ABSTRACT
Hypertrophy Cardiomyopathy (HCM) is the most prevalent hereditary cardiovascular disease - affecting >1:500 individuals. Advanced forms of HCM clinically present with hypercontractility, hypertrophy and fibrosis. Several single-point mutations in b-myosin heavy chain (MYH7) have been associated with HCM and increased contractility at the organ level. Different MYH7 mutations have resulted in increased, decreased, or unchanged force production at the molecular level. Yet, how these molecular kinetics link to cell and tissue pathogenesis remains unclear. The Hippo Pathway, specifically its effector molecule YAP, has been demonstrated to be reactivated in pathological hypertrophic growth. We hypothesized that changes in force production (intrinsically or extrinsically) directly alter the homeostatic mechano-signaling of the Hippo pathway through changes in stresses on the nucleus. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we asked whether homeostatic mechanical signaling through the canonical growth regulator, YAP, is altered 1) by changes in the biomechanics of HCM mutant cardiomyocytes and 2) by alterations in the mechanical environment. We use genetically edited hiPSC-CM with point mutations in MYH7 associated with HCM, and their matched controls, combined with micropatterned traction force microscopy substrates to confirm the hypercontractile phenotype in MYH7 mutants. We next modulate contractility in healthy and disease hiPSC-CMs by treatment with positive and negative inotropic drugs and demonstrate a correlative relationship between contractility and YAP activity. We further demonstrate the activation of YAP in both HCM mutants and healthy hiPSC-CMs treated with contractility modulators is through enhanced nuclear deformation. We conclude that the overactivation of YAP, possibly initiated and driven by hypercontractility, correlates with excessive CCN2 secretion (connective tissue growth factor), enhancing cardiac fibroblast/myofibroblast transition and production of known hypertrophic signaling molecule TGFß. Our study suggests YAP being an indirect player in the initiation of hypertrophic growth and fibrosis in HCM. Our results provide new insights into HCM progression and bring forth a testbed for therapeutic options in treating HCM.
ABSTRACT
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have advanced our ability to study the basic function of the heart and model cardiac diseases. Due to the complexities in stem cell culture and differentiation protocols, many researchers source their hiPSC-CMs from collaborators or commercial biobanks. Generally, the field has assumed the health of frozen cardiomyocytes is unchanged if the cells adhere to the substrate and commence beating. However, very few have investigated the effects of cryopreservation on hiPSC-CM's functional and transcriptional health at the cellular and molecular level. Here we review methods and challenges associated with cryopreservation, and examine the effects of cryopreservation on the functionality (contractility and calcium handling) and transcriptome of hiPSC-CMs from six healthy stem cell lines. Utilizing protein patterning methods to template physiological cell aspect ratios (7:1, length:width) in conjunction with polyacrylamide (PA) hydrogels, we measured changes in force generation and calcium handling of single hiPSC-CMs. We observed that cryopreservation altered the functionality and transcriptome of hiPSC-CMs towards larger sizes and contractile force as assessed by increased spread area and volume, single cell traction force microscopy and delayed calcium dynamics. hiPSC-CMs are broadly used for basic science research, regenerative medicine, and testing biological therapeutics. This study informs the design of experiments utilizing hiPSC-CMs to avoid confounding functional changes due to cryopreservation with other treatments.
