Adeno-Associated Virus Serotype 9–Driven Expression of BAG3 Improves Left Ventricular Function in Murine Hearts With Left Ventricular Dysfunction Secondary to a Myocardial Infarction

Figure 1. rAAV9-BAG3 Improves LV Function in Mice With LV Dysfunction Secondary to Ligation of the Left Coronary Artery (A) Western blot analysis of tissue obtained on various days from mice after retro-orbital injection of myc−-tagged adeno-associated virus serotype 9 expressing BAG3 (rAAV9-BAG3). (B) Eight-week-old C57BL/6J were subjected to ligation of the left coronary artery (myocardial infarction [MI]) or sham surgery (Sham) (week 0, arrow A). The first 1 week post-surgical echocardiogram is indicated by arrow B. At 8 weeks post-surgery (arrow C), the MI and Sham groups were randomly assigned to receive 60 to 80 μl of either rAAV9-GFP (3.1 × 1012 genome copies [GC]/ml; Sham- or MI-GFP) or rAAV9-BAG3 (5 to 6.5 × 1013 GC/ml; Sham- or MI-BAG3) by injection into the retro-orbital venous plexus. Serial echocardiography was performed to determine the ejection fraction (EF), including 1 week before and 9 days and 3 weeks (arrow D) after injection. Data was analyzed using a 2-way analysis of variance for repeated measures. n = 14 Sham-GFP, n = 12 Sham-BAG3, n = 12 MI-GFP, and n = 13 MI-BAG3. ∗MI-GFP versus -BAG3 at week 9, p < 0.0001; †MI-GFP versus -BAG3 at week 11, p < 0.0001. There was no…

Figure 2. BAG3 Overexpression Improves Myocyte Contractility Post-MI Myocytes were isolated from the left ventricles and septum of the 3 groups of mice (n = 3 mice each) used in Figure 1, with care being taken to excise the infarct scar in MI-GFP and -BAG3 hearts. In Sham-GFP hearts, the area corresponding to the infarct scar in MI hearts was excised before myocyte isolation. Representative contraction traces obtained in freshly isolated myocytes (2 Hz, 37°C, 1.8 mmol/l [Ca2+]o) (please see the Methods section) are shown at baseline (A) and after 1 μmol/l of isoproterenol (Iso) (B). Contraction amplitudes (C), maximal shortening (D), and re-lengthening velocities (E) are shown for Sham-GFP, MI-GFP, and MI-BAG3 myocytes, both at baseline and in the presence of isoproterenol. There are 17 Sham-GFP, 18 MI-GFP, and 25 MI-BAG3 myocytes at baseline and 21 Sham-GFP, 18 MI-GFP, and 17 MI-BAG3 myocytes after isoproterenol. Differences between groups are analyzed with 2-way analysis of variance are given in the Results section. RCL = resting call length; other abbreviations as in Figure 1.

Figure 3. BAG3 Overexpression Improves [Ca2+]i Dynamics Post-MI [Ca2+]i transients were measured in fura-2 loaded myocytes (2 Hz, 37°C, 1.8 mmol/l [Ca2+]o) and representative traces are shown both at baseline (A) and after isoproterenol (1 μmol/l) (B). Summaries of systolic and diastolic [Ca2+]i(C) and [Ca2+]i transient amplitudes (D) are shown. Error bars are not shown if they fall within the boundaries of the symbols. There are 15 Sham-GFP, 16 MI-GFP, and 21 MI-BAG3 myocytes at baseline, and 11 Sham-GFP, 13 MI-GFP, and 18 MI-BAG3 myocytes after isoproterenol. Differences between groups are analyzed with 2-way analysis of variance and are given in the Results section. There are no differences in [Ca2+]i transients between Sham-GFP and MI-BAG3 myocytes. Abbreviations as in Figure 1.

Figure 4. BAG3 Overexpression Increases Peak ICa Amplitudes Post-MI L-type Ca2+ current (ICa) ([Ca2+]o 2 mmol/l) in 8 Sham-GFP, 8 MI-GFP, and 6 MI-BAG3 myocytes (3 mice each group) are measured at baseline (A) and after 1 μmol/l isoproterenol (B). Error bars are not shown if they fall within the boundaries of the symbols. Abbreviations as in Figure 1.