46. In Vivo Adenoviral Gene Transfer of Activated PI3-Kinase Rescues Cardiac Dysfunction and Injury Induced by Ischemia-Reperfusion after Chronic Cardiac Akt Activation

Abstract

Acute activation of the serine-threonine kinase, Akt, is cardioprotective and reduces both infarction and dysfunction after ischemia-reperfusion injury (IRI). However, Akt is paradoxically and chronically activated in human advanced heart failure suggesting it may become maladaptive. To test the effects of chronic Akt activation, we generated transgenic mice (TG) with cardiac specific expression of either activated Akt (myr-Akt) or dominant-negative (dn) Akt and studied their response to global IRI in the ex vivo Langendorff perfusion model. We have previously reported that after IRI (20min Ischemia, 40min Reperfusion), none of the myr-Akt TG hearts recovered any function while littermate control (NTG) hearts recovered to 53.5|[plusmn]|3.5% of the baseline LV developed pressure (LVDP) (n=11 each, p<0.01). dn-Akt manifested a moderate reduction in functional recovery (34.4|[plusmn]|7.5%, n=5, p<0.001), consistent with a cardioprotective role for endogenous Akt. TTC staining and CPK release into the perfusate demonstrated dramatically increased infarct size in myr-Akt TG hearts (p<0.01). Biochemical analyses demonstrated that chronic Akt activation induces feedback inhibition of upstream activation of PI3Kinase (PI3K) through both proteasome-dependent degradation of insulin-receptor substrate-1 (IRS-1) and inhibition of IRS transcription. QRT-PCR demonstrated mRNA levels for both IRS-1 and IRS-2 were significantly reduced in myr-Akt TG hearts (68.7%, p<0.001 and 59.9%, p<0.005, respectively). To understand the functional significance of this, we performed in vivo cardiac gene transfer of constitutively active PI3K (Ad.BD110) that does not depend on IRS-1 for activation or control virus in myr-Akt TG mice. Three days after transfection, expression of BD110 was documented by immunoblotting and associated with a significant increase in PI3K activity (2.6 fold, p<0.05). No functional recovery was seen in myr-Akt TG mice transduced with control virus (Ad.GFP) after IRI. However, after Ad.BD110 infection, myr-Akt TG mice demonstrated significant functional recovering achieving a maximal LVDP of 34.5|[plusmn]|1.8% (n=7, p<0.01 compared to myr-Akt TG without BD110). CPK release from myr-Akt TG hearts was also dramatically reduced after IRI (0.015 fold, p<0.01 vs myr-Akt TG alone, p=NS vs NTG). Taken together, these data demonstrate that PI3K-dependent but Akt-independent effectors are required for full cardioprotection. These findings demonstrate a mechanism by which chronic Akt activation can become maladaptive in heart failure and suggest the importance of therapeutic strategies that target multiple PI3K-dependent pathways simultaneously.