Enhanced Cardiac Regenerative Ability of Stem Cells After Ischemia-Reperfusion Injury Role of Human CD34+ Cells Deficient in MicroRNA-377

AbstractBackgroundMicroRNA (miR) dysregulation in the myocardium has been implicated in cardiac remodeling after injury or stress.ObjectivesThe aim of this study was to explore the role of miR in human CD34+ cell (hCD34+) dysfunction in vivo after transplantation into the myocardium under ischemia-reperfusion (I-R) conditions.MethodsIn response to inflammatory stimuli, the miR array profile of endothelial progenitor cells was analyzed using a polymerase chain reaction–based miR microarray. miR-377 expression was assessed in myocardial tissue from human patients with heart failure (HF). We investigated the effect of miR-377 inhibition on an hCD34+ cell angiogenic proteome profile in vitro and on cardiac repair and function after I-R injury in immunodeficient mice.ResultsThe miR array data from endothelial progenitor cells in response to inflammatory stimuli indicated changes in numerous miR, with a robust decrease in the levels of miR-377. Human cardiac biopsies from patients with HF showed significant increases in miR-377 expression compared with nonfailing control hearts. The proteome profile of hCD34+ cells transfected with miR-377 mimics showed significant decrease in the levels of proangiogenic proteins versus nonspecific control–transfected cells. We also validated that serine/threonine kinase 35 is a target of miR-377 using a dual luciferase reporter assay. In a mouse model of myocardial I-R, intramyocardial transplantation of miR-377 silenced hCD34+ cells in immunodeficient mice, promoting neovascularization (at 28 days, post–I-R) and lower interstitial fibrosis, leading to improved left ventricular function.ConclusionsThese findings indicate that HF increased miR-377 expression in the myocardium, which is detrimental to stem cell function, and transplantation of miR-377 knockdown hCD34+ cells into ischemic myocardium promoted their angiogenic ability, attenuating left ventricular remodeling and cardiac fibrosis

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

The Essentials of Protein Import in the Degenerate Mitochondrion of Entamoeba histolytica

Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica

A Chaperone Trap Contributes to the Onset of Cystic Fibrosis

Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a ‘chaperone trap’. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF

Heat shock protein (HSP)-47 decreases in myocardium under LVAD-support

Objectives: Previously, we reported a change in phenotype of myocardial mast cells during LVAD-support. Such altered mast cells had modulating effects on collagen production in a co-culture system with fibroblasts. To support our findings, we determined the HSP-47 expression in the myocardium, a specific molecular chaperone for procollagen. Material and Methods: HSP-47 staining was performed on matched tissue slides from 10 patients with DCM at the time of LVAD implantation and of transplantation following LVAD support. Tissue slides from 5 donor hearts not used for transplantation served as a control group. Results: While slides of normal myocardium presented almost no staining for HSP-47 (1 positive cell per microscopy field), there was a highly significant increase in HSP-47 positive cells in the pre-LVAD DCM-tissue (34 positive cells per microscopy field) (p<0.01). Interestingly, the post-LVAD myocardium showed a significant decrease in HSP-47 positive cells (8 positive cells per microscopy fields) (p<0.01). All Hsp-47 positive cells stained also positive for the fibroblast marker vimentin. Conclusions: There was a decrease of HSP-47 positive cells following LVAD support compared to pre-LVAD DCM myocardium. This may confirm results of our previous study showing mast cells isolated from post-LVAD myocardium having an inhibiting effect on the fibroblastic protein production in vitro

Hirntodinduzierte Aktivierung proinflammatorischer Reaktionen in potentiellen Spenderorganen

Eine Serie unspezifischer inflammatorischer Ereignisse, ausgelöst durch explosiven Hirntod, scheint in potentiellen Spenderorganen eine Reihe von funktionellen und immunologischen Veränderungen zu verursachen. Ziel dieser Studie ist es, den Verlauf pro-inflammatorischer Zytokine in Herz, Lunge und Niere hirntoter Tiere aufzuzeigen.Bei 4 Hausschweinen wurde Hirntod durch akuten Druckanstieg über einen intrakraniell eingebrachten Foley- Katheter herbeigeführt. Weitere 4 Schweine wurden scheinoperiert und dienten somit als Kontrolltiere. Jedes Experiment wurde 6 Stunden nach Hirntodinduktion beendet und die Organe wurden entnommen. Wir bestimmten die mRNA-Expression für TNF-α, IL-1β und IL-6 mittels rt-PCR in Herz, Lunge und Niere. Die Ergebnisse werden als Relation der Dichtewerte der Zytokine zum Housekeeping-Gen β-actin in der Gelelektrophorese beschrieben.Die 6h-Werte für IL-1β- und IL-6-mRNA waren in allen untersuchten Organen von hirntoten Tieren signifikant höher als in den von scheinoperierten. TNF-α mRNA-Expression war in Lungegewebe hirntoter Tiere ebenfalls signifikant höher, wohingegen in Herz- und Nierengewebe die Werte für TNF-α mRNA in der Hirntod-Gruppe eher niedriger waren als in der Kontrollgruppe (statistisch nicht signifikant).Explosiver Hirntod führt zu Zytokininduktion in den peripheren Organen. Aufgrund unserer Ergebnisse schlussfolgern wir, dass es sich hierbei um eine organ-spezifische Regulation handelt.Organ dysfunction after explosive brain death has been well documented. In addition, a series of nonspecific inflammatory events may increase the intensity of the immunological host response. The aim of the present study was to monitor the course of proinflammatory cytokines in heart, lung and kidney after brain death induction.Brain death (BD) was induced in 4 pigs by inflation of an intracranial Foley catheter and separately 4 pigs were sham-operated. Each experiment was terminated 6 hours after brain death/sham operation and the organs were harvested. We determined the mRNA-expression for TNF-α, IL-1β and IL-6 in the heart, lung and kidney using rt-PCR techniques. The results are presented as absorbance intensity (Mean±SEM). β-actin was used for standardization.After 6 hours, IL-1β and IL-6 mRNA expression increased significantly in all investigated organs in the brain dead animals compared to sham-operated. TNF-α transcripts increased significantly only in lung, while in heart and kidney TNF-α mRNA tended even to lower values in brain dead animals.Brain death was associated with an increase in mRNA concentration of IL-1β and IL-6 in lung, heart and kidney, while TNF-α mRNA expression was only up-regulated in lung. We suggest that brain death initiates a cascade of organ-specific inflammatory events that may play a crucial role in early graft dysfunction and rejection via a priming effect on the transplanted organ