Genetically Engineered Mice for Combinatorial Cardiovascular Optobiology

Abstract

RationaleOptogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. The effective combination of these optical tools in mice requires designed genetic models that are optically compatible and efficiently assembled. We designed strains for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology.ObjectiveTo address this lack of optogenetic resources by combining lineage-specific expression of optogenetic effectors and sensors in single mouse lines and demonstrating their utility.Methods and ResultsTwenty-two lines of mice were created in which optogenetic effectors (11 lines) or Ca2+ sensors (11 lines) were expressed in a lineage-specific manner in cardiac pacemaker cells (SAN), cardiomyocytes, vascular endothelial and smooth muscle cells, as well as other cell types. We show functional responses associated with optical formation of the second messengers InsP3 (optoα1AR) and cAMP (optoβ2AR), or Ca2+-permeant membrane channels (CatCh2), in targeted cells in these mice. Green (GCaMP5, 8 and 8.1) and red (RCaMP 1.07) Ca2+ sensors were chosen to enable simultaneous fluorescent detection of cellular responses. Biallelic crosses were created with robust expression of sensor/effector pairs. We present examples of novel in vivo and ex vivo experiments in selected strains including cardiac pacing with stimulation of HCN4 conduction cells, intravital cardiac imaging of GCaMP8 with two-photon microscopy, optical vascular dilation/constriction by smooth muscle (Acta2) and vasodilation by light activation of endothelium (Cdh5). These experiments highlight the potential of these mice to uncover new insight into mechanisms regulating cardiovascular function.ConclusionsThese new mouse lines efficiently express optical effectors and sensors in heart, blood vessels, and other tissues and can be efficiently combined enable novel in vivo and ex-vivo studies and can be combined or crossed with relevant cardiovascular disease models, constituting a new toolbox for cardiovascular biology.