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Functional differences between dimeric and octameric mitochondrial creatine kinase.

ABSTRACT: Mitochondrial creatine kinase (Mi-CK) consists of octameric and dimeric molecules that are interconvertible. In the present study, the kinetic properties of purified chicken heart Mi-CK (Mib-CK) dimers and octamers were investigated separately under highly controlled conditions. Gel-permeation chromatography was performed before and after kinetic measurements in order to clearly define the proportions of octamers and dimers. 'Dimeric' Mi-CK solutions consisted of > or = 90% dimers throughout the experiment whereas 'octameric' Mi-CK solutions consisted in the beginning of 90% octamers, but upon measuring with the highest concentrations of creatine (Cr) and ATP approximately one-third of the octamers dissociated into dimers. These proper controls enabled us to pinpoint the observed kinetic differences between dimers and octamers solely to the oligomeric state of Mib-CK. Both dimeric and octameric Mi-CK displayed synergism in substrate binding (Kd values are higher than Km values), meaning that binding of the first substrate facilities subsequent binding of the second substrate. Most interestingly, Km(Cr) and Kd(Cr) values are both 2-3 times higher for octameric than for dimeric Mi-CK. Thus, at low Cr concentrations, the dimer is kinetically favoured for the forward direction of the reaction (phosphorylcreatine synthesis) compared with the octamer. The possible physiological significance of the lower Kd(Cr) value of dimeric versus octameric Mib-CK, as well as the apparent negative cooperativity of ATP binding at higher [Cr], are discussed within the context of a possible functional role for dimeric Mib-CK in vivo.

SUBMITTER: Kaldis P

PROVIDER: S-EPMC1136971 | biostudies-other | 1995 Jun

REPOSITORIES: biostudies-other

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Project description:BackgroundAbnormalities in cardiac energy metabolism occur in heart failure (HF) and contribute to contractile dysfunction, but their role, if any, in HF-related pathologic remodeling is much less established. CK (creatine kinase), the primary muscle energy reserve reaction which rapidly provides ATP at the myofibrils and regenerates mitochondrial ADP, is down-regulated in experimental and human HF. We tested the hypotheses that pathologic remodeling in human HF is related to impaired cardiac CK energy metabolism and that rescuing CK attenuates maladaptive hypertrophy in experimental HF.MethodsFirst, in 27 HF patients and 14 healthy subjects, we measured cardiac energetics and left ventricular remodeling using noninvasive magnetic resonance 31P spectroscopy and magnetic resonance imaging, respectively. Second, we tested the impact of metabolic rescue with cardiac-specific overexpression of either Ckmyofib (myofibrillar CK) or Ckmito (mitochondrial CK) on HF-related maladaptive hypertrophy in mice.ResultsIn people, pathologic left ventricular hypertrophy and dilatation correlate closely with reduced myocardial ATP levels and rates of ATP synthesis through CK. In mice, transverse aortic constriction-induced left ventricular hypertrophy and dilatation are attenuated by overexpression of CKmito, but not by overexpression of CKmyofib. CKmito overexpression also attenuates hypertrophy after chronic isoproterenol stimulation. CKmito lowers mitochondrial reactive oxygen species, tissue reactive oxygen species levels, and upregulates antioxidants and their promoters. When the CK capacity of CKmito-overexpressing mice is limited by creatine substrate depletion, the protection against pathologic remodeling is lost, suggesting the ADP regenerating capacity of the CKmito reaction rather than CK protein per se is critical in limiting adverse HF remodeling.ConclusionsIn the failing human heart, pathologic hypertrophy and adverse remodeling are closely related to deficits in ATP levels and in the CK energy reserve reaction. CKmito, sitting at the intersection of cardiac energetics and redox balance, plays a crucial role in attenuating pathologic remodeling in HF. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00181259.

Project description:Aims:Mitochondrial creatine kinase (MtCK) couples ATP production via oxidative phosphorylation to phosphocreatine in the cytosol, which acts as a mobile energy store available for regeneration of ATP at times of high demand. We hypothesized that elevating MtCK would be beneficial in ischaemia-reperfusion (I/R) injury. Methods and results:Mice were created over-expressing the sarcomeric MtCK gene with ?MHC promoter at the Rosa26 locus (MtCK-OE) and compared with wild-type (WT) littermates. MtCK activity was 27% higher than WT, with no change in other CK isoenzymes or creatine levels. Electron microscopy confirmed normal mitochondrial cell density and mitochondrial localization of transgenic protein. Respiration in isolated mitochondria was unaltered and metabolomic analysis by 1?H-NMR suggests that cellular metabolism was not grossly affected by transgene expression. There were no significant differences in cardiac structure or function under baseline conditions by cine-MRI or LV haemodynamics. In Langendorff-perfused hearts subjected to 20?min ischaemia and 30?min reperfusion, MtCK-OE exhibited less ischaemic contracture, and improved functional recovery (Rate pressure product 58% above WT; P?<?0.001). These hearts had reduced myocardial infarct size, which was confirmed in vivo: 55?±?4% in WT vs. 29?±?4% in MtCK-OE; P?<?0.0001). Isolated cardiomyocytes from MtCK-OE hearts exhibited delayed opening of the mitochondrial permeability transition pore (mPTP) compared to WT, which was confirmed by reduced mitochondrial swelling in response to calcium. There was no detectable change in the structural integrity of the mitochondrial membrane. Conclusions:Modest elevation of MtCK activity in the heart does not adversely affect cellular metabolism, mitochondrial or in vivo cardiac function, but modifies mPTP opening to protect against I/R injury and improve functional recovery. Our findings support MtCK as a prime therapeutic target in myocardial ischaemia.

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Functional differences between dimeric and octameric mitochondrial creatine kinase.

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