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, 175 (11), 2028-2045

Defining the Ionic Mechanisms of Optogenetic Control of Vascular Tone by channelrhodopsin-2

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Defining the Ionic Mechanisms of Optogenetic Control of Vascular Tone by channelrhodopsin-2

Nils J G Rorsman et al. Br J Pharmacol.

Abstract

Background and purpose: Optogenetic control of electromechanical coupling in vascular smooth muscle cells (VSMCs) is emerging as a powerful research tool with potential applications in drug discovery and therapeutics. However, the precise ionic mechanisms involved in this control remain unclear.

Experimental approach: Cell imaging, patch-clamp electrophysiology and muscle tension recordings were used to define these mechanisms over a wide range of light stimulations.

Key results: Transgenic mice expressing a channelrhodopsin-2 variant [ChR2(H134R)] selectively in VSMCs were generated. Isolated VSMCs obtained from these mice demonstrated blue light-induced depolarizing whole-cell currents. Fine control of artery tone was attained by varying the intensity of the light stimulus. This arterial response was sufficient to overcome the endogenous, melanopsin-mediated, light-evoked, arterial relaxation observed in the presence of contractile agonists. Ca2+ entry through voltage-gated Ca2+ channels, and opening of plasmalemmal depolarizing channels (TMEM16A and TRPM) and intracellular IP3 receptors were involved in the ChR2(H134R)-dependent arterial response to blue light at intensities lower than ~0.1 mW·mm-2 . Light stimuli of greater intensity evoked a significant Ca2+ influx directly through ChR2(H134R) and produced marked intracellular alkalinization of VSMCs.

Conclusions and implications: We identified the range of light intensity allowing optical control of arterial tone, primarily by means of endogenous channels and without substantial alteration to intracellular pH. Within this range, mice expressing ChR2(H134R) in VSMCs are a powerful experimental model for achieving accurate and tuneable optical voltage-clamp of VSMCs and finely graded control of arterial tone, offering new approaches to the discovery of vasorelaxant drugs.

Figures

Figure 1
Figure 1
Expression of ChR2(H134R) channels and current density in isolated VSMCs. (A) Representative confocal images of freshly isolated aortic, mesenteric and pulmonary VSMCs obtained from control and ChR2(H134R)‐SM mice, as indicated. Similar fluorescence distribution was observed in cells obtained from a total of five mice. (B) Normalized fluorescence intensity of cell cross sections of aortic, mesenteric and pulmonary VSMCs (cross‐section location indicated by white bars in A). (C) Average fluorescence density of aortic (N = 5, n = 61), mesenteric (N = 5, n = 49) and pulmonary artery (N = 5, n = 64) VSMCs isolated from ChR2(H134R)‐SM. (D) Whole‐cell currents recorded from aortic, mesenteric and pulmonary artery VSMCs isolated from control and ChR2(H134R)‐SM mice, as indicated. Currents were recorded at −80 mV. VSMCs isolated from control and ChR2(H134R)‐SM mice were exposed to periods (2 s) of blue light of various intensities, as indicated by the horizontal blue bars. The dashed lines represent the level of current in the absence of light stimulation. (E) Mean current density versus blue light intensity relationships for VSMCs obtained from control aortic (N = 5, n = 5), mesenteric (N = 5, n = 10) and pulmonary artery (N = 5, n = 6) VSMCs or ChR2(H134R)‐SM aortic (N = 10, n = 18), mesenteric (N = 6, n = 16) and pulmonary artery (N = 7, n = 23) VSMCs. The curves through the open symbols (control) were drawn by the eye. The curves through the filled symbols represent the best fit of the data with Equation (1). * P < 0.05, significantly different as indicated; one‐way ANOVA.
Figure 2
Figure 2
Effects of blue light on the tension of isolated artery rings. (A) Isometric tension recordings obtained from aortic, mesenteric and pulmonary artery rings isolated from control and ChR2(H134R)‐SM mice, as indicated. Artery rings were exposed to periods (7 min) of blue light of various intensities, as indicated by horizontal blue bars. Dashed lines represent baseline tension level. (B) Mean tension versus blue light intensity relationships for (i) control aortic (N = 5, n = 10), mesenteric (N = 5, n = 6) and pulmonary artery (N = 5, n = 6) rings or (ii) aortic (N = 32, n = 36), mesenteric (N = 10, n = 15) and pulmonary artery (N = 5, n = 9) artery rings obtained from ChR2(H134R)‐SM mice, as indicated. The curves through open symbols (control) were drawn by the eye. The curves through the filled symbols represent the best fit of the data with Equation (5). (C) Isometric tension recordings obtained from isolated aortic rings in response to periods of exposure to blue light (0.23 mW·mm−2) of different durations (2 s, 1 min and 7 min) or periods of exposure (1 or 7 min) to PSS supplemented with 50 mM KCl, as indicated by the horizontal bars. Traces are represented in different colours for clarity. (D) Mean tension developed by isolated aortic rings in response to 0.23 mW·mm−2 blue light or 50 mM KCl for periods of different duration (N = 5, n = 10 in each case). * P < 0.05, significantly different as indicated; one‐way ANOVA.
Figure 3
Figure 3
Effects of blue light on V m in isolated aortic VSMCs. (A) V m recordings of aortic VSMCs isolated from control and ChR2(H134R)‐SM mice in response to blue light stimulations (2 s) of various intensities, as indicated by the horizontal blue bars. The dashed lines indicate the zero voltage level. (B) Mean V m versus blue light intensity relationships for aortic VSMCs isolated from control (N = 5, n = 12) or ChR2(H134R)‐SM mice (N = 11, n = 17). The curve through open circles (control) was drawn by the eye. The curve through filled circles (ChR2(H134R)‐SM) represents the best fit of the data with Equation (2). (C) V m recording of an aortic VSMC isolated from ChR2(H134R)‐SM mouse in response to 7 min blue light stimulation (0.22 mW·mm−2). (D) Mean V m measured during the first and seventh minute of exposure to blue light (0.22 mW·mm−2) (N = 6, n = 15). * P < 0.05, significantly different from control; Student's t‐test.
Figure 4
Figure 4
Effect of blue light on tension of isolated aortic rings in the presence of phenylephrine. (A) Isometric tension recordings obtained from aortic rings isolated from control or ChR2(H134R)‐SM mice, as indicated. Artery rings were exposed to phenylephrine (PE; 1 μM) and blue light as indicated by the black and blue horizontal bars respectively. The dashed lines indicate the basal tension level. (B) Mean tension versus blue light intensity relationships for aortic rings obtained from control (N = 5, n = 9) or ChR2(H134R)‐SM mice (N = 5, n = 10), as indicated. The smooth curves through symbols represent the best fit of the data with Equation (5).
Figure 5
Figure 5
Effect of pharmacological ion channel modulation and ionic substitution on blue light‐induced tension of aortic rings obtained from ChR2(H134R)‐SM mice. (A) Top panels represent isometric tension recordings obtained from aortic rings isolated from ChR2(H134R)‐SM mice in response to 0.23 mW·mm−2 blue light in the presence of PSS (control) or a nominally Ca2+‐free PSS (0 Ca2+). The lower panel represents mean tension versus blue light intensity relationship for experiments conducted in Ca2+‐free PSS (N = 5, n = 10). The sigmoidal curve (dashed) is the Hill fit (Equation (5)) of the aortic ring tension versus blue light intensity relationship presented in Figure 2B. (B) Top panel represents isometric tension recordings obtained from an aortic ring isolated from ChR2(H134R)‐SM mice in response to 0.23 mW·mm−2 blue light in the presence of PSS supplemented with nifedipine (1 μM). The lower panel represents mean tension versus blue light intensity relationship for experiments conducted in PSS supplemented with nifedipine (1 μM) (N = 7, n = 8). Continuous sigmoidal curve through squares symbols indicates the best fit of the data with Equation (5). Dashed sigmoidal curve is the Hill fit (Equation (5)) of the aortic ring tension versus blue light intensity relationship presented in Figure 2B. (C) Mean tension in response to blue light stimulations in PSS supplemented with 3 μM ryanodine (N = 6, n = 8) or 60 μM 2‐APB (N = 6, n = 8), as indicated. Open circles refer to experiments conducted in control PSS solution and are replotted from Figure 2B. (D) Mean tension in response to blue light stimulations in PSS supplemented with either MONNA (3 μM) (N = 5, n = 10) or Ani9 (1 μM) (N = 5, n = 10). Open circles refer to experiments conducted in control PSS solution and are replotted from Figure 2B. (E) Mean tension in response to blue light stimulations in PSS supplemented with either clotrimazole (10 μM) (N = 5, n = 9) or 9‐phenanthrol (50 μM) (N = 5, n = 5). Open circles refer to experiments conducted in control PSS solution and are replotted from Figure 2B. * P < 0.05, significantly different from control; Student's t‐test.
Figure 6
Figure 6
Effect of blue light on pHi of aortic VSMCs. (A) pHi measurements in individual aortic VSMCs, isolated from control and ChR2(H134R)‐SM mice, before and after a 7 min exposure to blue light of different intensities, as indicated. The horizontal blue bars in each panel indicate the 7 min illumination period. (B) Mean blue light‐induced pHi change versus blue light intensity relationship for VSMCs isolated from ChR2(H134R)‐SM mice (N = 8, n = 201) and control mice (N = 5, n = 215). * P < 0.05, significantly different from control; Student's t‐test.
Figure 7
Figure 7
Comparison of blue light‐induced and agonist‐induced contraction of aortic rings obtained from ChR2(H134R)‐SM mice. (A) Representative isometric tension recordings obtained from aortic rings isolated from ChR2(H134R)‐SM mice. Aortic rings were exposed to blue light, phenylephrine (PE) or noradrenaline (NA) for 25 min, as indicated by the horizontal bars. (B) Mean stability of contraction expressed as the ratio between the maximal and minimal level of contraction elicited by blue light (N = 5, n = 8), PE (N = 5, n = 11) or NA (N = 5, n = 11). * P < 0.05, significantly different as indicated; one‐way ANOVA.

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