Imaging cellular signals in the heart in vivo : Cardiac expression of the high-signal Ca ²⁺ indicator GCaMP2

Abstract

Genetically encoded sensor proteins provide unique opportunities to advance the understanding of complex cellular interactions in physiologically relevant contexts; however, previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved Ca 2؉ sensor, GCaMP2, its inducible expression in the mouse heart, and its use to examine signaling in heart cells in vivo. The high brightness and stability of GCaMP2 enable the measurement of myocyte Ca 2؉ transients in all regions of the beating mouse heart and prolonged pacing and mapping studies in isolated, perfused hearts. Transgene expression is efficiently temporally regulated in cardiomyocyte GCaMP2 mice, allowing recording of in vivo signals 4 weeks after transgene induction. High-resolution imaging of Ca 2؉ waves in GCaMP2-expressing embryos revealed key aspects of electrical conduction in the preseptated heart. At embryonic day (e.d.) 10.5, atrial and ventricular conduction occur rapidly, consistent with the early formation of specialized conduction pathways. However, conduction is markedly slowed through the atrioventricular canal in the e.d. 10.5 heart, forming the basis for an effective atrioventricular delay before development of the AV node, as rapid ventricular activation occurs after activation of the distal AV canal tissue. Consistent with the elimination of the inner AV canal muscle layer at e.d. 13.5, atrioventricular conduction through the canal was abolished at this stage. These studies demonstrate that GCaMP2 will have broad utility in the dissection of numerous complex cellular interactions in mammals, in vivo. atrioventricular node ͉ Ca 2ϩ imaging ͉ genetic sensor ͉ heart development ͉ fluorescent Ca 2ϩ sensor T ransient, highly regulated elevations in cytosolic free Ca 2ϩ underlie numerous cellular processes that enable organ function (1-5). In the mammalian heart, for example, efficient function depends upon the coordinated release and reuptake of Ca 2ϩ ions from intracellular organelles in millions of cells, at rates between 0.5 and 15 Hz throughout life, and even subtle dysfunctions of this process can result in cardiac arrythmias and sudden death. Whereas fluorescent imaging using purpose-designed small Ca 2ϩ -binding indicator molecules has enabled important advances in the understanding of the regulatory processes underlying Ca 2ϩ signaling in single cells (6, 7), these approaches have significant limitations in the context of a complex, multicellular organ such as the beating heart. Thus, difficulties in obtaining an adequate and stable concentration of indicator molecules within cells deep in complex tissues, the incompatibility of loading procedures in the in vivo setting, and the inability to selectively load specific cell lineages constitute substantial experimental constraints on the study of multicellular, processive Ca 2ϩ signaling in complex organ function. Genetically encoded sensors of Ca 2ϩ signaling (7-13) hold great promise in this regard and have been used t...

Presynaptically expressed long-term depression at cerebellar parallel fiber synapses

“…The expression pattern of the sensor protein together with the pharmacological properties of fluorescence transients obtained upon stimulation PF demonstrated that they report PF Ca 2+ transients. These transients have slower kinetics than the actual (volumeaveraged) PF calcium transients because of reporter kinetics [42] and not because of adverse slice conditions as GCaMP2 signals with comparable kinetics have been observed in PFs of living mice [14]. Furthermore, the expression level of GCaMP is low enough as to not buffer the PF Ca 2+ transients to a functionally significant degree because significant Ca 2+ buffering affects PF PPR, and this parameter did not differ between control and GCaMP2 mice.…”

Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries

“…To determine whether TRPV4 sparklets potentiate Ca 2+ release from the ER in PAs, Ca 2+ signals were recorded in PAs from mice that express the Ca 2+ biosensor GCaMP2 selectively in ECs 27, 28. The effect of GSK101 on the activity of Ca 2+ release events and TRPV4 Ca 2+ sparklets was studied by utilizing the differences in kinetics between Ca 2+ release signals from the ER (Ca 2+ pulsars,2, 4 spikes with duration <300 ms, Figure S5A and S5B)4 and TRPV4 Ca 2+ sparklets (discrete, square amplitudes, duration >300 ms, Figure S5A and S5B).…”

“…Male C57BL6/J, transgenic GCaMP2 Cx40 , TRPV4 −/− , and eNOS −/− (The Jackson Laboratory, Bar Harbor, ME) mice (10–14 weeks old) were used for all the studies. GCaMP2 Cx40 mice express GCaMP2, a Ca 2+ ‐specific biosensor under the connexin 40 promoter, thereby limiting its expression to only ECs 27, 28. Mice were euthanized with pentobarbital (90 mg/kg; intraperitoneal) followed by decapitation.…”

Elsie Widdowson Lecture 2006 Mining the depths: metabolic insights into mineral nutrition

“…More recently, it has been recognised that in some situations circulating 25-hydroxyvitamin D is taken up by extrarenal target cells and acts as a substrate for intracellular 1-a-hydroxylase. This action enables the local production of 1,25-hydroxyvitamin Further information on this area of discussion is available (20,33,(49)(50)(51)(52)(53) .…”