Poster R16 - A Comparative Microstructural Study between Human Hypertrophied Septum and Healthy Septum

Introduction: Hypertrophic cardiomyopathy (HCM) is often caused by genetic mutations, resulting in abnormal thickening of ventricular muscle, particularly the septum, and causing left ventricular outflow tract (LVOT) obstruction. Hypertrophic cardiomyopathy can be microscopically assessed and diagnosed by enlarged cell size of the hypertrophic cardiomyocytes, disarray of those cells, and a dense abnormal fibrotic microenvironment [1]. The thickening of the ventricular wall and septum can cause poor cardiac outcomes, which consequently lead to heart failure and mortality [2]. The extent of cell hypertrophy and fibrosis can directly impact the mechanical behavior of septal tissues. Determining the microstructural abnormalities is hence essential to better understand the biomechanical properties of the hypertrophic septal tissues. In this study, we performed a comparative microstructural study between human hypertrophied septum and healthy septum using histology and light sheet images, with the goal of revealing the underlying structural causes of the poor mechanical performance of HCM tissues.

Materials and

Methods: Hypertrophied septal tissues were obtained as surgical waste following septal myectomy surgery with IRB approval. Healthy human septal tissues were obtained from a tissue bank under the same IRB protocol. For histology, 5 μm sections were cut, and a standard Masson’s trichrome staining protocol was performed. Masson’s trichrome protocol stains heart muscle red and collagen blue. Histological images were taken under a bright light field using a Nikon Eclipse Ti Microscope. The average cell size (area), approximate cell diameter, area percentage occupied by cells, nearest neighboring distance of cells, and cell size distribution histogram are quantified from the cross-sectional images of the muscle fibers, using Image J software. For light sheet imaging, the iDISCO method employs a three-step process comprising dehydration, dilapidation, and refractive index matching. The light sheet is created with a cylindrical lens, and ETL moves the focal region of the Gaussian beam which is synchronized with the active sensor on the camera. The auto-fluorescence emitting from the heart is captured by the detection objective lens and sCMOS camera. A customized LabVIEW control algorithm is used to synchronize the propagation of the laser, the stage movement, the sCMOS camera rolling shutter, and the electrically tunable lens (ETL). During the image acquisition process, the inherent trigger and rolling shutter on the sCMOS are employed to capture sequential uniform 2D images, while the stage continuously translates the tissue across the light sheet at a constant velocity that is determined by the step size and exposure time.

Results, Conclusions, and Discussions: Histological images (Figure 1-A,B) revealed that the HCM septal tissues had hypertrophic cardiomyocytes that were significantly larger and disarrayed when compared with the healthy cardiomyocytes (quantification ongoing). As shown by light sheet images (Figure 1-C,D), the healthy septum showed normal morphology with highly-aligned heart muscle fibers and evenly distributed small interfibrillar space, while the human hypertrophic septal tissues showed disarrayed muscle fiber configurations, with wider interfibrillar space that was unevenly distributed. In our mechanical study [3], we reported that human hypertrophic septal tissues had larger stress relaxation and larger creep and were stiffer at the initial phase of tissue loading, less nonlinear, less stiff in the linear region, and much weaker in mechanical strength. Both our histological images and light sheet images showed that the hypertrophic septal tissues had a higher degree of disarrayed, enlarged heart muscles and disorganized, higher amount of fibrotic collagen fibers in comparison to the healthy septum. The poorly-organized pathological compositions in the hypertrophic septum resulted in the loss of the benefits of native tissue design. For instance, the higher amount of fibrotic collagen could cause HCM tissues to be stiffer at the initial phase of mechanical loading; however, the pathologically-deposited fibrotic collagen and the disarrayed hypertrophic cardiomyocytes led to a less stiff behavior in the linear region and a much lower mechanical strength of the HCM tissues when compared to the healthy septal tissues [3]. That is to say, the inferior mechanical behavior of the HCM septal tissues can be attributed to microstructural abnormalities.

Acknowledgements (Optional): NIH R15HL159599, R01HL157050

[1] A. Varnava, et al., Heart (2000) 84(5): 476-482.
[2] R. Hinojar, et al., International journal of cardiology (2017) 249: 467-472.
[3] K. Copland, et al., SB3C Conference (2023).