Proteomic analysis of pharmacologically preconditioned cardiomyocytes reveals novel phosphorylation of myosin light chain 1

Proteomic analysis of rabbit ventricular myocytes revealed a novel posttranslational modification to myosin light chain I (MLCl), consisting of phosphorylation at two sites. Subproteomic extraction to isolate myofilament-enriched fractions enabled determination of the extent of phosphorylation, which increased from 25.7 +/- 1.6% to 34.0 +/- 2.7% (mean +/- SE. n=4; P < 0.05) after adenosine treatment at levels sufficient to pharmacologically precondition the myocytes (100 mu mol/L). Mass spectrometry of MLCl tryptic digests identified two peptide fragments modified by phosphorylation. These two phosphopeptides were characterized by peptide mass fingerprinting to determine the phosphorylation sites within rabbit ventricular MLCl, which correspond to Thr69 and Ser200 of rat MLCl, and to Thr64 and Ser194 or 195 of human MLCl. This proteomic analysis of preconditioned myocardium has revealed a previously unsuspected in vivo posttranslational modification to MLCl.