Mathematical modeling of the metabolic implications of the diabetic heart

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Cardiac metabolism needs to adapt to short-term and long-term variations in oxygen and nutrient supply and energy demand to ensure sufficient ATP supply for proper cardiac function. While short term adaptation is the result of kinetic regulation of metabolic enzymes by substrate availability, allosteric regulation and hormonal regulation of interconvertible enzymes, long term adaptation proceed by alterations of the metabolic capacity and the regulatory signaling pathways by gene expression. Especially Diabetes Mellitus Typ 2 (DMT2) is characterized by alterations in the blood plasma nutrient and hormone composition resulting in cardiac remodeling. Additional, alterations in ATP demanding processes (AP generation, heart power) and alterations in the cardiac vascularization can be observed. The associated adaptive processes can result in mitochondrial dysfunction, ATP deficiency and impaired cardiac functionality thereby contributing significantly to cardiac dysfunction and heart failure. The aim of this project is to develop a comprehensive kinetic model of the central metabolism of cardiomyocytes (carbohydrate metabolism, fatty acid metabolism, amino acid metabolism) and radical detoxification, including short term regulation of enzyme activities by variations of substrate concentrations, hormone-dependent reversible phosphorylation and allosteric regulation as well as long term adaptive changes in enzyme abundances (gene expression). Modeling will be based on a kinetic approach. Each enzymatic step will be described by an appropriate rate law. Enzymatic rate laws will be derived from the biochemistry literature compiled during the last six decades. We will use the model to predict the metabolic state of cardiomyocytes (metabolite concentrations, flux distributions, nutrient exchange, etc.) under different external and internal conditions. We will use protein abundance data obtained from diabetic animals during different disease stages for scaling of the metabolic processes. We will validate the model by measurements of metabolite concentrations, proteins and phosphorylated proteins. Phosphorylated proteins will be determined by immunoblotting, metabolites will be analyzed by HPLC or by using commercial kits for individual metabolites. In the case such kits are not-available or not applicable an HPLC/HPLC-MS method application will be established. Furthermore, mitochondrial function (O2 consumption rate) will be assessed by XF instrumentation (Seahorse Bioscience). Relevant exchange fluxes between cells and the external space will be measured by time-dependent detection of metabolites using LC-MS/MS. Additionally fluorescence microscopy and confocal-laser scanning microscope for the determination of intracellular states such as NAD(P)H redox state and multiphoton microscopy for the determination of cardiac vascularization will be used. Alterations in cardiac action potentials will be assessed juxtacellular recordings in vivo.