Analysis of WT and E34Q Lmod2 expression in rat neonatal cardiomyocytes. (A) Violin plots of average GFP intensities in cardiomyocytes expressing GFP (turquoise), GFP-Lmod2 WT (WT, blue), and GFP-Lmod2 E34Q (E34Q, red), used for Tmod1 and thin filament length assessment (as in Fig. 5). (B) Scatter plot showing relationship and variability between levels of GFP-tagged proteins (average GFP intensity per cell, X axis) and thin filament lengths (average per cell, Y axis) in cells expressing GFP (turquoise), GFP-Lmod2 WT (WT, blue), and GFP-Lmod2 E34Q (E34Q, red). Pearson tests show correlation between Lmod2 levels and thin filament length (both WT and E34Q, P < 0.0001), but not between GFP alone and thin filament length (P = 0.219). The intercepts of GFP-Lmod2 WT and GFP-Lmod2 E34Q significantly differ from each other (P = 0.0006), and their slopes differ from GFP alone (P = 0.0092, P = 0.0036). (C) Scatter plot showing relationship between sarcomere (X) and thin filament lengths (Y) in cells expressing GFP (turquoise), GFP-Lmod2 WT (WT, blue), and GFP-Lmod2 E34Q (E34Q, red). The slopes of all three linear regression lines are significantly nonzero (R² = 0.61, R² = 0.67, R² = 0.61, P < 0.0001) and significantly different, GFP and WT (slope P = 0.0064), GFP and E34Q (intercept P < 0.0001), and WT and E34Q (intercept P < 0.0001). (D) Representative images of relaxed sarcomeres stained for GFP (green), endogenous Tmod1 (blue), and F-actin (phalloidin, red). Yellow arrowheads indicate pointed ends of thin filaments, and red arrowheads show Z-lines. (E) Results of nested one-way ANOVA and Tukey multiple comparison tests of three independent thin filament measurement data. (F) Comparison of Tmod1 (red) intensities in cells expressing GFP-Lmod2 WT and E34Q (green, marked with *) to the neighboring non-transduced cardiomyocytes (no GFP-Lmod2, marked with**). Example of 60% reduction of Tmod1 levels in GFP-Lmod2 WT cardiomyocytes (left panels) and no change in GFP-Lmod2 E34Q cardiomyocytes (right panels). Scale bars are 1 μm.