Abstract 3668: PKN is Essential for Survival of Cardiac Myocytes During Ischemia/Reperfusion
PKN is a serine/threonine kinase whose catalytic domain is homologous to the catalytic domain of protein kinase C. We have shown previously that PKN stimulates hypertrophy in cardiac myocytes (CMs). Furthermore, PKN is activated in response to hypotonic swelling in CMs in vitro and ischemia/reperfusion (I/R) in the heart in vivo. However, the role of PKN in mediating death/survival of CMs during I/R is currently unknown. Adenovirus constructs harboring wild type PKN, dominant negative PKN (DNPKN) and shRNA-PKN were transduced into neonatal rat CMs. DN-PKN (1.9 fold) and shRNA-PKN (1.5 fold) induced apoptotic cell death in CMs, as determined by TUNEL assays. Furthermore, Ad-PKN (70% vs control 51%) increased, while Ad-DNPKN (31%) and Ad-shRNA-PKN (28%) decreased the viability of CMs after H2O2 treatment. Ad-PKN increased (1.50 fold), whereas Ad-shRNA-PKN decreased (0.58 fold) phosphorylation of αB-crystallin (Ser 59) in CMs. In order to evaluate the function of PKN in the heart in vivo, we generated transgenic mice with cardiac specific overexpression of constitutively active (CA) and DNPKN (Tg-CAPKN and Tg-DNPKN) and examined whether activation of PKN has a protective effect in the heart. Tg-CAPKN exhibited mild baseline hypertrophy but Tg-DN-PKN did not. Echocardiographic analysis indicated that cardiac function was comparable among Tg-CAPKN, Tg-DNPKN and non-transgenic (NTg) mice. Three month old mice were subjected to 45 min ischemia followed by 24 hr reperfusion. The size of myocardial infarction (MI)/area at risk (AAR) determined by Alcian blue and TTC staining showed that Tg-CAPKN hearts exhibited a smaller MI/AAR than NTg (15.1±5.2% vs 37.5±4.9%, p<0.01), whereas Tg-DNPKN had a greater MI/AAR (NTg vs. Tg-DNPKN, 62.5±9.3% vs 45.2±8.4%, p<0.05). TUNEL positive nuclei in the border of the ischemic area were significantly fewer in Tg-CAPKN (NTg vs. Tg-CAPKN, 0.96±0.24% vs. 0.30±0.15%, p<0.05), but more in Tg-DNPKN (NTg vs. Tg-DNPKN, 1.33±0.88% vs. 7.85±1.01%, p<0.001). Phosphorylation (Ser 45) of αB-crystallin was significantly increased in NTg hearts in response to I/R, but not in Tg-DNPKN hearts. Taken together, these results suggest that endogenous PKN plays a protective role in the mouse heart against I/R.