LMNA Mutations G232E and R482L Cause Dysregulation of Skeletal Muscle Differentiation, Bioenergetics, and Metabolic Gene Expression Profile

Figures

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  Figure 1. Validation of experimental model: (A) Verification of transgene hLMNA expression in C2C12 mouse myoblasts: the cells were stained with anti lamin A antibody recognizing only human lamin A. LMNA G232E/R482L shows aggregates and nuclear blebbing in some of the transduced cells as a result of the mutation; (B) stimulation of myogenic differentiation resulted in myotubes formation in all transgene lines (scale bar = 200 μm); (C) hLMNA mRNA expression tested in transgene C2C12 myoblasts and in differentiated myotubes (n = 3; * p < 0.05); (D) nuclei in myotubes demonstrate hLMNA-positive staining (scale bar = 100 μm).

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  Figure 2. G232E/R482L-LMNA—induced alterations in functional properties of C2C12 myoblasts: (A) Estimation of the cell population doubling time for C2C12 cell lines, transduced with WT-LMNA, G232E-LMNA, and R482L-LMNA at three time points until 72 h. Values are mean ± SEM (n = 4). ** p < 0.01; (B) mRNA expression of the genes that regulate the early steps of myogenic differentiation in C2C12 myoblasts. (n = 4; * p < 0.05; ** p < 0.01; *** p < 0.001); (C) GSEA enrichment plots of the hallmark myogenesis pathway over ranked genes between all possible pairs of C2C12 myoblasts: WT versus G232E-LMNA, WT versus R482L-LMNA, and R482L-LMNA versus G232E-LMNA. (p-adjusted < 0.001, normalized enrichment scores (NES) are shown); (D) number of up- and downregulated differentially expressed genes (DEGs) found in all three pairs (log2 fold change > 1, p-adj < 0.01); (E) Venn diagrams of upregulated DEGs.

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  Figure 3. Myogenesis in transgene cultures. (A) Fusion coefficient calculated as a percent of nuclei incorporated in differentiated myotube in independent photographs; n = 8; *** p < 0.001, t-test; (B) width of myotubes, measured in arbitrary units in 5–6 independent photographs; the number of myotubes on each photo was 10–20; *** p < 0.001, t-test; floating bars are shown with mean, minimum and maximum values; (C) diagram illustrate the fractions of “massive” and “overage” myotubes in cultures; * p < 0.05; (D–F) The mRNA expression dynamics of genes that regulate myoblast fusion (Mymk, Mymx) and of myogenic regulatory factor Myog (n = 4; mean + SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; Mann–Whitney test); (G–I) the mRNA expression dynamics of fiber-specific genes: fast glycolitic fiber Myh1 isoform; oxidative slow fiber Myh7 isoform; embryonic myosin isoform Myh3; (n = 4, mean + SEM, * p < 0.05; ** p < 0.01; Mann–Whitney test). (J) Western blotting analysis for total myosin (slow (antibody interact with slow Myh7) and fast (antibody interact with fast Myh1/Myh2) proteins in cultures stimulated with myogenic differentiation media at day 4 after induction; (K,L) Results of gene set…

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  Figure 4. Metabolic profiles of C2C12 myotubes bearing lentiviral WT/G232E/R482L-LMNA. (A–H): Determination of mitochondrial bioenergetic parameters from OCR profile. (A) OCR traces for myotubes expressing WT/G232E/R482L-LMNA; (B–G) Indices of mitochondrial respiratory function: (B) maximal respiratory capacity; (C) spare respiratory capacity; (D) ATP-linked respiration; (E) proton leak respiration; (F) coupling efficiency; (G) cell respiratory control ratio; (H) OCR/ECAR ratio at maximal respiration. OCR was measured at the same time as ECAR. (I–L): Determination of glycolytic function. (I) Kinetic profiles of ECAR in myotubes expressing WT/G232E-LMNA/R482L-LMNA. ECAR was measured in real time, as indicated. Indices of glycolytic pathway activation calculated from ECAR profile: (J) Maximal glycolysis and (K) glycolytic reserve capacity. All data are calculated from 6–8 Seahorse microplate wells and are normalized to total protein in each well. * p < 0.05; ** p < 0.01 *** p < 0.001; two-tailed Student’s t-test. All floating bars are shown with mean, minimum and maximum values. (L) Heat map of NES values after GSEA analysis represents metabolic pathways found up- or down-regulated in…