Zoxazolamine-induced stimulation of cardiomyogenesis from embryonic stem cells is mediated by Ca, nitric oxide and ATP release.

Ca-activated potassium (K) channels of small and intermediate conductance influence proliferation, apoptosis, and cell metabolism. We analysed whether prolonged activation of K channels by zoxazolamine (ZOX) induces differentiation of mouse embryonic stem (ES) cells towards cardiomyocytes. ZOX treatment of ES cells dose-dependent increased the number and diameter of cardiac foci, the frequency of contractions as well as mRNA expression of the cardiac transcription factor Nkx-2.5, the cardiac markers cardiac troponin I (cTnI), α-myosin heavy chain (α-MHC), ventricular myosin light chain-2 (MLC2v), and the pacemaker hyperpolarization-activated, cyclic nucleotide-gated 4 channel (HCN4). ZOX induced hyperpolarization of membrane potential due to activation of IK, raised intracellular Ca concentration ([Ca]) and nitric oxide (NO) in a Ca-dependent manner. The Ca response to ZOX was inhibited by chelation of Ca with BAPTA-AM, release of Ca from intracellular stores by thapsigargin and the phospholipase C (PLC) antagonist U73,122. Moreover, the ZOX-induced Ca response was blunted by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) as well as the specific P2Y antagonist MRS 2,179, suggesting purinergic receptor-stimulated signal transduction. Consequently, ZOX initiated ATP release from differentiating ES cells, which was inhibited by the chloride channel inhibitor NPPB and the gap junction inhibitor carbenoxolone (CBX). The stimulation of cardiomyogenesis by ZOX was blunted by the nitric oxide synthase (NOS) inhibitor l-NAME, as well as CBX and NPPB. In summary, our data suggest that ZOX enhances cardiomyogenesis of ES cells by ATP release presumably through gap junctional hemichannels, purinergic receptor activation and intracellular Ca response, thus promoting NO generation.