ ADP concentration ratio [2,3]. The roles of Mi-CK and M-CK in this CK ⁄ PCr shuttle model are to maintain a high local ADP ⁄ ATP concentra- tion ratio near the adenine nucleotide translocase (ANT) by transphosphorylation of mitochondrially generated ATP to PCr and a high local ATP ⁄ ADP ratio near extramitochondrial ATPases, respect- ively [4]. Keywords heart; metabolic control; mitochondrial respiration; skeletal muscle; transgenic mice Correspondence F. ter Veld, Laboratory for Biophysics and Cell Biology, Department of Epithelial Cell Physiology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany Fax: +49 231133 2299 Tel: +49 231133 2226 E-mail: frank.terveld@mpi-dortmund.mpg.de (Received 30 July 2004, revised 8 December 2004, accepted 14 December 2004) doi:10.1111/j.1742-4658.2004.04529.x Adaptations of the kinetic properties of mitochondria in striated muscle lacking cytosolic (M) and ⁄ or mitochondrial (Mi) creatine kinase (CK) iso- forms in comparison to wild-type (WT) were investigated in vitro. Intact mitochondria were isolated from heart and gastrocnemius muscle of WT and single- and double CK-knock-out mice strains (cytosolic (M-CK – ⁄ – ), mitochondrial (Mi-CK – ⁄ – ) and double knock-out (MiM-CK – ⁄ – ), respect- ively). Maximal ADP-stimulated oxygen consumption flux (State3 V max ; nmol O 2 Æmg mitochondrial protein )1 Æmin )1 ) and ADP affinity (K ADP 50 ; lm) were determined by respirometry. State 3 V max and K ADP 50 of M-CK – ⁄ – and MiM-CK – ⁄ – gastrocnemius mitochondria were twofold higher than those of WT, but were unchanged for Mi-CK – ⁄ – . For mutant cardiac mito- chondria, only the K ADP 50 of mitochondria isolated from the MiM-CK – ⁄ – phenotype was different (i.e. twofold higher) than that of WT. The implica- tions of these adaptations for striated muscle function were explored by constructing force-flow relations of skeletal muscle respiration. It was found that the identified shift in affinity towards higher ADP concentra- tions in MiM-CK – ⁄ – muscle genotypes may contribute to linear mitochond- rial control of the reduced cytosolic ATP free energy potentials in these phenotypes. Abbreviations ACR, acceptor control ratio; AT, atractyloside; CK, creatine kinase; Cr, creatine; CS, citrate synthase; EDL, extensor digitorum longus; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; LDH, lactate dehydrogenase; PCr, phosphocreatine; RCR, respiratory control ratio; VDAC, voltage-dependent anion channel. 956 FEBS Journal 272 (2005) 956–965 ª 2005 FEBS Loss of CK function either by depletion of Cr via beta-guanidinopropionic acid feeding [5,6] or by dele- tion of CK isoforms in striated muscle weakens con- trol of ATP ⁄ ADP concentration ratios in the cellular ATPase network [7–9]. Elevated ADP concentrations compared to wild-type (WT) have been measured at steady states set by comparable cytosolic ATPase rates in Mi-CK knockout hearts [8,9] and M-CK knockout fast-twitch gastrocnemius muscle [7] compared to WT. In the latter muscle type, this is the case both at rest as well as during contraction, in spite of phenotypic adaptations of the muscle at the protein level. For example, a shift in the myosin composition of the myofibrils towards slower, energetically more efficient isoforms has been documented for fast-twitch muscle in response to CK deletion [10]. The adaptive response of mitochondrial function in CK-deficient muscle cells is less well documented. Deletion of CK function leads to increased citrate syn- thase (CS) activity in skeletal muscle and an increased V max of ADP-stimulated respiration in gastrocnemius skinned-fibres [11]. Here we investigated if the ADP concentration increase found in CK-deficient muscle is accompanied by a compensatory, adaptive shift in mito- chondrial ADP affinity towards these higher ADP concentrations. We measured the ADP-stimulated V max of respiration and the affinity for ADP (K ADP 50 ) in isolated mitochondria from two extreme striated muscle phenotypes: slow-twitch heart and fast-twitch gastrocnemius muscle. Results Isolation of mouse heart and gastrocnemius mitochondria Percoll density gradient centrifugation was added as a final purification step to obtain a high quality mitoch- ondrial preparation. CS activity was increased in Percoll purified mitochondria when compared to the heart homogenate and the crude mitochondrial preparation, albeit not significantly (Table 1). Based on the activity of aryl esterase (AE) as a microsomal marker, 7% of the microsomal contamination remained in the final mito- chondrial preparation (on protein basis) when compared to the homogenate (Table 1). The final mitochondrial suspension was furthermore greatly deprived of lactate dehydrogenase (LDH) activity, as a cytosolic marker. One of the most important quality criteria for the final mitochondrial preparation is the respiratory control ratio (RCR). The crude mitochondrial fraction had a low RCR (2.6 ± 0.3) and a relatively high ATPase activity (Table 1). Considerable levels of contaminating ATPases remained in the final mitochondrial sample during isolation of mouse heart mitochondria when con- ventional differential centrifugation protocols were used (Table 1). The reduction of the ATPase activity in the final heart mitochondrial preparation was accompanied by a considerably higher RCR of 11.2 ± 1.7, using pyruvate ⁄ malate as substrate (Table 1). Percoll density gradient centrifugation also strongly increased the RCR of the gastrocnemius mitochondrial preparation, i.e. from 1.9 ± 0.4 to 5.9 ± 0.5, using succinate as sub- strate (data not shown). Creatine kinase activity Table 2 shows the specific activity of CK in mouse heart and gastrocnemius homogenates as well as in mitochondria isolated from these WT and CK-deficient mouse tissues. In agreement with the genotypes, the CK activities in mitochondria isolated from Mi-CK – ⁄ – and MiM-CK – ⁄ – mouse heart were negligible. Import- antly, the data show that there is no significant change in Mi-CK activity in the case of M-CK deficiency. The total CK activity was significantly lower in the heart homogenate of the three CK-deficient mice com- pared to WT mice. In the gastrocnemius homogenate the total CK activities of WT and Mi-CK – ⁄ – were not significantly different, which is in line with the low Mi-CK content in glycolytic gastrocnemius muscle. Isolated gastrocnemius mitochondria from WT and M-CK – ⁄ – mice displayed a relatively low specific Mi-CK activity, compared to heart mitochondria. In preparations of mitochondria isolated from Mi-CK – ⁄ – gastrocnemius the relatively high CK activity, com- pared to mitochondria from MiM-CK – ⁄ – muscle, is probably due to contamination with M-CK. Table 1. RCR, ATPase activity and marker enzyme activities for mouse heart homogenate, crude mitochondria and purified mitochondria. Number of preparations is shown in parentheses. Activities are shown for ATPase, CS, LDH and AE (mUÆmg protein )1 ). RCR ATPase CS LDH AE Heart homogenate (3) – – 711 ± 256 2031 ± 265 38 ± 13 Crude mitochondria (3) 2.6 ± 0.3 958 ± 16 944 ± 224 491 ± 18 16 ± 5 Purified mitochondria (3) 11.2 ± 1.7 477 ± 23 1094 ± 202 51 ± 18 3 ± 1 F. ter Veld et al. Kinetic properties of CK – ⁄ – mitochondria FEBS Journal 272 (2005) 956–965 ª 2005 FEBS 957 V max of heart and gastrocnemius mitochondrial respiration The basic respiratory rates for maximal ADP stimula- ted (State 3), the atractyloside inhibited (AT) state and the optimally uncoupled (FCCP) state were essentially identical across the different types of cardiac mito- chondria (Table 3, A). Interestingly, respiratory rates in State 3, AT state and FCCP state were significantly higher in isolated gastrocnemius mitochondria from M-CK – ⁄ – and MiM-CK – ⁄ – mice, compared to WT (Table 3, B). The respiratory rates of isolated gastroc- nemius mitochondria from WT and Mi-CK-deficient mice were not significantly different. An acceptor control ratio (ACR), and not an RCR, was calculated from ADP titration experiments using State 3 and AT state rates due to the limited amount of mitochondria obtained from gastrocnemius muscle. K ADP 50 of heart and gastrocnemius mitochondrial respiration In the presence of Cr, the concentration of ADP nee- ded to induce half-maximal respiration in isolated car- diac mitochondria, the apparent K 50 value for ADP (K ADP 50 ), was expectedly and significantly lowered from 21.3 ± 2.8 lm to 15.8 ± 1.6 lm and from 20.5 ± 1.7 lm to 14.5 ± 0.2 lm for mitochondria from WT and M-CK – ⁄ – myocardium, respectively (Table 3, A). For heart mitochondria from Mi-CK – ⁄ – and MiM-CK – ⁄ – mice, these values, in the presence of Cr, were 21.0 ± 4.7 lm and 32.2 ± 4.2 lm, respect- ively, and did not differ when Cr was omitted (Table 3, A). As such, the K ADP 50 in the presence of Cr, representative of the conditions in vivo, of heart mitochondria was twofold higher for MiM-CK – ⁄ – mice (P<0.05) and tended to be higher (1.3-fold; not sig- nificant) for Mi-CK – ⁄ – mice compared to WT. The apparent K 50 for ADP of gastrocnemius muscle mitochondria, in the absence of Cr, were 7.0 ± 1.0 lm and 7.3 ± 1.0 lm for M-CK – ⁄ – and MiM-CK – ⁄ – , respectively, vs. 2.4 ± 0.3 lm for WT, and 6.4 ± 0.8 lm and 5.7 ± 0.7 lm vs. 3.5 ± 0.3 lm, respectively, when Cr was present (Table 3, B). No differences were found between WT and Mi-CK – ⁄ – gastrocnemius mitochondria (Table 3, B). K ADP 50 was in all cases lower than for cardiac mitochondria Table 2. CK activities in muscle homogenates and isolated mito- chondria from WT and CK-deficient mice. Number of preparations is shown in parentheses. Creatine kinase activity (nmol ADPÆmg protein )1 Æmin )1 ) Heart Mitochondria WT (4) 9607 ± 1539 4889 ± 457 Mi-CK – ⁄ – (4) 5152 ± 365* 0.3 ± 0.3* M-CK – ⁄ – (4) 3332 ± 265* 3587 ± 239 MiM-CK – ⁄ – (4) 172 ± 14* 5 ± 3* Gastrocnemius Mitochondria WT (6) 9768 ± 960 693 ± 49 Mi-CK – ⁄ – (4) 13159 ± 1106 105 ± 52* M-CK – ⁄ – (6) 351 ± 93* 766 ± 178 MiM-CK – ⁄ – (4) 0 ± 32* 1 ± 1* *P < 0.05 compared to WT. Table 3. Kinetic characterization of succinate ⁄ rotenone-dependent respiration of isolated heart and gastrocnemius mitochondria from WT and CK-deficient mice. Respiratory rates of isolated mouse heart (A) and gastrocnemius (B) mitochondria (0.1 mgÆmL )1 ) were measured in mitochondrial medium (see Experimental procedures) containing succinate as substrate and rotenone. The RCR value (A) is the ratio of state 3 over state 4 (data not shown). The ACR value (B) is the ratio of state 3 over the atractyloside-inhibited state. For the determination of apparent K 50 steady-state respiratory rates were measured at increasing [ADP]. Mi-CK activity was induced by adding 25 mM Cr. Number of experiments is shown in parentheses. Respiratory Rate (nmol O 2 Æmg mitochondrial protein )1 Æmin )1 ) RCR (ACR) App. K 50 for ADP (lM) State 3 AT-State FCCP-State –Cr +Cr A WT (4) 152.0 ± 15.9 27.0 ± 2.5 130.3 ± 14.1 4.5 ± 0.1 21.3 ± 2.8 15.8 ± 1.6** Mi-CK – ⁄ – (4) 123.7 ± 19.9 23.3 ± 3.7 114.8 ± 23.1 3.7 ± 0.4 20.0 ± 4.3 21.0 ± 4.7 M-CK – ⁄ – (4) 130.5 ± 20.3 22.0 ± 2.8 119.0 ± 22.8 4.8 ± 0.1 20.5 ± 1.7 14.5 ± 0.2** MiM-CK – ⁄ – (4) 126.7 ± 14.5 20.9 ± 1.6 112.7 ± 13.5 4.7 ± 0.4 30.6 ± 3.6 32.2 ± 4.2* B WT (6) 45.5 ± 5.2 8.3 ± 1.5 43.0 ± 5.0 5.9 ± 0.5 2.4 ± 0.3 3.5 ± 0.4** Mi-CK – ⁄ – (6) 37.0 ± 2.3 6.4 ± 0.7 34.0 ± 3.6 6.0 ± 0.4 2.7 ± 0.4 4.2 ± 0.2** M-CK – ⁄ – (6) 80.1 ± 4.0* 15.9 ± 1.7* 75.2 ± 3.7* 5.2 ± 0.3 7.0 ± 1.0* 6.4 ± 0.8 MiM-CK – ⁄ – (6) 82.4 ± 8.2* 19.7 ± 1.9* 92.6 ± 4.8* 4.9 ± 0.3 7.3 ± 1.0* 5.7 ± 0.7* *P < 0.05 compared to WT. **P < 0.05 compared to minus Cr (–Cr). Kinetic properties of CK – ⁄ – mitochondria F. ter Veld et al. 958 FEBS Journal 272 (2005) 956–965 ª 2005 FEBS (Table 3, B). In addition, the sensitivity of WT mito- chondria to the presence of Cr in the medium differed between gastrocnemius and cardiac preparations: addi- tion of Cr to the medium significantly increased K ADP 50 of gastrocnemius mitochondria by 40% (Table 3, B). In contrast, the K ADP 50 of gastrocnemius mitochondria from M-CK – ⁄ – and MiM-CK – ⁄ – mice was not sensitive to the presence of Cr in the medium, and was significantly higher than WT in both condi- tions studied. Discussion In this study we compared the functional kinetic char- acteristics of mitochondria from WT and CK-deficient mice in fast-twitch gastrocnemius and slow-twitch heart muscle, which represent two very different stri- ated muscle phenotypes. Fast-twitch glycolytic skeletal muscle The main finding of our studies on mitochondria isola- ted from various CK genotypes of fast-twitch gastroc- nemius muscles was a twofold higher rate of endogenous and State3 respiration (V max ) and a two- fold higher apparent K 50 for ADP for M-CK – ⁄ – and MiM-CK – ⁄ – mice compared to WT mitochondria (Table 3, B). Mitochondria from Mi-CK – ⁄ – gastro- cnemius had essentially the same respiratory properties as WT mitochondria, being in line with previous reports [12] (Table 3, B). The finding of an adaptive increase in respiratory V max in M-CK – ⁄ – and MiM- CK – ⁄ – gastrocnemius mitochondria is in line with the results of previous studies on muscle homogenate that reported an increase of mitochondrial protein in these genotypes [10,13–15]. Also, the results of polarographic measurements of respiratory V max (but not K ADP 50 ; see [16]) in permeabilized M-CK – ⁄ – gastrocnemius fibres, which can be compared to our results in a straightfor- ward manner, are similar [11,17]. Our present investi- gations did not provide insight into the exact sites of V max up-regulation in M-CK – ⁄ – and MiM-CK – ⁄ – phe- notypes in terms of activities of individual components of the respiratory chain. However, an interesting, but speculative, scenario could be that the documented cal- cium homeostasis impairment due to CK deficiency [18], possibly resulting from loss of CK function [19], may have played a role in directing the increase in mitochondrial capacity via the recently discovered cal- modulin-kinase calcium-signalling pathway controlling mitochondrial biogenesis [20]. The K ADP 50 in the presence of Cr, representative of the conditions in living muscle, was 6.4 lm and 5.7 lm for M-CK – ⁄ – and MiM-CK – ⁄ – , respectively, compared to 3.5 lm for WT gastrocnemius mitochondria. This twofold-decrease in affinity for ADP in these two phenotypes is physiologically relevant in view of the reported twofold higher ADP concentration in resting MiM-CK – ⁄ – hindleg muscles [7] as will be discussed below. The apparent K ADP 50 is determined by the per- meability of the outer mitochondrial membrane to ADP via VDAC porins [21] and the affinities of ANT and F1-ATPase for ADP [22]. The latter also introduces a dependence on the mitochondrial membrane potential and thereby on the respiratory substrate [23]. The poss- ible role of mitochondrial adenylate kinase in setting the apparent K 50 ADP was not addressed in this study. However, the V max activities of mitochondrial adenylate kinase in isolated heart or gastrocnemius mitochondria from CK-deficient genotypes were not significantly dif- ferent compared to WT mitochondria (data not shown). This makes it unlikely that adenylate kinase is the source of the observed differences in K ADP 50 . Interestingly, recent experimental data reveal a relat- ive decrease in VDAC mRNA and protein expression compared to the expression of other mitochondrial proteins in M-CK – ⁄ – and MiM-CK – ⁄ – gastrocnemius muscle [13,24] suggesting a lower permeability of the outer mitochondrial membrane for adenine nucleotides. The decrease in ADP affinity of isolated M-CK – ⁄ – and MiM-CK – ⁄ – muscle mitochondria we found is therefore in line with these findings at the protein level. In addition, experiments on VDAC-1 deficient mouse gastrocnemius have clearly shown VDAC to be a important determinant in setting K ADP 50 , giving rise to twofold higher K ADP 50 values upon VDAC-1 deletion [25]. Slow-twitch oxidative cardiac muscle No significant differences in mitochondrial respiratory V max were found when comparing isolated mitochon- dria from heart muscle from Mi-CK – ⁄ – , M-CK – ⁄ – and MiM-CK – ⁄ – mice with heart mitochondria from WT mice (Table 3, A). These findings are in line with previous studies on skinned ventricular fibres from Mi-CK – ⁄ – and M-CK – ⁄ – mice that reported no differ- ence in respiratory V max compared to WT [11,12]. Our finding of twofold higher K ADP 50 of MiM-CK – ⁄ – heart mitochondria and the trend towards a higher K ADP 50 in the case of Mi-CK – ⁄ – mitochondria correlates well with recent studies on perfused hearts from CK mutant ani- mals. In these studies a compromised capacity for free energy homeostasis was demonstrated in isolated per- fused heart from Mi-CK – ⁄ – and MiM-CK – ⁄ – mice [8,9], but not M-CK – ⁄ – mice [8,26]. F. ter Veld et al. Kinetic properties of CK – ⁄ – mitochondria FEBS Journal 272 (2005) 956–965 ª 2005 FEBS 959 Integration of adapted mitochondrial function in the CK mutant striated muscle cell In this section we discuss the implications of the identi- fied V max and K ADP 50 adaptations of mitochondria with respect to the function of the integrated ATPase network of the active striated muscle cell in which spe- cific CK isoforms are absent. The role of mitochondria in the ATPase network of the cell is to both generate ATP synthase flux matching cytosolic ATPase flux as well as to control the extramitochondrial ATP ⁄ ADP free energy potential [27]. This is captured in Fig. 1 which shows respiratory flux of WT muscle as a func- tion of the extramitochondrial ATP ⁄ ADP free energy potential. This relationship is quasi-linear over 5–85% of respiratory V max in skeletal muscle [28], with the operational ATP synthase flux domain being able to maintain adequate control over cytosolic ATP ⁄ ADP [27,28]. Above this maximal operational ATP synthase rate, respiration can no longer control cytosolic ATP ⁄ ADP and the free energy potential rapidly deteri- orates. The kinetic graph format of Fig. 1 will now be used to qualitatively illustrate (i.e. focusing on trends rather than absolute numbers) the implications of the mitoch- ondrial V max and K ADP 50 adaptations to (Mi)M-CK- deficient skeletal muscle physiology. In order to do so, we first translated the relative change in K ADP 50 to in vivo conditions on basis of information in the litera- ture. This was necessary because K ADP 50 values for iso- lated mitochondria are typically lower than estimated in vivo values {5 lm (this study) vs. 23–44 lm [28–30], respectively, for skeletal muscle, and 20–30 lm [31,32] vs. 80 lm [33], respectively, in cardiac muscle oxidizing glucose}. For skeletal muscle, we thus obtained an in vivo K ADP 50 of 72 lm for MiM-CK-deficient skeletal muscle on basis of an in vivo K ADP 50 value for WT skeletal muscle of 44 lm [29] and the 1.6-fold increase in in vitro K ADP 50 for MiM-CK – ⁄ – compared to WT (Table 3, B). These translated K ADP 50 values together with measured in vitro mitochondrial V max rates were first converted to muscle V max rates assuming 10.3 mg mitochondrial proteinÆ g skeletal muscle tissue mass )1 [34] and then used to construct flow-force relations for three cases: (I) WT muscle characterized by V max ¼ (V max ) WT and K ADP 50 ¼ (K ADP 50 ) WT ; (II) MiM-CK – ⁄ – characterized by V max ¼ 2*(V max ) WT and K ADP 50 ¼ 2*(K ADP 50 ) WT ; (III) a hypothetical case characterized by V max ¼ 2*(V max ) WT and K ADP 50 ¼ (K ADP 50 ) WT (Fig. 1). In the final step, we calculated the ATP ⁄ ADP free energy potential in resting WT and MiM-CK defi- cient fast-twitch mouse extensor digitorum longus (EDL) muscle on the basis of reported PCr, Cr and ATP concentrations at 20 °C [35] and a value of 166 for CK-K eq [36] yielding ATP ⁄ ADP ratios of 533 and 163 for WT and MiM-CK – ⁄ – EDL, respectively. This approach was valid because at rest thermodynamic equilibrium is also established in MiM-CK – ⁄ – due to the presence of some remaining CK activity [18]. The free energy offset-points of the ATPase network for the two genotypes are indicated in Fig. 1 by broken lines. Clearly, the cytosolic ATP free energy potential in MiM-CK – ⁄ – fast-twitch muscle is compromised already under conditions of basal ATP demand. The flow–force relationship for WT muscle first of all shows that without any adaptation of V max or K ADP 50 , mitochondria in MiM-CK-deficient skeletal muscle would have a seriously compromised dynamic range to respond to cytosolic ATPase load increments. This is because the ATP ⁄ ADP free energy potential Fig. 1. Qualitative illustration of flow-force relations in fast-twitch skeletal muscle of WT and MiM-CK-deficient mice. Extramitochon- drial ATP free energy potential represented by the ATP ⁄ ADP ratio in skeletal muscle from WT and MiM-CK – ⁄ – mice is plotted against muscle respiratory flux (JO 2 in nmoles O 2 Æg muscle -1 Æmin -1 ), based on converted mitochondrial respiratory V max rates and extrapolated K 50 values from Table 3B (in the presence of Cr). Three cases are presented: (I) WT with V max ¼ (V max ) WT and K ADP 50 ¼ (K ADP 50 ) WT ; (II) MiM-CK – ⁄ – with V max ¼ 2*(V max ) WT and K ADP 50 ¼ 2*(K ADP 50 ) WT ; and (III) a hypothetical case with V max ¼ 2*(V max ) WT and K ADP 50 ¼ (K ADP 50 ) WT . The free energy ATP ⁄ ADP offset-points at rest of the ATPase network for the two genotypes (case I and II) are indicated by dashed lines. The arrows indicate the available dynamic range to respond to cellular ATPase load increments [with the WT (arrow A) and compromised MiM-CK – ⁄ – (arrows B, C and D) free energy ATP ⁄ ADP potential as offset-point]. The gray boxes indicate the quasi-linear domains of respiratory V max . Kinetic properties of CK – ⁄ – mitochondria F. ter Veld et al. 960 FEBS Journal 272 (2005) 956–965 ª 2005 FEBS offset-point has shifted in MiM-CK-deficient muscle from 533 to 163, giving rise to an increase in basal res- piratory rate from 25% to 60% WT V max (Fig. 1, case I, arrows A and B, respectively). This would pose a problem, as the absolute cytosolic ATPase load during contraction in MiM-CK-deficient muscle is higher than for WT because of an increased basal rate associated with the compromised Ca 2+ homeostasis, as observed in CK deficiency [18]. In addition, we recently obtained experimental proof for higher absolute respiration rates in MiM-CK-deficient mouse EDL muscles at one and the same contraction frequency compared to WT due to a significantly increased basal respiration rate (F. ter Veld, unpublished data). Secondly, the relation- ship for MiM-CK-deficient skeletal muscle (case II) shows that the increase in respiratory V max of mito- chondria in this genotype rescues the absolute capacity to generate ATP synthase flux, as compared to mito- chondria with WT V max (clearly illustrated by com- paring the dynamic range of arrows C and B, respectively). In addition, the observed increase in K ADP 50 shifts the dynamic range of ATP synthase flux in MiM-CK-deficient muscle (arrow C) to a more lin- ear range of respiratory flux (grey box, case II), com- pared to rather small linear range (grey box, case III) corresponding to the dynamic range in case III (arrow D). This hypothetical case III illustrates the import- ance of combining these two kinetic properties, in that while an increase of V max may be essential to restore one aspect of mitochondrial function, i.e. ATP syn- thase flux, a second crucial aspect has to be main- tained in addition, i.e. control of the cytosolic ATP free energy potential. This second aspect is resolved by an adaptive response of a twofold higher K ADP 50 in MiM-CK-deficient muscle. In this light, it is of interest that a doubling of K ADP 50 has also been found in skel- etal muscle of patients with mitochondrial lesions reducing V max by 50% [37,38]. In spite of the severely reduced capacity to generate ATP synthase flux, these muscles have residual capacity for contractile work accompanied by linear changes in cytosolic ATP free energy at low ATP ⁄ ADP potentials [37,38]. Analogously, we can now explain the benefit of an increased mitochondrial K ADP 50 in MiM-CK – ⁄ – hearts in which mitochondrial control of the cytosolic ATP free energy potential is compromised [8,26]. One would perhaps have expected also a higher mitochondrial V max in these cardiac muscle genotypes. An attractive, but speculative, explanation for the lack of any such V max increase is offered by Lindstedt et al. [39] who have pro- posed that the volume ratio of mitochondria, sarcoplas- mic reticulum and myofibrils in a striated muscle cell is optimized for the particular mechanical task of the muscle. Our results suggest that cardiac muscle may well be limited in its ability to increase mitochondrial volume without compromising mechanical function, at least in comparison to fast-twitch skeletal muscle. In conclusion, we propose that an increase in oxida- tive capacity and a reduction of the ADP affinity both constitute adaptations of mitochondrial function to alleviate compromised temporal and spatial buffering of the ATP free energy potential due to specific CK deletions. A specific mechanism for the regulation of mitochondrial capacity has recently been identified [20]. It remains to be determined which regulatory mechanisms are involved in setting the apparent mito- chondrial K ADP 50 . Experimental procedures Animals Adult WT C57BL ⁄ 6 mice were used as controls. Cytosolic muscle-type CK-deficient mice (M-CK – ⁄ – ), sarcomeric mit- ochondrial CK-deficient mice (Mi-CK – ⁄ – ) and double knock-out mice, deficient in both cytosolic muscle-type and sarcomeric mitochondrial CK (MiM-CK – ⁄ – ), were gener- ated in the laboratory of B. Wieringa (Nijmegen University, the Netherlands) by gene targeting as described previously [10,15]. Offspring obtained in the breeding program were genotyped by PCR analysis on a regular basis. All experi- mental procedures were approved by the Committee on Animal Experiments of the University Medical Center Utrecht and complied with the principles of good laborat- ory animal care. Biochemicals Percoll was from Pharmacia Biotech (Rosendaal, the Netherlands). Essentially fatty acid free BSA, lyophilized Leuconostoc mesenteroides glucose-6-phosphate dehydroge- nase (NAD + specific form) and lyophilized yeast HK (essentially salt free) were from Sigma (Zwijndrecht, the Netherlands). ATP and ADP were obtained from Roche Diagnostics (Almere, the Netherlands). All other chemicals used were of the highest grade available and were obtained from regular commercial sources. Preparation of heart muscle mitochondria The isolation of mitochondria from mouse heart was based on the procedure of Cairns et al. [40], which represents a modification of the technique of Sims [41]. For each prepar- ation, four mice were sedated with diethyl-ether and decap- itated after which beating hearts were removed. The hearts (approx. 500 mg total wet-weight) were quickly placed in isolation medium [IM, containing 250 mm mannitol, 10 mm F. ter Veld et al. Kinetic properties of CK – ⁄ – mitochondria FEBS Journal 272 (2005) 956–965 ª 2005 FEBS 961 Hepes, 0.5 mm EGTA and 0.1% (w ⁄ v) BSA, pH 7.4; adjus- ted with KOH]. Next, the ventricles were carefully freed of blood, minced intensively in 5 mL IM using scissors and homogenized in a 12 mL centrifuge tube by five strokes (up and down) using a loosely fitting Teflon pestle rotating at 1000 r.p.m. Large cell debris and nuclei were pelleted by centrifugation for 5 min at 500 g in a Sorvall SS34 rotor. Mitochondria were pelleted by centrifuging the supernatant for 5 min at 10 000 g in the same rotor. The mitochondrial pellet was resuspended in 2 mL 12% (v ⁄ v) Percoll in IM, loaded on a discontinuous density gradient consisting of 3 mL 26% (v ⁄ v) Percoll and 4 mL 40% (v ⁄ v) Percoll in IM and centrifuged for 5 min at 31 000 g in a Sorvall SS34 rotor. Three major bands were obtained and the purified mitochondria were collected from the bottom band contain- ing high-density mitochondria. Finally, the mitochondria were washed with IM by centrifuging twice for 5 min at 10 000 g and resuspended in 200 lL IM at a mitochondrial protein concentration of  12 mgÆmL )1 . The isolations typ- ically took 45 min and were carried out at a temperature of 0–4 °C. Preparation of gastrocnemius muscle mitochondria The isolation of mitochondria from mouse gastrocnemius was essentially the same as procedure described above for heart mitochondria, with some minor modifications. Four mice were sedated with diethyl-ether and decapitated after which hindleg gastrocnemius muscles were removed, placed in IM and freed of fat tissue. The muscle tissue was minced intensively in IM using scissors and homogenized in a cen- trifuge tube by five strokes (up and down) using a loosely fitting Teflon pestle rotating at 700 r.p.m. To obtain gas- trocnemius mitochondria, again a discontinuous density gradient was used. The 26% (v ⁄ v) Percoll layer was replaced with a 20% (v ⁄ v) Percoll layer. Two major bands were obtained and the purified mitochondria were collected from the bottom band containing high-density mitochon- dria. Finally, the mitochondria were washed with IM as described above and resuspended in IM to a mitochondrial protein concentration of approximately 5 mgÆmL )1 . Protein determination The protein concentration of the mitochondrial preparation was determined by the BCA assay (Pierce, Etten-Leur, the Netherlands). The BCA reagent was supplemented with 0.1% (w ⁄ v) SDS. BSA was used as standard. Measurements of respiratory parameters The rates of oxygen consumption (nmol O 2 Æmg mitochond- rial protein )1 Æmin )1 ) were determined at 25 °C, using a high-resolution oxygraph (Oroboros Oxygraph; Innsbruck, Austria) and 0.1 mg mitochondria in mitochondrial med- ium [containing 200 mm sucrose, 20 mm Hepes, 20 mm tau- rine, 10 mm KH 2 PO 4 ,3mm MgCl 2 , 0.5 m m EGTA, 0.1% (w ⁄ v) BSA, pH 7.4 adjusted with KOH]. The final volume of the oxygraph chamber was 2.0 mL. The oxygen solubil- ity of air-saturated mitochondrial medium was taken to be 221 nmol O 2 ÆmL )1 [42]. Substrates were 10 mm pyruvate plus 2 mm malate, or 10 mm succinate (in the presence of 10 lm rotenone). Respiratory assays were typically carried out in the following order. Endogenous respiration (State 2) was measured before the submaximal stimulation of oxida- tive phosphorylation using 0.1 mm ADP while maximal ADP stimulated respiration (State 3) was initiated by add- ing 0.25 mm ADP. After the resting state (State 4) had again been reached, 12.5 lm atractyloside was added to measure the rate of ANT-inhibited respiration. Finally, approximately 2 lm FCCP was titrated into the oxygraph chamber to induce maximally uncoupled respiration. The apparent K 50 values for ADP, i.e., the concentration of ADP needed to induce half-maximal respiration in isolated mitochondria, were determined by measuring respiration at increasing [ADP] in mitochondrial medium containing 10 mm succinate, 10 lm rotenone, 20 mm glucose and 0.3 IU Æ mL )1 yeast hexokinase (type VI), for depletion of mitochondrially formed ATP. The ADP concentration of stock solutions was determined enzymatically as described before [21]. To assess functional coupling of Mi-CK to oxi- dative phosphorylation, respiration was stimulated at increasing [ADP] in the presence of 25 mm Cr. To obtain the rate of ADP-stimulated respiration, the rates of respir- ation were corrected for ‘leak’ respiration based on a dynamic computer model of oxidative phosphorylation in muscle [43] according to [44]. Spectrophotometric determination of enzyme activities CK activity was measured at 25 °C on a Beckman DU65 spectrophotometer using coupled enzyme systems. Briefly, CK activity was assayed according to [45] in the forward direction in a medium containing 10 mm imidazole, 2 mm EDTA, 10 mm Mg-acetate, 2 mm ADP, 20 mm N-acetyl- cysteine, 20 mm glucose, 5 mm AMP, 1 mm NAD + ,50lm P 1 ,P 5 -di(adenosine-5¢)pentaphosphate, 25 mm PCr (pH 7.4, adjusted with acetic acid). Hexokinase and glucose-6-phos- phate dehydrogenase were added at 3 IUÆmL )1 and 2IUÆmL )1 , respectively. Pyruvate kinase and lactate dehy- drogenase were both added at 4.5 IUÆmL )1 . Lactate dehy- drogenase [46], citrate synthase [47] and aryl esterase [48] enzyme activities were measured at 37 °C and pH 7.4 according to published methods. The media used in the above assays were adjusted to 0.2% Triton X-100 to obtain maximal enzyme activities in muscle homogenates and Kinetic properties of CK – ⁄ – mitochondria F. ter Veld et al. 962 FEBS Journal 272 (2005) 956–965 ª 2005 FEBS mitochondrial fractions. Total ATPase activities in suspen- sions of intact mitochondria were measured as described previously [46,49]. Care was taken to avoid detergent con- tamination and no Triton X-100 was added. Data analysis and statistics Oxygraph data analysis was performed with high-resolution respirometry software (oroboros datlab 2.1; Innsbruck, Austria). 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