Sarcomeres Morphology and Z-Line Arrangement Disarray Induced by Ventricular Premature Contractions through the Rac2/Cofilin Pathway

Figures

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  Figure 1. Heart weight, left ventricular mass, left ventricular size, and left ventricular ejection fraction between the RVOT-VPC group and the sham control group at baseline and 6-month follow-up. Heart weight of the RVOT-VPC group was bigger than the sham control group at the 6-month follow-up (p < 0.001) (A). The left ventricular end-diastolic diameter (LVEDD) of the RVOT-PVC group at the 6-month follow-up was significantly larger than that of the RVOT-PVC group at the baseline (p = 0.0022) and that of the sham control group at the 6-month follow-up (p = 0.0101) (B). The LV mass index of the RVOT-VPC group was significantly higher than that of the sham control group at the 6-month follow-up (p = 0.041) (C). There was a trend toward lower LV ejection fraction (LVEF) in the RVOT-PVC group compared to the sham control group at the 6-month follow-up (D). *: p < 0.05; ***: p < 0.001. Blue line to express the trend of difference between Sham control and RVOT-PVC groups at baseline; Red line to express the trend of difference between Sham control and RVOT-PVC groups at the 6-month follow-up.

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  Figure 2. Elevated serum cardiac troponin I level and cardiomyocyte enlargement developed in left ventricular myocardium following 6-month RVOT bigeminy VPCs. (A) Before VPC creating, serum cardiac troponin I levels were similar between RVOT-VPC group and sham control group. However, the RVOT-VPC group had higher serum cardiac troponin I level than the sham control group at the 6-month follow-up, although the difference did not reach statistical significance. The changes in cardiac troponin I level in the RVOT VPC group and sham control group were illustrated by the slopes of the red line and blue line, respectively. (B) Immunofluorescence study with wheat germ agglutinin stain for the cell membrane of cardiomyocytes showed that the RVOT-VPC group had significantly larger cardiomyocyte size than the sham control group at both the left ventricular septum (LVS) (p = 0.0043) and LV free wall (LVFW) (p = 0.0041). Round dot: Sham control group; Square dot: RVOT-VPC group. Each datum in this figure represents the average of 1000 cells per sample. MCSA: myocyte cross-sectional area. Scale bar = 75 μm. *: p < 0.05; **: p < 0.01.

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  Figure 3. Sarcomere morphology, shear angle at the Z-line and the distance between two Z-lines following 6-month RVOT bigeminy VPCs. (A) Immunohistological studies of left ventricular septal and free wall myocardial tissue showed organized sarcomeres (homogenous distribution of F-actin in the sarcomeres, Z-lines in parallel, and equal distance between Z-lines (stained with α-actinin)) in the sham control group but showed that the sarcomere morphology and Z-line arrangement became disarrayed (non-homogenous distribution of F-actin in the sarcomeres and Z-lines not in parallel and different distances between two Z-lines) and spindle-shaped actin accumulations (white arrow) in the RVOT-VPC group. (B–D). The distance between two Z-lines (sarcomere length) and the shear angle at the Z-line (an indicator of reduced Z-direction contraction) were significantly increased in the cardiomyocytes of RVOT-VPC group compared to sham control group at both locations of left ventricle. The length of sarcomeres was measured as the distance between neighboring Z-lines, i.e., the distance between the neighboring peak immunofluorescent intensities of α-actinin (C). The shear angle at the Z-line was defined as…

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  Figure 4. Immunohistological and immunoblotting studies of the expression of β-catenin in the RVOT-VPC group and sham control group at left ventricular septum (LVS A,C) and left ventricular free wall (LVFW B,D. The expression of β-catenin at intercalated discs significantly increased in the RVOT-VPC group than in the sham control group at LVS (A,C) and LVFW (B,D). In addition, the spindle-shape actin accumulation was located at the intercalated discs with β-catenin (insert) (white arrow at right upper panel in (A,B). DAPI staining for nuclei. Scale bar = 10 μm. *: p < 0.05 and **: p < 0.01. PC: positive control.

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  Figure 5. Five differentially expressed genes related to actin cytoskeleton signaling induced by 6-month RVOT bigeminy VPCs in the septum and free wall of left ventricle. Five genes that were differentially upregulated or downregulated in the RVOT-VPC group compared to the sham control group according to next generation sequencing analysis results were selected and studied. The expressions of RAC2, ACTG2, and ITGA4 were downregulated while the expressions of CYFIP2 and FGF16 were upregulated in the RVOT-VPC group compared to sham control group. *: p < 0.05 and **: p < 0.01.

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  Figure 6. The expression of Rac2, phosphorylation of cofilin and F-actin/G-actin ratio in the LV myocardium following 6-month RVOT bigeminy VPC. Immunoblotting study showed that the expressions of (A) Rac family small GTPase 2 (Rac2), (B) Ratio of p-cofilin/cofilin, and (C) F-actin/G-actin ratio in left ventricular (LV) septum and LV free wall were significantly decreased in the RVOT bigeminy VPC group compared to the sham control group at the 6-month follow-up. *: p < 0.05; **: p < 0.01. PC: positive control.

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  Figure 7. The expression of the Rac2, ratio of p-cofilin/cofilin and F-actin in the rat ventricular cardiomyocytes after silencing Rac2 gene. After silencing Rac2, the expression of Rac2 protein in the rat ventricular cardiomyocytes was significantly decreased compared to the control group (siCTL) without silencing Rac2 by immunoblotting study (A). The ratio of p-cofilin/cofilin in the rat ventricular cardiomyocytes was significantly lower in the siRac2 rat ventricular cardiomyocytes than the siCTL group (B). Flow cytometric study showed the mean fluorescence intensity (MFI) of F-actin was significantly lower in the siRac2 rat ventricular cardiomyocytes compared to the siCTL group (C). *: p < 0.05 and ***: p < 0.001. PC: positive control.

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  Figure 8. The expression of actin filament in terms of intensity and number of actin filaments in the rat ventricular cardiomyocytes with and without silencing Rac2 gene by confocal immunofluorescence study. (A) Silencing Rac2 gene to suppress Rac2 expression significantly changed the actin distribution in the rat ventricular cardiomyocytes. ß-catenin staining for cell membrane; DAPI staining for nuclei. (B) Three 10-μm lines were drawn at equal distance at three separate parts of cardiomyocyte and the mean immunofluorescence intensity and number of immunofluorescence peaks were calculated by using ImageJ (spot function) and these values were measured from 100 individual cells per sample in each of the four repeated experiments. (C) The intensity of actin (F-actin) and number of actin myofilaments per cardiomyocyte were significantly decreased by Rac2 siRNA. Scale bar = 20 μm. ***: p < 0.001.