Propagated Endothelial Ca ²⁺ Waves and Arteriolar Dilation In Vivo

Abstract

Abstract-To study endothelial cell (EC)-specific Ca 2ϩ signaling in vivo we engineered transgenic mice in which the Ca 2ϩ 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 2ϩ 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 2ϩ fluorescence that propagated along the arteriole with an initial velocity of Ϸ116 m/s (nϭ28) and decayed over distances up to 974 m. The local rise in EC Ca 2ϩ was followed (delay, 830Ϯ60 ms; nϭ8) by vasodilation that conducted rapidly (mm/s), bidirectionally, and into branches for distances exceeding 1 mm. At intermediate distances (300 to 600 m), rapidly-conducted vasodilation occurred without changing EC Ca 2ϩ , and additional dilation occurred after arrival of a Ca 2ϩ wave. In contrast, focal delivery of sodium nitroprusside evoked similar local dilations without Ca 2ϩ 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 2ϩ but independent of EC Ca 2ϩ signaling at remote sites; and (2) a slower complementary dilation associated with a Ca 2ϩ wave that propagates along the endothelium. 2ϩ signaling is implicated in regulating the resistance microvasculature. [1][2][3][4][5] Conducted electrical signals travel for millimeters along the vessel wall, 6 mediate coordinated vasomotor responses to localized stimuli, 7-9 and involve distinct Ca 2ϩ signals within smooth muscle (SM) and ECs. For example, arteriolar dilation in response to the endothelium-dependent vasodilator acetylcholine (ACh) involves an increase in ECs 2 and decrease in SM 10 Ca 2ϩ . Although recent intravital studies have demonstrated a crucial role for the endothelium in conducted vasodilation, 9 the extent to which Ca 2ϩ signals are transmitted along the arteriolar wall and the mechanisms underlying Ca 2ϩ transmission are controversial. Some studies indicate that EC Ca 2ϩ responses to dilatory stimuli are localized, 1,2 whereas others indicate that Ca 2ϩ signals can travel along the endothelium for a millimeter or more. 5,11 The majority of information concerning microvascular Ca 2ϩ signaling has been derived from isolated vessels studied in vitro, 4,5,12 largely because of the difficulty of selectively loading ECs or SM cells with Ca 2ϩ sensitive dyes in vivo. 1,10 A fundamental limitation to isolated arterioles is their disconnection from networks in which they normally reside. The extent to which manipulation of arterioles to obtain dye loading and the loss of physiological parameters (eg, pressure or flow) alter Ca 2ϩ signaling is unknown. Therefore, alternative approaches are necessa...

Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries

“…To determine whether TRPV4 sparklets potentiate Ca 2+ release from the ER in PAs, Ca 2+ signals were recorded in PAs from mice that express the Ca 2+ biosensor GCaMP2 selectively in ECs 27, 28. The effect of GSK101 on the activity of Ca 2+ release events and TRPV4 Ca 2+ sparklets was studied by utilizing the differences in kinetics between Ca 2+ release signals from the ER (Ca 2+ pulsars,2, 4 spikes with duration <300 ms, Figure S5A and S5B)4 and TRPV4 Ca 2+ sparklets (discrete, square amplitudes, duration >300 ms, Figure S5A and S5B).…”

“…Male C57BL6/J, transgenic GCaMP2 Cx40 , TRPV4 −/− , and eNOS −/− (The Jackson Laboratory, Bar Harbor, ME) mice (10–14 weeks old) were used for all the studies. GCaMP2 Cx40 mice express GCaMP2, a Ca 2+ ‐specific biosensor under the connexin 40 promoter, thereby limiting its expression to only ECs 27, 28. Mice were euthanized with pentobarbital (90 mg/kg; intraperitoneal) followed by decapitation.…”

c-kit expression identifies cardiovascular precursors in the neonatal heart

“…C-kit expression appears to characterize a developmental stage of one subset of these cardiovascular precursor cells (1), but studies in mouse (4) and human (2, 3) ES cell-derived precursors suggest that a c-kit-negative stage precedes commitment. To examine the role of c-kit expression in CPcs, and to facilitate their isolation and analysis, we created BAC transgenic mice in which EGFP is placed under the transcriptional control of the c-kit locus to achieve high expression levels and outstanding transcriptional fidelity (20,21). Bernex et al (22) and Wouters et al (23) knocked reporters into the c-kit locus, but did not examine cardiac expression, and the dominant nature of the c-kit locus complicates this strategy.…”

Visualization of Calcium Dynamics in Kidney Proximal Tubules

“…The GCaMP2 protein has already been applied in various in vivo measurements within selected tissue preparations and in transgenic mice. [11][12][13][14] Although new versions of genetically engineered calcium indicators are also available, the GCaMP2 construct was reliably and efficiently working in our hands in various mammalian cellular systems. As reported, 15 in human stem cells calcium imaging could be performed without the need for potentially toxic dye loading.…”