Varikmaa 2013 Thesis

Varikmaa M (2013) Structural and Functional Studies of Mitochondrial Respiration Regulation in Muscle Cells. Thesis Tallinn University of Technology - Tallinn pp147.

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Varikmaa M (2013) Thesis Tallinn University of Technology - Tallinn

Abstract: Highly oxidative muscles, such as heart and soleus, rely almost entirely on mitochondrial ATP to support the work of contractile apparatus. It is essential thus, that ATP production rate in mitochondria would correspond to its turnover in cytosol. Yet, the cytosolic concentrations of relevant signal molecules, ATP and PCr, stay almost unaltered in highly oxidative muscles irrespective of muscle work-load. This puzzying discovery has obscured the understanding of how ATP production rate in mitochondria is matched with its turnover in cytosol.

The aim of this thesis was to study the mechanisms of mitochondrial respiration regulation in permeabilized cardiac cells and skeletal muscle fibers and to elucidate the role of tubulin in mitochondria functional regulation. To this end the kinetics of respiration regulation was analyzed by oxygraphy and the role of tubulin was examined by confocal microscopy, oxygraphy and by biochemical tools. Kinetic analysis of mitochondrial respiration in cardiac cells showed that the majority of ATP synthesized in mitochondrial matrix is exploited for PCr synthesis by MtCK (PCr/O2 ~6), showing thus that coupling between PCr synthesis and oxidative phosphorylation is highly efficient. Previous kinetic studies on cardiac cells have demonstrated, that limited permeability of mitochondrial outer membrane (mtOM) for ADP, is crucial for the efficiency of this coupling. To validate if tubulin participates in this regulation, reconstitution experiments with isolated cardiac mitochondria were performed. It was found that 1 μM tubulin decrease mitochondrial respiratory affinity for ADP over 20- fold (apparent Km ADP increased from 11±2 to 330±47 μM), which was reversed by the addition of creatine. Thus, these data clearly show that tubulin binding to mtOM induce selective permeability restrictions for ADP in cardiac cells. • Keywords: Bioenergetics, Muscle cells, Energy metabolism, Tubulin, Dissertations

Labels: MiParea: Respiration, Comparative MiP;environmental MiP 

Organism: Rat  Tissue;cell: Skeletal muscle  Preparation: Permeabilized tissue 

Pathway: N  HRR: Oxygraph-2k 

Competition assay of mitochondria and PK/PEP for endogneous ADP on skeletal muscles revealed, that similarly to cardiac cells, the prominent feedback signal in highly oxidative soleus muscle is creatine. The efficiency of this feedback was significantly lower (~50% decline in respiration rate upon PK/PEP addition) in glycolytic muscles (EDL, GW), where mtOM permeability for ADP was high and MtCK expression relatively modest. Thus, regulation of mtOM permeability for ADP appears to be a common mechanisms for muscle cells to finetune the coupling of oxidative phosphorylation with cytosolic ATPases via CK reactions. Analysis of the distribution of β-tubulin isoforms in cardiac cells, revealed that βII-tubulin participates in this regulation. Its expression was relatively high in oxidative, heart and soleus muscles, and low or entirely missing from muscles (GR, EDL, GW) and cells (NB HL-1), where restrictions at mtOM for ADP were absent. Furthermore, it was found that βII-tubulin belongs to a subset of β-tubulin isoforms which subcellular localization is remodelled in parallel with mitochondrial, suggesting that its subcellular localization could direct compartment specific organization of mitochondria in muscle cells. Finally MCA revealed that the sum of flux control coefficients largely exceed the unity in both soleus and GW muscles, suggesting that respiratory enzymes, ANT and VDAC, and in soleus, also MtCK, are organized into supramolecular complex that facilitate direct metabolite channeling and the efficiency of respiratory control. In all, these findings demonstrate that highly oxdative muscles, such as heart and soleus, rely on creatine mediated feedback signalling of mitochondrial respiration, which efficiency is determined by βII-tubulin induced permeability restrictions at mtOM for ADP. As a result, ATP/ADP remains cycling within mitochondria and cytosolic PCr reserves are efficiently replenished via coupled CK reaction and oxidative phoshorylation. This mechanism forms the basis for maintaining metabolic homeostastis in the cytosol and for achieving efficient creatine-dependent control of mitochondrial ATP production.