Südamerakkude sünnijärgse arengu bioenergeetilised aspektid: struktuuri ja funktsiooni vaheliste seoste väljakujunemine

Taust ja eesmärk. Täiskasvanud südamerakkude bioenergeetikas on valdavaks ATP genereerimisemehhanismiks mitokondriaalne oksüdatiivne fosforüülimine, mis katab tavatingimustel üle 90% südame energeetilisest vajadusest. Mitokondrid paiknevad kardiomüotsüütides korrapäraselt müofibrillide vahel, asetudes kohakuti libisevate filamentide kontaktalaga (sarkomeeri anisotroopne (A) vööt). Aktomüosiinisüsteem, mitokondrid, sarkoplasmaatiline võrgustik ja nendega seotud tsütoskeleti valgud moodustavad rakus ühtse struktuurse ja funktsionaalse terviku, nn energeetilise üksuse (EÜ), mis reguleerib efektiivselt energia tootmist ja fosforüülrühma ülekannet. Vahetult pärast sündi on mitokondrite paigutus ebakorrapärane, täiskasvanud kardiomüotsüüdiga võrreldes on oluliselt erinev ka südamerakkude metabolism ning energiaülekande regulatsioon. Töö eesmärgiks oli uurida südame mitokondriaalse hingamise regulatsiooni mehhanismide väljakujunemist südame sünnijargses arengus ning selle seotust mitokondrite ja tubuliini isovormide rakusisese paigutusega. Töö tulemused võimaldavad selgitada südamerakkude teatud patoloogiliste seisundite etioloogiat. Metoodika. Kardiomüotsüüdid isoleeriti, perfuseerides katseloomade (Wistari liini rotid) südant kollagenaas A lahusega. Skineeritud kiudude eraldamiseks kasutati meetodit, mille käigus lihaskiud eraldatakse õrnalt pintsettidega ja töödeldakse seejärel saponiiniga. Permeabiliseeritud kardiomüotsüütide ja skineeritud kiudude hapnikutarbimine registreeriti suure lahutusvõimega oksügraafil. Preparaatide visualiseerimiseks kasutati konfokaalmikroskoope Zeiss LSM 510 ja Olympus FluoView FV10i-W. Tulemused. Katseloomade sünni järel toimuvad esimese pooleteise kuu jooksul südamerakuenergiaülekande regulatsioonis kiired muutused: mitokondrite paigutus muutub korrapäraseks, toimub tsütoskeleti funktsionaalselt oluliste komponentide paigutumine mitokondrite lähedusse ja sellega samal ajal kasvavad oluliselt difusioonitakistused adenosiindifosfaadile (Km(ADP) väärtus suureneb 75,0 ・} 4,5 μM 3 päeva vanuste rottide kardiomüotsüütides kuni 317 ・} 29,5 μM vorreldes 84päevaste katseloomadega) ning käivitub kreatiinkinaasi-fosfokreatiini ülekandevõrgustik mitokondrite ja tsütosoolsete ATPaaside vahel. Järeldused. Katseloomade sünnijargse arengu käigus toimuvad dünaamilised muutusedkardiomüotsüütide struktuuris, millega kaasnevad muutused nende funktsioonis. Funktsionaalsete vastasmõjude tekkimine mitokondrite ja tsütoskeleti komponentide vahel on eelduseks täiskasvanud südamerakule omase energiametabolismi väljakujunemiseks. Eesti Arst 2013; 92(7):372–38

Uusi suundi kasvajate energiametabolismi uuringutes

Genoomika kiire arengu käigus on selgunud, et selle valdkonna meetoditega ei ole võimalik erinevaid metabolismihäireid terviklikult kirjeldada ning täiendavalt on vaja kasutusele võtta teisi meetodeid rakuenergeetikast ning proteoomikast. Äärmiselt huvitavaks kujuneb selline süsteemsem käsitlus ulatuslike patoloogiliste muutustega maliigses koes. Eelmise sajandi alguses kirjeldas Otto Warburg efekti, kus tuumorirakkudes toimus eelistatult glükolüüs isegi normoksiatingimustes. Tema esmane arvamus, et just see asjaolu ongi raku maliigsuse allikas, lükati järgnevatel aastatel uute avastuste valguses ümber. Lisaks ulatuslikele rakuenergeetilistele ümberkorraldustele maliigse raku sees (nt kärbitud Krebsi-tsükkel, hingamisahela superkompleksid) on viimastel aastatel erinevate vähipaikmete juures korduvalt tõestatud ka kahe kompartmendi olemasolu, kus maliigne rakk allutab ümbritseva strooma enda jaoks vajalikke metaboliite tootma. Maliigsuse täpsem olemus, paremad ravimisihtmärgid ning -strateegiad võivad peituda just kasvajate süsteemsemate uuringute tulemustes. Eesti Arst 2013; 92(5):261–26

Südamelihase rakkude struktuuri olulisus rakuhingamise regulatsioonis

Viimastel aastatel on järjest selgemaks saanud seos raku energeetilise ainevahetuse ja südamehaiguste vahel, mistõttu on oluline uurida seda mõjutavaid tegureid. Töös uuriti südamelihase rakkude mitokondriaalse hingamise regulatsiooni väga erineva rakustruktuuriga preparaatides: 1) permeabiliseeritud kardiomüotsüütides, kus mitokondrid on regulaarselt organiseeritud; 2) südamelihase fenotüübiga sarnastes kontraheeruvates HL-1 (B HL-1) rakkudes ja 3) HL-1 mittekontraheeruvates (NB HL-1) rakkudes. Nende preparaatide vahel esines suur erinevus mitokondriaalse hingamise regulatsioonis. Selline tulemus näitab raku struktuuri ja funktsiooni vaheliste seoste tähtsust südamelihase rakkudes ning võimaldab paremini mõista protsesse nii terves kui ka patoloogilises südamelihases. Eesti Arst 2008; 87(1):19−2

Adaptation of striated muscles to Wolframin deficiency in mice: Alterations in cellular bioenergetics

Background: Wolfram syndrome (WS), caused by mutations in WFS1 gene, is a multi-targeting disease affecting multiple organ systems. Wolframin is localized in the membrane of the endoplasmic reticulum (ER), influencing Ca2+ metabolism and ER interaction with mitochondria, but the exact role of the protein remains unclear. In this study we aimed to characterize alterations in energy metabolism in the cardiac and in the oxidative and glycolytic skeletal muscles in Wfs1-deficiency. Methods: Alterations in the bioenergetic profiles in the cardiac and skeletal muscles of Wfs1-knock-out (KO) male mice and their wild type male littermates were determined using high resolution respirometry, quantitative RT-PCR, NMR spectroscopy, and immunofluorescence confocal microscopy. Results: Oxygen consumption without ATP synthase activation (leak) was significantly higher in the glycolytic muscles of Wfs1 KO mice compared to wild types. ADP-stimulated respiration with glutamate and malate was reduced in the Wfs1-deficient cardiac as well as oxidative and glycolytic skeletal muscles. Conclusions: Wfs1-deficiency in both cardiac and skeletal muscles results in functional alterations of energy transport from mitochondria to ATP-ases. There was a substrate-dependent decrease in the maximal Complex I –linked respiratory capacity of the electron transport system in muscles of Wfs1 KO mice. Moreover, in cardiac and gastrocnemius white muscles a decrease in the function of one pathway were balanced by the increase in the activity of the parallel pathway. General significance: This work provides new insights to the muscle involvement at early stages of metabolic syndrome like WS as well as developing glucose intoleranc

Altered mitochondrial metabolism in the insulin-resistant heart.

Obesity-induced insulin resistance and type 2 diabetes mellitus can ultimately result in various complications, including diabetic cardiomyopathy. In this case, cardiac dysfunction is characterized by metabolic disturbances such as impaired glucose oxidation and an increased reliance on fatty acid (FA) oxidation. Mitochondrial dysfunction has often been associated with the altered metabolic function in the diabetic heart, and may result from FA-induced lipotoxicity and uncoupling of oxidative phosphorylation. In this review, we address the metabolic changes in the diabetic heart, focusing on the loss of metabolic flexibility and cardiac mitochondrial function. We consider the alterations observed in mitochondrial substrate utilization, bioenergetics and dynamics, and highlight new areas of research which may improve our understanding of the cause and effect of cardiac mitochondrial dysfunction in diabetes. Finally, we explore how lifestyle (nutrition and exercise) and pharmacological interventions can prevent and treat metabolic and mitochondrial dysfunction in diabetes.COST Action MitoEAGL

In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography.

International audienceA method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest

Studies of mitochondrial respiration in muscle cells in situ: use and misuse of experimental evidence in mathematical modelling.

International audienceApplications of permeabilized cell and skinned fiber techniques in combination with methods of mathematical modelling for studies of mitochondrial function in the cell are critically evaluated. Mathematical models may be useful tools for explaining biological phenomena, but only if they are selected by fitting the computing results with real experimental data. Confocal microscopy has been used in experiments with permeabilized cardiomyocytes and myocardial fibers to determine the maximal diffusion distance from medium to the core of cells, which is shown not to exceed 8-10 microm. This is a principal index for correctly explaining high apparent Km for exogenous ADP (200-300 microM) in regulation of mitochondrial respiration in oxidative muscle cells in situ. The best fitting of the results of in silico studies may be achieved by using of the compartmentalized energy transfer model. From these results, it may be concluded that in cardiac muscle cells the mitochondria and ATPases are organized into intracellular energetic units (ICEUs) separated from the bulk phase of cytoplasm by some barriers which limit the diffusion of adenine nucleotides. In contrast, alternative models based on the concept of the cell as homogenous system do not explain the observed experimental phenomena and have led to misleading conclusions. The various sources of experimental and conceptual errors are analyzed

Study of possible interactions of tubulin, microtubular network, and STOP protein with mitochondria in muscle cells.

International audienceWe studied possible connections of tubulin, microtubular system, and microtubular network stabilizing STOP protein with mitochondria in rat and mouse cardiac and skeletal muscles by confocal microscopy and oxygraphy. Intracellular localization and content of tubulin was found to be muscle type-specific, with high amounts in oxidative muscles, and much lower in glycolytic skeletal muscle. STOP protein localization and content in muscle cells was also muscle type-specific. In isolated heart mitochondria, addition of 1 microM tubulin heterodimer increased apparent K(m) for ADP significantly. Dissociation of microtubular system into free tubulin by colchicine treatment only slightly decreased initially high apparent K(m) for ADP in permeabilized cells, and diffusely distributed free tubulin stayed inside the cells, obviously connected to the intracellular structures. To identify the genes that are specific for oxidative muscle, we developed and applied a method of kindred DNA. The results of sequencing and bioinformatic analysis of isolated cDNA pool common for heart and m. soleus showed that in adult mice the beta-tubulin gene is expressed predominantly in oxidative muscle cells. It is concluded that whereas dimeric tubulin may play a significant role in regulation of mitochondrial outer membrane permeability in the cells in vivo, its organization into microtubular network has a minor significance on that process