Mechanobiology of myofibroblast adhesion in fibrotic cardiac disease.

Fibrotic cardiac disease, a leading cause of death worldwide, manifests as substantial loss of function following maladaptive tissue remodeling. Fibrosis can affect both the heart valves and the myocardium and is characterized by the activation of fibroblasts and accumulation of extracellular matrix. Valvular interstitial cells and cardiac fibroblasts, the cell types responsible for maintenance of cardiac extracellular matrix, are sensitive to changing mechanical environments, and their ability to sense and respond to mechanical forces determines both normal development and the progression of disease. Recent studies have uncovered specific adhesion proteins and mechano-sensitive signaling pathways that contribute to the progression of fibrosis. Integrins form adhesions with the extracellular matrix, and respond to changes in substrate stiffness and extracellular matrix composition. Cadherins mechanically link neighboring cells and are likely to contribute to fibrotic disease propagation. Finally, transition to the active myofibroblast phenotype leads to maladaptive tissue remodeling and enhanced mechanotransductive signaling, forming a positive feedback loop that contributes to heart failure. This Commentary summarizes recent findings on the role of mechanotransduction through integrins and cadherins to perpetuate mechanically induced differentiation and fibrosis in the context of cardiac disease.

Cadherin-11 blockade reduces inflammation-driven fibrotic remodeling and improves outcomes after myocardial infarction.

Over one million Americans experience myocardial infarction (MI) annually, and the resulting scar and subsequent cardiac fibrosis gives rise to heart failure. A specialized cell-cell adhesion protein, cadherin-11 (CDH11), contributes to inflammation and fibrosis in rheumatoid arthritis, pulmonary fibrosis, and aortic valve calcification but has not been studied in myocardium after MI. MI was induced by ligation of the left anterior descending artery in mice with either heterozygous or homozygous knockout of CDH11, wild-type mice receiving bone marrow transplants from Cdh11-deficient animals, and wild-type mice treated with a functional blocking antibody against CDH11 (SYN0012). Flow cytometry revealed significant CDH11 expression in noncardiomyocyte cells after MI. Animals given SYN0012 had improved cardiac function, as measured by echocardiogram, reduced tissue remodeling, and altered transcription of inflammatory and proangiogenic genes. Targeting CDH11 reduced bone marrow-derived myeloid cells and increased proangiogenic cells in the heart 3 days after MI. Cardiac fibroblast and macrophage interactions increased IL-6 secretion in vitro. Our findings suggest that CDH11-expressing cells contribute to inflammation-driven fibrotic remodeling after MI and that targeting CDH11 with a blocking antibody improves outcomes by altering recruitment of bone marrow-derived cells, limiting the macrophage-induced expression of IL-6 by fibroblasts and promoting vascularization.

Loss of CENP-F Results in Dilated Cardiomyopathy with Severe Disruption of Cardiac Myocyte Architecture.

Centromere-binding protein F (CENP-F) is a very large and complex protein with many and varied binding partners including components of the microtubule network. Numerous CENP-F functions impacting diverse cellular behaviors have been identified. Importantly, emerging data have shown that CENP-F loss- or gain-of-function has critical effects on human development and disease. Still, it must be noted that data at the single cardiac myocyte level examining the impact of CENP-F loss-of-function on fundamental cellular behavior is missing. To address this gap in our knowledge, we analyzed basic cell structure and function in cardiac myocytes devoid of CENP-F. We found many diverse structural abnormalities including disruption of the microtubule network impacting critical characteristics of the cardiac myocyte. This is the first report linking microtubule network malfunction to cardiomyopathy. Importantly, we also present data demonstrating a direct link between a CENP-F single nucleotide polymorphism (snp) and human cardiac disease. In a proximate sense, these data examining CENP-F function explain the cellular basis underlying heart disease in this genetic model and, in a larger sense, they will hopefully provide a platform upon which the field can explore diverse cellular outcomes in wide-ranging areas of research on this critical protein.

I-Wire Heart-on-a-Chip II: Biomechanical analysis of contractile, three-dimensional cardiomyocyte tissue constructs.

This companion study presents the biomechanical analysis of the "I-Wire" platform using a modified Hill model of muscle mechanics that allows for further characterization of construct function and response to perturbation. The I-Wire engineered cardiac tissue construct (ECTC) is a novel experimental platform to investigate cardiac cell mechanics during auxotonic contraction. Whereas passive biomaterials often exhibit nonlinear and dissipative behavior, active tissue equivalents, such as ECTCs, also expend metabolic energy to perform mechanical work that presents additional challenges in quantifying their properties. The I-Wire model uses the passive mechanical response to increasing applied tension to measure the inherent stress and resistance to stretch of the construct before, during, and after treatments. Both blebbistatin and isoproterenol reduced prestress and construct stiffness; however, blebbistatin treatment abolished subsequent force-generating potential while isoproterenol enhanced this property. We demonstrate that the described model can replicate the response of these constructs to intrinsic changes in force-generating potential in response to both increasing frequency of stimulation and decreasing starting length. This analysis provides a useful mathematical model of the I-Wire platform, increases the number of parameters that can be derived from the device, and serves as a demonstration of quantitative characterization of nonlinear, active biomaterials. We anticipate that this quantitative analysis of I-Wire constructs will prove useful for qualifying patient-specific cardiomyocytes and fibroblasts prior to their utilization for cardiac regenerative medicine. STATEMENT OF SIGNIFICANCE: Passive biomaterials may have non-linear elasticity and losses, but engineered muscle tissue also exhibits time- and force-dependent contractions. Historically, mathematical muscle models include series-elastic, parallel-elastic, contractile, and viscous elements. While hearts-on-a-chip can demonstrate in vitro the contractile properties of engineered cardiac constructs and their response to drugs, most of these use cellular monolayers that cannot be readily probed with controlled forces. The I-Wire platform described in the preceding paper by Sidorov et al. addresses these limitations with three-dimensional tissue constructs to which controlled forces can be applied. In this companion paper, we show how to characterize I-Wire constructs using a non-linear, active Hill model, which should be useful for qualifying cells prior to their use in cardiac regenerative medicine.

Serotonin 2B Receptor Antagonism Prevents Heritable Pulmonary Arterial Hypertension.

Serotonergic anorexigens are the primary pharmacologic risk factor associated with pulmonary arterial hypertension (PAH), and the resulting PAH is clinically indistinguishable from the heritable form of disease, associated with BMPR2 mutations. Both BMPR2 mutation and agonists to the serotonin receptor HTR2B have been shown to cause activation of SRC tyrosine kinase; conversely, antagonists to HTR2B inhibit SRC trafficking and downstream function. To test the hypothesis that a HTR2B antagonist can prevent BMRP2 mutation induced PAH by restricting aberrant SRC trafficking and downstream activity, we exposed BMPR2 mutant mice, which spontaneously develop PAH, to a HTR2B antagonist, SB204741, to block the SRC activation caused by BMPR2 mutation. SB204741 prevented the development of PAH in BMPR2 mutant mice, reduced recruitment of inflammatory cells to their lungs, and reduced muscularization of their blood vessels. By atomic force microscopy, we determined that BMPR2 mutant mice normally had a doubling of vessel stiffness, which was substantially normalized by HTR2B inhibition. SB204741 reduced SRC phosphorylation and downstream activity in BMPR2 mutant mice. Gene expression arrays indicate that the primary changes were in cytoskeletal and muscle contractility genes. These results were confirmed by gel contraction assays showing that HTR2B inhibition nearly normalizes the 400% increase in gel contraction normally seen in BMPR2 mutant smooth muscle cells. Heritable PAH results from increased SRC activation, cellular contraction, and vascular resistance, but antagonism of HTR2B prevents SRC phosphorylation, downstream activity, and PAH in BMPR2 mutant mice.

Network Modeling Approach to Predict Myofibroblast Differentiation.

Fibrotic disease is a major cause of morbidity and mortality and is characterized by the transition of resident fibroblast cells into active myofibroblasts, identified by their expression of alpha smooth muscle actin. Myofibroblast differentiation is regulated by growth factor signaling and mechanical signals transduced through integrins, which converge at focal adhesion proteins (Src and FAK) and MAPK signaling, but lead to divergent outcomes. While details are known about individual pathways, little is known about their interactions. To this end, an ODE-based model of this cell signaling network was developed in parallel with in vitro experiments to analyze potential mechanisms of crosstalk and regulation of αSMA production. We found that cells lacking Src or FAK produce significantly less or more αSMA than wild type cells, respectively. Transforming growth factor beta 1 and fibroblast growth factor signal through ERK and MAPK p38 with different dynamic profiles to increase or decrease αSMA expression, respectively. Our model effectively recreated αSMA expression levels across a set of 22 experimental conditions and matched some features of transient phosphorylation of ERK and p38. These results support a potential mechanism for regulation of fibroblast differentiation: αSMA production is promoted by active p38 and Src and opposed by ERK.

The passive properties of muscle fibers are velocity dependent.

The passive properties of skeletal muscle play an important role in muscle function. While the passive quasi-static elastic properties of muscle fibers have been well characterized, the dynamic visco-elastic passive behavior of fibers has garnered less attention. In particular, it is unclear how the visco-elastic properties are influenced by lengthening velocity, in particular for the range of physiologically relevant velocities. The goals of this work were to: (i) measure the effects of lengthening velocity on the peak stresses within single muscle fibers to determine how passive behavior changes over a range of physiologically relevant lengthening rates (0.1-10Lo/s), and (ii) develop a mathematical model of fiber viscoelasticity based on these measurements. We found that passive properties depend on strain rate, in particular at the low loading rates (0.1-3Lo/s), and that the measured behavior can be predicted across a range of loading rates and time histories with a quasi-linear viscoelastic model. In the future, these results can be used to determine the impact of viscoelastic behavior on intramuscular stresses and forces during a variety of dynamic movements.