Spatiotemporal and Multicellular Intravital Microscopy Analysis during Cardiac Injury and Repair
Authors: Small David M. , Allan‐Rahill Nathaniel H. , Jhala Marvarakumari G. , Nishimura Nozomi 
Published: May 2022

Background

Understanding the response to cardiac injury across multiple cell types and timescales has relied on techniques that reduce the complexity of the in vivo environment to provide a step‐by‐step picture of heart function. These models do not recapitulate the temporal and spatial complexities of the true in vivo environment. We have developed a suite of in vivo imaging methods that enable the study of cardiac injury, and here demonstrate intravital cardiac multiphoton microscopy (MPM) to assess cardiomyocyte function and the dynamics of heart and inflammatory cell types in response to cardiac injury and repair.

Methods

Intravital cardiac MPM of male and female mice was performed in the anesthetized mechanically ventilated mouse following left thoracotomy and placement of a customized imaging window on the left ventricle. Fast image acquisition (30 frame/sec) and simultaneous electrocardiogram and respiratory pressure recording enable volumetric image reconstruction throughout the cardiac cycle and quantitative measures of tissue motion and deformation. To demonstrate tracking of acute cellular changes over 1‐2 h, we performed focal sterile injury (FSI; non‐scanning high‐power laser exposure causing capillary and cell disruption) and measured cardiomyocyte calcium dynamics, as well as neutrophil and macrophage/monocyte trafficking in αMHC‐GCaMP8, Rosa26‐Cre‐Ly6G+/TdTom, and Cx3Cr1+/GFP‐CCR2+/RFP mice, respectively. To demonstrate monitoring of inflammatory cell interactions and tissue function in actively repairing heart, we performed left ventricle cryoinfarction (CI) or sham in the same mouse strains and imaged after 7‐days. To explore the behavior of a recently focused upon cell class expressing c‐Kit, we performed FSI and CI in c‐KitBAC‐EGFP and c‐Kit‐rtTA/H2B‐EGFP mice. Intravenous injection of fluorescent dyes labelled vasculature (Texas Red or fluoresceine conjugated 70kDa‐dextran, or Q‐dot 655 or 705) and nuclei (Hoechst).

Results

Intravital cardiac MPM and FSI, enabled the visualization and quantification of non‐cyclical and uncoordinated cardiomyocyte calcium activity associated with a 40% reduction in cell motion and deformation, increased Ly6G+ neutrophil trafficking and Cx3Cr+ resident macrophage polarity over 2 h (p<0.05). Resident c‐Kit+ cell morphology and position did not change over 150 min in both c‐KitBAC‐EGFP and c‐Kit‐rtTA/H2B‐EGFP mouse strains. Imaging 7‐days after‐CI, our approaches were able to identify reduced cardiomyocyte calcium activity within the infarct zone associated with a 90% reduction in tissue motion and deformation, morphological alterations in the microvasculature (wider, reduced bifurcations; p<0.05), increased tissue Cx3Cr1+ macrophage number, Ly6G+ neutrophil arrest in vasculature (p<0.05), and punctate perivascular c‐kit expression that shifts to a vascular phenotype.

Conclusion

We have demonstrated the ability to image and quantify the spatiotemporal and multicellular dynamics of cardiac injury and repair using intravital cardiac MPM. These techniques provide a powerful tool to investigate active phases of cardiac repair post‐injury in the true in vivo environment.