Propagated Endothelial Ca 2+ Waves and Arteriolar Dilation In Vivo: Measurements in Cx40 BAC –GCaMP2 Transgenic Mice

Abstract

To study endothelial cell (EC)– specific Ca 2+ signaling in vivo we engineered transgenic mice in which the Ca 2+ sensor GCaMP2 is placed under control of endogenous connexin40 (Cx40) transcription regulatory elements within a bacterial artificial chromosome (BAC), resulting in high sensor expression in arterial ECs, atrial myocytes, and cardiac Purkinje fibers. High signal/noise Ca 2+ signals were obtained in Cx40 BAC -GCaMP2 mice within the ventricular Purkinje cell network in vitro and in ECs of cremaster muscle arterioles in vivo. Microiontophoresis of acetylcholine (ACh) onto arterioles triggered a transient increase in EC Ca 2+ fluorescence that propagated along the arteriole with an initial velocity of ≈116 μm/s (n=28) and decayed over distances up to 974 μm. The local rise in EC Ca 2+ was followed (delay, 830±60 ms; n=8) by vasodilation that conducted rapidly (mm/s), bidirectionally, and into branches for distances exceeding 1 mm. At intermediate distances (300 to 600 μm), rapidly-conducted vasodilation occurred without changing EC Ca 2+ , and additional dilation occurred after arrival of a Ca 2+ wave. In contrast, focal delivery of sodium nitroprusside evoked similar local dilations without Ca 2+ signaling or conduction. We conclude that in vivo responses to ACh in arterioles consists of 2 phases: (1) a rapidly-conducted vasodilation initiated by a local rise in EC Ca 2+ but independent of EC Ca 2+ signaling at remote sites; and (2) a slower complementary dilation associated with a Ca 2+ wave that propagates along the endothelium.