We studied how deficiency in lamins A/C and lamina-associated polypeptide 2α (Lap2α) affects DNA repair after irradiation. A-type lamins and Lap2α were not recruited to local DNA lesions and did not accumulate to γ-irradiation-induced foci (IRIF), as it is generally observed for well-known marker of DNA lesions, 53BP1 protein. At micro-irradiated chromatin of lmna double knockout (dn) and Lap2α dn cells, 53BP1 protein levels were reduced, compared to locally irradiated wild-type counterpart. Decreased levels of 53BP1 we also observed in whole populations of lmna dn and Lap2α dn cells, irradiated by UV light. We also studied distribution pattern of 53BP1 protein in a genome outside micro-irradiated region. In Lap2α deficient cells, identical fluorescence of mCherry-tagged 53BP1 protein was found at both microirradiated region and surrounding chromatin. However, a well-known marker of double strand breaks, γH2AX, was highly abundant in the lesion-surrounding genome of Lap2α deficient cells. Described changes, induced by irradiation in Lap2α dn cells, were not accompanied by cell cycle changes. In Lap2α dn cells, we additionally performed analysis by FLIM (Fluorescence Lifetime Imaging Microscopy) that showed different dynamic behavior of mCherry-tagged 53BP1 protein pools when it was compared with wild-type (wt) fibroblasts. This analysis revealed three different fractions of mCherry-53BP1 protein. Two of them showed identical exponential decay times (τ1 and τ3), but the decay rate of τ2 and amplitudes of fluorescence decays (A1-A3) were statistically different in wt and Lap2α dn fibroblasts. Moreover, γ-irradiation weakened an interaction between A-type lamins and Lap2α. Together, our results demonstrate how depletion of Lap2α affects DNA damage response (DDR) and how chromatin compactness is changed in Lap2α deficient cells exposed to radiation.

• MeSH
• 53BP1 genetika   metabolismus   MeSH
• chromatin chemie   účinky záření   ultrastruktura   MeSH
• DNA vazebné proteiny nedostatek   genetika   MeSH
• embryo savčí MeSH
• fibroblasty cytologie   metabolismus   účinky záření   MeSH
• FRAP MeSH
• histony genetika   metabolismus   MeSH
• lamin typ A nedostatek   genetika   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• membránové proteiny nedostatek   genetika   MeSH
• myši MeSH
• oprava DNA * MeSH
• poškození DNA MeSH
• regulace genové exprese MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• reportérové geny MeSH
• signální transdukce MeSH
• transformované buněčné linie MeSH
• ultrafialové záření MeSH
• záření gama MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Telomeres are specialized chromatin structures that are situated at the end of linear chromosomes and play an important role in cell senescence and immortalization. Here, we investigated whether changes in histone signature influence the nuclear arrangement and positioning of telomeres. Analysis of mouse embryonic fibroblasts revealed that telomeres were organized into specific clusters that partially associated with centromeric clusters. This nuclear arrangement was influenced by deficiency of the histone methyltransferase SUV39h, LMNA deficiency, and the histone deacetylase inhibitor Trichostatin A (TSA). Similarly, nuclear radial distributions of telomeric clusters were preferentially influenced by TSA, which caused relocation of telomeres closer to the nuclear center. Telomeres also co-localized with promyelocytic leukemia bodies (PML). This association was increased by SUV39h deficiency and decreased by LMNA deficiency. These differences could be explained by differing levels of the telomerase subunit, TERT, in SUV39h- and LMNA-deficient fibroblasts. Taken together, our data show that SUV39h and A-type lamins likely play a key role in telomere maintenance and telomere nuclear architecture.

• MeSH
• DNA vazebné proteiny metabolismus   MeSH
• epigeneze genetická MeSH
• fibroblasty metabolismus   MeSH
• genová přestavba MeSH
• intranukleární inkluzní tělíska metabolismus   MeSH
• lamin typ A metabolismus   MeSH
• lidé MeSH
• methyltransferasy metabolismus   MeSH
• myši MeSH
• protein TRF1 metabolismus   MeSH
• průtoková cytometrie MeSH
• rap1 proteiny vázající GTP metabolismus   MeSH
• represorové proteiny metabolismus   MeSH
• telomerasa metabolismus   MeSH
• telomery genetika   metabolismus   MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Recent studies have shown that histone code dictates the type and structure of chromatin. Bearing in mind the importance of A-type lamins for chromatin arrangement, we studied the effect of trichostatin A (TSA)-induced histone hyperacetylation in lamin A/C-deficient (LMNA-/-) fibroblasts. Lamin A/C deficiency caused condensation of chromosome territories and the nuclear reorganization of centromeric heterochromatin, which was accompanied by the appearance of a chain-like morphology of HP1beta foci. Conversely, histone deacetylase (HDAC) inhibition induced de-condensation of chromosome territories, which compensated the effect of lamin A/C deficiency on chromosome regions. The amount of heterochromatin in the area associated with the nuclear membrane was significantly reduced in LMNA-/- cells when compared with lamin A/C-positive (LMNA+/+) fibroblasts. TSA also decreased the amount of peripheral heterochromatin, similarly as lamin A/C deficiency. In both LMNA+/+ and LMNA-/- cells, physically larger chromosomes were positioned more peripherally as compared with the smaller ones, even after TSA treatment. Our observations indicate that lamin A/C deficiency causes not only reorganization of chromatin and some chromatin-associated domains, but also has an impact on the extent of chromosome condensation. As HDAC inhibition can compensate the lamin A/C-dependent chromatin changes, the interaction between lamins and specifically modified histones may play an important role in higher-order chromatin organization, which influences transcriptional activity.

• MeSH
• acetylace účinky léků   MeSH
• buněčné jádro metabolismus   MeSH
• centromera metabolismus   MeSH
• chromatin metabolismus   účinky léků   MeSH
• fibroblasty metabolismus   MeSH
• financování organizované MeSH
• heterochromatin metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• inhibitory histondeacetylas MeSH
• jaderné proteiny metabolismus   MeSH
• kyseliny hydroxamové farmakologie   MeSH
• lamin typ A genetika   metabolismus   nedostatek   MeSH
• lamin typ B metabolismus   MeSH
• myši MeSH
• restrukturace chromatinu účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH

BACKGROUND INFORMATION: Promyelocytic leukaemia (PML) bodies are specific nuclear structures with functional significance for acute promyelocytic leukaemia. In this study, we analysed the trajectories of PML bodies using single-particle tracking. RESULTS: We observed that the recovery of PML protein after photobleaching was ATP dependent in both wild-type (wt) and A-type lamin-deficient cells. The movement of PML bodies was faster and the nuclear area occupied by particular PML bodies was larger in A-type lamin-deficient fibroblasts compared with their wt counterparts. Moreover, dysfunction of the LMNA gene increased the frequency of mutual interactions between individual PML bodies and influenced the morphology of these domains at the ultrastructural level. As a consequence of A-type lamin deficiency, PML protein accumulated in nuclear blebs and frequently appeared at the nuclear periphery. CONCLUSIONS: We suggest that the physiological function of lamin A proteins is important for events that occur in the compartment of PML bodies. This observation was confirmed in other experimental models characterised by lamin changes, including apoptosis or the differentiation of mouse embryonic stem cells.

• MeSH
• akutní promyelocytární leukemie metabolismus   MeSH
• apoptóza MeSH
• embryo savčí cytologie   MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• fibroblasty cytologie   metabolismus   ultrastruktura   MeSH
• FRAP MeSH
• intranukleární inkluzní tělíska metabolismus   ultrastruktura   MeSH
• kinetika MeSH
• lamin typ A nedostatek   metabolismus   MeSH
• myši MeSH
• reprodukovatelnost výsledků MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

• MeSH
• dilatační kardiomyopatie genetika   metabolismus   patologie   MeSH
• Duchennova muskulární dystrofie genetika   metabolismus   patologie   MeSH
• dystrofin nedostatek   genetika   MeSH
• kardiomyocyty metabolismus   patologie   MeSH
• kardiovaskulární systém metabolismus   patologie   MeSH
• kmenové buňky metabolismus   patologie   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myokard metabolismus   patologie   MeSH
• myši inbrední mdx genetika   MeSH
• myši MeSH
• poškození DNA genetika   MeSH
• protoonkogenní proteiny c-kit genetika   MeSH
• regulace genové exprese genetika   MeSH
• stárnutí genetika   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

RATIONALE: Cardiac ECM (extracellular matrix) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. OBJECTIVES: We aimed to (1) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (2) determine the underlying molecular mechanisms. METHODS AND RESULTS: We first performed decellularization of human and murine ECM (decellularized ECM) and then analyzed the pathological changes occurring in decellularized ECM during HF by atomic force microscopy, 2-photon microscopy, high-resolution 3-dimensional image analysis, and computational fluid dynamics simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts based on YAP (yes-associated protein)-transcriptional enhanced associate domain (TEAD) mechanosensing activity and collagen contraction assays. The analysis of HF decellularized ECM resulting from ischemic or dilated cardiomyopathy, as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3-dimensional topography. As compared with healthy heart, HF ECM exhibited aligned, flat, and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF cardiac fibroblasts highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFβ1 (transforming growth factor β1), interleukin-1, TNF-α, and BDNF signaling pathways. Functional tests performed on HF cardiac fibroblasts pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. CONCLUSIONS: Our multiparametric approach has highlighted repercussions of ECM remodeling on cell homing, cardiac fibroblast activation, and focal adhesion protein expression via hyperactivated YAP signaling during HF.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• buněčný převod mechanických signálů MeSH
• dilatační kardiomyopatie genetika   metabolismus   patologie   patofyziologie   MeSH
• extracelulární matrix genetika   metabolismus   ultrastruktura   MeSH
• fibroblasty metabolismus   ultrastruktura   MeSH
• funkce levé komory srdeční * MeSH
• infarkt myokardu genetika   metabolismus   patologie   patofyziologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myokard metabolismus   ultrastruktura   MeSH
• myši inbrední C57BL MeSH
• pohyb buněk MeSH
• remodelace komor * MeSH
• srdeční selhání genetika   metabolismus   patologie   patofyziologie   MeSH
• studie případů a kontrol MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH