Biosensor Mice Research Articles

O2 affects mitochondrial functionality ex vivo

Mitochondria have originated in eukaryotic cells by endosymbiosis of a specialized prokaryote approximately 2 billion years ago. They are essential for normal cell function by providing energy through their role in oxidizing carbon substrates. Glutathione (GSH) is a major thiol-disulfide redox buffer of the cell including the mitochondrial matrix and intermembrane space. We have generated cardiomyocyte-specific Grx1-roGFP2 GSH redox potential (EGSH) biosensor mice in the past, in which the sensor is targeted to the mitochondrial matrix. Using this mouse model a distinct EGSH of the mitochondrial matrix (−278.9 ± 0.4 mV) in isolated cardiomyocytes is observed. When analyzing the EGSH in isolated mitochondria from the transgenic hearts, however, the EGSH in the mitochondrial matrix is significantly oxidized (−247.7 ± 8.7 mV). This is prevented by adding N-Ethylmaleimide during the mitochondria isolation procedure, which precludes disulfide bond formation. A similar reducing effect is observed by isolating mitochondria in hypoxic (0.1–3% O2) conditions that mimics mitochondrial pO2 levels in cellulo. The reduced EGSH is accompanied by lower ROS production, reduced complex III activity but increased ATP levels produced at baseline and after stimulation with succinate/ADP. Altogether, we demonstrate that oxygenation is an essential factor that needs to be considered when analyzing mitochondrial function ex vivo.

Imaging sympathetic neurogenic Ca2+ signaling in blood vessels.

We review the information that has been provided by optical imaging experiments directed at understanding the role and effects of sympathetic nerve activity (SNA) in the functioning of blood vessels. Earlier studies utilized electric field stimulation of nerve terminals (EFS) in isolated arteries and vascular tissues (ex vivo) to elicit SNA, but more recently, imaging studies have been conducted in vivo, enabling the study of SNA in truly physiological conditions. Ex vivo: In vascular smooth muscle cells (VSMC) of isolated arteries, the three sympathetic neurotransmitters, norepinephrine (NE), ATP and neuropeptide Y (NPY), elicit or modulate distinct patterns of Ca2+ signaling, as revealed by confocal imaging of exogenous fluorescent Ca2+ indicators. Purinergic junctional Ca2+ transients (jCaTs) arise from Ca2+ influx during excitatory junction potentials (eJPs), and are associated with the initial neurogenic contraction. Adrenergic Ca2+ waves and oscillations cause contraction while SNA-induced endothelial Ca2+ 'pulsars' cause relaxation. In vivo: optical biosensor mice, which express genetically encoded Ca2+ indicators (GECI's) specifically in smooth muscle, combined with non-invasive imaging techniques has enabled imaging SNA-induced Ca2+ signaling and arterial diameter in vivo. SNA induces Ca2+ oscillations in intact arteries. [Ca2+] of arterial smooth muscle cells increased in hypertension, in association with increased SNA. High resolution imaging has revealed local sympathetic, neurogenic Ca2+ signaling within smooth muscle and endothelial cells of the vasculature. The ongoing development of in vivo imaging together with an expanding availability of different biosensor animals promises to enable the further assessment of SNA and its effects in the vasculature of living animals.

Redox Imaging Using Cardiac Myocyte-Specific Transgenic Biosensor Mice.

Changes in redox potentials of cardiac myocytes are linked to several cardiovascular diseases. Redox alterations are currently mostly described qualitatively using chemical sensors, which however do not allow quantifying redox potentials, lack specificity, and the possibility to analyze subcellular domains. Recent advances to quantitatively describe defined redox changes include the application of genetically encoded redox biosensors. Establishment of mouse models, which allow the quantification of the glutathione redox potential (EGSH) in the cytoplasm and the mitochondrial matrix of isolated cardiac myocytes and in Langendorff-perfused hearts based on the use of the redox-sensitive green fluorescent protein 2, coupled to the glutaredoxin 1 (Grx1-roGFP2). We generated transgenic mice with cardiac myocyte-restricted expression of Grx1-roGFP2 targeted either to the mitochondrial matrix or to the cytoplasm. The response of the roGFP2 toward H2O2, diamide, and dithiothreitol was titrated and used to determine the EGSH in isolated cardiac myocytes and in Langendorff-perfused hearts. Distinct EGSH were observed in the cytoplasm and the mitochondrial matrix. Stimulation of the cardiac myocytes with isoprenaline, angiotensin II, or exposure to hypoxia/reoxygenation additionally underscored that these compartments responded independently. A compartment-specific response was also observed 3 to 14 days after myocardial infarction. We introduce redox biosensor mice as a new tool, which allows quantification of defined alterations of EGSH in the cytoplasm and the mitochondrial matrix in cardiac myocytes and can be exploited to answer questions in basic and translational cardiovascular research.

380. Use of Somatotransgenic Bioimaging as a Platform for Studying NFkB Pathway in a Tissue-Specific Manner in Two Mouse Models of Inflammation: Rheumatoid Arthritis and Contact Dermatitis

Somatotransgenic bioimaging is a technology for in vivo imaging using lentiviral mediated delivery of a luciferase construct into neonatal mice. This luciferase marker, under the control of transcription factor binding elements or surrogate promoters, can be activated during physiological and pathological processes and can be quantified as photonic readout by whole-body bioluminescence imaging technology. The technique permits lifelong expression with immune tolerance to the transgenic proteins.In this study, we show the utility of somatotransgenic bioimaging using NFkB signaling as a model of inflammation in mouse models of rheumatoid arthritis and contact dermatitis.An NFkB transcription factor response element was inserted upstream of a mouse minimal promoter and the genes encoding firefly luciferase and green fluorescent protein. The cassette was inserted into a lentivirus vector.VSV-G-pseudotyped lentivirus vector was injected subcutaneously into CD1 outbread neonatal (P0) mice either in the footpad or in the abdominal skin to generate paw or skin specific light emitting transgenic mice.Canonical NFkB signaling (through TLR4) was first validated using ultrapure LPS in both the paw and skin of somatic transgenic mice. Following intraperitoneal LPS injection we observed upregulation of luciferase activity in the paw or the skin which returned to background levels within a few days.As a model of inflammatory mediated rheumatoid arthritis, we injected zymosan, which activates NFkB via TLR2 into the mouse paw fat pad. Induction of contact dermatitis was achieved by applying topically oxazolone, which plays an indirect activator role in the NFkB pathway. Luciferase expression was then monitored by whole body bioluminescence.Stimulation with Zymosan of the paw biosensor mice induced a prolonged upregulation of luciferase expression, specifically into the mouse paw which peaked at day 3 post injection and gradually returned to background level after twenty days.Similarly, after topical application of oxazolone onto the skin biosensor mice, we observed a specific, localised and increased luciferase activity which peaked at day 2 and returned to baseline by day 5.We have therefore shown that targeted gene expression can be achieved in two mouse models of inflammation. This also allowed us to quantify NFkB-mediated response to pro-inflammatory stimuli in a disease- and tissue-specific manner. Applied to collagen-induced arthritis, this technique could improve quantitation of disease severity compared to subjective scoring.

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

Two-photon fluorescence microscopy and conscious, restrained optical biosensor mice were used to study smooth muscle Ca2+ 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 Ca2+ 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 [Ca2+] oscillations from ~0.1 to 0.5 μM, with maximum [Ca2+] occurring immediately before the rapid decrease in diameter. Very rapid, spatially uniform “Ca2+ flashes” were also observed but not asynchronous propagating Ca2+ waves. In contrast, vasomotion and dynamic Ca2+ 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 Ca2+ transients.Summary: MAP and heart rate (HR) were normal during high-resolution Ca2+ imaging of conscious, restrained mice. SNA induced continuous vasomotion and irregular vasoconstrictions via spatially uniform Ca2+ signaling within the arterial wall. FRET-based biosensor mice and two-photon imaging provided the first measurements of [Ca2+] in vascular smooth muscle cells in arterioles of conscious animals.

A method for noninvasive longitudinal measurements of [Ca2+] in arterioles of hypertensive optical biosensor mice.

We used two-photon (2-p) Förster resonance energy transfer (FRET) microscopy to provide serial, noninvasive measurements of [Ca(2+)] in arterioles of living "biosensor" mice. These express a genetically encoded Ca(2+) indicator (GECI), either FRET-based exMLCK or intensity-based GCaMP2. The FRET ratios, Rmin and Rmax, required for in vivo Ca(2+) calibration of exMLCK were obtained in isolated arteries. For in vivo experiments, mice were anesthetized (1.5% isoflurane), and arterioles within a depilated ear were visualized through the intact skin (i.e., noninvasively), by 2-p excitation of exMLCK (at 820 nm) or GCaMP2 (at 920 nm). Spontaneous or agonist-evoked [Ca(2+)] transients in arteriolar smooth muscle cells were imaged (at 2 Hz) with both exMLCK and GCaMP2. To examine changes in arteriolar [Ca(2+)] that might accompany hypertension, five exMLCK mice were implanted with telemetric blood pressure transducers and osmotic minipumps containing ANG II (350 ng·kg(-1)·min(-1)) and fed a high (6%)-salt diet for 9 days. [Ca(2+)] was measured every other day in five smooth muscle cells of two to three arterioles in each animal. Prior to ANG II/salt, [Ca(2+)] was 246 ± 42 nM. [Ca(2+)] increased transiently to 599 nM on day 2 after beginning ANG II/salt, then remained elevated at 331 ± 42 nM for 4 more days, before returning to 265 ± 47 nM 6 days after removal of ANG II/salt. In summary, two-photon excitation of exMLCK and GCaMP2 provides a method for noninvasive, longitudinal quantification of [Ca(2+)] dynamics and vascular structure in individual arterioles of a particular animal over an extended period of time, a capability that should enhance future studies of hypertension and vascular function.

Two‐photon microscopy enables non‐invasive, in vivo, longitudinal study of arterial Ca2+ and diameter in optical biosensor mice during the development of hypertension (856.7)

Here, we report that use of two‐photon microscopy of ear arteries in exMLCK biosensor mice enables non‐invasive, in vivo, serial examination of vascular smooth muscle Ca2+ during the development of hypertension. ExMLCK mice express a FRET‐type biosensor of Ca2+/Calmodulin based on smooth muscle myosin light chain kinase (MLCK) and its ratiometric properties permit direct quantification of intracellular Ca2+ concentration. Anesthetized biosensor mice were positioned on a custom platform, facilitating imaging of selected arterial branches within the intact depilated ear, and studied before and during chronic infusion of angiotensin‐II (ANGII; 800 µg/g/day) via mini‐osmotic pump. Acute responsiveness to i.p. phenylephrine (PE; 30µg/g) was determined in a separate group of mice equipped with arterial blood pressure radio telemetry. Eight days of chronic ANGII increased intracellular Ca2+ from 294±47nM to 501±92nM (P<0.05) and decreased artery diameter by ‐14±2% (P<0.05). Acute PE induced robust vasomotion and 2‐3 fold oscillations in spatially averaged Ca2+. In summary, this new methodology permits for the first time the simultaneous study of Ca2+ signaling, structural remodeling and vascular reactivity in the intact living mouse during hypertension development.Grant Funding Source: Supported by NHLBI RO1091969, RO1HL107654, RO1DK077140, and T32HL072751.

Immunization alters body odor

Infections have been shown to alter body odor. Because immune activation accompanies both infection and immunization, we tested the hypothesis that classical immunization might similarly result in the alteration of body odors detectable by trained biosensor mice. Using a Y-maze, we trained biosensor mice to distinguish between urine odors from rabies-vaccinated (RV) and unvaccinated control mice. RV-trained mice generalized this training to mice immunized with the equine West Nile virus (WNV) vaccine compared with urine of corresponding controls. These results suggest that there are similarities between body odors of mice immunized with these two vaccines. This conclusion was reinforced when mice could not be trained to directly discriminate between urine odors of RV- versus WNV-treated mice. Next, we trained biosensor mice to discriminate the urine odors of mice treated with lipopolysaccharide (LPS; a general elicitor of innate immunological responses) from the urine of control mice. These LPS-trained biosensors could distinguish between the odors of LPS-treated mouse urine and RV-treated mouse urine. Finally, biosensor mice trained to distinguish between the odors of RV-treated mouse urine and control mouse urine did not generalize this training to discriminate between the odors of LPS-treated mouse urine and control mouse urine. From these experiments, we conclude that: (1) immunization alters urine odor in similar ways for RV and WNV immunizations; and (2) immune activation with LPS also alters urine odor but in ways different from those of RV and WNV.

Intravital Förster resonance energy transfer imaging reveals elevated [Ca2+]i and enhanced sympathetic tone in femoral arteries of angiotensin II‐infused hypertensive biosensor mice

Artery narrowing in hypertension can only result from structural remodelling of the artery, or increased smooth muscle contraction. The latter may occur with, or without, increases in [Ca(2+)]i. Here, we sought to measure, in living hypertensive mice, possible changes in artery dimensions and/or [Ca(2+)]i, and to determine some of the mechanisms involved. Ca(2+)/calmodulin biosensor (Förster resonance energy transfer-based) mice were made hypertensive by s.c. infusion of angiotensin II (Ang II, 400 ng kg(-1) min(-1), 2-3 weeks). Intravital fluorescence microscopy was used to determine [Ca(2+)]i and outer diameter of surgically exposed, intact femoral artery (FA) of anaesthetized mice. Active contractile FA 'tone' was calculated from the basal-state diameter and the passive (i.e. Ca(2+)-free) diameter (PD). Compared to saline control, FAs of Ang II-infused mice had (1) ∼21% higher active tone and (2) ∼78 nm higher smooth muscle [Ca(2+)]i, but (3) the same PDs. The local Ang II receptor (AT1R) blocker losartan had negligible effect on tone or [Ca(2+)]i in control FAs, but reduced the basal tone by ∼9% in Ang II FAs. Both i.v. hexamethonium and locally applied prazosin abolished the difference in FA tone and [Ca(2+)]i, suggesting a dominant role of sympathetic nerve activity (SNA). Changes in diameter and [Ca(2+)]i in response to locally applied phenylephrine, Ang II, arginine vasopressin, elevated [K(+)]o and acetylcholine were not altered. In summary, FAs of living Ang II hypertensive mice have higher [Ca(2+)]i, and are more constricted, due, primarily, to elevated SNA and some increased arterial AT1R activation. Evidence of altered artery reactivity or remodeling was not found.

Abstract 534: Increased in vivo Sympathetic Tone in Femoral Arteries of MLCK Biosensor Mice Exposed to Chronic Intermittent Hypoxia

Increased sympathetic nerve activity (SNA) has been implicated in hypertension induced by chronic exposure to intermittent hypoxia (CIH) in both human and rodents. We have previously reported that the basal femoral arterial tone in vivo was primarily mediated by α1-adrenergic SNA, and hence was useful for assessing SNA in living animals in disease models, such as hypertension. While SNA hyperactivity is believed to be region specific in certain types of hypertension, such as that induced by angiotensin II plus salt, the aim of this study is to determine whether sympathetic tone is increased in femoral arteries in CIH-induced hypertension and to characterize vascular reactivity in CIH animals. Mice with smooth muscle-specific FRET-based Ca/calmodulin sensitive myosin light chain kinase biosensor were exposed to CIH (5% nadir O 2 for 1 min, followed by 21% O 2 for 1 min) or sham exposure (21% O 2 constantly) 8 hrs/day for 5 weeks. At the end of the exposure, blood pressure, measured with an intra-carotid artery catheter (1.5% isoflurane), was greatly increased in CIH group (91 ± 4 mm Hg, n = 8, vs. 83 ± 2 mm Hg in sham control, n = 6, P < 0.05). Mid-thigh femoral arteries were slightly dissected and superfused with physiological saline solution. Simultaneous arterial diameter and quantitative cytoplasmic [Ca 2+ ] in smooth muscle cells within intact arteries were measured with Intra-Vital FRET microscopy. Under resting condition, femoral arteries from CIH-treated mice exhibited significantly increased basal tone (38 ± 4% vs 29 ± 4% of PD in control group, n = 5/group) and [Ca 2+ ] (280 ± 27 vs 205 ± 27 nM). Local application, to the exposed portion of arteries, of phentolamine (0.01-3 μM) and prazosin (1 μM), caused more relaxation and [Ca 2+ ] reduction in CIH than in handled control group. Dose-dependent vasoconstriction induced by phenylephrine, however, was similar, suggesting that the expression or sensitivity of the adrenoceptor was not altered. Passive outer diameter, an indicator of arterial remodeling, measured in Ca 2+ -free solution, were not different between control and CIH groups. These results indicate that the increased femoral arterial tone in vivo was also neurogenic, primarily due to hyper-(central) SNA in mice that developed hypertension following CIH.

Vascular Tone and Ca2+ Signaling in Murine Cremaster Muscle Arterioles In Vivo

We sought to determine some of the molecular requirements for basal state "tone" of skeletal muscle arterioles in vivo, and whether asynchronous Ca(2+) waves are involved or not. Cremaster muscles of anesthetized exMLCK and smGCaMP2 biosensor mice were exteriorized, and the fluorescent arterioles were visualized with wide-field, confocal or multiphoton microscopy to observe Ca(2+) signaling and arteriolar diameter. Basal state tone of the arterioles was ~50%. Local block of Ang-II receptors (AT1 ) or α1 -adrenoceptors (α1 -AR) had no effect on diameter, nor did complete block of sympathetic nerve activity (SNA). Inhibition of phospholipase C caused dilation nearly to the Ca(2+) -free (passive) diameter, as did exposure to nifedipine or 2-APB. Arterioles were also dilated when treated with SKF96365. High-resolution imaging of exMLCK fluorescence (ratio) or GCaMP2 fluorescence in smooth muscle cells failed to reveal Ca(2+) waves (although Ca(2+) waves/transients were readily detected by both biosensors in small arteries, ex vivo). Arterioles of cremaster muscle have vascular tone of ~50%, which is not due to α1 -AR, AT1 R, or SNA. PLC activity, L-type Ca(2+) channels, 2-APB- and SKF96365-sensitive channels are required. Propagating Ca(2+) waves are not present. A key role for PLC and InsP3 R in vascular tone in vivo, other than producing Ca(2+) waves, is suggested.

Quantitative Intra-Vital FRET Imaging Reveals Enhanced Sympathetic Adrenergic Tone in Femoral Arteries in Chronic Angiotensin Ii-Infused Biosensor Mice

Hypertension only exists in living animals. Administration of Angiotensin II (Ang II) to animals increases blood pressure. One of the major effects of Ang II is to cause vasoconstriction via directly activating the Ang II type-1 receptor (AT1R) in blood vessels. The aim of this study is to determine whether or not chronic Ang II infusion evokes conduit arterial constriction in vivo, and if so what the underlying mechanisms are. Ang II (400 ng/kg/min) or vehicle (saline) was continuously infused for 2 weeks by subcutaneously implanted osmotic mini-pumps in exogenous myosin light chain kinase FRET biosensor mice. Blood pressure, measured with an intra-carotid artery catheter under isoflurane anesthesia, was significantly elevated in Ang II-infused mice. Mid-thigh femoral arteries were slightly dissected and superfused with physiological saline solution. Simultaneous in vivo arterial diameter and quantitative cytoplasmic [Ca2+] in smooth muscle cells within intact arteries were measured. Under resting condition, femoral arteries from Ang II-infused mice exhibited significantly increased basal tone and cytoplasmic [Ca2+]. Local application, in the superfusion solution, of phentolamine (1 μM), a non-selective α adrenoceptor antagonist, and prazosin (100 nM), a selective α1 adrenoceptor antagonist, caused significantly more relaxation and [Ca2+] reduction in Ang II than in vehicle group. Dose-dependent vasoconstriction induced by an α adrenoceptor agonist, phenylephrine, however, was similar, suggesting a lack of expression or sensitivity change of the local adrenoceptor. In contrast, acute Ang II-induced vasoconstriction was reduced in chronic Ang II-infused mice, suggesting a possible down-regulation of AT1R. These results indicate that, in chronic Ang II-infused mice, albeit the reduced acute vasoconstrictor response to Ang II, there is an increased sympathetic adrenergic tone of femoral arteries, which correlates with blood pressure elevation. (AHA SDG0735461N, NIH R01 HL107654).

Abstract 469: Intra-vital FRET Imaging Reveals Basal α-adrenergic Dominated Tone in Mouse Femoral Conduit Arteries

Enhanced vascular tone is always observed and responsible for the blood pressure elevation in hypertension. The cellular mechanisms that control vascular tone in vivo, particularly in hypertension, however, are not known with certainty. Here, using intra-vital FRET imaging in smooth muscle-specific exogenous myosin light chain kinase biosensor mice, we aimed to study the mechanism(s) that control the basal tone in femoral conduit arteries under normal physiological conditions (Zhang et al., Am J Physiol 299: H946-56, 2010). Quantification of arterial [Ca 2+ ] i and diameter revealed that, in the basal state, femoral arteries exhibit 33 ± 3% tone (percentage of passive diameter, 400 ± 30 μm, obtained in Ca 2+ -free solution), [Ca 2+ ] i is 233 ± 18 nM. Acute ganglionic blockade with hexamethonium (30 mg/kg wt, i.p.) lowered [Ca 2+ ] i by 216 ± 23 nM, reduced the basal tone by 67 ± 12%, and decreased mean blood pressure by ∼60 mm Hg, suggesting a dominant autonomic/sympathetic control of the basal tone. To assess contribution of the major sympathetic neurotransmitters, noradrenaline (NE), ATP and neuropeptide Y (NPY), to the basal tone in vivo , respective agonists and antagonists were applied locally to segments of exposed arteries. Prazosin (0.01-3 μM), an α1 adrenoceptor blocker, dose-dependently decreased [Ca 2+ ] i and basal tone, without affecting blood pressure or heart rate. Yohimbine (0.1-1 μM), an α2 adrenoceptor blocker, reduced tone with negligible effect on [Ca 2+ ] i ( n = 4). These effects were abolished by chemical destruction of sympathetic nerves locally with hydroxydopamine (2 mM), indicating that the basal tone is maintained by neurotransmitters released from nerve terminals, but not activated by circulating neurotransmitter-like factors. Suramin (a purinergic receptor blocker), BIBO 3304 (a NPY receptor blocker), and losartan (angiotensin II AT1 receptor blocker), had negligible effect on either [Ca 2+ ] i or diameter. Our results indicate that femoral arteries possess basal neurogenic tone, primarily α-adrenergic, under normal physiological condition. This basal tone observed in femoral arteries could be particularly useful for assessing sympathetic nerve activity in disease models, such as hypertension.

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

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.

Quantification of Mlck Activation in Arteries of Living Mice That Express a Genetically Encoded Fret-Based Biosensor

FRET (Forster resonance energy transfer)-based sensors are powerful tools for understanding physiological mechanisms at the molecular level. Here, we used transgenic mice that express, specifically in smooth muscle, a FRET-based, exogenous myosin light chain kinase biosensor (‘exMLCK’) to observe for the first time changes in [Ca2+/Calmodulin] and MLCK activation within arteries in vivo (‘intra-vital FRET imaging’). exMLCK biosensor mice were anesthetized (1.5% isoflurane) and placed on the stage of a ‘macro’ epi-fluorescence microscope (Olympus MVX 10) equipped with an image splitter and a digital CCD camera. Arterial blood pressure (AP) was recorded. Femoral or mesenteric arteries were exposed through cutaneous incisions. Quantification of exMLCK FRET in vivorequires precise assessment and removal of tissue intrinsic fluorescence and extraneous sources of light. Intrinisic fluorescence (of isolated arteries) was negligible in comparison to exMLCK fluorescence. In vivo, background fluorescence was minimized by dissection of surrounding tissues and the use of an appropriate field diaphragm. Artery diameter was determined using edge detection and spatially averaged exMLCK FRET ratio (CFP/YFP) was determined from regions of interest. In the basal state (mean AP of ∼90 mm Hg), average exMLCK FRET ratio in femoral artery walls was 1.8 to 1.9, similar to that obtained by us in isolated arteries. Application of the α1-adrenoceptor agonist, phenylephrine (PE) directly to an artery increased exMLCK FRET ratio transiently to a peak of about 2.2 and caused a local vasoconstriction of ∼33%, without changing AP. Intra-venous application of PE elevated AP and caused smaller changes in exMLCK FRET ratio and artery diameter (than direct application). In conclusion, quantitative intra-vital FRET imaging in arteries of transgenic animals is feasible and will permit observation of specific molecular events in tissues of living animals. (AHA, NIH-HL078870).

Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse.

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) initiates smooth muscle contraction and regulates actomyosin-based cytoskeletal functions in nonmuscle cells. The net extent of RLC phosphorylation is controlled by MLCK activity relative to myosin light chain phosphatase activity. We have constructed a CaM-sensor MLCK where Ca(2+)-dependent CaM binding increases the catalytic activity of the kinase domain, whereas coincident binding to the biosensor domain decreases fluorescence resonance energy transfer between two fluorescent proteins. We have created transgenic mice expressing this construct specifically in smooth muscle cells to perform real-time evaluations of the relationship between smooth muscle contractility and MLCK activation in intact tissues and organs. Measurements in intact bladder smooth muscle demonstrate that MLCK activation increases rapidly during KCl-induced contractions but is not maximal, consistent with a limiting amount of cellular CaM. Carbachol treatment produces the same amount of force development and RLC phosphorylation, with much smaller increases in [Ca(2+)](i) and MLCK activation. A Rho kinase inhibitor suppresses RLC phosphorylation and force but not MLCK activation in carbachol-treated tissues. These observations are consistent with a model in which the magnitude of an agonist-mediated smooth muscle contraction depends on a rapid but limited Ca(2+)/CaM-dependent activation of MLCK and Rho kinase-mediated inhibition of myosin light chain phosphatase activity. These studies demonstrate the feasibility of producing transgenic biosensor mice for investigations of signaling processes in intact systems.