Frequency-Dependent Activation of a Constitutive Nitric Oxide Synthase and Regulation of Contractile Function in Adult Rat Ventricular Myocytes

Abstract

Cardiac myocytes have recently been shown to express a constitutive Ca²⁺-sensitive isoform of NO synthase (NOS3), although the mechanism(s) responsible for activation of NOS3 and its physiological function remain to be determined. Since the activity of NOS3 is known to be regulated in part by the intracellular Ca²⁺ activity ([Ca²⁺]_i) in endothelial cells, we determined whether increasing myocyte [Ca²⁺]_i by uniform electric field pacing was accompanied by an increase in NOS3 activity, detected as nitrite accumulation in the medium. A higher [Ca²⁺]_i with increasing pacing frequencies was shown to be accompanied by a time-dependent accumulation of nitrite in medium that bathed adult rat ventricular myocytes stimulated at 3 Hz. Nitrite release by paced cells was significantly attenuated by treatment with either the NO synthase inhibitor nitro-l-arginine (L-NA, 1 mmol/L) or the intracellular Ca²⁺ chelator BAPTA-AM (20 μmol/L). Paced myocytes also exhibited a frequency- and time-dependent increase in intracellular cGMP content that could be inhibited significantly by either L-NA or the soluble guanylate cyclase inhibitor LY83583 (5 μmol/L). To determine whether the increase in NOS3 activity with pacing affected contractile function, myocytes were sequentially paced at frequencies from 0.5 to 3 Hz. Methylene blue, L-NA, and LY83583 all increased the amplitude of shortening of myocytes paced at 3 Hz. Furthermore, a significantly greater positive inotropic response to high extracellular Ca²⁺ (3 mmol/L) was demonstrated by myocytes pretreated with L-NA compared with control cells. These data indicate that myocyte NOS3 activity is regulated in part by [Ca²⁺]_i, whether induced by changes in pacing frequency or [Ca²⁺]_o, and depresses myocyte contractile responsiveness to higher stimulation frequencies.