Fig. 6: Hemodynamic analysis of VHH2 B4B4-T2A-ZsGreen AAV9-treated mice versus control (ZsGreen AAV9).
From: Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure
a AAV9 vector design. Left ITR and Right ITR refer to the left and right inverted terminal repeat, TNT455 promoter is the Troponin-T heart-specific promoter, VHH B4 is the VHH B4 intrabody gene sequence, GS is a DNA sequence that encodes a glycine-serine linker peptide (GGGSGGGSGGGSGGGS), T2A is the T2A peptide encoded gene sequence, ZsGreen represents the ZsGreen reporter gene sequence. b Overview of the procedure: MLP−/− mice were injected with vehicle ZsGreen AAV9 (n = 7) or VHH2 B4B4-T2A-ZsGreen AAV9 (n = 5) and hemodynamic parameters were assessed in acute terminal experiments 14 days later. c–e Individual data points (open circles) and average values (bar graph) of contractility (pressure increase velocity (dP/dTmax), relaxation (pressure decrease velocity dP/dTmin), and relaxation time (τ). Significant differences are indicated and evaluated using two-tailed student t-test. Error bars represent the standard error of the mean (SEM). Significance level between groups are indicated: *P-value < 0.05, **P-value < 0.01. (exact P-values in Supplementary Table 5). f Western blot analysis of heart samples of AAV9 ZsGreen control mice (ZsG) (n = 7) and the AAV9 VHH2 B4B4-T2A-ZsGreen (VHH2 B4B4-T2A-ZsG) group (n = 7). Membranes were stained with anti-VHH and anti-GAPDH, (Top) and with anti-PLN and anti-GAPDH (bottom). Pentameric PLN is indicated as PLN5. g Liver (top) and quadriceps (bottom) tissue samples from mice of the VHH2 B4B4-T2A-ZsGreen group (VHH2 B4B4-T2A-ZsG). A liver (top) or quadriceps (bottom) sample of mouse #7 of the control group (ZsG) was added as negative control and a heart sample from mouse #7 of the AAV9 VHH2 B4B4-T2A-ZsGreen treated group was used as a positive control (indicated above the lane). Arrows labelled “VHH2 B4B4” and “NS”, mark the bands for VHH2 B4B4 and a faint non-specific band present in all samples including the negative control. Sample identities are indicated above the lanes for all blots. The sizes of the molecular weight marker “M” are noted on the right side of the membrane with the primary antibody used to stain the membrane. Source data are provided as a Source Data file.
