Neuropeptide B (NPB) and neuropeptide W (NPW) are neuropeptides, which constitute NPB/W signaling systems together with G-protein coupled receptors NPBWR1. The location and function of NPB/W signaling systems have been predominantly detected and mapped within the CNS, including their role in the modulation of inflammatory pain, neuroendocrine functions, and autonomic nervous systems. The aim of the study is to investigate the impact of diabetes on the neuropeptide B/W signaling system in different heart compartments and neurons which innervates it. In the RT-qPCR analysis, we observed the upregulation of mRNA for preproNPB in RV, for preproNPW in LA, and for NPBWR1 in DRG in diabetic rats. On the contrary, the expression of mRNA for NPBWR1 was downregulated in LV in diabetic rats. In the WB analysis, significant downregulation of NPBWR1 in LV (0.54-fold, p = 0.046) in diabetic rats was observed at the proteomic level. The presence of NPBWR1 was also confirmed in a dissected LCM section of cardiomyocytes and coronary arteries. The positive inotropic effect of NPW described on the diabetic cardiomyocytes in vitro could point to a possible therapeutic target for compensation of the contractile dysfunction in the diabetic heart. In conclusion, the NPB/W signaling system is involved in the regulation of heart functions and long-term diabetes leads to changes in the expression of individual members of this signaling system differently in each cardiac compartment, which is related to the different morphology and function of these cardiac chambers.
- MeSH
- experimentální diabetes mellitus * genetika MeSH
- krysa rodu rattus MeSH
- messenger RNA genetika MeSH
- proteomika MeSH
- receptory neuropeptidů * genetika metabolismus MeSH
- receptory spřažené s G-proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Mitochondria are considered central regulator of the aging process; however, majority of studies dealing with the impact of age on mitochondrial oxygen consumption focused on skeletal muscle concluding (although not uniformly) a general declining trend with advancing age. In addition, gender related differences in mitochondrial respiration have not been satisfactorily described yet. The aim of the present study was to evaluate mitochondrial oxygen consumption in various organs of aging male and female Fischer 344 rats at the ages of 6, 12 and 24 months. Mitochondrial respiration of homogenized (skeletal muscle, left and right heart ventricle, hippocampus, cerebellum, kidney cortex), gently mechanically permeabilized (liver) tissue or intact cells (platelets) was determined using high-resolution respirometry (oxygraphs O2k, Oroboros, Austria). The pattern of age-related changes differed in each tissue: in the skeletal muscle and kidney cortex of both sexes and in female heart, parameters of mitochondrial respiration significantly declined with age. Resting respiration of intact platelets displayed an increasing trend and it did not correlate with skeletal muscle respiratory states. In the heart of male rats and brain tissues of both sexes, respiratory states remained relatively stable over analyzed age categories with few exceptions of lower mitochondrial oxygen consumption at the age of 24 months. In the liver, OXPHOS capacity was higher in females than in males with either no difference between the ages of 6 and 24 months or even significant increase at the age of 24 months in the male rats. In conclusion, the results of our study indicate that the concept of general pattern of age-dependent decline in mitochondrial oxygen consumption across different organs and tissues could be misleading. Also, the statement of higher mitochondrial respiration in females seems to be conflicting, since the gender-related differences may vary with the tissue studied, combination of substrates used and might be better detectable at younger ages than in old animals.
- MeSH
- anestezie MeSH
- buněčné dýchání MeSH
- dýchání MeSH
- kosterní svaly metabolismus MeSH
- krysa rodu rattus MeSH
- mitochondrie * MeSH
- spotřeba kyslíku fyziologie MeSH
- stárnutí MeSH
- svalové mitochondrie * metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Five-sixths nephrectomy is a widely used experimental model of chronic kidney disease (CKD) that is associated with severe mitochondrial dysfunction of the remnant tissue. In this study, we assessed the effect of CKD on mitochondrial respiration separately in the rat kidney cortex and medulla 10 weeks after induction of CKD by subtotal 5/6 nephrectomy (SNX). Mitochondrial oxygen consumption was evaluated on mechanically permeabilized samples of kidney cortex and medulla using high-resolution respirometry and expressed per mg of tissue wet weight or IU citrate synthase (CS) activity. Mitochondrial respiration in the renal cortex of SNX rats was significantly reduced in all measured respiratory states if expressed per unit wet weight and remained lower if recalculated per IU citrate synthase activity, i.e. per mitochondrial mass. In contrast, the profound decrease in the activity of CS in SNX medulla resulted in significantly elevated respiratory states expressing the OXPHOS capacity when Complexes I and II or II only are provided with electrons, LEAK respiration after oligomycin injection, and Complex IV-linked oxygen consumption per unit CS activity suggesting compensatory hypermetabolic state in remaining functional mitochondria that is not sufficient to fully compensate for respiratory deficit expressed per tissue mass. The results document that CKD induced by 5/6 nephrectomy in the rat is likely to cause not only mitochondrial respiratory dysfunction (in the kidney cortex), but also adaptive changes in the medulla that tend to at least partially compensate for mitochondria loss.
- MeSH
- chronická renální insuficience * MeSH
- citrátsynthasa MeSH
- krysa rodu rattus MeSH
- kůra ledviny MeSH
- ledviny * metabolismus MeSH
- mitochondrie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm2 with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA integration. We detect signals ranging from a highly-abundant protein, β-actin, to a low-abundance protein, LC-3AB, using 0.125 mm2 LCM section from rat kidney, but, so far, a relatively large section is required for good quality of results. This integration is applicable for a highly-sensitive and accurate assessment of microdissected tissue sections to decipher hidden proteomic information of pure targeted cells. To validate this integration, PCK2 protein expression is studied within Langerhans islets of normal and diabetic rats. Our results show significant overexpression of PCK2 in Langerhans islets of rats with long-term diabetes.
- Publikační typ
- časopisecké články MeSH
Sarcomas are a heterogeneous group of mesenchymal tumours, with a great variability in their clinical behaviour. While our knowledge of sarcoma initiation has advanced rapidly in recent years, relatively little is known about mechanisms of sarcoma progression. JUN-murine fibrosarcoma progression series consists of four sarcoma cell lines, JUN-1, JUN-2, JUN-2fos-3, and JUN-3. JUN-1 and -2 were established from a single tumour initiated in a H2K/v-jun transgenic mouse, JUN-3 originates from a different tumour in the same animal, and JUN-2fos-3 results from a targeted in vitro transformation of the JUN-2 cell line. The JUN-1, -2, and -3 cell lines represent a linear progression from the least transformed JUN-2 to the most transformed JUN-3, with regard to all the transformation characteristics studied, while the JUN-2fos-3 cell line exhibits a unique transformation mode, with little deregulation of cell growth and proliferation, but pronounced motility and invasiveness. The invasive sarcoma sublines JUN-2fos-3 and JUN-3 show complex metabolic profiles, with activation of both mitochondrial oxidative phosphorylation and glycolysis and a significant increase in spared respiratory capacity. The specific transcriptomic profile of invasive sublines features very complex biological relationships across the identified genes and proteins, with accentuated autocrine control of motility and angiogenesis. Pharmacologic inhibition of one of the autocrine motility factors identified, Ccl8, significantly diminished both motility and invasiveness of the highly transformed fibrosarcoma cell. This progression series could be greatly valuable for deciphering crucial aspects of sarcoma progression and defining new prognostic markers and potential therapeutic targets.
- Publikační typ
- časopisecké články MeSH
Members of neuropeptide B/W signaling system have been predominantly detected and mapped within the CNS. In the rat, this system includes neuropeptide B (NPB), neuropeptide W (NPW) and their specific receptor NPBWR1. This signaling system has a wide spectrum of functions including a role in modulation of inflammatory pain and neuroendocrine functions. Expression of NPB, NPW and NPBWR1 in separate heart compartments, dorsal root ganglia (DRG) and stellate ganglia was proven by RT-qPCR, Western blot (WB) and immunofluorescence. Presence of mRNA for all tested genes was detected within all heart compartments and ganglia. The presence of proteins preproNPB, preproNPW and NPBWR1 was confirmed in all the chambers of heart by WB. Expression of preproNPW and preproNPB was proven in cardiac ganglionic cells obtained by laser capture microdissection. In immunofluorescence analysis, NPB immunoreactivity was detected in nerve fibers, some nerve cell bodies and smooth muscle within heart and both ganglia. NPW immunoreactivity was present in the nerve cell bodies and nerve fibers of heart ganglia. Weak nonhomogenous staining of cardiomyocytes was present within heart ventricles. NPBWR1 immunoreactivity was detected on cardiomyocytes and some nerve fibers. We confirmed the presence of NPB/W signaling system in heart, DRG and stellate ganglia by proteomic and genomic analyses.
- MeSH
- exprese genu MeSH
- fluorescenční protilátková technika MeSH
- ganglion stellatum metabolismus MeSH
- myokard metabolismus MeSH
- neuropeptidy genetika imunologie metabolismus MeSH
- potkani Zucker MeSH
- receptory neuropeptidů genetika imunologie metabolismus MeSH
- receptory spřažené s G-proteiny genetika imunologie metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- signální transdukce MeSH
- spinální ganglia metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Spinocerebellar ataxia 1 (SCA1) is a devastating neurodegenerative disease associated with cerebellar degeneration and motor deficits. However, many patients also exhibit neuropsychiatric impairments such as depression and apathy; nevertheless, the existence of a causal link between the psychiatric symptoms and SCA1 neuropathology remains controversial. This study aimed to explore behavioral deficits in a knock-in mouse SCA1 (SCA1154Q/2Q) model and to identify the underlying neuropathology. We found that the SCA1 mice exhibit previously undescribed behavioral impairments such as increased anxiety- and depressive-like behavior and reduced prepulse inhibition and cognitive flexibility. Surprisingly, non-motor deficits characterize the early SCA1 stage in mice better than does ataxia. Moreover, the SCA1 mice exhibit significant hippocampal atrophy with decreased plasticity-related markers and markedly impaired neurogenesis. Interestingly, the hippocampal atrophy commences earlier than the cerebellar degeneration and directly reflects the individual severity of some of the behavioral deficits. Finally, mitochondrial respirometry suggests profound mitochondrial dysfunction in the hippocampus, but not in the cerebellum of the young SCA1 mice. These findings imply the essential role of hippocampal impairments, associated with profound mitochondrial dysfunction, in SCA1 behavioral deficits. Moreover, they underline the view of SCA1 as a complex neurodegenerative disease and suggest new avenues in the search for novel SCA1 therapies.
- MeSH
- atrofie metabolismus patologie MeSH
- biologické markery metabolismus MeSH
- duševní poruchy metabolismus patologie MeSH
- hipokampus metabolismus patologie MeSH
- mitochondrie metabolismus patologie MeSH
- modely nemocí na zvířatech MeSH
- mozeček metabolismus patologie MeSH
- myši MeSH
- spinocerebelární ataxie metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Burden of obesity is increasing in the contemporary world. Although multifactorial in origin, appropriate mitochondrial function of adipocytes emerges as a factor essential for healthy adipocyte differentiation and adipose tissue function. Our study aimed to evaluate mitochondrial functions of human adipose-derived mesenchymal stem cells committed to adipogenesis. On days 0, 4, 10, and 21 of adipogenesis, we have characterized adipocyte proliferation and viability, quantified lipid accumulation in maturing cells, performed qualitative and quantitative analysis of mitochondria, determined mitochondrial respiration of cells using high-resolution respirometry, and evaluated mitochondrial membrane potential. In the course of adipogenesis, mitochondrial oxygen consumption progressively increased in states ROUTINE and E (capacity of the electron transfer system). State LEAK remained constant during first days of adipogenesis and then increased probably reflecting uncoupling ability of maturing adipocytes. Citrate synthase activity and volume of mitochondrial networks increased during differentiation, particularly between days 10 and 21. In addition, lipid accumulation remained low until day 10 and then significantly increased. In conclusion, during first days of adipogenesis, increased mitochondrial respiration is needed for transition of differentiating cells from glycolytic to oxidative metabolism and clonal expansion of preadipocytes and then more energy is needed to acquire typical metabolic phenotype of mature adipocyte.
- MeSH
- adipogeneze * MeSH
- buněčné dýchání MeSH
- kultivované buňky MeSH
- lidé MeSH
- membránový potenciál mitochondrií MeSH
- metabolismus lipidů MeSH
- mezenchymální kmenové buňky fyziologie MeSH
- mitochondrie metabolismus MeSH
- tukové buňky metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The study aim was to compare molecular-level effects (blood-dialyzer interactions) of heparin and citrate anticoagulation using proteome-wide analysis of biofilm adsorbed to dialysis membrane. Ten patients receiving maintenance hemodialysis were examined in a crossover design under three different anticoagulation regimens, namely citrate, heparin, and anticoagulation-free (control). Following a regular hemodialysis session (4 hours, polysulfone membrane), dialyzers were flushed and the surface biofilm eluted by acetic acid. Protein composition of the eluates was determined by 2-dimensional gel electrophoresis and resulting patterns compared between regimens. Proteins responsible for the difference were identified by mass spectrometry. Citrate anticoagulation was associated with significantly less protein adsorption to the membrane than heparin (2.2 [1.1-2.9] mg vs. 6.5 [2.9-11.6] mg, P = 0.009). Among the proteins identified as major discriminators between citrate and the other regimens, fibrin α-chain fragments of molecular weight below 40 kDa prevailed. In these fragments, an analysis of the amino acid sequence has been performed by comparison with the UniProt database. It showed missing α-chain cross-links. On the contrary, heparin prevented adsorption and cleavage of several heparin-binding proteins; especially complement factor H-related protein 3, insulin-like growth factor binding proteins (2, 4, and 5), and chemerin. Compared to heparin, citrate is associated with less protein adsorption and imperfectly crosslinked fibrin clot formation. Membrane adsorptive properties are significantly modified by the anticoagulation regimen.
- MeSH
- adsorpce MeSH
- antikoagulancia farmakologie MeSH
- dialýza ledvin přístrojové vybavení metody MeSH
- hemokoagulace účinky léků MeSH
- heparin farmakologie MeSH
- krevní proteiny analýza MeSH
- kyselina citronová farmakologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- membrány umělé MeSH
- polymery chemie MeSH
- proteomika MeSH
- senioři MeSH
- sulfony chemie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The complex pathogenesis of sepsis and septic shock involves myocardial depression, the pathophysiology of which, however, remains unclear. In this study, cellular mechanisms of myocardial depression were addressed in a clinically relevant, large animal (porcine) model of sepsis and septic shock. Sepsis was induced by fecal peritonitis in eight anesthetized, mechanically ventilated, and instrumented pigs of both sexes and continued for 24 h. In eight control pigs, an identical experiment but without sepsis induction was performed. In vitro analysis of cardiac function included measurements of action potentials and contractions in the right ventricle trabeculae, measurements of sarcomeric contractions, calcium transients and calcium current in isolated cardiac myocytes, and analysis of mitochondrial respiration by ultrasensitive oxygraphy. Increased values of modified sequential organ failure assessment score and serum lactate levels documented the development of sepsis/septic shock, accompanied by hyperdynamic circulation with high heart rate, increased cardiac output, peripheral vasodilation, and decreased stroke volume. In septic trabeculae, action potential duration was shortened and contraction force reduced. In septic cardiac myocytes, sarcomeric contractions, calcium transients, and L-type calcium current were all suppressed. Similar relaxation trajectory of the intracellular calcium-cell length phase-plane diagram indicated unchanged calcium responsiveness of myofilaments. Mitochondrial respiration was diminished through inhibition of Complex II and Complex IV. Defective calcium handling with reduced calcium current and transients, together with inhibition of mitochondrial respiration, appears to represent the dominant cellular mechanisms of myocardial depression in porcine septic shock.
- Publikační typ
- časopisecké články MeSH
