In vivo assessment of artery smooth muscle [Ca2+]i and MLCK activation in FRET-based biosensor mice

Abstract

The cellular mechanisms that control arterial diameter in vivo, particularly in hypertension, are uncertain. Here, we report a method that permits arterial intracellular Ca(2+) concentration ([Ca(2+)](i)), myosin light-chain kinase (MLCK) activation, and artery external diameter to be recorded simultaneously with arterial blood pressure (BP) in living mice under 1.5% isofluorane anesthesia. The method also enables an assessment of local receptor activity on [Ca(2+)](i), MLCK activity, and diameter in arteries, uncomplicated by systemic effects. Transgenic mice that express, in smooth muscle, a Ca(2+)/calmodulin-activated, Förster resonance energy transfer (FRET)-based "ratiometric", exogenous MLCK biosensor were used. Vasoactive substances were administered either intravenously or locally to segments of exposed femoral or cremaster arteries. In the basal state, mean BP was approximately 90 mmHg, femoral arteries were constricted to 65% of their passive diameter, MLCK fractional activation was 0.14, and [Ca(2+)](i) was 131 nM. Phenylephrine (300 ng/g wt iv) elevated mean BP transiently to approximately 110 mmHg, decreased heart rate, increased femoral artery [Ca(2+)](i) to 244 nM and fractional MLCK activation to 0.24, and decreased artery diameter by 23%. In comparison, local application of 1.0 muM phenylephrine raised [Ca(2+)](i) to 279 nM and fractional MLCK activation to 0.26, and reduced diameter by 25%, but did not affect BP or heart rate. Intravital FRET imaging of exogenous MLCK biosensor mice permits quantification of changes in [Ca(2+)](i) and MLCK activation that accompany small changes in BP. Based on the observed variance of the FRET data, this method should enable the detection of a difference in basal [Ca(2+)](i) of 29 nM between two groups of 12 mice with a significance of P < 0.05.

Fig. 1.

Artery constriction and internal blood perfusion do not directly affect the biosensor (exogenous myosin light chain kinase, exMLCK) FRET (Forster resonance energy transfer) ratio. All arteries were superfused with a Ca²⁺-free solution and pressurized internally. Top: solid traces are the cyan fluorescent protein (CFP) fluorescence; shaded traces are the yellow fluorescent protein (YFP) fluorescence corrected for spectral overlap. Middle: exMLCK FRET ratio. Bottom: outer diameter. A: as indicated, the internal perfusion solution was changed from albumin-free dissection solution to heparinzed blood. Insets show that the CFP fluorescence in the artery is visibly decreased by the presence of blood inside, compared with saline. Both CFP and YFP fluorescence decreased substantially. The exMLCK FRET ratio and artery diameter were unaffected. B: same artery, again initially in Ca²⁺-free solution, then exposed, at the time indicated, to 117 mM external K⁺ concentration ([K⁺]_o) with 2.5 mM Ca²⁺ solution. This caused a large contraction and large changes in CFP and YFP fluorescence and the exMLCK FRET ratio. C: effect of changing internal pressure from 120 to 0 mmHg in a different artery. The artery collapsed when internal pressure was dropped to 0 mmHg. This resulted in large changes in CFP and YFP fluorescence and diameter, but no substantial change in the exMLCK FRET ratio.

Fig. 2.

In vivo images of a segment of femoral artery in an anesthetized exMLCK FRET biosensor mouse, in the basal state. A: broad spectrum (470–720 nm) image obtained with a spectral imaging camera. Excitation light wavelength: 436 ± 10 nm. Prominent fluorescence within the smooth muscle cells of the artery arises from the exogenous CFP/YFP containing MLCK biosensor molecule (exMLCK). Spectrally “pure” images of CFP (B), YFP (C), and tissue intrinsic (D) fluorescence were created by linear spectral unmixing of image (A), using reference spectra for CFP, YFP, and tissue intrinsic fluorescence. D: image has been scaled up by a factor of 10.0 to facilitate visualizing the low levels of tissue intrinsic fluorescence. E: exMLCK FRET ratio image, CFP/YFP (B/C), in basal state. F: exMLCK FRET ratio image of the same artery segment during exposure of the segment to high [K⁺]_o superfusion solution.

Fig. 3.

In vivo imaging of a segment of small cremaster artery in an anesthetized exMLCK FRET biosensor mouse during local application (superfusion) of phenylephrine (PE; 10.0 μM) to the artery. A: unprocessed images from the “dual view” image splitter. a: CFP and YFP channel images at 25 s after beginning of recording. b: Corresponding images at 90 s, at the peak of the contraction elicited by PE. B: colored, processed images illustrating the definition of artery walls from the images shown in A. Gray-scale and color bars indicate fluorescence intensity levels from 0 to 4,095 in all cases. 250 frame movies of the images in A and B are available in the Supplemental Data. C: spatially averaged exMLCK FRET ratio was computed from the summed fluorescence of the artery walls, after correction for spectral overlap and tissue intrinsic fluorescence (see methods for details). PE elicited a large decrease in diameter with some small oscillations. Oscillations in the exMLCK FRET ratio, indicative of Ca²⁺ oscillations, are evident.

Fig. 4.

In vivo recording of exMLCK FRET ratio, femoral artery external diameter, arterial pressure, and heart rate during systemic administration (intravenous) of PE in an exMLCK biosensor mouse. PE was administered at a constant rate via a syringe pump connected to a catheter in the jugular vein, as indicated. At total doses of 100 and 300 ng/g body wt, PE increased the exMLCK FRET ratio, constricted the femoral artery, increased mean arterial pressure, and decreased the heart rate. bpm, Beats/min.

Fig. 5.

In vivo recording of exMLCK FRET ratio, femoral artery external diameter, arterial pressure, and heart rate during local administration (superfusion) of PE and prazosin in an exMLCK biosensor mouse. A: PE and prazosin were administered locally to an exposed segment of femoral artery. PE increased the exMLCK FRET ratio and decreased diameter, but had no effect on mean arterial pressure or heart rate. Prazosin elicited a small decrease in exMLCK FRET ratio and vasodilation. B: summary of exMLCK FRET ratio (left) and diameter changes (Δ) as a percentage of passive diameter (PD; right) in response to local application of PE (1.0 μM) in the absence (open bars, n = 8) and presence (solid bars, n = 4) of 100 nM prazosin. Ctrl, control PE response. **P < 0.01; ***P < 0.001. C: summary of local application of PE (3.0 μM) on exMLCK FRET ratio (left) and diameter (right) in the absence (n = 6) and presence (n = 3) of 100 nM prazosin. *P < 0.05. Thus locally applied prazosin blocks local α₁-adrenoceptors, but does not produce any systemic hemodynamic effects.

Fig. 6.

In vivo exMLCK calibration procedure. Segments of femoral artery, in vivo, are exposed locally to superfusing solution containing 1.0 μM PE, then to “Ca²⁺-free (+0.5 mM EGTA)” (“0Ca”) solution, and, finally, to high [K⁺]_o solution with 2.5 mM Ca²⁺. A: representative experiments show changes in exMLCK FRET ratio (a) and arterial diameter (b) during standard calibration procedure. Record of (outer) diameter has been retouched slightly to remove an artifact that occurred during exposure to high [K⁺]_o (+Ca²⁺). PE elicited a transient increase in exMLCK FRET ratio and constricted the artery. Ca²⁺-free solution reduced the exMLCK FRET ratio and induced a slow vasodilatation, abolishing the basal vascular tone. High [K⁺]_o (+Ca²⁺) caused a large increase in the exMLCK FRET ratio and a maximal vasoconstriction. B: summary of exMLCK FRET ratios (n = 5 arteries), as measured from records such as in A. C: histogram of R_Basal (basal state exMLCK FRET ratio) values measured from 12 arteries. The smooth curve is a three-parameter Gaussian distribution fit to the data (correlation coefficient, R² = 0.8223).

Fig. 7.

Estimation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and fractional activation of MLCK in arteries in vivo. Smooth curve is a graph of the Hill equation with EC₅₀ at pCa 6.1 and Hill coefficient (n) of 1.0. Curve was calculated on the assumption that arterial [Ca²⁺]_i achieved during exposure to Ca²⁺-free/0.5 mM EGTA solution was 63 nM (pCa 7.2), as described in the text. Inset: normalized exMLCK FRET ratio (a) and calculated [Ca²⁺]_i (b) under “basal” physiological conditions (open bars) and in response to locally applied 1.0 μM PE (solid bars). Normalized basal exMLCK FRET ratio, R_Basal,Norm = (R_Basal − R_min)/(R_max − R_min). For R_Basal, n = 12. R_min and R_max, minimum and maximum exMLCK FRET ratios that correspond to Ca²⁺/calmodulin-free (Ca²⁺-free/EGTA solution) and Ca²⁺/calmodulin-saturated (high [K⁺]_o solution) exMLCK, respectively. R_PE,Norm (normalized PE exMLCK FRET ratio) was calculated similarly (n = 8).