Genetically engineered mice for combinatorial cardiovascular optobiology

Abstract

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

Adenylyl cyclase isoform 1 contributes to sinoatrial node automaticity via functional microdomains

“…SAN-specific CRISPR/Cas9-mediated gene silencing of AC I . A transgenic mouse model expressing a fluorescent Ca 2+ indicator (GCaMP8) under the control of the Hcn4 promoter was previously generated and used for the study (71). CRISPR/Cas9 system containing 3× sgRNA (GeneCopoeia) was used to specifically target the AC I isoform, followed by in vivo delivery using liposome and SAN painting technique (27,28).…”

Cardiogenic control of affective behavioural state

“…Precise modulation of electrochemical signals in the heart and other peripheral organs in vivo would enable fundamental studies of physiology and interoceptive signalling [15][16][17][18][19] , but stimulation methods that operate with high spatial and temporal precision in highly dynamic environments such as the beating heart [20][21][22][23][24][25][26][27][28] are limited. Electrical pacemakers require invasive surgical implantation to deliver local indiscriminate stimulation that lacks cell-type specificity [20][21][22][23] .…”

“…Electrical pacemakers require invasive surgical implantation to deliver local indiscriminate stimulation that lacks cell-type specificity [20][21][22][23] . Optogenetics might in principle facilitate cardiomyocyte-specific control with high spatial and temporal precision 14 , but existing optogenetic methods have been limited to acute demonstrations that require exposure or even excision of the heart to deliver light [23][24][25][26][27][28] , all of which are incompatible with freely moving studies of behaviour. Thus far, to our knowledge, no study beyond the brain has achieved precise and noninvasive organ-level control of behavioural or physiological function.…”

“…Conventional microbial opsins have not been sensitive enough to control a large organ such as the heart with the requisite power to facilitate behavioural studies within intact animals [23][24][25][26][27][28] , but the discovery of the highly sensitive and red-shifted pump-like channelrhodopsin ChRmine led us to consider the potential for noninvasive optogenetic control of deep tissue with minimal irradiance 29 . We previously found that ChRmine enabled neuromodulation of deep brain circuits without intracranial surgery 30 , which raised the possibility that this optogenetic tool might be broadly applicable to modulating biological processes across the entire body of large organisms such as mammals.…”

Arbitrary Ca2+ regulation for endothelial nitric oxide, NFAT and NF-κB activities by an optogenetic approach

“…We attempted to manipulate cellular Ca 2+ levels in cultured vascular endothelial cells, and consequently NO production and changes in gene expression by means of an optogenetic technique, which is widely available in neurobiological fields. Recently, studies using this technique for the cardiovascular system, have mainly focused on cardiac or vascular smooth muscle cells ( Lee et al, 2021 ; Tong et al, 2021 ); to our knowledge, there is no literature focusing on vascular endothelial cells. As some optogenetic tools that can regulate Ca 2+ levels are known, we employed a BACCS system which utilizes store-operated calcium entry, the ORAI1-STIM1 machinery, that is present endogenously in vascular endothelial cells.…”