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. 2011 May-Jun;2(3):195-207.
doi: 10.4161/nucl.2.3.15731.

Post-natal myogenic and adipogenic developmental: defects and metabolic impairment upon loss of A-type lamins

Nard Kubben  1 , Jan Willem Voncken, Gonda Konings, Michel van Weeghel, Maarten Mg van den Hoogenhof, Marion Gijbels, Arie van Erk, Kees Schoonderwoerd, Bianca van den Bosch, Vivian Dahlmans, Chantal Calis, Sander M Houten, Tom Misteli, Yigal M Pinto
Affiliations

Post-natal myogenic and adipogenic developmental: defects and metabolic impairment upon loss of A-type lamins

Nard Kubben et al. Nucleus. 2011 May-Jun.

Abstract

A-type lamins are a major component of the nuclear lamina. Mutations in the LMNA gene, which encodes the A-type lamins A and C, cause a set of phenotypically diverse diseases collectively called laminopathies. While adult LMNA null mice show various symptoms typically associated with laminopathies, the effect of loss of lamin A/C on early post-natal development is poorly understood. Here we developed a novel LMNA null mouse (LMNA(GT-/-)) based on genetrap technology and analyzed its early post-natal development. We detect LMNA transcripts in heart, the outflow tract, dorsal aorta, liver and somites during early embryonic development. Loss of A-type lamins results in severe growth retardation and developmental defects of the heart, including impaired myocyte hypertrophy, skeletal muscle hypotrophy, decreased amounts of subcutaneous adipose tissue and impaired ex vivo adipogenic differentiation. These defects cause death at 2 to 3 weeks post partum associated with muscle weakness and metabolic complications, but without the occurrence of dilated cardiomyopathy or an obvious progeroid phenotype. Our results indicate that defective early post-natal development critically contributes to the disease phenotypes in adult laminopathies.

Keywords: LMNA; cardiac hypertrophy; differentiation; knock-out mouse; lamin A; laminopathies; muscular dystrophy.

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Figures

Figure 1
Figure 1
General phenotypical characterization of the LMNAGT−/− model. (A) Overview of the ROSAFARY genetrap (GT) sequence insertion in the LMNA gene. The GT consists of a splice acceptor (SA) containing βgeo-reporter with a polyA signal (bpA) and a PGK promoter driven hygromycin antibiotics resistance cassette surrounded by FRT sites and harbouring a splice donor (SD) site. 5′ and 3′ ends of the GT sequence are demarcated by long terminal repeats (LTR). FRT sites were not used for recombination in ES cells and the LMNAGT mice. Arrows indicate PCR primers used for detection of full length lamin A, lamin C and the LMNA-βgeo splicing product (See Fig. 1B). (B) Relative mRNA expression levels for full length lamin A, C and the LMNA-βgeo splicing product (n = 3). Levels are normalized to HPRT mRNA. (C) Western blot using polyclonal antibodies against LacZ (Abcam, Ab616), Lamin A/C (SantaCruz, Sc-6215), and a monoclonal antibody against α-Actin antibodies (Sigma, A781) on WT, LMNAGT+/− and LMNAGT−/− mouse embryonic fibroblasts protein extracts. (D) Immonofluorescence microscopy on WT, LMNAGT+/− and LMNAGT−/− mouse embryonic fibroblasts with antibodies against lamin A/C (SantaCruz, Sc-6215), emerin (Leica, cl.4G5), Lamin B1 (SantaCruz, Sc6217) and various NPC proteins (Abcam, mAB 414). DNA is stained with DAPI. Arrows indicate a local loss of lamin B1 and NPC proteins.
Figure 2
Figure 2
Embryonic and postnatal phenotypical characterization of the LMNAGT−/− mouse. (A) LMNA promoter activity is visualized by β-galactosidase staining in LMNAGT+/− embryo's (E8.0, E9.0, E11.0). LMNAGT+/− placental tissue is indicated by an asterisk. (B) β-galactosidase stained LMNAGT+/− embryo E11.0 tissue section (7 µm) counterstained with Azo Phloxine (magnification 2.5×), including a close-up image (magnification 5.0×) of the heart, the heart's outflow tract (OFT), liver and dorsal aorta (D. Aorta). (C) Macroscopical view of WT, LMNAGT+/− and LMNAGT−/− siblings 12 days post partum (PP12). (D) Body weight over time graph (PP2–PP18). Asterisks indicate a significant difference for LMNAGT−/− to LMNAGT+/− and WT littermates (N = 10, p < 0.05). (E) Survival curves for all three genotypes (N = 10) during the first 3 weeks post partum.
Figure 3
Figure 3
Transcriptome analysis of the LMNAGT−/− mouse. (A) mRNA expression levels of left ventricle cardiac tissue were analyzed by microarrays at PP5 and PP13. Scatter plots indicate logarithmic expression levels of mRNA of LMNAGT−/− mice (N = 2) vs. WT littermates (N = 2) at both time points. Twofold up- and downregulation borders are indicated by blue lines within the scatter plots. (B) A Venn diagram of >1.2 fold up or down regulated genes in LMNAGT−/− mice compared (N = 2) to WT siblings (N = 2) at PP5 and PP13. The number of genes and direction of change are indicated for all deregulated genes at PP5, PP13 and genes which are deregulated at both time points. (C) Heatmaps of mRNA expression levels of the most deregulated genes at PP5 and PP13 within the biological pathways of striated muscle contraction, hypertrophy, fatty acid beta oxidation, adipogenesis and electron transport chain. WT expression levels for each gene were set at 1.0 (black color code). Relative differences in expression levels for LMNAGT−/− mice are indicated as fold change values and color coded.
Figure 4
Figure 4
Post-natal cardiac hypertropy in the LMNAGT−/− mouse. (A) Haematoxylin and eosin (H&E) staining on left ventricle sections of WT and LMNAGT−/− mice (PP15). (B) Bar graph indicating left ventricle weight (LVW) standardized to tibia length over time (PP5–PP18) for all three genotypes (N = 5 per time point). (C) Bar graph of cardiac myocytes cross sectional area over time (PP2–PP18) for LMNAGT−/− and WT siblings (N = 5 per time point). (D) mRNA levels of proliferative markers Ki67 and PCNA and hypertrophic markers ANF and BNP, standardized for GAPDH. Asterisks in Figure 4 indicate significant differences for LMNAGT−/− vs. both LMNAGT+/− and WT values (N = 5, p < 0.05).
Figure 5
Figure 5
Post-natal skeletal muscle morphology and functioning in the LMNAGT−/− mouse. (A) Typical example of a haematoxylin and eosin (H&E) staining on quadriceps skeletal muscle sections of LMNAGT−/− and WT sibling (PP15). (B) Total quadriceps muscle weight normalized to tibia length assessed at PP9 and PP15 in LMNAGT−/−, LMNAGT+/− and WT mice (N = 8 each time point). (C) Quantification of the cross sectional area of quadriceps skeletal muscle myocytes for all 3 genotypes (PP9, PP15) (N = 8 each time point). (D) Assessment of skeletal muscle strength: time (in seconds) indicates the lag time until a mouse, hanging upside down, releases the grip; Asterisks in Figure 5 indicate significant differences for LMNAGT−/− vs. both LMNAGT+/− and WT values (p < 0.05; N = 4).
Figure 6
Figure 6
Adipogenic capacity of cells of the LMNAGT−/− mouse. (A) Quantification of 5 and 14 day old subcutaneous adipose tissue deposits normalized to tibia length (See Material & Methods; N = 5 per genotype for each timepoint). (B) Representative examples of Oil Red O staining at 16 days of differentiation for all genotypes. (C) Quantification of Oil Red O staining at 6, 10, and 16 days post induction. Asterisks in Figure 6 indicate significant differences for LMNAGT−/− vs. both LMNAGT+/− and WT values (p < 0.05; N = 3).
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