A de novo Mutation in the MTUS1 Gene Decreases the Risk of Non-compaction of Ventricular Myocardium via the Rac1/Cdc42 Pathway

Figure 1. Familial pedigrees of NVM and information of the de novo mutation of MTUS1 c. 2617A->C. (A) Pedigree showing individuals with non-compaction of ventricular myocardium (NVM) and Rheumatic Heart Disease (RHD). The arrow shows the index patient with NVM carrying the MTUS1 mutation (c. 2617A->C). Squares, men; circles, women; black figures, individuals with NVM; oblique line figures, individuals with RHD; oblique line figures with a black question mark in the middle, suspicious RHD patient. (B) The echocardiographic image showing that the ratio of the thickness of the non-compact endocardial layer to the thickness of the compact epicardial layer was >2.0 (0.791/0.381). (C) Backward sequencing chromatogram of propositus and her family (sister, mother, and aunt). Parts of the nucleotide sequence are given below. The mutation site is pointed by an arrow (the position means c. 2617A->C). (D) Evolutionary conservation of c. 2617A->C mutation in all species. (E) Schematic representation of the structural organization shows human MTUS1 gene and ATIP3 (encoded by MTUS1 gene) protein regions. The de novo mutation of MTUS1 c. 2617A->C located in exon 6. Position of the mutant amino-acid…

Figure 2. Lentiviral vector validation and transfection. (A) Co-transfect with lentiviruses containing GFP (c. 2617A->C mutation, wild type, vector, respectively), 48 h after transfection, cell state and transfection efficiency of each group were observed under ordinary microscope and fluorescence microscope. (B) After 48 h transfection, the MTUS1 mRNA level expression was increased in the mutation and wild type group. (C) When GFP fluorescence occurs in mutation, wild type and vector group, the expression of flag-MTUS1 protein was detected to verify successful overexpression in HEK293 cells. FG-MT: flag-c. 2617A->C mutation, FG-WT: flag-wild type, FG-EV: flag-vector, Scale bar = 500 μm.

Figure 3. The stability of microtubules in HEK293 cells by immunofluorescence. (A) Images showing α-tubulin expression in four groups. The expression of α-tubulin in c. 2617A->C mutation group was significantly decreased than wild type group. (B) Images showing α/β-tubulin expression in four groups. Increased expression levels of α/β-tubulin in c.2617A->C mutation group were observed compared with wild type group. (C) Quantification of fluorescence intensity measurements of α-tubulin in four groups under the conditions described in (A). (D) The quantification of fluorescence intensity of α/β-tubulin in four groups under the conditions described in (B). ***P < 0.001; scale bar = 50 μm. Error bars show mean ± standard deviation in (C,D).

Figure 4. The c. 2617 A->C mutation increased PAR6 protein expression. (A) Images showing PAR6 protein (red) expression and location in four groups in cell division. Nuclei were stained by DAPI (blue). In Merged images, the location of PAR6 protein in c.2617A->C mutation group was abnormal, more partial to the side of daughter cells, compared with wild type group. (B) Quantification of fluorescence intensity of PAR6 in four groups under the conditions described in A. ***P < 0.001, Scale bar = 10 μm. Error bars show mean ± standard deviation.

Figure 5. The c. 2617 A->C mutation regulated PAR6 protein expression in association with Rac1/Cdc42 signaling. (A) Western blotting analysis of phosphorylated Rac1/Cdc42 protein in four groups. Total Rac1/cdc42 is shown as a loading control. In cell division, the expression level of phosphorylated Rac1/Cdc42 was decreased in c. 2617A->C mutation than that in wild type group. (B) Quantification of western blot measurements of p-Rac1/Cdc42 level (total Rac1/Cdc42 as control) in four groups under the conditions described in A. **P < 0.01; Error bars show mean ± standard deviation in (B).

Figure 6. The morphology of daughter cells in HEK293 cells transfected with c. 2617A->C mutation. (A) In the merge images, the arrows (yellow) represent the distance from the spindle to the daughter cell membrane respectively at the stage of cell division. (B) Quantification of the distance from the spindle to the daughter cell membrane respectively at the stage of cell division in four groups under the conditions described in (A). (C) The area of daughter cells in mutation group was obviously significantly larger than other groups. Blue, DAPI; Red, α-tubulin; Green, GFP. **P < 0.01; ***P < 0.001. Scale bar = 10 μm. Error bars show mean ± standard deviation in (B,C).