Titin is a new factor regulating arterial stiffness through vascular smooth muscle cell tone in male rats

Figures

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  FIGURE 1. Titin expression in aortic smooth muscle cells. (a) Titin immunostaining in aorta from wildtype (WT) rats. Titin (red), α‐Actin (green), and DAPI (blue). (b) Titin immunostaining in vascular smooth muscle cells (VSMCs) isolated from WT rats. Titin (red), α‐Actin (green), and DAPI (blue). (c) Titin immunostaining in isolated VSMCs. Titin (green) and DAPI (blue). Scale bars: Aorta, 50 μm; VSMC, 100 μm. (d) Smooth muscle titin different mRNA splicing patterns between WT and Rbm20 knockout (KO) detected by RT‐PCR. (e) Western blot analysis of titin isoform expression in aortic smooth muscle. The T2 band corresponds to a fragment of titin encompassing titin's A‐band section and ~ 100–200 kDa its distal I‐band section (Opitz et al., 2004).

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  FIGURE 2. Rbm20 KO reduces stiffness of VSMCs. (a) Computed elastic stiffness of individual VSMCs, as determined by atomic force microscopy (AFM) measurements in WT and Rbm20 KO rats. n = 40 total cells from 4 rats per group. (b) Distribution of force as a function of indentation in WT and Rbm20 KO VSMCs measured by AFM. n = 35–43 cells from 4 rats per group. (c) Elastic stiffness of individual VSMC after re‐expression of RBM20 in Rbm20 KO VSMCs by adenovirus‐RBM20 infection. Adenovirus‐GFP served as a negative control. n = 35–68 cells per group. Bar graphs show mean ± SD. Data points represent measurements from individual cells. Significance determined by pairwise comparisons with unpaired Student's t test.

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  FIGURE 3. Rbm20 KO ameliorates angiotensin II (Ang II)‐induced hypertension and aortic stiffness. (a) Pulse wave velocity (PWV) measured by Doppler System. n = 5–10 animals per group. Diastolic blood pressure (b), systolic blood pressure (c), and mean blood pressure (d). n = 4–9 animals per group. Bar graphs show mean ± SD. Data points represent measurements from individual animals. Significance determined by one‐way ANOVA followed by the Tukey post hoc test.

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  FIGURE 4. Rbm20 KO does not affect vascular remodeling in response to Ang II treatment. (a) Schematic showing a simplified artery structure. Aorta sections (b) were stained and quantified for media‐to‐lumen ratio (c). Scale bar, 500 μm. n = 4–8 animals per group. Aorta sections were stained for fibrosis with trichrome staining (d) and quantified (e, f). Scale bar, 100 μm. n = 3–6 animals per group. Bar graphs show mean ± SD. Data points represent measurements from individual animals. Significance determined by one‐way ANOVA followed by the Tukey post hoc test.

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  FIGURE 5. Rbm20 Ko has little effect on the expression of contractile proteins in aorta. (a–e) Expression of phosphorylated myosin light chain (p‐MLC), myosin light chain (MLC), and α‐smooth muscle Actin in WT and KO control (saline) and Ang II‐treated groups with quantification. n = 3 animals per group. Bar graphs show mean ± SD. Data points represent measurements from individual animals. Significance determined by one‐way ANOVA followed by the Tukey post hoc test.

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  FIGURE 6. Rbm20 KO alleviates Ang II‐induced cardiac hypertrophy. (a) Representative echocardiographic pictures. Quantification of EF (ejection fraction) (b), FS (fractional shortening) (c), LVID;d (left ventricular end diastolic internal diameter) (d), LVID;s (left ventricular end systolic internal diameter) (e), IVS;d (interventricular septum dimension in diastole) (f), IVS;s (interventricular septum dimension in systole) (g), LVPW;d (left ventricular posterior wall thickness in diastole) (h), and LVPW;s (left ventricular posterior wall thickness in systole) (i). n = 5–22 animals per group. Bar graphs show mean ± SD. Data points represent measurements from individual animals. Significance determined by one‐way ANOVA followed by the Tukey post hoc test.

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  FIGURE 7. Rbm20 KO attenuates Ang II‐induced cardiac remodeling. (a) Wheat germ agglutinin staining of heart sections for cardiomyocyte size measurement. Scale bar, 50 μm. (b) Quantification of cell size. n = 106–119 total cells from 3 animals per group. (c) Trichrome staining of heart sections. Scale bar, 100 μm. (d) Quantification of fibrosis area from trichrome staining. n = 3–4 animals per group. (e) Hydroxyproline assay to measure the collagen content in hearts. n = 4–6 animals per group. Bar graphs show mean ± SD. Data points represent measurements from individual animals. Significance determined by one‐way ANOVA followed by the Tukey post hoc test.