Combined in vivo and in silico investigations of activation of glycolysis in contracting skeletal muscle

Abstract

The hypothesis was tested that the variation of in vivo glycolytic flux with contraction frequency in skeletal muscle can be qualitatively and quantitatively explained by calciumcalmodulin activation of phosphofructokinase (PFK-1). Ischemic rat tibialis anterior muscle was electrically stimulated at frequencies between 0 and 80 Hz to covary the ATP turnover rate and calcium concentration in the tissue. Estimates of in vivo glycolytic rates and cellular free energetic states were derived from dynamic changes in intramuscular pH and phosphocreatine content, respectively, determined by phosphorus magnetic resonance spectroscopy ( 31 P-MRS). Computational modeling was applied to relate these empirical observations to understanding of the biochemistry of muscle glycolysis. Hereto, the kinetic model of PFK activity in a previously reported mathematical model of the glycolytic pathway (Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. J Physiol 588: 1961-1983) was adapted to contain a calcium-calmodulin binding sensitivity. The two main results were introduction of regulation of PFK-1 activity by binding of a calcium-calmodulin complex in combination with activation by increased concentrations of AMP and ADP was essential to qualitatively and quantitatively explain the experimental observations. Secondly, the model predicted that shutdown of glycolytic ATP production flux in muscle postexercise may lag behind deactivation of PFK-1 (timescales: 5-10 s vs. 100 -200 ms, respectively) as a result of accumulation of glycolytic intermediates downstream of PFK during contractions. glycolysis; skeletal muscle; calcium regulation; 31 P-MRS; computational modeling; systems biology GLYCOLYSIS PLAYS A CENTRAL role in catabolism and anabolism for all cell types (20; 44). Identification of regulatory mechanisms has been important to many areas of biomedical research, ranging from basic understanding of the biochemistry of carbohydrate utilization to applications in biotechnology (48) and drug development for cancer therapies (39). In mammalian cells, skeletal muscle has been a key experimental model to study the regulation of glycolysis and glycogenolysis. It can increase the glycogenolytic ATP production flux by two orders of magnitude during rest to work transitions on a timescale of seconds (56). This exceptionally broad and dynamic operational range of glyco(genol)ytic flux puts a high duty cycle upon the control mechanism(s) of this pathway.Several approaches have been used to elucidate the underlying regulatory mechanisms including physical isolation and in vitro kinetic characterization of individual enzymes from skeletal muscle providing a wealth of information on the individual components of this pathway (3). The application of noninvasive, nondestructive investigative techniques such as in vivo nuclear magnetic resonance spectroscopy (MRS) have since allowed studying the behavior of the intact pathway in muscle (8). For example, it has been demonstrated that glycolytic flux rapidly shuts down in the absence of mu...

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