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Cardiomyocytes (CMs), the contractile heart cells that can be derived from human induced pluripotent stem cells (hiPSCs). These hiPSC derived CMs can be used for cardiovascular disease drug testing and regeneration therapies, and they have therapeutic potential. Currently, hiPSC-CM differentiation cannot yet be controlled to yield specific heart cell subtypes consistently. Designing differentiation processes to consistently direct differentiation to specific heart cells is important to realize the full therapeutic potential of hiPSC-CMs. A model that accurately represents the dynamic changes in cell populations from hiPSCs to CMs over the differentiation timeline is a first step towards designing processes for directing differentiation. This paper introduces a microsimulation model for studying temporal changes in the hiPSC-to-early CM differentiation. The differentiation process for each cell in the microsimulation model is represented by a Markov chain model (MCM). The MCM includes cell subtypes representing key developmental stages in hiPSC differentiation to early CMs. These stages include pluripotent stem cells, early primitive streak, late primitive streak, mesodermal progenitors, early cardiac progenitors, late cardiac progenitors, and early CMs. The time taken by a cell to transit from one state to the next state is assumed to be exponentially distributed. The transition probabilities of the Markov chain process and the mean duration parameter of the exponential distribution were estimated using Bayesian optimization. The results predicted by the MCM agree with the data.
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Cronos Titin Is Expressed in Human Cardiomyocytes and Necessary for Normal Sarcomere Function
Background: The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin ( TTN ) are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within TTN motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function. Methods: To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z −/− ) and A-band (TTN-A −/− ) regions of the TTN gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements. Results: After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z −/− -CMs had sarcomeres and visibly contracted, whereas TTN-A −/− -CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z −/− -CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z −/− -CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin. Conclusions: We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to human cardiac disease.
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- PAR ID:
- 10253900
- Date Published:
- Journal Name:
- Circulation
- Volume:
- 140
- Issue:
- 20
- ISSN:
- 0009-7322
- Page Range / eLocation ID:
- 1647 to 1660
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1161/CIRCULATIONAHA.119.039521
