α B Crystallin Translocation and Phosphorylation: Signal Transduction Pathways and Preconditioning in the Isolated Rat Heart

In this program of studies we have characterized in detail the translocation (assessed by Triton-insolubility) and phosphorylation (using serine-45 or -59 phosphospecific antibodies) of α B crystallin during myocardial ischemia [both with or without ischemic preconditioning (IPC)]. Pharmacological activators and inhibitors allowed us to characterize the signaling pathways involved in α B crystallin phosphorylation during ischemia. Ischemic preconditioning alone caused 30% of the heartʹs α B crystallin pool to translocate, providing a significant translocation «head-start» in protected tissue. This enhanced translocation is coupled with increased (3-fold) α B crystallin phosphorylation at both serine residues. The possible role of α B crystallin in the protection afforded by ischemic preconditioning is supported by the signal transduction data; which showed preconditioning-induced α B crystallin phosphorylation can be blocked by tyrosine kinase inhibition (using genistein) and by p38 MAP kinase or PKC inhibition (using SB203580 or bisindolylmaleimide, respectively). The activation of both p38 MAP kinase and PKC are recognized requirements for the induction of preconditioning and their inhibition is known to block protection. Western immunoblotting analysis after isoelectric focusing electrophoresis, confirmed the observations made with the phosphospecific antibodies; but also showed that 27±4% of total cardiac crystallin was phosphorylated after 30 min of ischemia. α B crystallin exists as large polymeric aggregates in cardiac tissue under basal conditions (≅1 MDa as determined by gel filtration chromatography). We induced phosphorylation of α B crystallin during aerobic perfusion by the administration of phenylephrine. However this treatment did not alter the molecular aggregate size of α B crystallin. It appears that α B crystallin molecular aggregate size is not simply regulated by phosphorylation.α B crystallin may have a role to play in the myocardial protection induced by ischemic preconditioning, as both translocation and phosphorylation are both accelerated and enhanced by ischemic preconditioning.