Abstract Body: Chaperone proteins such as αB-Crystallin (CRYAB) are critical for maintaining protein quality in cardiomyocytes. CRYAB mutations provoke aggregation of CRYAB and its client protein, Desmin, resulting in cardiomyopathy in humans. Most prior studies of CRYAB-linked desmin dysfunction have relied on mouse models with overexpression of mutant protein, as human iPSC-cardiomyocytes (iPS-CMs) are structurally immature. We hypothesized that culturing iPS-CM in 3D micro-heart tissues (μHT) improves structural maturity, permitting further examination of the mechanisms underlying cardiomyopathy caused by CRYAB mutations.
To test our hypothesis, we engineered the first in vitro μHT platform from iPS-CMs expressing physiological levels of aggregate-prone CRYAB with knock-in of arginine-to-glycine mutation at position 120 (CRYAB-R120G) (Fig. 1a). At day 10, CRYAB-R120G μHT were analyzed for desmin expression, sarcomere organization, contractility, and action potential waveforms. To investigate protein quality control, μHTs were treated for 48 hours with proteasome inhibitor Bortezomib (100nM) or autophagy inhibitor Bafilomycin (10nM) followed by contractility and structural analysis.
Culturing iPSC-CMs within 3D μHT significantly improved desmin expression compared to 2D (Fig. 1b, c, d). CRYAB-R120G-μHT showed similar action potential waveforms and desmin expression level as control μHT but increased desmin aggregation and diminished contractility (Fig. 1e). Finally, CRYAB-R120G-μHT were more sensitive to disruptions in protein quality control pathways, showing greater contractile deficit and desmin disorganization with Bortezomib (Fig. 1f).
In conclusion, our findings demonstrate that 3D μHT culture of iPS-CM significantly improved desmin maturation, and CRYAB mutant protein expressed at physiological levels in μHT impairs cardiomyocyte contractility and disrupts desmin organization providing a model system for mechanistic studies.