| 1. |
- Rohner, Eduarde, et al.
(författare)
-
An mRNA assay system demonstrates proteasomal-specific degradation contributes to cardiomyopathic phospholamban null mutation
- 2021
-
Ingår i: Molecular Medicine. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1528-3658 .- 1076-1551.
-
Tidskriftsartikel (refereegranskat)abstract
- Background: The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics. Methods: A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing. Results: Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes. Conclusion: Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.
|
|
| 2. |
- Sahara, Makoto, et al.
(författare)
-
Population and single-cell analysis of human cardiogenesis reveals unique LGR5 ventricular progenitors in embryonic outflow tract
- 2019
-
Ingår i: Developmental Cell. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1534-5807 .- 1878-1551.
-
Tidskriftsartikel (refereegranskat)abstract
- The morphogenetic process of mammalian cardiac development is complex and highly regulated spatiotemporally by multipotent cardiac stem/progenitor cells (CPCs). Mouse studies have been informative for understanding mammalian cardiogenesis; however, similar insights have been poorly established in humans. Here, we report comprehensive gene expression profiles of human cardiac derivatives from multipotent CPCs to intermediates and mature cardiac cells by population and single-cell RNA-seq using human embryonic stem cell-derived and embryonic/fetal heart-derived cardiac cells micro-dissected from specific heart compartments. Importantly, we discover a uniquely human subset of cono-ventricular region-specific CPCs, marked by LGR5. At 4 to 5 weeks of fetal age, the LGR5+ population appears to emerge specifically in the proximal outflow tract of human embryonic hearts and thereafter promotes cardiac development and alignment through expansion of the ISL1+TNNT2+ intermediates. The current study contributes to a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations.
|
|
| 3. |
- Santoro, Federica, et al.
(författare)
-
Isolation of human ESC-derived cardiac derivatives and embryonic heart cells for population and single-cell RNA-seq analysis
- 2021
-
Ingår i: STAR PROTOCOLS. - : Elsevier. - 2666-1667. ; 2:1
-
Tidskriftsartikel (refereegranskat)abstract
- The combination of population and single-cell RNA sequencing analysis using hu-man embryonic stem cell (hESC) differentiation and developmental tissues is a powerful approach to elucidate an organ-specific cellular and molecular atlas in human embryogenesis. This protocol describes (1) cardiac-directed differentia-tion and isolation of hESC-derived cardiac derivatives with fluorescence -acti-vated cell sorting, (2) isolation of human embryonic heart-derived single cardiac cells, and (3) construction of cDNA libraries with Smart-seq2. These allow for the preparation of human developmental samples for comprehensive transcriptional analysis. For complete details on the use and execution of this protocol, please refer to Sahara et al. (2019).
|
|
| 4. |
- Witman, Nevin, et al.
(författare)
-
Cardiac progenitors and paracrine mediators in cardiogenesis and heart regeneration
- 2020
-
Ingår i: Seminars in Cell and Developmental Biology. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1084-9521 .- 1096-3634.
-
Tidskriftsartikel (refereegranskat)abstract
- The mammalian hearts have the least regenerative capabilities among tissues and organs. As such, heart regeneration has been and continues to be the ultimate goal in the treatment against acquired and congenital heart diseases. Uncovering such a long-awaited therapy is still extremely challenging in the current settings. On the other hand, this desperate need for effective heart regeneration has developed various forms of modern biotechnologies in recent years. These involve the transplantation of pluripotent stem cell-derived cardiac progenitors or cardiomyocytes generated in vitro and novel biochemical molecules along with tissue engineering platforms. Such newly generated technologies and approaches have been shown to effectively proliferate cardiomyocytes and promote heart repair in the diseased settings, albeit mainly preclinically. These novel tools and medicines give somehow credence to breaking down the barriers associated with re-building heart muscle. However, in order to maximize efficacy and achieve better clinical outcomes through these cell-based and/or cell-free therapies, it is crucial to understand more deeply the developmental cellular hierarchies/paths and molecular mechanisms in normal or pathological cardiogenesis. Indeed, the morphogenetic process of mammalian cardiac development is highly complex and spatiotemporally regulated by various types of cardiac progenitors and their paracrine mediators. Here we discuss the most recent knowledge and findings in cardiac progenitor cell biology and the major cardiogenic paracrine mediators in the settings of cardiogenesis, congenital heart disease, and heart regeneration. Corrigendum in Semin Cell Dev Biol. 2021 Jan:109:151. DOI: 10.1016/j.semcdb.2020.09.008
|
|
| 5. |
- Xu, Jiejia, et al.
(författare)
-
Genome‐wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors
- 2020
-
Ingår i: Stem Cells. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1066-5099 .- 1549-4918.
-
Tidskriftsartikel (refereegranskat)abstract
- Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two-stage genome-wide CRISPR-knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNA-Seq profiling of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps in human mesoderm precursor cell- and heart progenitor cell-fate determination during in vitro hPSC cardiogenesis.
|
|
