Establishment of a Biaxial Testing System for Characterization of Right Ventricle Viscoelasticity Under Physiological Loadings.

PURPOSE: Prior studies have indicated an impact of cardiac muscle viscoelasticity on systolic and diastolic functions. However, the studies of ventricular free wall viscoelasticity, particularly for that of right ventricles (RV), are limited. Moreover, investigations on ventricular passive viscoelasticity have been restricted to large animals and there is a lack of data on rodent species. To fill this knowledge gap, this study aims to develop a biaxial tester that induces high-speed physiological deformations to characterize the passive viscoelasticity of rat RVs. METHODS: The biaxial testing system was fabricated so that planar deformation of rat ventricle tissues at physiological strain rates was possible. The testing system was validated using isotropic polydimethylsiloxane (PDMS) sheets. Next, viscoelastic measurements were performed in healthy rat RV free walls by equibiaxial cyclic sinusoidal loadings and stress relaxation. RESULTS: The biaxial tester's consistency, accuracy, and stability was confirmed from the PDMS samples measurements. Moreover, significant viscoelastic alterations of the RV were found between sub-physiological (0.1 Hz) and physiological frequencies (1-8 Hz). From hysteresis loop analysis, we found as the frequency increased, the elasticity and viscosity were increased in both directions. Interestingly, the ratio of storage energy to dissipated energy (Wd/Ws) remained constant at 0.1-5 Hz. We did not observe marked differences in healthy RV viscoelasticity between longitudinal and circumferential directions. CONCLUSION: This work provides a new experimental tool to quantify the passive, biaxial viscoelasticity of ventricle free walls in both small and large animals. The dynamic mechanical tests showed frequency-dependent elastic and viscous behaviors of healthy rat RVs. But the ratio of dissipated energy to stored energy was maintained between frequencies. These findings offer novel baseline information on the passive viscoelasticity of healthy RVs in adult rats.

Role of Microtubule Network in the Passive Anisotropic Viscoelasticity of Healthy Right Ventricle.

Cardiomyocytes are viscoelastic and key determinants of right ventricle (RV) mechanics. Intracellularly, microtubules are found to impact the viscoelasticity of isolated cardiomyocytes or trabeculae; whether they contribute to the tissue-level viscoelasticity is unknown. Our goal was to reveal the role of the microtubule network in the passive anisotropic viscoelasticity of the healthy RV. Equibiaxial stress relaxation tests were conducted in healthy RV free wall (RVFW) under early (6%) and end (15%) diastolic strain levels, and at sub- and physiological stretch rates. The viscoelasticity was assessed at baseline and after the removal of microtubule network. Furthermore, a quasi-linear viscoelastic (QLV) model was applied to delineate the contribution of microtubules to the relaxation behavior of RVFW. After removing the microtubule network, RVFW elasticity and viscosity were reduced at the early diastolic strain level and in both directions. The reduction in elasticity was stronger in the longitudinal direction, whereas the degree of changes in viscosity were equivalent between directions. There was insignificant change in RVFW viscoelasticity at late diastolic strain level. Finally, the modeling showed that the tissue's relaxation strength was reduced by the removal of the microtubule network, but the change was present only at a later time scale. These new findings suggest a critical role of cytoskeleton filaments in RVFW passive mechanics in physiological conditions.

Role of the microtubule network in the passive anisotropic viscoelasticity of right ventricle with pulmonary hypertension progression.

Cardiomyocytes are viscoelastic and contribute significantly to right ventricle (RV) mechanics. Microtubule, a cytoskeletal protein, has been shown to regulate cardiomyocyte viscoelasticity. Additionally, hypertrophied cardiomyocytes from failing myocardium have increased microtubules and cell stiffness. How the microtubules contribute to the tissue-level viscoelastic behavior in RV failure remains unknown. Our aim was to investigate the role of the microtubules in the passive anisotropic viscoelasticity of the RV free wall (RVFW) during pulmonary hypertension (PH) progression. Equibiaxial stress relaxation tests were conducted in the RVFW from healthy and PH rats under early (6%) and end (15%) diastolic strains, and at sub- (1Hz) and physiological (5Hz) stretch-rates. The RVFW viscoelasticity was also measured before and after the depolymerization of microtubules at 5Hz. In intact tissues, PH increased RV viscosity and elasticity at both stretch rates and strain levels, and the increase was stronger in the circumferential than longitudinal direction. At 6% of strain, the removal of microtubules reduced elasticity, viscosity, and the ratio of viscosity to elasticity in both directions and for both healthy and diseased RVs. However, at 15% of strain, the effect of microtubules was different between groups - both viscosity and elasticity were reduced in healthy RVs, but in the diseased RVs only the circumferential viscosity and the ratio of viscosity to elasticity were reduced. These data suggest that, at a large strain with collagen recruitment, microtubules play more significant roles in healthy RV tissue elasticity and diseased RV tissue viscosity. Our findings suggest cardiomyocyte cytoskeletons are critical to RV passive viscoelasticity under pressure overload. STATEMENT OF SIGNIFICANCE: This study investigated the impact of microtubules on the passive anisotropic viscoelasticity of the right ventricular (RV) free wall at healthy and pressure-overloaded states. We originally found that the microtubules contribute significantly to healthy and diseased RV viscoelasticity in both (longitudinal and circumferential) directions at early diastolic strains. At end diastolic strains (with the engagement of collagen fibers), microtubules contribute more to the tissue elasticity of healthy RVs and tissue viscosity of diseased RVs. Our findings reveal the critical role of microtubules in the anisotropic viscoelasticity of the RV tissue, and the altered contribution from healthy to diseased state suggests that therapies targeting microtubules may have potentials for RV failure patients.

Alterations of biaxial viscoelastic properties of the right ventricle in pulmonary hypertension development in rest and acute stress conditions.

Introduction: The right ventricle (RV) mechanical property is an important determinant of its function. However, compared to its elasticity, RV viscoelasticity is much less studied, and it remains unclear how pulmonary hypertension (PH) alters RV viscoelasticity. Our goal was to characterize the changes in RV free wall (RVFW) anisotropic viscoelastic properties with PH development and at varied heart rates. Methods: PH was induced in rats by monocrotaline treatment, and the RV function was quantified by echocardiography. After euthanasia, equibiaxial stress relaxation tests were performed on RVFWs from healthy and PH rats at various strain-rates and strain levels, which recapitulate physiological deformations at varied heart rates (at rest and under acute stress) and diastole phases (at early and late filling), respectively. Results and Discussion: We observed that PH increased RVFW viscoelasticity in both longitudinal (outflow tract) and circumferential directions. The tissue anisotropy was pronounced for the diseased RVs, not healthy RVs. We also examined the relative change of viscosity to elasticity by the damping capacity (ratio of dissipated energy to total energy), and we found that PH decreased RVFW damping capacity in both directions. The RV viscoelasticity was also differently altered from resting to acute stress conditions between the groups-the damping capacity was decreased only in the circumferential direction for healthy RVs, but it was reduced in both directions for diseased RVs. Lastly, we found some correlations between the damping capacity and RV function indices and there was no correlation between elasticity or viscosity and RV function. Thus, the RV damping capacity may be a better indicator of RV function than elasticity or viscosity alone. These novel findings on RV dynamic mechanical properties offer deeper insights into the role of RV biomechanics in the adaptation of RV to chronic pressure overload and acute stress.

Strain-dependent stress relaxation behavior of healthy right ventricular free wall.

The increasing evidence of stress-strain hysteresis in large animal or human myocardium calls for extensive characterizations of the passive viscoelastic behavior of the myocardium. Several recent studies have investigated and modeled the viscoelasticity of the left ventricle while the right ventricle (RV) viscoelasticity remains poorly understood. Our goal was to characterize the biaxial viscoelastic behavior of RV free wall (RVFW) using two modeling approaches. We applied both quasi-linear viscoelastic (QLV) and nonlinear viscoelastic (NLV) theories to experimental stress relaxation data from healthy adult ovine. A three-term Prony series relaxation function combined with an Ogden strain energy density function was used in the QLV modeling, while a power-law formulation was adopted in the NLV approach. The ovine RVFW exhibited an anisotropic and strain-dependent viscoelastic behavior relative to anatomical coordinates, and the NLV model showed a higher capacity in predicting strain-dependent stress relaxation than the QLV model. From the QLV fitting, the relaxation term associated with the largest time constant played the dominant role in the overall relaxation behavior at most strains from early to late diastole, whereas the term associated with the smallest time constant was pronounced only at low strains at early diastole. From the NLV fitting, the parameters showed a nonlinear dependence on the strain. Overall, our study characterized the anisotropic, nonlinear viscoelasticity to capture the elastic and viscous resistances of the RVFW during diastole. These findings deepen our understanding of RV myocardium dynamic mechanical properties. STATEMENT OF SIGNIFICANCE: Although significant progress has been made to understand the passive elastic behavior of the right ventricle free wall (RVFW), its viscoelastic behavior remains poorly understood. In this study, we originally applied both quasi-linear viscoelastic (QLV) and nonlinear viscoelastic (NLV) models to published experimental data from healthy ovine RVFW. Our results revealed an anisotropic and strain-dependent viscoelastic behavior of the RVFW. The parameters from the NLV fitting showed nonlinear relationships with the strain, and the NLV model showed a higher capacity in predicting strain-dependent stress relaxation than the QLV model. These findings characterize the anisotropic, nonlinear viscoelasticity of RVFW to fully capture the total (elastic and viscous) resistance that is critical to diastolic function.

Multiscale Contrasts Between the Right and Left Ventricle Biomechanics in Healthy Adult Sheep and Translational Implications.

Cardiac biomechanics play a significant role in the progression of structural heart diseases (SHDs). SHDs alter baseline myocardial biomechanics leading to single or bi-ventricular dysfunction. But therapies for left ventricle (LV) failure patients do not always work well for right ventricle (RV) failure patients. This is partly because the basic knowledge of baseline contrasts between the RV and LV biomechanics remains elusive with limited discrepant findings. The aim of the study was to investigate the multiscale contrasts between LV and RV biomechanics in large animal species. We hypothesize that the adult healthy LV and RV have distinct passive anisotropic biomechanical properties. Ex vivo biaxial tests were performed in fresh sheep hearts. Histology and immunohistochemistry were performed to measure tissue collagen. The experimental data were then fitted to a Fung type model and a structurally informed model, separately. We found that the LV was stiffer in the longitudinal (outflow tract) than circumferential direction, whereas the RV showed the opposite anisotropic behavior. The anisotropic parameter K from the Fung type model accurately captured contrasting anisotropic behaviors in the LV and RV. When comparing the elasticity in the same direction, the LV was stiffer than the RV longitudinally and the RV was stiffer than the LV circumferentially, suggesting different filling patterns of these ventricles during diastole. Results from the structurally informed model suggest potentially stiffer collagen fibers in the LV than RV, demanding further investigation. Finally, type III collagen content was correlated with the low-strain elastic moduli in both ventricles. In summary, our findings provide fundamental biomechanical differences between the chambers. These results provide valuable insights for guiding cardiac tissue engineering and regenerative studies to implement chamber-specific matrix mechanics, which is particularly critical for identifying biomechanical mechanisms of diseases or mechanical regulation of therapeutic responses. In addition, our results serve as a benchmark for image-based inverse modeling technologies to non-invasively estimate myocardial properties in the RV and LV.

Pro-angiogenic Potential of Mesenchymal Stromal Cells Regulated by Matrix Stiffness and Anisotropy Mimicking Right Ventricles.

Capillary rarefaction is a hallmark of right ventricle (RV) failure. Mesenchymal stromal cell (MSC)-based therapy offers a potential treatment due to its pro-angiogenic function. However, the impact of RV tissue mechanics on MSC behavior is unclear, especially when referring to RV end-diastolic stiffness and mechanical anisotropy. In this study, we assessed MSC behavior on electrospun scaffolds with varied stiffness (normal vs failing RV) and anisotropy (isotropic vs anisotropic). In individual MSCs, we observed the highest vascular endothelial growth factor (VEGF) production and total tube length in the failing, isotropic group (2.00 ± 0.37, 1.53 ± 0.24), which was greater than the normal, isotropic group (0.70 ± 0.15, 0.55 ± 0.07; p < 0.05). The presence of anisotropy led to trends of increased VEGF production on normal groups (0.75 ± 0.09 vs 1.20 ± 0.17), but this effect was absent on failing groups. Our findings reveal synergistic effects of RV-like stiffness and anisotropy on MSC pro-angiogenic function and may guide MSC-based therapies for heart failure.

The Interventricular Septum Is Biomechanically Distinct from the Ventricular Free Walls.

The interventricular septum contributes to the pumping function of both ventricles. However, unlike the ventricular wall, its mechanical behavior remains largely unknown. To fill the knowledge gap, this study aims to characterize the biaxial and transmural variation of the mechanical properties of the septum and compare it to the free walls of the left and right ventricles (LV/RV). Fresh hearts were obtained from healthy, adult sheep. The septal wall was sliced along the mid-line into two septal sides and compared to the epicardial layers of the LV- and RV-free walls. Biaxial tensile mechanical tests and constitutive modeling were performed to obtain the passive mechanical properties of the LV- and RV-side of the septum and ventricular walls. We found that both sides of the septum were significantly softer than the respective ventricular walls, and that the septum presented significantly less collagen than the ventricular walls. At low strains, we observed the symmetric distribution of the fiber orientations and a similar anisotropic behavior between the LV-side and RV-side of the septum, with a stiffer material property in the longitudinal direction, rather than the circumferential direction. At high strains, both sides showed isotropic behavior. Both septal sides had similar intrinsic elasticity, as evidenced by experimental data and constitutive modeling. These new findings offer important knowledge of the biomechanics of the septum wall, which may deepen the understanding of heart physiology.