Clin Res Cardiol 98, Suppl 1, April 2009

V56 - Dose-dependent AAV9-S100B mediated antihypertrophic heart failure gene therapy
E. Gao1, G. Qiu1, J. E. Rabinowitz1, S. T. Pleger2, M. Voelkers2, H. A. Katus2, N. Frey3, P. Most1
1Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, USA; 2Medizinische Klinik III, Kardiologie, Angiologie, Pulmonolgie, Universitätsklinikum Heidelberg, Heidelberg; 3Klinik für Kardiologie und Angiologie, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Kiel;
Introduction: Failing human myocardium exhibits increased cardiomyocyte S100B protein levels and transgenic cardiac S100B overexpression in mice protects from catecholamine mediated cardiac hypertrophy and dysfunction. We were therefore interested in the therapeutic potential of the antihypertrophic cardiac factor S100B to prevent cardiac remodeling in the context of postischemic heart failure development.
Methods and Results:
 Adult C57/B6 mice were subjected to cardiac ischemia-reperfusion (I/R) injury by transient LAD ligation resulting in progressive cardiac hypertrophy and contractile dysfunction (HF) with increased S100B expression similar to human HF. Cardiac S100B gene transfer was performed by intravenous application of AAV9-S100B and control AAV9-GFP virus 2 days post-I/R, each transgene under the control of a cardiomyocyte-specific promoter (n=10 animals per group). I.v. application of 1*1011, 3*1011 and 6*1011 total viral particles both of AAV9-S100B and AAV9-GFP resulted in a dose-dependent cardiac overexpression of S100B (+3.2-, 10.8 and 16.7-fold above the HF group) and homogenous left ventricular (LV) GFP expression as shown by Western blotting and immunofluorescence 6 weeks after gene delivery. Serial echocardiography (2, 4 and 6 weeks post-I/R) showed similar progressive LV remodeling and deterioration of LV function in all AAV9-GFP groups versus sham animals (data 6 weeks post-I/R: +2.4-fold enddiastolic diameter (EDD) and +2.1-fold posterior wall thickness (PWT) increase, +40% HW/BW ratio, -45% reduction in LV FS%, 30% mortality, n=7, P<0.05 vs. sham, P=n.s. among GFP groups). In contrast, 1*1011AAV9-S100B effectively prevented LV hypertrophy and attenuated the decline in LV function (+1.3-fold EDD*,† and +1.5-fold PWT*,† increase, +11% HW/BW*,†, -17% reduction in LV FS%*,‡ 20% mortality, n=8, *P<0.05 vs. GFP groups, P=n.s. vs. sham, P<0.05 vs.sham). In line with these results, 3-fold S100B overexpression significantly reduced LV and serum BNP mRNA and protein compared with AAV9-GFP groups. However, a higher S100B gene dosage resulted in excessive mortality (80%, 3*1011; 90%, 6*1011) and severely impaired LV function in surviving animals compared with AAV9-GFP and low-dose AAV9-S100B treated HF animals. Immunohistochemical TUNEL and cleaved caspase-3 western blotting 3-weeks post-I/R revealed significantly less apoptotic cardiomyocytes in the low-dose AAV9-S100B group compared with all AAV9-GFP animals but an excessive increase in apoptotic cardiomyocytes both in the intermediate and high S100B gene-dosage group (n=6). Analysis of PKCalpha and calcineurin activity, HDAC4 localization and apoptosis in angiotensin-II and phenylephrin treated AAV9-S100B and control virus incubated neonatal rat cardiomyocytes assessed by PKCalpha phosphorylation, MCIP mRNA RT-PCR analysis, HDAC4 cytosolic/nuclear fractionation and nuclear DNA fragmentation revealed a dose-dependent inhibition of PKC-alpha phosphorylation, MCIP mRNA and nuclear HDAC4 export. However, high S100B expression (>4-fold above AT-II and PE induced increase) resulted in excessive apoptosis and enhanced atrogin expression as a marker of cardiac atrophy.
Our study identifies S100B as an attractive target for antihypertrophic HF gene therapy but highlights a delicate balance between dosage of a potent antihypertrophic gene and therapeutic outcome manipulating cardiac trophism in the context of post-MI HF development.