Effect of bortezomib on the efficacy of AAV9.SERCA2a treatment to preserve cardiac function in a rat pressure-overload model of heart failure

Abstract

Adeno-associated virus (AAV)-based vectors are promising vehicles for therapeutic gene delivery, including for the treatment for heart failure. It has been demonstrated for each of the AAV serotypes 1 through 8 that inhibition of the proteasome results in increased transduction efficiencies. For AAV9, however, the effect of proteasome inhibitors on in vivo transduction has until now not been evaluated. Here we demonstrate, in a well-established rodent heart failure model, that concurrent treatment with the proteasome inhibitor bortezomib does not enhance the efficacy of AAV9.SERCA2a to improve cardiac function as examined by echocardiography and pressure volume analysis. Western blot analysis of SERCA2a protein and reverse transcription-PCR of SERCA2a mRNA demonstrated that bortezomib had no effect on either endogenous rat SERCA2a levels nor on expression levels of human SERCA2a delivered by AAV9.SERCA2a. Similarly, the number of AAV9 genomes in heart samples was unaffected by bortezomib treatment. Interestingly, whereas transduction of HeLa cells and neonatal rat cardiomyocytes by AAV9 was stimulated by bortezomib, transduction of adult rat cardiomyocytes was inhibited. These results indicate an organ/cell-type-specific effect of proteasome inhibition on AAV9 transduction. A future detailed analysis of the underlying molecular mechanisms promises to facilitate the development of improved AAV vectors.

Figure 1. Echocardiography Reveals No Differences in Cardiac Function among Animals with Heart Failure that Were Treated with AAV9.SERCA2a Alone or with AAV9.SERCA2a and Bortezomib

A: Schematic illustration of the study design. B: There were no significant differences in septal and posterior wall thickness among the AAV9.Empty and AAV9.SERCA2a with and without bortezomib treated groups. C: There were significant decreases in LV end diastolic volume and LV end systolic volume in the AAV9.SERCA2a with and without bortezomib treatment, * = P<0.05.

Figure 2. Pressure-Volume Analysis Reveals No Differences in Cardiac Function among Animals with Heart Failure that Were Treated with AAV9.SERCA2a Alone or with AAV9.SERCA2a and Bortezomib

A and B: P-V loop tracings at baseline and during inferior vena cava occlusion. C: LV ejection fraction significantly decreased in systolic HF, #P<0.05 vs. sham. C and D: Gene transfer of AAV9.SERCA2a with and without bortezomib significantly increased LV ejection fraction and LV contractility, * = P<0.05. The measurements were done at two months post vector injection.

Figure 3. SERCA2a Protein Levels Are Not Different among Animals with Heart Failure that Were Treated with AAV9.SERCA2a Alone or with AAV9.SERCA2a and Bortezomib

A: Western blot analysis of SERCA2a expression. B: Quantification of the results in A. * = p<0.05. The measurements were done at two months post vector injection.

Figure 4. Bortezomib Treatment Does Not Affect Endogenous SERCA2a Expression and Fails to Increase Transduction by AAV9.SERCA2a

A: Rat SERCA2a and GAPDH expression were measured by RT-PCR with specific primers against rat SERCA2a and GAPDH. B: Human SERCA2a expression was measured with specific primers against human SERCA2a and normalized to rat GAPDH expression. C: AAV9 vector genomes were normalized to diploid genomes. The measurements were done at two months post vector injection.

Figure 5. Bortezomib Enhances AAV9 Transduction in HeLa Cells and Neonatal Rat Cardiomyocytes but Decreases Transduction in Adult Rat Cardiomyocytes

A: HeLa cells were pre-treated for 30 minutes with DMSO or bortezomib (1.25 μM) and infected with AAV2-Luciferase or AAV9-Luciferase (1e4 vg/cell). After 24h cells were lysed and firefly luciferase activity was measured. B: Neonatal Rat Cardiomyocytes were infected with AAV2-Luciferase, AAV6-Luciferase or AAV9-Luciferase (1e4 vg/cell) in the presence of DMSO or bortezomib (1 μM). Luciferase activity was measured 24h post-infection, C: Adult Rat Cardiomyocytes were infected with AAV2-Luciferase, AAV6-Luciferase or AAV9-Luciferase (1e4 vg/cell) in the presence of DMSO or bortezomib (1 μM). Luciferase activity was measured 48h post-infection. * = p<0.05.