X-ray diffraction and second harmonic imaging reveal new insights into structural alterations caused by pressure-overload in murine hearts

We demonstrate a label-free imaging approach to study cardiac remodeling of fibrotic and hypertrophic hearts, bridging scales from the whole organ down to the molecular level. To this end, we have used mice subjected to transverse aortic constriction and imaged adjacent cardiac tissue sections by microfocus X-ray diffraction and second harmonic generation (SHG) imaging. In this way, the acto-myosin structure was probed in a spatially resolved manner for entire heart sections. From the recorded diffraction data, spatial maps of diffraction intensity, anisotropy and orientation were obtained, and fully automated analysis depicted the acto-myosin filament spacing and direction. X-ray diffraction presented an overview of entire heart sections and revealed that in regions of severe cardiac remodeling the muscle mass is partly replaced by connective tissue and the acto-myosin lattice spacing is increased at these regions. SHG imaging revealed sub-cellular structure of cardiac tissue and complemented the findings from X-ray diffraction by revealing micro-level distortion of myofibrils, immune cell infiltration at regions of cardiac remodeling and the development of fibrosis down to the scale of a single collagen fibril. Overall, our results show that both X-ray diffraction and SHG imaging can be used for label-free and high-resolution visualization of cardiac remodeling and fibrosis progression at different stages in a cardiac pressure-overload mouse model that cannot be achieved by conventional histology.