The cellular bioenergetic status, often compromised in a wide range of disease states, may play a more active role in the regulation of cellular processes than previously thought. The state of muscle energetics, which can be defined by the chemical potential of ATP (muATP), is viewed as the medium through which two important cell functions are regulated: mitochondrial respiration and muscle phenotypic expression. Contractile activity induces changes in muATP and thus the position of equilibrium of the creatine kinase (CK) reaction. Cytoplasmic signals, regulating the mitochondria in the steady state, match the rate of oxygen consumption to the rate of ATP utilization. The signal was thought to be the concentration of ADP in a negative feedback manner, but recent data indicate the regulation of oxidative phosphorylation in skeletal muscle and other cells is more involved both in detail and concept. Our recent results indicate that altered bioenergetic states induced by competitively inhibiting CK activity in vivo also alter skeletal muscle phenotype. The new concept imagined is that muATP remains high when contractile activity is low, and this physiological state requires only a simple feedback regulation of respiration and leads to a fast muscle phenotype. In contrast, high contractile activity increases the ATPase drain on muATP and influences an additional feedforward respiratory control mechanism; this physiological state leads to a slow muscle phenotype. This view leads to several new hypotheses and will require a more rigorous test of CK function. It offers the possibility of a synthesis of cellular bioenergetics in that the cytoplasmic content of """"""""high energy phosphates"""""""" of a skeletal muscle cell may regulate cellular respiration and also play an important role in longer term adaptations. We set our specific aims as three hypotheses that define and test these concepts: 1. We propose to test the hypothesis that the kinetics under homogenous in vitro conditions are different from those in isolated mouse extensor digitorum longus (EDL) and soleus (SOL) muscles by measuring the forward and reverse fluxes between PCr and gammaATP catalyzed by CK in homogeneous solutions of reactants and enzyme designed to mimic the cytoplasmic milieu. 2. We propose to test that the negative feedback by ADP on mitochondria predicts the kinetics of recovery metabolism (31P NMR for high-energy phosphates and polarography for O2 consumption) in fast-twitch skeletal muscle, but that additional mechanisms are involved in slow-twitch muscle. Secondly, we will test the hypothesis that these kinetics are influenced by the type of metabolic substrate such that ADP acts as a fine control (which may be sufficient for certain cell types or physiological situations) with an additional feedforward control in certain cells. Thus these experiments introduce the concept of feedforward regulation of oxidative phosphorylation in addition to the classical feedback regulation by [ADP]. 3. We propose to test the hypothesis that mechanism of phenotype change induced by incorporation of creatine analogs into muscle is due to altered energetic state. We will use several analogs to test the generality of the induction of phenotype adaptations, and to test whether the phenotypic alterations are specific to cell types. These experiments address fundamental and new issues on the bases of regulation of cellular respiration and phenotype plasticity, and are designed to formulate a more integrative view of energetics than previously achieved.
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