Cellular and genetic approaches to myocardial regeneration
Tuyn, J. van
Citation
Tuyn, J. van. (2008, January 9). Cellular and genetic approaches to myocardial
regeneration. Department of Cardiology and Department of Molecular Cell Biology (MCB), Faculty of Medicine, Leiden University Medical Center (LUMC), Leiden University.
Retrieved from https://hdl.handle.net/1887/12548
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Chapter
2
Editorial by Carlo Ventura
Cardiovascular Research 2005;67(2):182-183
Forced myocardin expression primes cardiac and smooth muscle transcription patterning in human mesenchymal stem cells
Appendix
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Loss of cardiomyocytes during myocardial infarction or hereditary cardiomyopathies may represent a major determinant in the progression toward heart failure.
The reported ability of haematopoietic bone marrow cells to afford myocardial regeneration after direct injection in experimental animals1 has triggered a number of clinical trials in humans. Nevertheless, the initial observations and the scientific underpinning of the human trials have been challenged by a number of studies using a foreign gene, LacZ or GFP, to track the fate of haematopoietic stem cells after transplant into normal and/or injured mouse hearts2, 3. In these studies, no transdifferentiation into cardiomyocytes was detectable, and stem- cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, raising a cautionary note for clinical studies of infarct repair. These controversial results emphasize the need to assess whether cardiac lineage-committable cells may be comprised within the bone marrow, and prompt further studies to dissect the signals that govern stem cell cardiogenesis. To this end, human mesenchymal stem cells (hMSCs) represent a bone marrow population with high expansion and repair potential in many vital tissues4. Moreover, hMSCs have been shown to differentiate into cardiomyocytes when injected into healthy murine hearts5 or co-cultured with adult rat ventricular cardiomyocytes6, suggesting that hMSCs may afford highly effective cell therapy for the rescue of failing hearts. In this issue of Cardiovascular Research, van Tuyn et al.7 used hMSCs as an in vitro model for the analysis of the expression of muscle-specific genes in response to a defined orchestrating primer. For this purpose, primary hMSCs were transduced with human adenovirus vectors expressing the longest splice variant of the human myocardin gene. Myocardin is a recently discovered transactivator of the ubiquitous transcription factor (TF) serum response factor8, which regulates the expression of many growth-related and muscle-restrict ed genes by binding to CC(A/T)6GG and closely related nucleotide sequence motifs (also known as CArG boxes) in their promoters.
One week after transduction, forced expression of myocardin induced consistent amounts of cardiac mRNAs encoding cardiac troponin T, the atrial and ventricular forms of myosin light chain 2 (Mlc2a and Mlc2v, respectively), cardiac a-and h- myosin heavy chain (MHC), eHand, GATA4, ANF, and SERCA2a. Transduction of the hMSCs also led to the accumulation of smooth muscle MHC-specific transcripts, while failing to trigger the expression of skeletal muscle genes. These findings seem to exclude a generalized activation of repressed genes by myocardin, and suggest that its forced expression may involve mechanisms pertaining to the orchestration of targeted lineage commitments. Double-labelling experiments with monoclonal antibodies directed against sarcomeric a-actin, sarcomeric a- actinin, SERCA2a, or ANF in combination with polyclonal antibodies recognizing smMHC showed that the smMHC-specific antibodies labelled all hMSCs that stained positive with the above markers. These results led to the conclusion that transduced hMSCs had acquired mixed characteristics of both cardiac and smooth muscle cells. Nonetheless, forced myocardin expression could not ensue in the complete execution of a cardiomyocyte lineage. Within this context, myocardin overexpression failed to prime both Nkx-2.5 and cardiac troponin I gene expression. Concerted activation of the zinc finger-containing GATA-4 and the
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Chapter 2 - AppendixForced myocardin expression primes cardiac and smooth muscle transcription patterning in human mesenchymal stem cells
homeodomain Nkx-2.5 represents a mandatory requirement for cardiomyocyte specification from multipotent cells in different animal species, including humans9,
10. Failure to observe cardiomyocytes following hMSC transduction with myocardin may therefore depend on the lack of myocardin-related recruitment of and/or interplay with Nkx-2.5. It is now becoming evident that cell lineage specification is fashioned at multiple interconnected levels and is controlled by a complex interplay between cell signalling, nucleosomal assembly, the establishment of multifaceted transcriptional motifs, and the temporal and spatial organization of chromatin in loops and domains. Recent developments in stem cell research have been boosted by an increasing understanding of transcriptional regulation and epigenetic modifications, including histone acetylation, DNA methylation, and chromatin remodelling. In this regard, newly synthesized hyaluronan mixed esters with butyric and retinoic acid have been shown to act transcriptionally in mouse ES cells, affording both selective over-expression of cardiogenic genes and dramatic increase in the yield of ES-derived cardiomyocytes11. This finding indicates that the gene program of stem cells can be chemically modified turning the low- yield process of cardiogenesis into a high-throughput morphogenetic response.
The study by van Tuyn et al. shows that cardiogenesis can also be primed in a multipotent population of human stem cells. The observation that smooth muscle genes were also induced by myocardin overexpression led the authors to envision that forced myocardin expression in hMSCs before transplantation may enhance their propensity to differentiate into cardiac and smooth muscle cells in vivo.
On the other hand, the co-existence of a mixed program of cardiogenesis and smooth muscle specification within the same hMSC-derived population prompts further studies to assess whether additional gene transcription and signalling patterning may be recruited in transduced cells to afford a complete execution of both lineages in vitro. Even in the affirmative, the possibility that divergent cardiogenic and angiogenic fates may be brought to completion in a recipient damaged heart remains an open issue. Direct functional in vivo approaches should then be designed in experimental models of myocardial infarction or failure to assess whether myocardin-transduced cells may contribute to replenish lost cardiomyocytes and improve myocardial vascularization.
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Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature. 2004;428(6983):668-673.
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10. Schott JJ, Benson DW, Basson CT, Pease W, Silberbach GM, Moak JP, Maron BJ, Seidman CE, Seidman JG. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science. 1998;281(5373):108-111.
11. Ventura C, Maioli M, Asara Y, Santoni D, Scarlata I, Cantoni S, Perbellini A. Butyric and retinoic mixed ester of hyaluronan. A novel differentiating glycoconjugate affording a high throughput of cardiogenesis in embryonic stem cells. J Biol Chem. 2004;279(22):23574-23579.
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Chapter 2 - AppendixForced myocardin expression primes cardiac and smooth muscle transcription patterning in human mesenchymal stem cells
