Abstract 446: Synergistic Rescue of Failing Ventricular Cardiomyocytes through Dual S100A1/BARKct Adenoviral Gene Transfer
Background: Cardiac S100A1 and BARKct viral gene transfer have been shown to rescue failing myocardium in vitro and in vivo through restoration of defective sarcoplasmic reticulum calcium cycling and β-adrenergic signaling, respectively. In the current study we tested the hypothesis whether dual S100A1/BARKct viral gene transfer might exert synergistic therapeutic effects in failing ventricular cardiomyocytes in vitro.
Methods and Results: Ventricular cardiomyocytes were isolated from failing (FCM) and non-failing (NFCM’) adult rat hearts (n=3). FCM’s were transfected in culture either with Ad-S100A1-GFP (A1) and Ad-BARKct-GFP (B) or each virus alone for 6 hours with 10 pfu whereas Ad-GFP (G) transfected FCM’s and NFCM’s served as controls. Western blotting confirmed 2-fold overexpression S100A1 protein levels in A1- and A1/B-FCM’s and robust BARKct overexpression in B- and A1/B-FCM’s. Analysis of field-stimulated steady-state contractility by VED (n=30 cells in each group) showed normalized fractional shortening in A1-FCM’s but still impaired contractility in B-FCM’s compared to G-FCM’s and G-NFCM’s under basal conditions (2Hz, 2mM extracellular calcium). In contrast, A1-FCM’s showed only partial restoration of defective β-adrenergic (βAR) responsiveness after isoproterenol (10–6M) stimulation that was completely rescued in B-FCM’s compared to G-NFCM’s. Similar results were obtained for the assessment of calcium transient amplitudes in field-stimulated FURA2-AM loaded A1- and B-FCM’s. Importantly, only A1/B-FCM’s showed a complete rescue of contractility and calcium-cycling under basal and βAR-stimulated conditions compared with A1-, B-FCM’s and G-NFCM’s. Accordingly, analysis of βAR-stimulated (isoproterenol 10–6M) dependent PLB- and TnI-phosphorylation confirmed normalization of βAR downstream signaling only in B- and A1/B-FCM’s but not in A1-FCM’s compared to G-NFCM’s.
Conclusion: Dual S100A1/BARKct gene transfer exerts synergistic therapeutic effects in failing cardiomyocytes most likely due to the different molecular mechanisms of both molecules. Hence, dual S100A1/βARKct gene transfer might be a promising approach for in vivo gene therapy of heart failure.