title: A method for noninvasive longitudinal measurements of [Ca2+] in arterioles of hypertensive optical biosensor mice.
abstract: 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.
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland.
authors.forename: Joseph R H
authors.initials: JR
authors.lastname: Mauban
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland.
authors.forename: Seth T
authors.initials: ST
authors.lastname: Fairfax
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Rizzo
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland gwier001@gmail.com.
authors.forename: Withrow Gil
authors.initials: WG
authors.lastname: Wier
mesh_terms: D000804:Angiotensin II; D000818:Animals; D062186:Arterial Pressure; D001160:Arterioles / Q000378:metabolism / Q000503:physiopathology; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D004195:Disease Models, Animal; D031541:Fluorescence Resonance Energy Transfer; D006973:Hypertension / Q000139:chemically induced / Q000378:metabolism* / Q000503:physiopathology; D051379:Mice; D008822:Mice, Transgenic; D036641:Microscopy, Fluorescence, Multiphoton; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000503:physiopathology; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000098:blood supply*; D017673:Sodium Chloride, Dietary; D013997:Time Factors
chemical_list: D017673:Sodium Chloride, Dietary; D000804:Angiotensin II; D009219:Myosin-Light-Chain Kinase; D002118:Calcium
keywords: FRET; calcium; hypertension; two photon; vascular