(175w) Applying Engineered Microenvironments to Assess Structural and Mechanical Remodeling of the Cytoskeleton in Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disorder frequently caused by mutations in sarcomeric genes that are responsible for contractility. It is the leading cause of sudden cardiac death in young adults, with an estimated prevalence of 0.2% (1 in 500 adults). HCM is typically characterized by cardiomyocyte hypertrophy, hypercontractility, and diastolic dysfunction. Over 1000 mutations have been found, with a third of the mutations being identified in β-myosin, the dominant myosin isoform in the mature human heart. The condition presents heterogeneously, both genetically and clinically, making it challenging to diagnose and therefore treat. Further, the altered mechanics of the heart’s mechanosensing machinery in HCM are poorly understood. Costameres, which are rib-like structures aligned predominantly with z disks in cardiac muscle, are made up of a complex network of mechanosensitive proteins that are crucial for lateral force transmission and coordinated contraction of the heart muscle. One of these proteins is vinculin, which has historically been used as a distinguishing marker of costameres. It is a ubiquitously expressed protein that binds to talin and α-actinin, and functions as a linker between the extracellular matrix and the cytoskeleton. While mutations related to vinculin rarely cause hypertrophic cardiomyopathy, it has been found to be implicated in a missense mutation in obstructive hypertrophic cardiomyopathy¹. Its expression levels have also been found to be elevated in diseased myocardium tissue. These key findings suggest the involvement of vinculin in cardiomyopathies either directly through mutations, or indirectly as a compensatory mechanism for cardiac failure.

Here, we aim to understand how a point mutation from proline to arginine at the P710 residue (P710R) in β-cardiac myosin impacts force sensitivity by leveraging the structure and molecular dynamics of vinculin, along with Forster Resonance Energy Transfer (FRET). The P710 residue is located at the proximal edge of the converter domain, which is crucial for communication between the catalytic motor and the lever arm in myosins, and thus myosin’s force-producing power stroke². We investigate how this P710R mutation alters force dynamics at cell-cell and cell-matrix junctions and how pharmacologics affect these dynamics by studying vinculin’s force-sensitive responses. Structurally, vinculin has a globular head linked to a flexible tail by a proline-rich hinge region. It has been described as a “clutch” system, such that when engaged, the head binds to talin, while the tail binds to a-actinin, causing it to be in an open conformation. Here, CRISPR-edited P710R hiPSC-CMs were micropatterned on polyacrylamide gel at 10 kPa, which closely mimics the physiological stiffness, to allow for the alignment of myofibrils. Our image-splitting optical system allows for high-speed ratio metric imaging for quantification of sensitive, dynamically changing protein interactions. Computational segmentation of the vinculin structures in cardiomyocytes suggests that when P710R cells are treated with mavacamten, a novel drug used to treat obstructive hypertrophic cardiomyopathy, force is reduced. Our FRET metrics suggest that altered force generation in live micropatterned P710R hiPSC-CMs affects the tension-sensing dynamics of vinculin in the myocardium. Taken together, our findings suggest that vinculin is crucial for force stabilization in HCM, and understanding its interactions with cardiac myofilament as well as other costameric proteins could elucidate its role in HCM.

1. Vasile, Vlad C., et al. "A missense mutation in a ubiquitously expressed protein, vinculin, confers susceptibility to hypertrophic cardiomyopathy." Biochemical and biophysical research communications (2006)
2. Vander Roest, Alison, et al. "Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state." Proceedings of the National Academy of Sciences (2021)