Cardiac Overexpression of XIN Prevents Dilated Cardiomyopathy Caused by TNNT2 ΔK210 Mutation

Figures

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  FIGURE 1. Cardiac differentiation of TNNT2-ΔK210 mutant hESCs and the expression of XIN. (A) Schematics of optimized chemically defined cardiac differentiation protocol. (B) Quantitative PCR verification of XIRP1 expression in wild type (WT/WT), TNNT2-ΔK210 (heter: ΔK210/WT, homo: ΔK210/ΔK210) hESC-cardiomyocytes at day35 post cardiac differentiation. (C) Detection of XIN protein expression at day35 post differentiation by Western blotting. GAPDH was used as the loading control. (D), Immunostaining detection of XIN (green) and cTnT (red) in cultured WT/WT, WT/ΔK210 and ΔK210/ΔK210 cardiomyocytes. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01.

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  FIGURE 2. Overexpression of XINB partially rescued the DCM phenotype of TNNT2-ΔK210 cardiomyocytes. (A) Brightfield and fluorescence images of hESC-cardiomyocytes illustrating green fluorescence protein (GFP) expression after XINB-lentivirus transfection. Scale bars, 200 mm. (B) Relative XINB expression in WT/WT, WT/ΔK210, and ΔK210/ΔK210 cardiomyocytes determined by quantitative PCR. (C) Representative immunostaining of cTnT (red) in cultured WT/WT, WT/ΔK210, and ΔK210/ΔK210 cardiomyocytes after XINB overexpression. Cells transfected with lentivirus expressing GFP were used as control. Scale bars, 50 mm. (D) Quantification of relative cell size and (E) statistics of the relative disorganized sarcomeric pattern for WT/WT, WT/ΔK210, and ΔK210/ΔK210 cardiomyocytes after overexpression of XINB. (F) Percent of multinuclear cells increased after XINB overexpression in TNNT2-ΔK210 cardiomyocytes. n > 30, 3 lines in each group; data are represented as mean ± SEM. *P < 0.05.

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  FIGURE 3. XINB increased contractility of TNNT2-ΔK210 cardiomyocytes. (A) Representative video recording traces of spontaneous contraction of single WT/WT. (B) ΔK210/WT and (C) ΔK210/ΔK210 cardiomyocytes in control or XINB overexpression group. (D) Statistics of video recording intensities showed increased contractile forces after XINB overexpression in WT/WT (n = 29), ΔK210/WT (n = 30), and ΔK210/ΔK210 (n = 30) cardiomyocytes from 3 independent lines in each group. Relative expression of cardiac muscle contraction related genes (E) TNNT2, (F) ACTN2, and (G) MYH7 increased after XINB overexpression. Data are represented as mean ± SEM. *P < 0.05.

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  FIGURE 4. Expression of Xirp1 in the TNNT2-ΔK210 mice hearts. (A) Changes in temporal mouse Xirp1 expression in wild type and TNNT2-ΔK210 mutant mice. (B) Western blot analysis showed decreased level of XINprotein in Day 7 TNNT2-ΔK210 mice hearts. (C), Quantification of western blot signals in (B) and normalized to the loading control (β-Tubulin). Data are represented as mean ± SEM. *P < 0.05.

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  FIGURE 5. Cardiac-specific expression of XINB improved cardiac function of TNNT2-ΔK210 mice. (A) Schematic illustration of the design of XINB delivery through AAV9. (B) In vivo bioluminescence imaging of mice tested the efficiencies of AAV9-Luci with the cTnT promoter for cardiomyocyte-specific gene expression. 3-day-old neonatal mice were intraperitoneally injected with AAV9-Luci viruses (1 × 1012 viral genomes per mouse). Mouses without AAV injection were used as control. High: high dose. Low: low dose. (C) Representative M-mode echocardiography recordings of 3-months-old WT mice, AAV9-Luci injected TNNT2-ΔK210 mice, and AAV9-XINB injected TNNT2-ΔK210 mice. (D) Echocardiographic analysis of ejection fraction (EF), (E) fractional shortening (FS), (F) left ventricular end systolic internal diameter (LVID; s), and (G) left ventricular end diastolic internal diameter (LVID; d) of TNNT2-ΔK210 mice after XINB treatment (n = 5 per group). Data are represented as mean ± SEM. *P < 0.05, **P < 0.01.

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  FIGURE 6. Morphological studies showed cardiac-specific expression of XINB in TNNT2-ΔK210 mice rescued DCM phenotypes. (A), Representative H&E staining of the hearts from 3-months old control and AAV9-XINB injected TNNT2-ΔK210 mice. Masson’s trichrome staining showed less fibrosis in the mutant LV tissue when compared to control. (B) Quantification of interstitial fibrosis area in each group. (C,D) Wheat germ agglutinin (WGA; as an indicator of cardiomyocyte size) staining of left ventricle sections and quantification of myocyte sizes. Scale bars, 50 mm. (E) Ultrastructure of the left ventricular tissue sections from 3-months old mice revealed by transmission electron microscopy at ×6,000 magnification. AAV9-XINB treatment significantly improved the sarcomeric structure of TNNT2-ΔK210 mice. Data are represented as mean ± SEM. *P < 0.05, ***P < 0.001.