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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2003 as doi:10.1096/fj.02-0684fje. |
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* U-446 INSERM, Université Paris-Sud, 92296 Châtenay-Malabry, France; and
Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic
4Correspondence: U-446 INSERM, Université Paris-Sud, 92296 Châtenay-Malabry, France. E-mail: Renee.Ventura{at}cep.u-psud.fr
SPECIFIC AIMS
Fast-twitch skeletal muscle of mice deficient in both cytosolic and mitochondrial creatine kinase isoforms (CK-/-) lack burst activity, but can sustain prolonged contractile activity, suggesting that adaptive mechanisms can regulate local adenine nucleotide turnover near the main cellular ATPases (sarcoplasmic reticulum and myofibrils). We thus hypothesized that if, in normal fast-twitch twitch muscle, bound CK is necessary to provide efficient control of adenine nucleotides near the active sites of the ATPases in the CK-/- mice, the remodeling of mitochondrial network would ensure the rescue of calcium uptake and myofibrillar function by interacting directly with myofibrils and SR.
PRINCIPAL FINDINGS
1. Cytoarchitecture remodeling in fast skeletal muscle of CK-/- mice
Engineered mice where a kind gift from Drs. B. Wieringa and F. Oerlemans (University of Nijmegen, Netherlands). Mitochondria are sparse and mainly present under the sarcolemma and close to the Z-line in control mice. Electron microscopy study shows a remodeling of the mitochondrial network in the gastrocnemius muscle of CK-/- mice. Mitochondria are arranged in rows between myofilaments. New mitochondria variable in size and shape are observed along the myosin filaments and the longitudinal SR.
2. Oxidative capacity are enhanced in fast skeletal muscle of CK-/- mice
Regulation of mitochondrial respiration was studied in saponin permeabilized fibers. Oxidative capacity of gastrocnemius fibers of CK-/- mice (expressed as µmol O2·min-1·g dw-1) was increased by 90%, and the regulation of respiration by ADP was modified toward more oxidative muscle. A shift from glycolytic to oxidative metabolism was confirmed by measurements of enzymatic markers.
3. Direct interaction between sarcoplasmic reticulum (SR) and mitochondria in fast skeletal muscle of CK-/- mice
We investigated whether a direct channeling of ATP and ADP between energy-producing mitochondria and energy-consuming SR ATPase could exist in fast-twitch skeletal muscles. Saponin permeabilized fibers of gastrocnemius muscle were incubated for 1 min with calcium (pCa 7) and different energy sources to allow loading of the SR with calcium (Fig. 1
). Calcium release was induced by 20 mM caffeine. When the SR was loaded with 3.16 mM external ATP as substrate in control mice (Fig. 1A
), only a small calcium transient could be elicited due to the lack of a local ADP regenerating system. When mitochondrial respiration was stimulated in the presence of ATP, with glutamate and malate as substrates, the resulting tension transient was higher than with ATP alone. However, the loading was considerably more effective in the presence of PCr. These results show that there is no efficient channeling of adenine nucleotides between SR and mitochondria in control gastrocnemius and that, in this muscle, bound CK controls the local ATP/ADP ratio. In CK-/- gastrocnemius muscle when the SR was loaded with ATP alone, again the tension transient elicited by caffeine was of small amplitude. However, a strong tension transient could be elicited when mitochondrial respiration was activated (Fig. 1B
). As expected, PCr was without effect. These results show that in CK-/- muscle mitochondria could locally rephosphorylate ADP and yield ATP for efficient calcium uptake by the SR. Total calcium release was estimated by calculating calcium-time integral from the calcium-tension relationship as an internal calibration for each fiber. Mean results of five control and six CK-/- muscles are averaged in Fig. 1C
. In CK-/- fibers (n=6), switching on mitochondria led to a 12-fold increase in released calcium compared with external ATP only, close to the levels obtained in control fibers when both mitochondria and CK support energy regeneration. These data show that whereas a direct energy cross-talk between mitochondria and SR could not provide energy to SR in control gastrocnemius, this could be achieved in CK-deficient animals.
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4. Direct interaction between myofibrils and mitochondria in fast skeletal muscle of CK-/- mice
We checked whether a similar transition from local control of adenine nucleotides by bound CK to control by mitochondria in response to CK deficiency occurs in myofilaments, where ADP accumulation also impairs myofibrillar ATPase activity. This was achieved by using the approach of rigor tension, which develops in the absence of ATP. Rigor tension was induced by exchanging all MgATP with 1 mM MgADP in the absence of calcium (Fig. 2
). In control fibers (Fig. 2A
), stimulation of mitochondrial respiration was without effect on the relaxation of rigor tension. However, addition of PCr strongly and quickly relaxed rigor tension due to the local ATP regeneration by bound CK.
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In CK-/- mice (Fig. 2B
), a fast and complete relaxation of rigor tension could be obtained by activating mitochondrial respiration. Mean values of rigor tension for 7 control and 5 CK-/- fibers are presented in Fig. 2C
. These results show that the control of local ATP/ADP ratio shifts from bound CK in control mice to mitochondria in CK-/- mice.
5. ATPase-driven ATP production by mitochondria is enhanced in fast skeletal muscle of CK-/- mice
The relative contribution of mitochondria and bound CK in ATP production in control and CK-/- muscle fibers was estimated by measuring creatine (Cr) formation and oxygen consumption in permeabilized fibers, taking respectively an ATP/Cr ratio of 1 and an ATP/O ratio of 3. In control muscles, when both CK and mitochondria were working, the rate of ATP formation from CK was 72 ± 8 nmol·min-1·mg dw-1 and ATP formation from mitochondria was only 16 ± 1 nmol·min-1·mg dw-1. Thus, >80% of the ATP consumed came from the CK reaction and 
20% from mitochondria. In CK-/- mice, no creatine was produced and mitochondria-produced ATP amounted to 59 ± 8 µmol·min-1·g dw-1, a value lower than the total ATP production in control muscle (86.8±5.1 µmol·min-1·g dw-1, P<0.006). This shows that ATPase-driven ATP production by mitochondria is fourfold higher in CK-/- mice but does not reach the values of control muscle.
CONCLUSIONS
Among the different cell types, muscle is a paradigm of cell complexity and efficiency, in which cytoarchitecture plays a key role. The diffusion of metabolites close to the active sites of ATPases represents a limitation for efficient ATPase activity. Due to the low Km of the ATPases for ATP and the low Ki for ADP, ADP accumulation rather than ATP depletion is the rate-limiting step for ATPase activity, leading to slowing of myosin ATPase and contraction rates or impairment of SERC-ATPase and calcium uptake. Thus different systems exist to control the local ATP/ADP ratio. Among them, creatine kinase plays an important part in energy transfer in muscle cells. The family of CK isoenzymes catalyzes the reversible transfer of a phosphate moiety between creatine and ATP. Part of MM-CK is structurally associated with myofibrils and SR membranes and is functionally coupled to ATPases for optimal function of contractile machinery and SR calcium uptake. Gastrocnemius muscle of mice is a fast-twitch muscle comprising mainly fast fibers with a high content of CK (almost exclusively the MM isoform) and a small amount of mitochondria. In fast-twitch skeletal muscle, myofibrillar ATP consumption can exhibit a 100-fold increase within few milliseconds and thus is critically dependent on a fast process to deliver energy and withdraw inhibitory by-products of ATPase activity.
The main result of this study is that direct energy cross-talk between mitochondria and SR or myofibrils, which is weak in control mice, is clearly in play in the CK-/- mice so that the control of local ATP/ADP ratio passes from bound CK to mitochondria in these muscles (Fig. 3
). These results extend far beyond the adaptation process in CK-/- mice as they highlight the importance of ATP/ADP ratio for efficient functioning of ATPases and the importance of cytoarchitecture in the control of energy fluxes. Fast skeletal muscle of CK-deficient mice exhibits a morphological and functional remodeling aimed at rescuing contractile function and calcium homeostasis. There is an increase in mitochondrial number close to the myofibrils, increased oxidative capacity, changes in mitochondrial regulation, and increased aerobic metabolism. The most striking consequence of this remodeling, was a complete change in the interaction between mitochondria and other subcellular organelles. Mitochondria strongly stimulated calcium uptake and induced a fast and complete rigor tension relaxation. This suggests that newly formed mitochondria localized close to the longitudinal SR and myofilaments interact with the SERC-ATPase and myofibrillar ATPases in order to control the local adenine nucleotides and overcome the lack of CK. This morphological and functional remodeling takes place in the fast-twitch skeletal muscle where PCr/CK is the main system responsible for energy provision during contraction. The gastrocnemius muscle shifted to a new muscle phenotype: the very fast oxidative muscle. However, whether these adaptive mechanisms are sufficient to fully overcome the lack of CK is doubtful. The total ATP production was lower in CK-/- mice than in control muscle, showing the limited capacity of this adaptation. Moreover, important muscle atrophy is present in CK-/- mice and calcium handling and contractile function are impaired. Conversely, however, the increased oxidative capacity may explain part of the increased resistance to fatigue of the fast skeletal muscle of CK-/- mice.
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In general, these results strongly support our proposal for a direct energy cross-talk between organelles as one of the mechanisms by which energy is channeled from sites of production to sites of utilization in oxidative muscles. In addition, it shows that when CK is absent, direct energy transfer from mitochondria is a necessary rescue system to locally control adenine nucleotides. In other words, fast-twitch skeletal muscle switches from an economy of storage (with high available PCr content) to a direct flow economy (through mitochondrial ADP sensing and ATP production and distribution).
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0684fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0684fje 
2 Present address: Department of Pharmacology, University of Tartu, Tartu, Estonia.
3 Present address: Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford, OX37BN, UK.
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