Poster U8 - Cellular Dynamics in Hypertrophic Cardiomyopathy: Mechanobiology and Morphology in Mutant and Control Stem Cell Derived Cardiomyocytes

Introduction: Hypertrophic Cardiomyopathy (HCM) is the most common inherited cardiovascular disease, which affects every 1 in 500 individuals. HCM is characterized by hypercontractility and hypertrophy of heart muscle cells and tissue. It can be caused by genetic mutation in beta-myosin (MYH7). Effects of the H251N mutation in beta-myosin is studied with human stem-cell derived cardiomyocytes (CMs). Many groups have developed protocols for differentiating cardiomyocytes from stem cells which may have implications for cardiomyocyte development and function. We have previously seen changes in traction force generation and protein ratio through traction force microscopy and immunostaining but have not compared this mutation and differentiation methods directly in the past. In this study, cell size, aspect ratio, and protein ratio of CMs with and without H251N mutation are analyzed through immunostaining, western blot, and qPCR. This study aims to understand the morphology and mechanobiology of the CMs affected by the mutation and differentiation method and how it might further lead to the development of HCM.

Materials and

Methods: Induced pluripotent stem cells (iPSCs) were differentiated into cardiomyocytes (CMs) under two different protocols: standard and baker/helms. Both CMs with H251N mutation (C3) and an isogenic control line (C4) were obtained from collaborators at the Allen Institute. At day 30 of differentiation, these CMs were lysed for western blot and qPCR and replated to coverslips coated with Matrigel for immunostaining. At two timepoints, 48 hours and 1 week after replating, the cells were lysed for immunostaining. Samples were stained with primary and secondary antibodies for alpha and beta myosin and imaged. Potential impacts of differentiation protocols and mutation on cell size, aspect ratio, protein ratio/fluorescence from imaged samples were analyzed through Cell Profiler. Western blot samples were also stained with alpha-, beta-myosin, Erk, Akt and GAPDH house gene. qPCR was also performed to analyze the genetic expression of alpha- and beta-myosin.

Results, Conclusions, and Discussions: Analysis on the immunostaining samples indicated that there is a statistically significant difference in the cell size between C3 and C4 cardiomyocytes, varied by two protocols and two timepoints of immunostaining after replating the cells (Figure 1). Under the standard protocol, C3 cell size is significantly greater than C4 in both timepoints. However, when differentiated by the baker/helms protocol, the control line has a greater cell size than the mutant line. There is a statistically significant difference in cell size within C3 between two protocols. Between two timepoints, there is a difference in the cell size, cells being larger at 48 hours than at 1-week timepoint. This may imply changes in morphology and the importance in choice of timepoints in the future for analyzing repeated batches for cellular heterogeneity. There is also a significant difference in aspect ratio between mutant and control lines at 1-week timepoint in both protocols. Western blot and qPCR results confirmed that the expression level of proteins is consistent and there is no drastic change in the magnitude of expression.
These results show that H251N mutation in C3 line led to a hypertrophic condition, possibly leading to the hypertrophic cardiomyopathy. It is also shown that the protocols have a great impact on the morphology of the CMs, both in cell size and aspect ratio. Future work will compare contractility and mechanobiology and seek to understand the signaling through growth pathways to better understand the disease development.

Acknowledgements (Optional): We would like to acknowledge the funding sources, the NIH NHLBI K99/R00 Pathway to Independence, NIH NIGMS RM1 Early Stage Investigator Development grants. Additionally, we would like to thank the entire Vander Roest Cardiac Mechanobiology Laboratory and the Department of Biomedical Engineering for their support.