In this study the function of the two isoforms of creatine kinase (CK; EC 2.7.3.2) in myocardium is investigated. The 'phosphocreatine shuttle' hypothesis states that mitochondrial and cytosolic CK plays a pivotal role in the transport of high-energy phosphate (HEP) groups from mitochondria to myofibrils in contracting muscle. Temporal buffering of changes in ATP and ADP is another potential role of CK. With a mathematical model, we analyzed energy transport and damping of high peaks of ATP hydrolysis during the cardiac cycle. The analysis was based on multiscale data measured at the level of isolated enzymes, isolated mitochondria and on dynamic response times of oxidative phosphorylation measured at the whole heart level. Using 'sloppy modeling' ensemble simulations, we derived confidence intervals for predictions of the contributions by phosphocreatine (PCr) and ATP to the transfer of HEP from mitochondria to sites of ATP hydrolysis. Our calculations indicate that only 15±8% (mean±SD) of transcytosolic energy transport is carried by PCr, contradicting the PCr shuttle hypothesis. We also predicted temporal buffering capabilities of the CK isoforms protecting against high peaks of ATP hydrolysis (3750 µM*s(-1)) in myofibrils. CK inhibition by 98% in silico leads to an increase in amplitude of mitochondrial ATP synthesis pulsation from 215±23 to 566±31 µM*s(-1), while amplitudes of oscillations in cytosolic ADP concentration double from 77±11 to 146±1 µM. Our findings indicate that CK acts as a large bandwidth high-capacity temporal energy buffer maintaining cellular ATP homeostasis and reducing oscillations in mitochondrial metabolism. However, the contribution of CK to the transport of high-energy phosphate groups appears limited. Mitochondrial CK activity lowers cytosolic inorganic phosphate levels while cytosolic CK has the opposite effect.

MOTIVATION: Quantitative determination of metabolic fluxes in single tissue biopsies is difficult. We report a novel analysis approach and software package for in vivo flux quantification using stable isotope labeling. RESULTS: We developed a protocol based on brief, timed infusion of (13)C isotope-enriched substrates for the tricarboxylic acid (TCA) cycle followed by quick freezing of tissue biopsies. NMR measurements of tissue extracts were used for flux estimation based on a computational model of carbon transitions between TCA cycle metabolites and related amino acids. To this end, we developed a computational framework in which metabolic systems can be flexibly assembled, simulated and analyzed. Flux parameters were quantified from NMR multiplets by a partial grid search followed by repeated Nelder-Mead optimizations implemented on a computer grid. We implemented a model of the TCA cycle and showed by extensive simulations that the timed infusion protocol reliably quantitates multiple fluxes. Experimental validation of the method was done in vivo on hearts of anesthetized pigs under two different conditions: basal state (n = 7) and cardiac stress caused by infusion of dobutamine (n = 7). About nine tissue samples (40-200 mg dry-weight) were taken per heart. TCA cycle flux was 6.11 +/- 0.28 (SEM) micromol/min x gdw at baseline versus 9.29 +/- 1.03 micromol/min x gdw for dobutamine stress. Oxygen consumption calculated from the TCA cycle flux and from 'gold standard' blood gas-based measurements were close, correlating with r=0.88 (P < 10(-4)). Spatial heterogeneity in metabolic fluxes is detectable amongst the small samples. We propose that our novel isotope snapshot methodology is suitable for flux measurements in biopsies in vivo. AVAILABILITY: Non-profit organizations will, upon request, be granted a non-exclusive license to use the software for internal research and teaching purposes at no charge. A web interface for using the software on our computer grid is available under http://www.ibi.vu.nl/programs/

Background Distinct functions have been hypothesized for the creatine kinase (CK) enzyme catalyzing the reversible transfer of the high-energy phosphate group of ATP to creatine. In muscle cells, two CK isoforms might mediate (i) temporal energy buffering to maintain ATP homeostasis and (ii) energy transport from mitochondria to myofibrils via the “phosphocreatine shuttle” mechanism. Here we investigate the relative importance of the two roles using a computational model [1]. Model simulations predict a contribution of CK to the net transcytosolic energy transport of less than 1/3.

The cells of the human body each contain thousands of molecular species,the dynamic interactions of which constitute the biomolecular system underlying cellular function. Many thousands of expressed genes, proteins, and metabolites can be measured simultaneously in a tissue or blood sample, making a scan of most biomolecules possible. This wealth of information causes the bottleneck in biomedical research to shift from making measurements to data integration and analysis. Management of biomolecular information requires efficient data storage and retrieval from integrated databases. Subsequent data analysis has three components: 1) identification of broad molecular "fingerprints" useful for early disease diagnosis and treatment selection; 2) identification of the relatively few molecular signals which change significantly above the high noise level formed by biological variation amongst individuals; and 3) development of a mechanistic understanding of the system by capturing its characteristics in computational models. At present, extensive parts of the biomolecular system are still uncharted. Representation of the entire system in silico presents a great challenge, made more challenging still by great interindividual variation at the molecular level. The prospect of much better control of human disease makes this gigantic enterprise worthwhile.