Comparative Study of Injury Models for Studying Muscle Regeneration in Mice

Background

A longstanding goal in regenerative medicine is to reconstitute functional tissus or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised.

Methods

We have used transgenic Tg:Pax7nGFP and Flk1^GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex^®.

Results

We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl₂), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a “dead zone” devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl₂ but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.

Conclusions

Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Furthermore, we show that histological parameters are not wholly sufficient to declare that regeneration is complete as molecular alterations (e.g. cycling SCs, cytokines) could have a major persistent impact.     

Phenotypic plasticity in life‐history traits of Daphnia galeata in response to temperature – a comparison across clonal lineages separated in time

Climatic changes are projected to result in rapid adaptive events with considerable phenotypic shifts. In order to reconstruct the impact of increased mean water temperatures during past decades and to reveal possible thermal micro‐evolution, we applied a resurrection ecology approach using dormant eggs of the freshwater keystone species Daphnia galeata. To this end, we compared the adaptive response of D. galeata clones from Lake Constance of two different time periods, 1965–1974 (“historical”) versus 2000–2009 (“recent”), to experimentally increased temperature regimes. In order to distinguish between genetic versus environmentally induced effects, we performed a common garden experiment in a flow‐through system and measured variation in life‐history traits. Experimental thermal regimes were chosen according to natural temperature conditions during the reproductive period of D. galeata in Central European lakes, with one additional temperature regime exceeding the currently observable maximum (+2°C). Increased water temperatures were shown to significantly affect measured life‐history traits, and significant “temperature × clonal age” interactions were revealed. Compared to historical clones, recent clonal lineages exhibited a shorter time to first reproduction and a higher survival rate, which may suggest temperature‐driven micro‐evolution over time but does not allow an explicit conclusion on the adaptive nature of such responses.     

Cryptosporidium parvum increases intestinal permeability through interaction with epithelial cells and IL‐1β and TNFα released by inflammatory monocytes

Intestinal epithelial cells form a single layer separating the intestinal lumen containing nutriments and microbiota from the underlying sterile tissue and therefore play a key role in maintaining homeostasis. We investigated the factors contributing to the alteration of the epithelial barrier function during Cryptosporidium parvum infection. Infected polarized epithelial cell monolayers exhibit a drop in transepithelial resistance associated with a delocalization of E‐cadherin and β‐catenin from their intercellular area of contact, the adherens junction complex. In neonatal mice infected by C. parvum, the increased permeability is correlated with parasite development and with an important recruitment of Ly6c⁺ inflammatory monocytes to the subepithelial space. TNFα and IL‐1β produced by inflammatory monocytes play a key role in the loss of barrier function. Our findings demonstrate for the first time that both the parasite and inflammatory monocytes contribute to the loss of intestinal barrier function during cryptosporidiosis.     

Determination of protein‐only RNase P interactome in Arabidopsis mitochondria and chloroplasts identifies a complex between PRORP1 and another NYN domain nuclease

The essential type of endonuclease that removes 5′ leader sequences from transfer RNA precursors is called RNase P. While ribonucleoprotein RNase P enzymes containing a ribozyme are found in all domains of life, another type of RNase P called ‘PRORP’, for ‘PROtein‐only RNase P’, is composed of protein that occurs only in a wide variety of eukaryotes, in organelles and in the nucleus. Here, to find how PRORP functions integrate with other cell processes, we explored the protein interaction network of PRORP1 in Arabidopsis mitochondria and chloroplasts. Although PRORP proteins function as single subunit enzymes in vitro, we found that PRORP1 occurs in protein complexes and is present in high‐molecular‐weight fractions that contain mitochondrial ribosomes. The analysis of immunoprecipitated protein complexes identified proteins involved in organellar gene expression processes. In particular, direct interaction was established between PRORP1 and MNU2 a mitochondrial nuclease. A specific domain of MNU2 and a conserved signature of PRORP1 were found to be directly accountable for this protein interaction. Altogether, results revealed the existence of an RNA maturation complex in Arabidopsis mitochondria and suggested that PRORP proteins cooperated with other gene expression factors for RNA maturation in vivo.     

Long‐term evolution of the natural isolate of Escherichia coli 536 in the mouse gut colonized after maternal transmission reveals convergence in the constitutive expression of the lactose operon

In vitro experimental evolution has taught us many lessons on the molecular bases of adaptation. To move towards more natural settings, evolution in the mice gut has been successfully performed. Yet, these experiments suffered from the use of laboratory strains as well as the use of axenic or streptomycin‐treated mice to maintain the inoculated strains. To circumvent these limitations, we conducted a one‐year experimental evolution in vivo using a natural isolate of E. coli, strain 536, in conditions mimicking as much as possible natural environment with mother‐to‐offspring microbiota transmission. Mice were then distributed in 24 independent cages and separated into two different diets: a regular one (chow diet, CD) and high‐fat and high‐sugar one (Western Diet, WD). Genome sequences revealed an early and rapid selection during the breastfeeding period that selected the constitutive expression of the well‐characterized lactose operon. E. coli was lost significantly more in CD than WD; however, we could not detect any genomic signature of selection, nor any diet specificities during the later part of the experiments. The apparently neutral evolution presumably due to low population size maintained nevertheless at high frequency the early selected mutations affecting lactose regulation. The rapid loss of lactose operon regulation challenges the idea that plastic gene expression is both optimal and stable in the wild.     

Role of HIV-1 Vpr-induced apoptosis on the release of mitochondrial lysyl-tRNA synthetase

Mitochondrial lysyl-tRNA synthetase (LysRS) is thought to be involved in the specific packaging of tRNA(3)(Lys) into HIV-1 viral particles. The HIV-1 auxiliary viral protein Vpr is an apoptogenic protein that affects the integrity of the mitochondrial membrane and has also been reported to interact with LysRS. In the present study, we show that HIV-1 Vpr expressed in E. coli and purified to homogeneity does not interact specifically with LysRS and does not impact its aminoacylation activity. However, we also show that the mitochondrial localization of LysRS in HeLa cells is altered after addition of Vpr in the culture medium. These results suggest that HIV-1 Vpr fulfills an essential role in the process of packaging of mitochondrial LysRS.     

Folding and turnover of human iron regulatory protein 1 depend on its subcellular localization

Aconitases are iron-sulfur hydrolyases catalysing the interconversion of citrate and isocitrate in a wide variety of organisms. Eukaryotic aconitases have been assigned additional roles, as in the case of the metazoan dual activity cytosolic aconitase-iron regulatory protein 1 (IRP1). This human protein was produced in yeast mitochondria to probe IRP1 folding in this organelle where iron-sulfur synthesis originates. The behaviour of human IRP1 was compared with that of genuine mitochondrial (yeast or human) aconitases. All enzymes were functional in yeast mitochondria, but IRP1 was found to form dense particles as detected by electron microscopy. MS analysis of purified inclusion bodies evidenced the presence of human IRP1 and alpha-ketoglutarate dehydrogenase complex component 1 (KGD1), one of the subunits of alpha-ketoglutarate dehydrogenase. KGD1 triggered formation of the mitochondrial aggregates, because the latter were absent in a KGD1(-) mutant, but it did not efficiently do so in the cytosol. Despite the iron-binding capacity of IRP1 and the readily synthesis of iron-sulfur clusters in mitochondria, the dense particles were not iron-rich, as indicated by elemental analysis of purified mitochondria. The data show that proper folding of dual activity IRP1-cytosolic aconitase is deficient in mitochondria, in contrast to genuine mitochondrial aconitases. Furthermore, efficient clearance of the aggregated IRP1-KGD1 complex does not occur in the organelle, which emphasizes the role of molecular interactions in determining the fate of IRP1. Thus, proper folding of human IRP1 strongly depends on its cellular environment, in contrast to other members of the aconitase family.