Mathematical modeling of the metabolic implications of the diabetic heart

心脏代谢需要适应短期和长期的氧气和营养供应以及能量需求的变化，以确保足够的ATP供应以维持正常的心脏功能。短期适应是代谢酶通过底物利用率、变构调节和相互转换酶的激素调节进行动力学调节的结果，而长期适应则是通过基因表达改变代谢能力和调节信号通路进行的。特别是2型糖尿病（DMT2）的特点是血浆营养和激素成分的改变导致心脏重塑。此外，可以观察到ATP需求过程（AP生成，心脏功率）和心脏血管化的改变。相关的适应性过程可导致线粒体功能障碍、ATP缺乏和心功能受损，从而导致心功能障碍和心力衰竭。本项目旨在建立心肌细胞中心代谢（碳水化合物代谢、脂肪酸代谢、氨基酸代谢）和自由基解毒的综合动力学模型，包括底物浓度变化对酶活性的短期调节、激素依赖性可逆磷酸化和变构调节以及酶丰度的长期适应性变化（基因表达）。建模将基于动力学方法。每个酶的步骤都可以用一个适当的速率定律来描述。酶的速率定律将从过去六十年来编写的生物化学文献中得到。我们将利用该模型预测不同内外条件下心肌细胞的代谢状态（代谢物浓度、通量分布、营养交换等）。我们将使用从糖尿病动物在不同疾病阶段获得的蛋白质丰度数据来衡量代谢过程。我们将通过测量代谢物浓度、蛋白质和磷酸化蛋白质来验证模型。磷酸化蛋白将通过免疫印迹法测定，代谢物将通过高效液相色谱法或使用单个代谢物的商业试剂盒进行分析。如果这些试剂盒不可用或不适用，将建立HPLC/HPLC- ms方法应用程序。此外，线粒体功能（氧气消耗率）将通过XF仪器（Seahorse Bioscience）进行评估。细胞与外部空间之间的相关交换通量将通过使用LC-MS/MS对代谢物进行时间依赖性检测来测量。此外，荧光显微镜和共聚焦激光扫描显微镜用于测定细胞内状态，如NAD(P)H氧化还原状态和多光子显微镜用于测定心脏血管化。心脏动作电位的改变将通过体内细胞旁记录来评估。

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.