Effects of collagen on the passive biaxial viscoelasticity of healthy right ventricle

Introduction: Right ventricle (RV) failure, often precipitated by increased afterload in conditions like pulmonary hypertension (PH), is involved with alterations of the RV free wall (RVFW) biological and viscoelastic properties. It is accepted that the low-strain elasticity of RV is dominated by myofibers whereas the high-strain elasticity is dominated by collagen fibers. The accumulation of collagenis identified in PH ovine RVs, and it is negatively correlated with stroke volume and positively correlated with RV hypertrophy indices. Therefore, collagen deposition during PH progression contributes to the RV mechanical changes and may play a role in regulating RV performance.  While the role of collagen in cardiac tissue biomechanics has been extensively investigated, its impact on RV biaxial viscoelasticity remains largely unknown. To our knowledge, the only study investigating the role of collagen in RV viscoelasticity is performed in papillary muscles, but only one dimensional passive viscoelasticity was characterized. Therefore, the role of collagen in the passive, biaxial viscoelasticity of RVFW in physiological and pathological states is unknown.

In this study, we aimed to elucidate the role of collagen in the passive, biaxial viscoelasticity of the healthy RVFW. Equibiaxial stress relaxation was conducted on RVFW tissues from healthy rats before and after collagenase treatment. Our findings reveal that collagen not only contributes to RVFW viscoelasticity differentially in different directions, but also alters the the degree of anisotropy as well as the relative strength of viscosity and elasticity.  These results provides new insights into the role of collagen in RV dynamic mechanical behavior.

Materials and

Methods: All procedures were approved by Colorado State University IACUC. Following euthanasia, the RVFW from healthy, male rats (n=4) was excised and immersed in a cardioplegic solution containing 30 mM 2,3-butanedione monoxime (BDM) maintained at 37°C. After tissue mounting, equibiaxial stress relaxation was performed. The longitudinal (L) direction was defined as the outflow tract direction. The tissue was preloaded to 0.025N, followed by 15 cycles of preconditioning cycles and then subjected to a strain to 20%, at physiological stretch rates of 5Hz. Following baseline mechanical characterization, the tissues were treated with collagenase (6 mg/mL, Sigma-Aldrich) at 37°C for 1 hour to enzymatically degrade the collagen fibers. Subsequent mechanical testing was performed under the same protocol as used in the baseline measurements. A rest period of 10 times that of the testing period was applied after each test to ensure complete recovery of the tissue.

The Second Piola-Kirchhoff stress and Green strain were calculated. Next, the relaxation modulus, stored energy (WS) and dissipated energy (Wd) were computed at 1s and 100s post peak stress to quantify the elasticity and viscosity of the RV myocardium at early and late relaxation stages. The V/E ratio was derived as Wd/WS, and the damping was derived as Wd/(Wd+WS). These methods have been established previously. Unpaired and paired student t-tests were used to compare data between groups and before and after collagenase treatment at each direction. Data is shown as mean ± SD. A p-value of less than 0.05 was considered statistically significant.

Results, Conclusions, and Discussions: Before collagen removal, the RVFW exhibited strong anisotropic behavior in most viscoelastic parameters, with the longitudinal direction presenting stronger viscosity and elasticity. The anisotropy behavior maintained from early to late relaxation, except that the V/E ratio was anisotropic at 1s after peak stress (early relaxation) but not at 100s after peak stress (late relaxation). After the removal of collagen, the tissue became less anisotropic (i.e., with a larger but still significant p value), indicating an alteration of tissue structure following the enzymatic degradation. The anisotropic behavior of RVFW is a combined influence of myofiber and collagen fibers, and our results suggest that collagen play a critical role in the anisotropic behavior of intact RV.

Next, we examind the effect of collagen on individual viscoelastic parameters. The collagen removal leads to reduced viscous and elastic parameters in the longitudinal direction (p < 0.05) (Figure 1). This trend was not observed in the circumferential direction. This suggest that the collagen’s contribution to RVFW viscoelasticity is different in different directions. Similar findings were observed in late relaxation stage. Moreover, we observed a reduction in V/E ratio in the longitudinal direction after collagne removal (Figure 1), and the change was only significant at early relaxation (p < 0.05). In the circumferential direction, collagen removal resulted in a trend of increased V/E ratio (p=0.09) at early relaxation. A complete summary of all viscoelastic parameters is presented in Table 1. These data suggest that the effect of collagen on tissue elasticity is stronger than that on the viscosity, and the impact of collagen is stronger in early than late relaxation stages.

In summary, our results revealed new findings on the effects of collagen on passive, biaxial viscoelastic properties of the healthy RVFW. The differential influences of collagen between viscosity and elasticity and between directions are critical to the overall dynamic mechanical behavior of the tissue.

Acknowledgements (Optional):