Maintenance of the metabolic homeostasis of the heart: developing a systems analysis approach

Abstract

The heart is almost unique in the body with a constant requirement to conduct work well beyond the normal maintenance of cellular integrity. With this constant workload, it is not surprising that cardiac energy conversion is highly specialized to maintain a constant supply of energy. This maintenance of cellular metabolites during alterations in workload has been termed metabolic homeostasis. Here we discuss our efforts to understand the cellular and mitochondrial control network that orchestrates the metabolic homeostasis of the heart. This begins with a better definition of the metabolic pathways, acute posttranslational control sites, and proper kinetic evaluation of the reaction steps in the intact mitochondrial environment. First, a quantitative model of mitochondrial energy conversion is presented and demonstrates several serious gaps in our knowledge of this process. Toward filling these gaps, screens of the entire mitochondrial proteome have been conducted to establish the metabolic pathways that need to be considered. In addition, the dynamic phosphoproteome of intact mitochondria, using 2D gel electrophoresis coupled to (32)P labeling, has revealed a remarkably extensive protein phosphorylation network throughout the mitochondrial metabolic network that has essentially been overlooked. Initial studies on evaluating the functional significance of these protein phosphorylations and the kinase-phosphatase system involved will be reviewed. One of the major deficits in the consensus quantitative model of oxidative phosphorylation to explain intact mitochondria activities is in complex I, where even the initiation of Nicotinamide Adenine Dinucleotide (reduced) (NADH) oxidation is problematical using in vitro kinetic data. Studies will be described where the NADH binding and oxidation kinetics at complex I in the intact mitochondria were determined using fluorescence lifetime and enzyme dependent-fluorescence recovery after photo-oxidation (ED-FRAP) techniques. These later studies suggest that matrix NADH binding characteristics are much different (>10(3) binding constant errors) than isolated proteins. In addition, complex I is far from equilibrium and may play an important role in regulating the rate of reducing equivalent delivery to the cytochromes.