Abstract 236: Phosphorylation is a Critical Step in the Cardioprotection by Intracellular Delivery of Hsp27 in Ischemia/Reperfusion Heart Injury
Background: The purpose of the present study was to examine whether direct intracellular delivery of Hsp27 using protein transduction domain (PTD) could preserve cardioprotective effect and to determine the role of the phosphorylation of delivered Hsp27 in ischemia/ reperfusion (I/R) model.
Methods: Wild-type Hsp27 (Hsp27-wt) and its mutant proteins (Hsp27–3D and Hsp27–3A) in which specific serine phosphorylation residues (S15, S78, S82) were substituted with aspartic acids or alanines were expressed in E. coli and purified as TAT-fusion protein containing the TAT-PTD. The rat cardiomyocytes were treated with recombinant proteins and exposed to hypoxic stress. Cell viability and apoptotic cell death were examined. Using the left anterior descending coronary artery ligation model of Sprague-Dawley rats, fusion proteins (5 mg/kg) were administered by i.p. injection at 30 min before reperfusion and 3 h, 12 h, 24 h after surgery. Fractional shortening and 2D strain were measured by echocardiography. LV infarct was also determined from histological sections stained with Masson trichrome.
Results: Hsp27-wt transduction showed cytoprotective effect against the hypoxic stress and attenuated hypoxia-induced apoptosis and caspase-3 activity. Phosphory-lation of Hsp27-wt delivered into cells was markedly increased. A p38 MAPK inhibitor (SB203580) inhibited the phosphorylation of Hsp27-wt. While Hsp27–3D (mimics the phos-phorylated form) reduced caspase activity and apoptosis, Hsp27–3A (nonphosphorylative form) could not protect against hypoxic stress effectively. The infarct area was significantly different between control and Hsp27-wt-treated rats (control vs Hsp27-wt, 39.1% vs 29.5%, p<0.05). There was significant improvement of cardiac function with Hsp27-wt transduction (FS 15.6% vs 33.4%, p<0.05; 2D strain of myocardium 4.83 vs 9.59, p<0.05), with no significant difference between Hsp27-wt and Hsp27–3D, determined at 7 d after I/R.
Conclusion: In conclusion, the intracellular delivery of Hsp27 as therapeutic protein may represent a novel strategy of protein therapy for ischemic heart diseases. Furthermore, phosphorylation of Hsp27 may influence its protective effect against I/R heart injury.