Abstract
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.
Highlights
It is well established that creatine kinase (CK) catalyzes the reversible transfer of phosphate from ATP to creatine (Cr): ATPzCr /CK? ADPzPCr ð1Þhow this biochemical function plays a role in cell functioning has been the subject of intense controversy [1]
This led to the idea of the ‘phosphocreatine shuttle’, proposed by Bessman and Geiger [2]: PCr formation from adenine nucleotide and creatine in the inner membrane space (IMS) is catalyzed by the mitochondrial isoform of CK, Mi-CK, located in the IMS
It catalyzes the reversible transfer of highenergy phosphate from ATP to creatine, facilitating storage of energy in the form of phosphocreatine. This extra energy buffer plays a pivotal role in maintaining ATP homeostasis. Another proposed function of CK is the transport of energy from ATP producing to ATP consuming sites via a shuttle mechanism involving a mitochondrial and a myofibrillar isoform of CK
Summary
It is well established that creatine kinase (CK) catalyzes the reversible transfer of phosphate from ATP to creatine (Cr): ATPzCr /CK? ADPzPCr ð1Þhow this biochemical function plays a role in cell functioning has been the subject of intense controversy [1]. It is remarkable that two distinct isoforms of CK are expressed in muscle cells, one in the mitochondrial inner membrane space (IMS) and one in the cytosol where the contractile elements are located. This led to the idea of the ‘phosphocreatine shuttle’, proposed by Bessman and Geiger [2]: PCr formation from adenine nucleotide and creatine in the IMS is catalyzed by the mitochondrial isoform of CK, Mi-CK, located in the IMS. Transfer of HEP was argued to be accomplished either by direct diffusion of ATP through the mitochondrial outer membrane (MOM) and cytosol or indirectly via the ‘phosphocreatine shuttle’. The phosphocreatine shuttle hypothesis has led to extensive scientific debates on the role of CK, e.g. [1,3,4]
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