Regulatory myosin light chain 2 (MLC2) is a 20 kDa protein located in the hinge region of the myosin molecule. MLC2 in smooth muscle must be phosphorylated in order to induce contraction. In cardiac muscle, MLC2 phosphorylation facilitates contraction and mediates inotropic responses to various neurohumoral stimuli. The aim of this study was to determine the relative roles of the Ca²⁺ -dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) in regulating the phosphorylation state of MLC2 in the heart. Cardiomyocytes were isolated from rat hearts by collagenase perfusion before stimulation. Phosphorylation of MLC2 was determined using charge gel electrophoresis and anti-ventricular MLC2 monoclonal antibody. The phosphorylation status of MLC2 is reported as phosphorylated MLC2 as percent of total MLC2. Data are presented as mean ± SEM. Calyculin A (MLCP inhibitor) increased MLC2-phosphorylation dose-dependently from 14.0%± 1.6% (0 M) to 39.8%± 3.8% (10^-7 M), p < 0.001, n = 6, with a log EC50 of -7.96, and time-dependently from 15.7%± 1.3% at 0 min to 39.4%± 1.8% at 80 min, p < 0.001, n = 6, with half maximal phosphorylation at 25 min and reaching a plateau at about 40 min. Preincubating cardiomyocytes with ML-9 (60 mM, 45 min; MLCK and

Rho kinase inhibitor) reduced basal MLC2 phosphorylation, but the effect was reversed by subsequent treatment with calyculin A (10^-7 M). Preincubating with staurosporine (10 mM, 45 min;a broad spectrum protein kinase inhibitor) removed all kinase activity. In conclusion, quiescent cardiomyocytes possess a staurosporine sensitive and ML-9 insensitive kinase, probably being Ca²⁺ -independent, phosphorylating MLC2 in addition to the Ca²⁺ -dependent MLCK.