Biology subjects

Malayath, G.

Publications and source records attributed to Malayath, G..

5 recordsLinked to original sources

bioRxiv2026

Tunable Magnetorheological Elastomer Substrates for Studying Mechano-Genetic Interactions in Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. While sarcomeric genomic variants drive HCM, a role for mechanical stress in disease pathogenesis is increasingly appreciated. However, existing in vitro models of HCM rely on model systems (e.g., hydrogels) with static mechanical properties, precluding investigation of how dynamic mechanical loading interacts with genetic susceptibility during disease initiation. Here, we leveraged a dynamically tunable magnetorheological elastomer (MRE) platform to model progressive mechanical stiffening in order to study mechano-genetic interactions in human induced pluripotent stem cell (hiPSC) derived cardiomyocytes harboring an HCM-associated MYBPC3 frameshift (MYBPC3fs) loss-of-function variant. Dynamic stiffening selectively unmasked pathological structural remodeling in MYBPC3fs cardiomyocytes while producing only modest changes in isogenic controls, suggesting that MYBPC3fs confers heightened mechanosensitivity. In MYBPC3fs cardiomyocytes, mechanical stiffening drove hypertrophic remodeling, sarcomere disorganization, nuclear remodeling, dysregulated NFAT3 signaling, and aberrant cell-cycle activity. MRE stiffening also drove fibroblast-to-myofibroblast transition in primary cardiac fibroblasts, recapitulating early fibrotic remodeling. Together, these findings position dynamically tunable MRE substrates as a human-relevant platform for dissecting mechano-genetic disease mechanisms and evaluating therapies targeting mechanotransduction pathways in HCM.

bioengineering↗

bioRxiv2026

Mechanical history shapes phenotypic plasticity and fibroblast-autonomous 3D remodeling in iPSC-derived cardiac fibroblasts

Human induced pluripotent stem cell-derived cardiac fibroblasts (iPSC-cFBs) offer a renewable, genetically defined stromal cell source for studying cardiac fibrosis and cardiomyocyte-fibroblast crosstalk during development and disease. Because cardiac fibroblasts are proliferative, expansion of iPSC-cFBs in culture is attractive for applications requiring large cell numbers, including drug screening and tissue engineering. However, phenotypic drift during expansion, particularly premature fibroblast-to-myofibroblast transition (FMT), remains a concern. Here, we investigated how differentiation protocol, mechanical culture history, and serial expansion shape iPSC-cFB phenotype, transforming growth factor beta 1 (TGF-beta1) responsiveness, and fibroblast-autonomous remodeling in three-dimensional (3D) tissues. Initial studies of iPSC-cFBs expanded on conventional tissue-culture polystyrene (TCP) revealed passage-associated increases in projected cell area and alpha-smooth muscle actin (alpha-SMA) expression, two markers associated with FMT. These findings prompted us to examine how the stiffness of poly(dimethylsiloxane) (PDMS) substrates (15 kPa or 2 MPa) used during iPSC-cFB derivation and expansion influences basal phenotype and response to TGF-beta1. Across two independent differentiation protocols, mechanical culture history shaped passage-dependent basal phenotypes and responses to TGF-beta1. Notably, its effects on collective TGF-beta1 responses in three-dimensional cultures differed from those observed after replating cells onto two-dimensional substrates. Together, these findings demonstrate that mechanical culture history modulates iPSC-cFB responsiveness and tissue-remodeling behavior without irreversibly determining fibroblast state. Subsequent passage and microenvironmental conditions can attenuate or re-express these history-dependent phenotypes. This work supports the use of iPSC-cFBs in basic biological discovery and future tissue engineering applications.

bioengineering↗

bioRxiv2026

Xeno-Free Peptide-Functionalized Hydrogels Support hiPSC Encapsulation and In Situ Differentiation into Structurally Mature Cardiomyocytes

While defined synthetic substrates can replace Matrigel for human induced pluripotent stem cell (hiPSC) culture and hiPSC-derived cardiomyocyte (hiPSC-CM) production, existing approaches culture cells on two-dimensional surfaces and yield structurally immature cardiomyocytes, limiting their use in disease modeling and regenerative medicine. Here, we developed a xeno-free, fully-defined cyclic RGD (cRGD)-functionalized alginate platform in which we encapsulated hiPSCs to support their expansion and in situ cardiac differentiation. cRGD functionalization was essential for hiPSC survival and pluripotency, with maximal support achieved at a low ligand density (25 {micro}M). In the presence of cRGD, hiPSC encapsulation into softer gels made from lower molecular weight alginates led to enhanced hiPSC expansion and improved cardiogenesis. Strikingly, differentiation in situ with 3D gels led to hiPSC-CM with higher structural maturity, including a markedly increased proportion of Desmin-positive cardiomyocytes. Finally, after enzymatic retrieval from hydrogels, cardiomyocytes derived from softer gels formed tissue-engineered myocardium with superior contractile force compared to tissue fashioned from hiPSC-CM derived from more rigid gels. Together, these results demonstrate the promise of this defined, tunable platform for biomanufacturing of structurally mature cardiomyocytes from hiPSC.

bioengineering↗

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↗

bioRxiv2025

Rapid thermoforming of polycarbonate cell culture accessories from 3D printed molds

Bespoke cell culture devices are essential for tissue engineering applications. Traditional manufacturing methods for cell culture accessories involve injection molding and machining, which are too costly and time-consuming to implement for producing custom designs in small batches, and/or while testing the usefulness of a new design before mass producing it. Materials typically used for rapid design iteration, like poly(dimethylsiloxane) (PDMS) elastomers, surmount this issue but present new challenges of affinity for hydrophobic small molecules and sub-optimal interactions with sensitive cell types. Here, we propose polycarbonate (PC) thermoforming as a solution for creating customized transparent and autoclavable accessories. We demonstrate that optimized preheating of PC overcomes issues with bubbling during thermoforming. The use of high heat deflection temperature (HDT) resins allows these PC devices to be thermoformed off molds created by Digital Light Processing (DLP) 3D prints, enabling rapid prototyping of the PC. Using this approach, we fabricated custom PC well plate inserts. These inserts combine many advantages of tissue culture polystyrene (negligible absorption of hydrophobic molecules, transparency, rigidity) and elastomers (ease of creating bespoke devices, ability to be sterilized by autoclaving) and are compatible with a variety of cell biology applications, including human induced pluripotent stem cells (iPSC) culture. PC inserts also supported iPSC differentiation into cardiomyocytes (iPS-CM) and micro-patterning of iPS-CM into cardiospheres. This low cost, customizable approach holds promise for a variety of bioengineering applications.

bioengineering↗