Ca(2+) signaling in arterioles and small arteries of conscious, restrained, optical biosensor mice

Abstract

Two-photon fluorescence microscopy and conscious, restrained optical biosensor mice were used to study smooth muscle Ca(2+) signaling in ear arterioles. Conscious mice were used in order to preserve normal mean arterial blood pressure (MAP) and sympathetic nerve activity (SNA). ExMLCK mice, which express a genetically-encoded smooth muscle-specific FRET-based Ca(2+) indicator, were equipped with blood pressure telemetry and immobilized for imaging. MAP was 101 ± 4 mmHg in conscious restrained mice, similar to the freely mobile state (107 ± 3 mmHg). Oscillatory vasomotion or irregular contractions were observed in most arterioles (71%), with the greatest oscillatory frequency observed at 0.25 s(-1). In a typical arteriole with an average diameter of ~35 μm, oscillatory vasomotion of a 5-6 μm magnitude was accompanied by nearly uniform [Ca(2+)] oscillations from ~0.1 to 0.5 μM, with maximum [Ca(2+)] occurring immediately before the rapid decrease in diameter. Very rapid, spatially uniform "Ca(2+) flashes" were also observed but not asynchronous propagating Ca(2+) waves. In contrast, vasomotion and dynamic Ca(2+) signals were rarely observed in ear arterioles of anesthetized exMLCK biosensor mice. Hexamethonium (30 μg/g BW, i.p.) caused a fall in MAP to 74 ± 4 mmHg, arteriolar vasodilation, and abolition of vasomotion and synchronous Ca(2+) transients.

Summary: MAP and heart rate (HR) were normal during high-resolution Ca(2+) imaging of conscious, restrained mice. SNA induced continuous vasomotion and irregular vasoconstrictions via spatially uniform Ca(2+) signaling within the arterial wall. FRET-based biosensor mice and two-photon imaging provided the first measurements of [Ca(2+)] in vascular smooth muscle cells in arterioles of conscious animals.

Keywords: Calcium; arteriole; imaging; sympathetic nervous system; two-photon.

Figure 1

Mean arterial pressure (MAP) and smooth muscle [Ca²⁺] increase in arterioles of exMLCK mice during chronic infusion of Angiotensin II and a high salt diet. MAP (top panel) in conscious state (open squares) and in anesthetized state (closed diamonds), during simultaneous measurements of [Ca²⁺] (lower panel; n = 5 animals). For [Ca²⁺] measurements, each point represents a mean ± s.e.m of 2 or 3 mice, because mice were imaged in 2 groups on alternating days. Missing data are from days in which imaging or MAP measurements were not performed. Gray lines indicate the mean values over the corresponding time periods. MAP increased by 26 mmHg during AngII/salt (shaded area) when animals were conscious and by 11 mmHg when anesthetized. During the last 4 days of the AngII/salt period, mean [Ca²⁺] was increased by 85 nM. Removal of AngII/salt reduced both MAP and arteriolar [Ca²⁺] (reproduced with Permission from AJP, Heart and Circulatory Physiology).

Figure 2

Preparation of exMLCK mouse ear vasculature for in vivo, longitudinal, 2-photon imaging. (A) Dorsal surface of the depilated ear of an anesthetized mouse. The ear is positioned within a recording chamber constructed of a polypropylene ring (arrowhead) pinned to a silicone block. Veins are readily visible and provide clear landmarks for repeated imaging. The ear is adhered via adhesive tape, pinned to a silicone block. The polypropylene ring retains water for the 20× dipping objective lens and has a cut-out region on its lower surface to ensure that blood flow is not occluded. The ear is thus positioned securely without any perturbation. (B) Confocal image (single photon excitation of YFP, YFP emission; bar = 800 μm) of the ear vasculature reveals the architecture of arteries and arterioles. Such low magnification images are used to precisely map the location of arterial sections used for longitudinal measurements. (C) Multiphoton excitation of CFP allows for FRET imaging of arterioles beneath light scattering tissue acquisition of multiple arterial sections (Z stack) enabling orthogonal view reconstruction. The blue and red lines denote the optical section shown in the paired panels. (D) 3D representation of a separate arteriole, in vivo, demonstrates the resolution of single smooth muscle cells (numbers denote axis scale in μm). (E) CFP channel images of the same arteriolar branch, ~18 μm in diameter, acquired on 6 separate occasions in a 13 day period. Scale bar = 100 μm. The data demonstrate the ability to locate particular sites in an individual animal, as required for longitudinal studies (reproduced with permission from AJP, Heart and Circulatory Physiology).

Figure 3

Effect of anesthesia and conscious restraint on heart rate and blood pressure. (A) Telemetric recordings of one mouse [Mean arterial pressure (MAP), closed circles; heart rate (HR), solid line; pulse pressure (PP), open diamonds] during preparation for ear arteriole imaging. Recording began under freely mobile conditions, after which the animal was restrained under a brief period of isoflurane anesthesia (1.5%). Anesthesia depressed all variables, which were restored upon removal of anesthesia. (B) Summary data (n = 4) demonstrate that all variables significantly decreased during isoflurane anesthesia (^*P < 0.05) and were unaltered during experimental restraint.

Figure 4

Observation of intracellular [Ca²⁺] and diameter during oscillatory vasomotion. (A) Left image shows an ear arteriole using CFP fluorescence (0–2500). Scale bar is 35 μm and applies to all images. White triangles denote the 5 ROIs used to measure [Ca²⁺]. Rainbow-colored images to the right demonstrate the range of calculated [Ca²⁺] (0–1.5 μM) over the time-course of spontaneous oscillatory vasomotion, as indicated by the small letters above each image, relating to (B). (B) Average [Ca²⁺] and change in wall position (change in diameter) from the 5 ROIs labeled in (A). Peak oscillations in [Ca²⁺] are indicated by gray diamonds in the diameter trace, and correspond with the rapid decrease in diameter during vasomotion. See Supplemental Video.

Figure 5

Observation of intracellular [Ca²⁺] and diameter during a spontaneous vasoconstriction. (A) CFP channel fluorescence, calculated [ca²⁺], (B) mean [Ca²⁺] from 5 ROIs and diameter in an exMLCK ear arteriole during 80 s of conscious restraint. Imaging scales are presented in grayscale to show CFP fluorescence (0–1400) and rainbow to show calculated [Ca²⁺] (0–1.1 μM). The 5 rainbow images show the frames immediately surrounding the transient increase in [Ca²⁺] occurring at 45.1 s. Vasoconstriction occurred in the frame following the peak Ca²⁺ and then immediately returned to basal diameter.

Figure 6

Effect of hexamethonium on [Ca²⁺] oscillations, vasomotion, and diameter in exMLCK ear arterioles. (A) Mean arterial pressure (MAP) before, during and following recovery of i.p. hexamethonium injection (30 μg/g BW). Brief attempts of activity by the mouse are indicated by transient changes in MAP. Small letters indicate the time point of the corresponding CFP images shown in (B). (B) Selected images demonstrate that arteriolar vasomotion (a) is eliminated by hexamethonium treatment (b). Recovery of tone (c) occurred ~30 min after hexamethonium injection. (C) Transient increases in [Ca²⁺] and the corresponding decreases in diameter during irregular vasomotion during conscious, restrained conditions (left). Hexamethonium eliminated vasomotion, [Ca²⁺] transients, and caused vasodilation (right). Baseline [Ca²⁺] was not affected by hexamethonium.

Figure 7

Temporal relationships between [Ca²⁺] oscillations and diameter in exMLCK ear arterioles. (A) Vasomotor activity from Figures 4b, 5, 6c, illustrating [Ca²⁺] and the rate of change in diameter for regular and irregular oscillatory vasomotion, and a spontaneous vasoconstriction. Black traces represent [Ca²⁺] and colored traces represent the time derivative (dx/dt) of arterial diameter. Decreasing arterial diameter produces negative derivative values. (B) Cross correlation of [Ca²⁺] and dx/dt. The strongest correlation between [Ca²⁺] and dx/dt occurs at a lag of ~0.3 s, indicating that the maximum rate of vasoconstriction follows the peak [Ca²⁺] with a delay of ~0.3 s.