Biology subjects

Mayourian, J.

Publications and source records attributed to Mayourian, J..

4 recordsLinked to original sources

bioRxiv2026

Rapid functional classification of cardiac genetic variants directly informs precision cardiology

Large-scale clinical genome sequencing yields vast numbers of variants of unknown significance (VUSs). The high frequency of VUSs and the paucity of platforms to characterize their functional impact pose significant challenges for clinical decision making. Here, we present an integrated end-to-end platform, REVi-SCOPE (Rapid evaluation of variants in single cells by optogenetics and prime editing), for characterization of the impact of VUSs on cardiac physiology. Our strategy consists of (1) introduction of variants directly into wild-type (WT) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) via prime editing; (2) optogenetic assessment of calcium and membrane voltage dynamics in single hiPSC-CMs within the pool of edited and unedited cells; and (3) in situ single-cell genotyping of the phenotyped hiPSC-CMs with single-allele resolution. By optimizing and integrating each of these steps, we created a platform that enables VUS characterization in 10 days. We validated the REVi-SCOPEs capabilities by analyzing the properties of established arrhythmogenic variants. We then used REVi-SCOPE to reveal the functional impact of a VUS, TRPM4A320V, identified in a child with a conduction block. Together, our results show that REVi-SCOPE enables functional characterization of VUSs linked to cardiac arrhythmias with unprecedented throughput.

bioengineering↗

bioRxiv2024

Cardiac Applications of CRISPR/AAV-Mediated Precise Genome Editing

The ability to efficiently make precise genome edits in somatic tissues will have profound implications for gene therapy and basic science. CRISPR/Cas9 mediated homology-directed repair (HDR) is one approach that is commonly used to achieve precise and efficient editing in cultured cells. Previously, we developed a platform capable of delivering CRISPR/Cas9 gRNAs and donor templates via adeno-associated virus to induce HDR (CASAAV-HDR). We demonstrated that CASAAV-HDR is capable of creating precise genome edits in vivo within mouse cardiomyocytes at the neonatal and adult stages. Here, we report several applications of CASAAV-HDR in cardiomyocytes. First, we show the utility of CASAAV-HDR for disease modeling applications by using CASAAV-HDR to create and precisely tag two pathological variants of the titin gene observed in cardiomyopathy patients. We used this approach to monitor the cellular localization of the variants, resulting in mechanistic insights into their pathological functions. Next, we utilized CASAAV-HDR to create another mutation associated with human cardiomyopathy, arginine 14 deletion (R14Del) within the N-terminus of Phospholamban (PLN). We assessed the localization of PLN-R14Del and quantified cardiomyocyte phenotypes associated with cardiomyopathy, including cell morphology, activation of PLN via phosphorylation, and calcium handling. After demonstrating CASAAV-HDR utility for disease modeling we next tested its utility for functional genomics, by targeted genomic insertion of a library of enhancers for a massively parallel reporter assay (MPRA). We show that MPRAs with genomically integrated enhancers are feasible, and can yield superior assay sensitivity compared to tests of the same enhancers in an AAV/episomal context. Collectively, our study showcases multiple applications for in vivo precise editing of cardiomyocyte genomes via CASAAV-HDR.

genetics↗

bioRxiv2024

Efficient and reproducible generation of human iPSC-derived cardiomyocytes using a stirred bioreactor

In the last decade human iPSC-derived cardiomyocytes (hiPSC-CMs) proved to be valuable for cardiac disease modeling and cardiac regeneration, yet challenges with scale, quality, inter-batch consistency, and cryopreservation remain, reducing experimental reproducibility and limiting clinical translation. Here, we report a robust cardiac differentiation protocol that uses Wnt modulation and a stirred suspension bioreactor to produce on average 124 million hiPSC-CMs with >90% purity using a variety of hiPSC lines (19 differentiations; 10 iPSC lines). After controlled freeze and thaw, bioreactor-derived CMs (bCMs) showed high viability (>90%), interbatch reproducibility in cellular morphology, function, drug response and ventricular identity, which was further supported by single cell transcriptomes. bCMs on microcontact printed substrates revealed a higher degree of sarcomere maturation and viability during long-term culture compared to monolayer-derived CMs (mCMs). Moreover, functional investigation of bCMs in 3D engineered heart tissues showed earlier and stronger force production during long-term culture, and robust pacing capture up to 4 Hz when compared to mCMs. bCMs derived from this differentiation protocol will expand the applications of hiPSC-CMs by providing a reproducible, scalable, and resource efficient method to generate cardiac cells with well-characterized structural and functional properties superior to standard mCMs.

cell biology↗

bioRxiv2021

PPARdelta signaling activation improves metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease. A major caveat however is that they remain functionally and structurally immature in culture, limiting their potential for disease modeling and regenerative approaches. Here, we address the question of how different metabolic pathways can be modulated in order to induce efficient hPSC-CM maturation. We show that PPAR signaling acts in an isoform-specific manner to balance glycolysis and fatty acid oxidation (FAO). PPARD activation or inhibition results in efficient respective up- or down-regulation of the gene regulatory networks underlying FAO in hPSC-CMs. PPARD induction further increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation and augments FAO flux. Lastly PPARD activation results in enhanced myofibril organization and improved contractility. Transient lactate exposure, commonly used in hPSC-CM purification protocols, induces an independent program of cardiac maturation, but when combined with PPARD activation equally results in a metabolic switch to FAO. In summary, we identify multiple axes of metabolic modifications of hPSC-CMs and a role for PPARD signaling in inducing the metabolic switch to FAO in hPSC-CMs. Our findings provide new and easily implemented opportunities to generate mature hPSC-CMs for disease modeling and regenerative therapy.

cell biology↗