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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License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Chapter
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Summary, conclusions and future directions
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Summary and conclusions
The purpose of this thesis was to develop new tools for myocardial repair by gene- and/or cell-based approaches. Cell therapy entails the injection of (stem) cells into the bloodstream or locally at a site of damage, as evidence suggests that this can have a positive effect on the damaged tissue and may contribute to tissue regeneration. Because the human heart has very limited regenerative capabilities, regeneration of damaged myocardium (e.g. after myocardial infarction) has great therapeutic potential, and is currently under investigation in numerous clinical settings. Stem cell therapy has been found to exert a beneficial effect on the damaged myocardium, although without significant myocardial regeneration. In this thesis we investigated two different strategies for improvement of myocardial regeneration. The first is to induce cardiomyogenic differentiation by genetically engineering cells to express the transcription factor myocardin, which is a key regulator of the cardiomyocyte differentiation pathway. Secondly, we hypothesized that myocardial regeneration might be enhanced by including novel cell types with supportive functions in heart development in cell therapy strategies. To this end we investigated the characteristics and the differentiation potential of epicardium- derived cells (EPDCs), which during embryogenesis induce growth and compaction of the embryonic myocardium and contribute to the development of the coronary arteries and connective tissues of the heart.
The mesenchymal stem cell (MSC) is a commonly used cell type in cell therapy studies aimed at repairing damaged myocardium. However, spontaneous differentiation of MSCs into cardiomyocytes following injection into damaged hearts appears to be a sporadic event. For this reason we tested whether cardiomyocyte differentiation could be induced in MSCs, by transducing them with a constitutively active copy of the myocardin gene. Myocardin is a recently identified transcription factor, which plays a pivotal role in the development of both cardiac and smooth muscle cells. We hypothesized that forced expression of myocardin might therefore induce a muscle phenotype in non-muscle cells. In chapter 2 we show that forced expression of myocardin in MSCs of human adults induces synthesis of various cardiac muscle sarcomeric components, cardiac transcription factors and the cardiac hormone atrial natriuretic factor. This indicates that a cardiomyocyte-like phenotype was induced by myocardin, although myocardin-transduced cells did not spontaneously contract, which suggests that differentiation was incomplete.
Aside from activating cardiac genes, we found that forced expression of myocardin also activated several smooth muscle-specific genes. Both cardiac and smooth muscle marker proteins were co-expressed in single myocardin-transduced cells, indicating that myocardin aberrantly induced both cardiac and smooth muscle differentiation pathways at the same time. Interestingly, we also found evidence that myocardin induces cardiac and smooth muscle genes in dermal fibroblasts and therefore we concluded that its functionality is not restricted to stem cells.
We hypothesized that an alternative approach to improve the function of damaged hearts would be (re)introduction of specific cell types that provide key supportive functions during normal heart development. Epicardium-derived cells (EPDCs) are
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multipotent cells derived from the outer layer of the heart (i.e. the epicardium), which during embryonic heart development produce the smooth muscle and connective tissue cells of coronary arteries and the cardiac interstitial fibroblasts.
EPDCs also provide inductive signals to the myocardium, allowing development of the Purkinje system and compaction of the developing myocardium. The first requirement for the potential therapeutic application of these cells was to establish that they were present in, and could therefore be isolated from, the epicardium of hearts from adult patients. In chapter 3 we show that the mesothelial cells of the human epicardium, like embryonic EPDCs, can undergo an epithelial-to- mesenchymal transition, resulting in fibroblast-like cells. Furthermore, these cells can differentiate into smooth muscle cells when subjected to appropriate cytokines, in vitro. We concluded that EPDCs from human adults recapitulate at least part of the differentiation potential of their embryonic counterparts. The EPDC cultures developed within the framework of this PhD project represent an excellent model system to explore the biological properties and therapeutic potential of these cells.
Myocardial scar formation after myocardial infarction impairs cardiac function by inducing cardiac remodeling, reducing cardiac compliance and compromising normal electrical conduction across the heart. In situ conversion of scar fibroblasts into cardiomyocytes would be an elegant method to accomplish myocardial regeneration. Such a strategy would circumvent known difficulties with cell therapy strategies such as poor survival and integration of injected cells after local intramyocardial injection. In chapter 4 we demonstrate that like the MSCs described in chapter 2, ventricular scar fibroblasts adopt a cardiomyocyte-like phenotype after infection with a vector encoding human myocardin. Cardiomyocyte-specific sarcomeric components, transcription factors, hormones and channel proteins can be readily detected in myocardin-transduced cells. Furthermore, we show that treatment with myocardin, allows cultured scar fibroblasts to transmit an action potential and to repair an artificially created conduction block in cardiomyocyte cultures. Although the myocardin-transduced ventricular scar fibroblasts did not spontaneously contract and displayed a disorganized contractile apparatus, the ability to endow them with cardiomyocyte-like electrical conduction may form the basis for a new therapy to treat arrhythmias.
Many of the clinical arrhythmias result from local slow electrical conduction caused by the presence of scar fibroblasts. In addition, failure to respond to resynchronization therapy in patients with severe heart failure is associated with the presence of scar tissue at the site of the left ventricular pacing lead of biventricular pacemaker devices. In chapter 5 we show that slow conduction across human ventricular scar fibroblasts can be recapitulated in an in vitro model and that infection of ventricular scar fibroblasts with a myocardin-encoding vector restores conduction velocities to levels close to those present in native cardiomyocytes.
Furthermore, ventricular scar fibroblasts can be paced after transduction with a myocardin-encoding vector, e.g. they can be electrically stimulated by an electrode and can pass this electrical signal to distant fields of cardiomyocytes. These data suggest that in situ myocardin gene delivery into scar fibroblasts may be used to
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improve conduction of an action potential over the damaged myocardium and may allow capture of electrical pacing pulses from biventricular pacemaker devices at areas of fibrosis in the heart. Both phenomena are expected to decrease left ventricular dyssynchrony, which is known to improve cardiac performance and patient outcome.
In chapter 2 we established that MSCs obtain characteristics of cardiomyocytes after infection with a vector encoding myocardin. In chapter 6 we compared the therapeutic effect of myocardin-transduced MSCs from ischemic heart disease patients to that of untreated MSCs from the same patients in a mouse model of myocardial infarction. Two weeks after local intramyocardial injection of untreated MSCs a modest improvement in cardiac function, but no reduction of cardiac remodeling was observed. However, injection of myocardin-transduced MSCs resulted in a significant positive effect on cardiac function and slowed down cardiac remodeling. Histological analysis confirmed that myocardin-transduced MSCs expressed cardiomyocyte marker genes in vivo, whereas control MSCs did not. However, myocardin-transduced MSCs did not become fully differentiated cardiomyocytes in vivo as evidenced by the absence of several typical cardiac muscle markers. The mechanistic basis for the observed greater beneficial effects on cardiac function and remodeling of myocardin-transduced MSCs over untreated MSCs remains to be determined. However, quantitative assessment revealed a significant higher engraftment rate of myocardin-transduced MSCs as compared to untreated MSCs, indicating that transduction with myocardin enhances cell survival and/or integration in the host myocardium.
Altogether, these results demonstrate that the phenotypic changes induced by forced expression of myocardin in MSCs allow these cells to improve cardiac function to a greater extent than untreated MSCs when injected in an area of myocardial damage in a mouse model of myocardial infarction.
In chapter 7 we used micro-array analysis to directly compare the gene expression programs induced by the cardiac and smooth muscle-enriched isoforms of myocardin to that of a functional cardiomyocyte. To this end, we utilized isolated human cardiac progenitor cells (CMPCs) as a model system of early human cardiomyocyte differentiation as these cells can be differentiated into spontaneously contracting cardiomyocytes in vitro by growing them in 5’-azacytidine-containing medium.
Forced synthesis of the cardiomyocyte-enriched isoform of myocardin (CM-MYOCD) in CMPCs resulted in a gene expression program that was remarkably similar to that of a cardiomyocyte. However, two clear differences were apparent. Although CM-MYOCD-transduced cells contain many sarcomeric components, CM-MYOCD does not upregulate nebulin, nebulette, titin cap, and myosin binding protein C3 gene expression. Consistent with the absence of organized sarcomeres in CM- MYOCD-transduced CMPCs, each of these four proteins has been described to be essential for sarcomere assembly and organization. Furthermore, as compared to cardiomyocytes the CM-MYOCD-transduced CMPCs contained much lower levels of transcripts encoding several cardiac ion channels such as ryanodine receptor 2 and various inwardly rectifying potassium channels. Comparison of the genes activated by the cardiac and smooth muscle-enriched isoforms of myocardin
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revealed that the former protein can activate a subset of cardiomyocyte-specific genes to a significantly greater extent, while both isoforms of myocardin activate other cardiomyocyte-specific genes and smooth muscle genes equally well. These findings show that the cardiac muscle-enriched isoform of myocardin is a much more potent activator of cardiac gene expression than the myocardin isoform found predominantly in smooth muscle. However, forced expression of CM-MYOCD alone is not sufficient to endow cells with a complete cardiomyocyte phenotype.
Conclusions and future directions
Reprogramming of somatic cells by means of genetic modification is a novel technology, which nonetheless holds great promise for future therapeutic application. With sufficient understanding of genetic circuitries controlling development, genetic modification can be a very powerful and precise means of inducing cellular differentiation. Conversely, increasing evidence shows that spontaneous differentiation of stem cells into (for example) cardiomyocytes in response to local environment or damage appears to be at best a very rare event.
Isolated myocardial scar fibroblasts could be endowed with fast electrical conduction by introduction of a permanently active copy of the myocardin gene. Restoration of fast conduction through fibrotic areas in the heart could be of great therapeutic use for treating arrhythmias, and for improving the success of resynchronization of contraction in a scarred heart. However, direct genetic modification of resident scar fibroblasts in the heart will require an efficient gene delivery system (vector).
Recent studies suggest that especially vectors based on adeno-associated virus (AAV) are very useful for myocardial gene therapy. The serotypes of AAV most potent at transducing the heart, e.g. serotype 1, 6, 8, and 9, have been most exhaustively studied in the rat. Therefore, testing this concept in a rat model of myocardial infarction would be the logical choice for future experiments.
Although myocardin is the only factor identified to date that can by itself activate an extensive cardiomyocyte gene expression program in somatic cells, forced expression of myocardin in naive target cells does not induce a complete cardiomyocyte phenotype. To achieve a (more) complete cardiomyocyte phenotype different factors or additional factors besides myocardin will have to be employed. The identification of such factors will require novel fundamental insights into the genetic program governing cardiomyocyte differentiation. A model of cardiomyocyte differentiation, such as the CMPCs, will prove invaluable for this research.
In a mouse model of myocardial infarction, injection of MSCs transduced with a myocardin-encoding vector could improve heart function more than untreated MSCs. These findings suggest that pretreatment of stem cells with a myocardin- encoding vector may enhance future stem cell therapy strategies. However, several issues remain to be resolved. First, the observed findings should be confirmed to be long-term effects. Second, the mechanistic basis whereby the genetically modified MSCs exert their beneficial effects on myocardial function should be elucidated.
Like EPDCs during embryonic development, EPDCs isolated from the epicardium
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of adult patients were reported to differentiate into fibroblasts and smooth muscle cells. These findings indicate that adult EPDCs may be able to contribute to healing of damaged hearts by restoring myocardial architecture and vascularization, and may therefore become a beneficial addition to cell therapy strategies targeting the damaged heart. Studies investigating long-term effects of EPDCs on cardiac function in a mouse model of myocardial infarction are currently in progress.
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