Date of Award
Doctor of Philosophy (PhD)
Kenneth B. Margulies
The development of myocardial hypertrophy and fibrosis are central pathological processes that are common features resulting from many types of cardiac diseases. Moreover, a wide variety of inputs and interactions contribute to pathological hypertrophy and fibrosis. For example, changes in biomechanical stress on the myocardium, as occur during chronic pressure or volume overload, is a fundamental trigger for hypertrophy and fibrosis. In addition, crosstalk between myocytes and fibroblasts contributes to the structural, mechanical, and electrical remodeling in the pathogenesis of various heart conditions that lead to heart failure. During the development of pathological hypertrophy and fibrosis, many agonists such as endothelin (ET)-1, angiotensin (Ang) II, and transforming growth factor (TGF)-β are activated in parallel, obscuring attribution of their individual, synergistic or subordinate effects. Finally, the development of hypertrophy and fibrosis themselves contribute to load changes in the heart, further complicating mechanistic interpretation. One impediment to further progress has been the lack of model systems that allow the experimental control required to draw definitive mechanistic conclusions of each of its components, yet retain the essential features of the in vivo environment.
Accordingly, a major focus of this thesis is to examine the ability of a myocardial tissue engineering platform to decouple the effects of biochemical, mechanical and cell-specific inputs on microtissue auxotonic contractility. The model employed is based on microfabricated polydimethylsiloxane (PDMS) templates that generate arrays of 3D cardiac microtissues (CMT). Cantilevers within templates provide physiologically relevant auxotonic loading to the CMTs, promote the appropriate 3D organization of neonatal rat cardiac myocytes and fibroblasts, and report resting and twitch force generation in real time. Additionally, we evaluated the correlation between sarcomere length and microtissue length, and developed twitch forces of these microtissues in an auxotonic preparation.
While the role of known hypertrophic factors has been extensively studied using conventional cell culture and integrated in vivo models, few studies have used engineered cardiac tissues to examine how key hypertrophic agonists, alone or in combination, affect contractile parameters, including resting and twitch force as well as rates of force generation and relaxation. We found that the pathological mediators, endothelin (ET)-1, angiotensin (Ang) II, and transforming growth factor (TGF)-β, altered contractility with different magnitudes. Differences in contractile responses led us to further investigate the length-tension relationship in the microtissues. We further investigated how sarcomere length related to tissue length and contractile properties. Interestingly, we identified differential sarcomere lengths upon stimulation with different hypertrophic factors. ET-1 in particular, led to the largest changes in contractile properties. These results are described in greater detail in Chapter 2.
Recognizing that biomechanical load acts in concert with pathological mediators in the development if cardiac hypertrophy, we utilized this cardiac microtissue model to generate templates with cantilevers with increased stiffness (Legant et al. 2009). The cantilever stiffness represent the resistance against which the engineered CMTs needs to contract, and mimics increased afterload as might occur during hypertension. We also studied the effect of increased afterload in combination with ET-1, Ang II, TGF-β upon force generation, and cell and tissue morphology. Interestingly, our data shows that cell area is altered only in the presence of increased afterload combined with hypertrophic factors, but not with the hypertrophic factors alone. These results are described in greater detail in Chapter 3.
While many studies have focused on the interactions of nonmyocytes and myocytes, few studies have looked at the role of nonmyocytes in engineered tissue contractile function. Our studies focus on the role of nonmyocytes in contractile function. Our results suggest that myocyte enrichment (nonmyocyte depletion) leads to decreased contractile function, suggesting that nonmyocytes are required for proper contractile function. We also evaluated how nonmyocytes within engineered tissues contribute to the ET-1-induced changes in contractility. These results are described in greater detail in Chapter 4.
Collectively, these studies have provided insights as to how cardiac microtissues can be employed to both isolate and integrate the biochemical and mechanical signals that contribute to changes in contractile function in the context of myocardial hypertrophy and disease. Continued work and future directions is discussed in Chapter 5.
Alvarez Lopez, Rosa Maria, "Structural And Functional Analysis Of Engineered Cardiac Tissues In Response To Hypertrophic Growth Factors" (2016). Publicly Accessible Penn Dissertations. 2163.