(B-50) Investigating Elastin Fiber Kinematics in Porcine Heart Epicardial Interface

Introduction:: Current research increasingly indicates the importance of structural proteins in the function of highly dynamic tissues in the body [1]. Specifically, elastin provides resilience and unique kinematic capabilities to many components within the respiratory and cardiovascular systems. Recently, our lab reported that the epicardial layer, rich in elastin, acts like a prestrained ‘balloon’ that wraps around the heart and functions as an extra confinement and protective interface for the ventricle [2]. This novel discovery alone indicates the necessity of elastin as an integral component of overall cardiac output. Moreover, these conclusions emphasize the importance of a full biomechanical characterization of the epicardial layer, which has vastly been ignored compared to the myocardial layer. For instance, how the elastin fibers arrange themselves into epicardium interface is still a missing component of our current understanding. Though we now know this intricate fiber network enables a significant dynamic deformation during the ventricular diastolic expansion and systolic contraction, we have limited knowledge on how this is so. To begin answering some of these questions, we designed a custom miniature uniaxial stretcher compatible for imaging under a laser scanning confocal microscope (LSCM) to reveal the epicardial elastin kinematics under mechanical loading. Using this technique will enable us to explore the microstructural arrangement and dynamic behavior of the elastin fibers responsible for maintaining cardiac output.

Materials and Methods:: Fresh adult epicardial layer samples were dissected from the porcine left ventricle. The epicardial layers were cleaned of most excess muscular tissue (remaining sample ~1 mm thick). The samples were decellularized for 24 hours in 0.1% SDS and rinsed with distilled water overnight. The decellularized samples were treated with glycerol to replace the water content in a gradient fashion. The glycerol has an optical index very close to collagen fibers and thus make the collagenous tissue transparent. The transparency of the tissue sample enables a deeper laser penetration, allowing for LSCM imaging of the 3D elastin fiber matrix. Glycerol-treated samples were mounted onto a custom-made uniaxial stretcher, which was designed to fit onto a LSCM sample stage for imaging. Markers were used to indicate the position of the sample stretching so deformation could be tracked. The following step strains were applied: 0%, 10%, and 20%. Note that the loading direction for this experiment is along the heart longitudinal direction. A Nikon LSCM (Nikon A1R HD25) was used for 3D elastin fiber imaging. The tissue was imaged using the CY3 channel, Ex 543 nm to obtain 2D images and z-stacks of the elastin at different tissue strain levels.

Results, Conclusions, and Discussions:: The 3D elastin fiber structure at different step strain levels were captured by LSCM imaging. The real time elastin fiber structure tracking showed unique engagement of fiber kinematics at different stages of the tissue’s loading conditions (Figure 1). We were able to image elastin fibers under different step strains to reveal the engagement of epicardial elastin fibers in global tissue deformations. At 0% global tissue strain, elastin fibers exhibited a high degree of waviness and fibers bound closely. Elastin fibers appeared relaxed and entangled, resulting in a less distinguished 3D fiber image due to autofluorescence signal disbursement. Under 10% global tissue strain, the elastin fibers were pulled apart and the mesh-like elastin architecture were shown with the 3D reconstructed LSCM image. This uncrimping, fiber rotation and mesh pulling kinematics corresponded well with the mechanical behavior in soft tissue toe region. Additionally, under 20% global tissue strain, elastin fibers indicted more fiber rotation towards the external loading direction with a taught, web-like mesh morphology. The distribution of fiber alignment reflected this fiber dynamic, with the alignment distribution shifting from a unimodal to a flattened distribution. Though there are still many gaps in our understanding elastin kinematics, our approach opens a new avenue to understand the structural mechanism of epicardium under dynamic, complex large deformations that associated with vigorous cyclic pumping of the heart.

Acknowledgements (Optional): : NIH R15HL159599, R01HL157050

References (Optional): : [1] H. M. Langevin et al., BMC Systems Biology, vol. 1, no. 1, pp. 1-12, 2007.

[2] X. Shi et al., Journal of the Royal Society Interface, vol. 16, no. 152, p. 20190028, 2019.