Kargar Gaz Kooh, Y. | bioRxiv Science

bioRxiv2025

A Micro-Engineered Heart Tissue Model of Desmin-related Cardiomyopathy Caused by Mutant αB Crystalin

Protein quality control (PQC) is essential for maintaining sarcomere integrity in cardiomyocytes. Crystallin B chain (CRYAB) R120G mutation disrupts CRYABs chaperone activity, leading to aggregation of CRYAB and its client proteins (including Desmin), leading to Desmin-related cardiomyopathy (DRM). Prior experimental systems for modeling DRM linked to CRYAB require massive overexpression of CRYAB mutant isoforms, raising questions about translational relevance. Here, we establish the first model of CRYAB-linked DRM that uses genome-edited hiPSC together with isogenic controls, allowing us to study the impact of mutant CRYAB expressed at near endogenous levels. Within micro-engineered heart tissues (HT), CRYAB-R120G mutant hiPSC-derived cardiomyocytes recapitulated key DRM hallmarks, including Desmin and CRYAB aggregation, contractile dysfunction, and increased vulnerability to PQC pathway inhibition. CRYAB-R120G mutant HT also exhibited dysfunctional calcium-contraction coupling, which exacerbated contractile deficits at higher pacing frequencies. JAK1 inhibition with Itacitinib partially restored contractile function at higher pacing frequencies, suggesting JAK1 inhibition as a viable therapeutic strategy. By preserving human-specific structural and functional features, our {micro}HT platform enables mechanistic characterization of proteotoxic cardiomyopathies and offers a scalable system for targeted drug screening.

bioengineering↗

bioRxiv2024

Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

BackgroundProtein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiac myocytes, and mutations in PQC pathway proteins, such as CRYAB (arginine to glycine at position 120, R120G) and BAG3 (proline to lysine at position 209, P209L) induce protein aggregate pathology with cardiomyopathy in humans. Novel observations in yeast and mammalian cells demonstrate mitochondrial uptake of cytosolic protein aggregates. We hypothesized that mitochondrial uptake of cytosolic protein aggregates and their removal by mitophagy, a lysosomal degradative pathway essential for myocardial homeostasis, facilitates cytosolic protein quality control in cardiac myocytes. MethodsMice with inducible cardiac myocyte specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assessed for protein aggregates with biochemical fractionation and super-resolution imaging in comparison to floxed controls. Induced pluripotent stem cell (iPSC)-derived cardiac myocytes with R120G knock-in to the CRYAB locus were assessed for localization of the CRYAB protein. Transgenic mice expressing R120G CRYAB protein (R120G-TG) were subjected to both TRAF2 gain-of-function (with AAV9-cardiac Troponin T promoter-driven TRAF2 transduction) and TRAF2 loss-of-function (with tamoxifen-inducible ablation of one Traf2 allele) in cardiac myocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology. ResultsCardiomyocyte-specific ablation of TRAF2 results accumulation of mitochondrial and cytosolic protein aggregates and DESMIN mis-localization to protein aggregates. Isolated mitochondria take up cardiomyopathy-associated aggregate-prone cytosolic chaperone proteins, namely arginine to glycine (R120G) CRYAB mutant and proline to lysine (P209L) BAG3 mutant. R120G-CRYAB mutant protein increasingly localizes to mitochondria in human and mouse cardiomyocytes. R120G-TG mice demonstrate upregulation of TRAF2 in the mitochondrial fraction with increased mitophagy as compared with wild type. Adult-onset inducible haplo-insufficiency of TRAF2 resulted in accelerated mortality, impaired left ventricular systolic function and increased protein aggregates in R120G-TG mice as compared with controls. Conversely, AAV9-mediated TRAF2 transduction in R120G-TG mice reduced mortality and attenuated left ventricular systolic dysfunction, with reduced protein aggregates and restoration of normal localization of DESMIN, a cytosolic scaffolding protein chaperoned by CRYAB, as compared with control AAV9-GFP group. ConclusionsTRAF2-mediated mitophagy in cardiac myocytes facilitates removal of cytosolic protein aggregates and can be stimulated to ameliorate proteotoxic cardiomyopathy.

cell biology↗