In vivo multiphoton microscopy of cardiomyocyte calcium dynamics in the beating mouse heart

Abstract

We demonstrated intravital multiphoton microscopy in the beating heart in an intact mouse and optically measured action potentials with GCaMP6f, a genetically-encoded calcium indicator. Images were acquired at 30 fps with spontaneous heart beat and continuously running ventilated breathing. The data were reconstructed into threedimensional volumes showing tissue structure, displacement, and GCaMP activity in cardiomyocytes as a function of both the cardiac and respiratory cycle. Main textIn vivo multiphoton microscopy (MPM) was recently demonstrated within the heart of rodent models [1][2][3][4][5][6] . However, the combined use of intravital MPM and genetically encoded calcium indicators, which has revolutionized brain studies by enabling in vivo measurement of action potentials from individual neurons within a genetically-defined cell population 7-9 , has not been achieved in the heart. As a result, little is known about calcium dynamics at the level of single cardiomyocytes in the in vivo environment. Previous in vivo studies of calcium dynamics utilized wide-field fluorescence imaging that primarily reports surface calcium transients averaged across many cells 10,11 , while cell-resolved calcium imaging has relied on reduced preparations such as ex vivo Langendorf perfusion models, which eliminate both blood flow and cardiomyocyte contraction 12,13 . Here, we demonstrated methods for imaging activity of the genetically encoded calcium indicator, GCaMP6f 7 , in the beating heart within a living mouse with the capability to resolve single cardiomyocytes, measure calcium dynamics as a function of depth into the ventricle wall, and characterize the dependence on both cardiac and respiratory cycles.In anesthetized, mechanically ventilated mice, we acquired ~100-µm thick image stacks with 2-µm step size and 50-100 images per plane through a window mounted to a stabilized probe glued to the left ventricle ( Fig. 1a and b, Supple. Fig.1) while recording the electrocardiogram (ECG) and ventilator pressure (Fig. 1c). This preparation caused minimal tissue damage (Supple. Fig. 2a) and heart rate was stable throughout the imaging session (Supple. Fig. 2b). High frame rate imaging (30 fps), using resonant scanners, produced clear images in real time throughout the cardiac cycle, with less distortion due to tissue motion than with slower scanning with . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/251561 doi: bioRxiv preprint first posted online Jan. 22, 2018; 3 galvanometers (Supple. Fig. 3). In contrast to previous approaches 2,3 , breathing was not paused during measurement and image acquisition and heart beat were not synchronized. Instead, the effects of breathing and heart beat were decoupled by reconstructing 3D volumes from smaller image segments sorted by both the cardiac and respiratory pha...