Deteriorated biomechanical properties of human hypertrophied septum in response to cardiomyocyte enlargement, overexpressed collagen, and disarrayed microstructures

Abstract

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 and inferior cardiac performance. The cell and microstructural abnormalities are believed to be the cause of the altered tissue mechanical properties and inferior performance. However, there is a lack of detailed biomechanical assessments of human hypertrophied septum and a lack of understanding of the structural-mechanical relationship between altered biomechanical properties and cellular hypertrophy, fibrotic overexpression, and microstructural disruptions. In this study, we performed thorough biomechanical and microstructural characterizations on the human hypertrophied septum and compared this with healthy septum. We found that the hypertrophied human septum was stiffer at the initial phase of tissue loading, but less nonlinear, less stiff in the linear region, and much weaker in mechanical strength when compared to the healthy human septum. The fibrosis-induced initial stiffening in the hypertrophied septum paradoxically coexists with compromised mechanical strength and integrity under physiological demands, correlating with the clinical observations of diastolic dysfunction and susceptibility to myocardial damage in HCM patients despite ventricular wall thickening. We also discovered that the human hypertrophied septum had significantly larger stress relaxation and slightly larger creep when compared to healthy septum. Moreover, the abnormal, disorganized cell-collagen microstructures in the hypertrophied septum make short-term stress release more difficult and require longer relaxation times to reach equilibrium. Biaxial testing performed at the initial phase of tissue loading showed that both the healthy septum and hypertrophied septum had nonlinear anisotropic stress-strain behavior and confirmed that, in the longitudinal direction, the hypertrophied septum was stiffer than the healthy septum. Our microstructural quantifications via histology and light-sheet microscopy revealed that (i) the heterogeneous cardiomyocyte enlargement and disarray, combined with disorganized collagen overexpression, create a mechanically inefficient tissue architecture in the hypertrophied septum, and (ii) the observed cell-collagen microstructural disruptions provide mechanistic explanations for the deteriorated biomechanical properties. Our viscoelastic mechanical data and microstructural characterizations build a strong foundation to understand the altered tissue behavior of the hypertrophied septum, the degree of deviation from the normal septum, and the underlying structural mechanisms.

Keywords: and applications in multi-scale mechanobiology; biomechanical deterioration; collagen overexpression; concepts; emerging tools; human hypertrophied septum; hypertrophic cardiomyopathy; microstructural abnormalities.

FIGURE 1

(A) Average stress-strain behavior of the healthy septum and HCM septum. (B) Average stress relaxation curves for the healthy septum and HCM septum; the final amount of stress decay at 15 min was 56.63% ± 6.29% for the healthy septum and 68.36% ± 9.85% for the HCM septum. (C) Average creep curves for healthy septum and HCM septum. The final amount of creep at 15 min reached 9.1% ± 1.0% for the healthy septum and 9.8% ± 4.2% for the HCM septum. Note that blue curves are for healthy septum and red curves are for HCM septum.

FIGURE 2

(A) Average biaxial mechanical behavior of the healthy septum. (B) Average biaxial mechanical behavior of the HCM septum.

FIGURE 3

Masson’s trichrome staining revealed that the HCM septum had hypertrophic cardiomyocytes that were significantly larger and more disarrayed (B) when compared with the healthy cardiomyocytes (A). The hypertrophic septum also had a higher amount of fibrotic collagen fibers (B). For both panel (A,B), the image on the left is a representative histology of a cross-sectional section, and the image on the right is a representative histology of a longitudinal section.

FIGURE 4

(A) Average cell size (cross-sectional area); HCM muscle fibers showed increased cell size. (B) Percentage of area occupied by heart muscle cells; smaller area percentage occupied by HCM muscle cells is consistent with the fact of overexpression of fibrotic ECM. (C) Nearest neighbor distance (NND): Higher NND in HCM reflects excess fibrotic ECM that further separates individual heart muscle cells, causing disorganization.

FIGURE 5

(A) Heart muscle fiber alignment quantified from the longitudinal view; the alignment of muscle fibers in HCM tissue was significantly more dispersed when compared with the healthy tissue. The loss of heart muscle alignment is one of the microstructural abnormalities of HCM. (B) Cell size distribution in frequency; HCM has a broader distribution, extending up to 12,000 μm², reflecting a portion of normal-sized cells and pathologically enlarged cells due to hypertrophic growth.

FIGURE 6

Light-sheet imaging of healthy human septum and hypertrophied septum. 2D section images of the healthy human septum (A) and hypertrophied septum (B), scale bar: 500 μm; 3D reconstructed images of the healthy human septum (C) and hypertrophied septum (D), scale bar: 200 µm.