Genetic mutations to the Lamin A/C gene (LMNA) can cause heart disease, but the mechanisms making cardiac tissues uniquely vulnerable to the mutations remain largely unknown. Further, patients with LMNA mutations have highly variable presentation of heart disease progression and type. In vitro patient-specific experiments could provide a powerful platform for studying this phenomenon, but the use of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) introduces heterogeneity in maturity and function thus complicating the interpretation of the results of any single experiment. We hypothesized that integrating single cell RNA sequencing (scRNA-seq) with analysis of the tissue architecture and contractile function would elucidate some of the probable mechanisms. To test this, we investigated five iPSC-CM lines, three controls and two patients with a (c.357-2A>G) mutation. The patient iPSC-CM tissues had significantly weaker stress generation potential than control iPSC-CM tissues demonstrating the viability of our in vitro approach. Through scRNA-seq, differentially expressed genes between control and patient lines were identified. Some of these genes, linked to quantitative structural and functional changes, were cardiac specific, explaining the targeted nature of the disease progression seen in patients. The results of this work demonstrate the utility of combining in vitro tools in exploring heart disease mechanics.
According to numerous fundamental studies, a mutated Lamin A/C gene ( LMNA ) can cause dilated cardiomyopathy, arrhythmia, and heart failure, yet the practical diagnosis and treatment for them is scarce. The LMNA encodes Lamin A/C proteins that make mesh like nuclear lamina under the nuclear envelope, and they interact to impact the nuclear shape, stability, protein transduction, and DNA replication. Although, almost all nucleated cells in the human body have Lamin A/C in the nuclear lamina, we study patients who have LMNA mutation with the main pathologies in the heart. Therefore, our goal is to understand the mechanisms of how an LMNA mutation can cause exclusively heart diseases. We hypothesize that an in vitro model using induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes can differentiate between a population of patients whose major phenotype presentation is heart disease due to an LMNA mutation from the control population in structural and functional mechanisms. We have used in vitro tissue engineering techniques to interrogate the iPSC-derived cardiomyocytes tissue structures and function. PATIENT’s and CONTROL’s skin fibroblasts were reprogrammed to iPSCs and differentiated to cardiomyocytes. The PATIENT and CONTROL iPSC-derived cardiomyocytes were immunostained, imaged and quantified. Moreover, for the functional assay, iPSC- derived cardiomyocytes were evaluated by the “Heart-on-a-Chip” device based on the muscular thin film technology. The dynamics of the PATIENTs and CONTROLs tissue constructs were recorded and analyzed for frequency and stress as a function of time. Our results demonstrate that it is possible to construct an iPSC-derived cardiomyocyte in vitro model that differentiates between our CONTROL and PATIENT population and shows significant differences between iPSC-derived cardiomyocytes of PATIENTs and CONTROLs in frequency and active stress. By further investigating the structural and functional differences between PATIENTs and CONTROLs iPSC-derived cardiomyocytes, we can find the downstream mechanisms of LMNA mutation consequences in heart and find the proper treatments through drug screening and the other techniques by monitoring the function and structure of the iPSC-derived cardiomyocytes.
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