Resumen
2. La sostenibilidad fiscal en el enfoque tradicional
Figure 1 Absence of LAP2α in mice causes deregulation of the muscle stem cell pool. (a) Immunostainig of isolated satellite cells reveals LAP2α expression in Pax7-positive cells (bar = 20 μm). (b) Flow cytometric analysis of skeletal muscle tissue shows an increase in the number of myofibre-associated CD45-Sca1-Mac1- and CXCR4+β1-integrin+ (CSM4B) cells within the parent CD45-Mac1-Sca1- satellite cell population in young Lap2α-/- mice (aged 10 weeks) compared to their wild type (WT) littermates (n = 5 WT + 6 knockout mice). The number of CSM4B satellite cells in Lap2α-/- skeletal muscle levels down to WT values by the age of 6 months (adult mice) and continues to decrease with age at a rate similar to the WT (n = 5 littermate pairs of each age, p<0.05 ANOVA for (a) WT young vs. KO young, (b) WT young vs. WT old, (c) KO young vs. KO adult and old; bars represent standard error (SE)).
Figure 2 Loss of LAP2α in primary myoblasts predominantly causes downregulation of genes.
Volcano plot of genes differentially expressed in proliferating Lap2α-/- and WT primary myoblasts shows that only a small number of genes other than Lap2 itself is affected by the loss of LAP2α.
Figure 3 Lap2α-/- myoblasts exhibit delayed differentiation. Protein (a) and mRNA (b) analyses of sequentially expressed differentiation markers in LAP2α-deficient cells show delayed myoblast differentiation. The increase in Pax7 and decrease in MyoD protein levels (upper panels) are followed by the diminished expression of MyoD target genes and myogenic interaction partners such as MEF2c, desmin and myosin heavy chain (MyHC), as well as upregulation of the alternative basic myogenic factor Myf5 (lower panels). In contrast, the expression level of lamin A/C, the major binding partner of LAP2α, is not altered (upper panels). (c) Although Lap2α-/- myoblasts were able to differentiate into myotubes (left panels; bright field images, bar = 100 μm), immunostaining of differentiating myoblasts shows a slight delay in the expression of myosin heavy chain (right panels; confocal images, bar = 20 μm). Data were normalized to endogenous levels of actin in Western blot, and the housekeeping gene Hprt (Hypoxanthine-Guanine Phosphoribosyl Transferase) according to the Pfaffl method (39) in quantitative PCR analyses. WT mRNA levels at different time points were compared to the Day0 stage and each knockout sample was normalized to its corresponding WT time point (error bars represent SE of the WT/KO ratio, *p<0.05 ANOVA).
Means of 3-4 independent experiments are shown.
Figure 4 Lap2α-/- mice exhibit normal skeletal muscle regeneration. (a) H & E staining (Haematoxylin and eosin; left panels) and immunofluorescence analysis of notexin-injured LAP2α-deficient skeletal muscle, using embryonic MyHC (EMyHC – red) antibody (right panels), showing normal in vivo muscle regeneration in Lap2α-/- mice (bar = 20 μm). (b) The efficiency of muscle regeneration quantified at day 4 post injury by measuring the size of embryonic MyHC-containing myofibres within the damaged region and calculating the corresponding fibrotic indexes (n = 4
littermate pairs of each age; p<0.05 ANOVA for (a) young vs. old knockout mice, bars represent SE).
Figure 5 Absence of LAP2α in mice promotes fast myofibre phenotype in predominantly slow muscles. Soleus muscle of Lap2α-/- mice, (a) immunostained with anti-slow MyHC antibody (type I – depicted in brown, bar = 50 μm), reveals a myofibre shift towards faster phenotypes (b), without a change in fibre size distribution (c) (n = 8 littermate pairs, *p<0.05, ANOVA, bars represent SE).
Figure 6 LAP2α is required during initial phases of myoblast differentiation as well as adult muscle remodelling. (a) Semi-quantitative PCR analysis of cell and tissue lysates showing a downregulation of LAP2α in the presence of MCK and Cre recombinase, which, in contrast to adult skeletal muscle, are not expressed in proliferating myoblasts and non-muscle tissues such as spleen. (Samples were normalized to endogenous levels of GAPDH). (b) CD45-Sca1-Mac1 -CXCR4+β1-integrin+ (CSM4B) muscle fibre-associated satellite cell numbers in Lap2αNeo-fl/Neo-fl /Mck-Cre+ mice are similar to WT at all ages. (FACS analysis of whole muscle digests, n = 5 littermate pairs of each age; p<0.05 ANOVA for (a) young vs. old Lap2αNeo-fl/Neo-fl/Mck-Cre+ mice, bars represent SE). (c) Bright field images of Lap2αNeo-fl/Neo-fl/Mck-Cre+ myoblasts showing a lag at day 3 of the in vitro muscle differentiation (bar = 100 μm). (d) As a consequence of Mck promoter activation and Cre recombinase expression, LAP2α mRNA levels do not increase during differentiation in Lap2αNeo-fl/Neo-fl/Mck-Cre+ myoblasts, causing a delay in the expression of MyHC.
Due to high variability in Cre recombinase expression and the consecutive substantial variation in LAP2α levels, which hindered the statistical analysis of qPCR data, representative images from 3 separate experiments are shown. Data were analyzed as described in Figure 3. (e) Quantification of muscle tissue sections immunostained with anti-type I and anti-type II (a + x/d) MyHC-specific antibodies showing a myofibre shift towards an intermediate (type I and type II (a, x/d) negative) phenotype in Lap2αNeo-fl/Neo-fl/Mck-Cre+ soleus muscle. (n = 3 littermate pairs, *p<0.05, paired Students’ t-test, bars represent SE).
Table 1 Changes in gene expression associated with LAP2α loss in primary mouse myoblasts – selected GO-enriched microarray candidates.
Category Gene Symbol
Probe Set ID Name/GO-annotation Fold change p value (BayesT)
-203.53 0.00004 1RT/qRT/Ab 1421237_at -12.53 0.0004 1RT/qRT/Ab
Syne1 1421545_a_at synaptic nuclear envelope 1 -2.00 0.01 - Muscle stem cell determinants 1438946_at marker for paraxial mesoderm
derived ES cells with muscle regeneration potential
-1.50 0.03 -
Gja1 1415801_at gap junction protein, alpha
1;connexin protein family -1.50 0.009 Grem1 1425357_a_at gremlin 1; sonic hedgehog
signaling;BMP4 signaling
-1.80 0.01 -
Prrx1 1425526_a_at paired related homeobox 1 -1.60 0.02 - FGF, sonic hedgehog
signaling
Hey1 1415999_at hairy/enhancer-of-split related with YRPW motif 1; Notch signaling
1.80 0.03 1RT
Igf2r 1424112_at insulin-like growth factor 2 receptor
-1.50 0.03 - Map3k2 1438719_at mitogen-activated protein
kinase kinase kinase 2
-1.50 0.04 -
Extracellular matrix
Capg 1450355_a_at capping protein (actin filament), gelsolin-like
-1.40 0.02 - Coch 1423285_at coagulation factor C homolog
(Limulus polyphemus)
Pcolce 1448433_a_at procollagen C-endopeptidase enhancer protein
-1.60 0.01 -
1437165_a_at -1.20 0.04 -
Smoc2 1415935_at SPARC related modular
calcium binding 2 -3.80 0.05 - Timp2 1454677_at tissue inhibitor of
metalloproteinase 2
-1.30 0.02 - 1RT (semi-quantitative reverse transcriptase PCR), qRT (quantitative reverse transcriptase PCR), Ab (western blot and/or immunofluorescence).