Varikmaa 2013 Thesis: Difference between revisions
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The aim of this thesis was to study the mechanisms of mitochondrial | The aim of this thesis was to study the mechanisms of mitochondrial | ||
respiration regulation in permeabilized cardiac cells and skeletal muscle fibers | 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 ( | 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 ฮผM tubulin decrease mitochondrial respiratory affinity for ADP over 20- fold (apparent Km ADP increased from 11ยฑ2 to 330ยฑ47 ฮผM), 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 | |keywords=Bioenergetics, Muscle cells, Energy metabolism, Tubulin, Dissertations | ||
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highly oxidative soleus muscle is creatine. The efficiency of this feedback was | highly oxidative soleus muscle is creatine. The efficiency of this feedback was | ||
significantly lower (~50% decline in respiration rate upon PK/PEP addition) in | significantly lower (~50% decline in respiration rate upon PK/PEP addition) in | ||
glycolytic muscles (EDL, GW), where | glycolytic muscles (EDL, GW), where mtOM permeability for ADP was high and | ||
MtCK expression relatively modest. Thus, regulation of | MtCK expression relatively modest. Thus, regulation of mtOM permeability for | ||
ADP appears to be a common mechanisms for muscle cells to finetune the | ADP appears to be a common mechanisms for muscle cells to finetune the | ||
coupling of oxidative phosphorylation with cytosolic ATPases via CK reactions. | coupling of oxidative phosphorylation with cytosolic ATPases via CK reactions. | ||
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ฮฒII-tubulin participates in this regulation. Its expression was relatively high in | ฮฒII-tubulin participates in this regulation. Its expression was relatively high in | ||
oxidative, heart and soleus muscles, and low or entirely missing from muscles | oxidative, heart and soleus muscles, and low or entirely missing from muscles | ||
(GR, EDL, GW) and cells (NB HL-1), where restrictions at | (GR, EDL, GW) and cells (NB HL-1), where restrictions at mtOM for ADP were | ||
absent. Furthermore, it was found that ฮฒII-tubulin belongs to a subset of ฮฒ-tubulin | absent. Furthermore, it was found that ฮฒII-tubulin belongs to a subset of ฮฒ-tubulin | ||
isoforms which subcellular localization is remodelled in parallel with | isoforms which subcellular localization is remodelled in parallel with | ||
Line 46: | Line 46: | ||
heart and soleus, rely on creatine mediated feedback signalling of mitochondrial | heart and soleus, rely on creatine mediated feedback signalling of mitochondrial | ||
respiration, which efficiency is determined by ฮฒII-tubulin induced permeability | respiration, which efficiency is determined by ฮฒII-tubulin induced permeability | ||
restrictions at | restrictions at mtOM for ADP. As a result, ATP/ADP remains cycling within | ||
mitochondria and cytosolic PCr reserves are efficiently replenished via coupled | mitochondria and cytosolic PCr reserves are efficiently replenished via coupled | ||
CK reaction and oxidative phoshorylation. This mechanism forms the basis for | CK reaction and oxidative phoshorylation. This mechanism forms the basis for | ||
maintaining metabolic homeostastis in the cytosol and for achieving efficient | maintaining metabolic homeostastis in the cytosol and for achieving efficient | ||
creatine-dependent control of mitochondrial ATP production. | creatine-dependent control of mitochondrial ATP production. |
Latest revision as of 08:23, 20 October 2017
Varikmaa M (2013) Structural and Functional Studies of Mitochondrial Respiration Regulation in Muscle Cells. Thesis Tallinn University of Technology - Tallinn pp147. |
ยป Tallinn University of Technology; Thesis Open Access
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 ฮผM tubulin decrease mitochondrial respiratory affinity for ADP over 20- fold (apparent Km ADP increased from 11ยฑ2 to 330ยฑ47 ฮผM), 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.
โข 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 ฮฒ-tubulin isoforms in cardiac cells, revealed that
ฮฒII-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 ฮฒII-tubulin belongs to a subset of ฮฒ-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 ฮฒII-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.