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.
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Chapter
1
John van Tuyn
General introduction
8
General introduction Cardiovascular disease
Cardiovascular disorders are currently the leading cause of death in the Western world. Despite advances in pharmacotherapy, heart and blood vessel surgery and the use of vascular stents and cardiac assist devices, mortality rates as a result of these diseases are rising1. Much effort is therefore invested in the prevention and early detection of cardiovascular disorders and in limiting the amount of tissue damage after a cardiovascular event.
A major problem in cardiovascular medicine is the absence of significant cardiomyoregenerative capacity in human adults. Whereas acute skeletal and smooth muscle injury typically induce a hyperplasic response resulting in the formation of new myocytes and the repair of the damaged tissue, myocardial infarctions (MIs) or other types of acute injury to the human heart usually lead to the irreversible loss of cardiac muscle cells. The lost cardiomyocytes are replaced by myocardial scar tissue composed of (myo)fibroblasts and large amounts of extracellular matrix. This does not only compromise the contractile function of the heart but may also cause arrhythmias2. Furthermore, the increased hemodynamic stress that is often experienced by surviving cardiomyocytes in damaged hearts triggers a hypertrophic response in these cells. The fibrotic and hypertrophic changes in infarcted myocardium leads to remodeling causing a further decline in cardiac performance and the development of congestive heart failure (CHF).
Although heart transplantation has been shown to be an effective therapy for CHF, it is only applied to a limited number of patients due to a large shortage of donor hearts, the complications associated with long-term immunosuppression, the risk of rejection of the transplanted organ, and the frequent development of allograft coronary vasculopathy3. Most CHF patients therefore only receive palliative treatment, which includes the administration of drugs to reduce cardiac fibrosis, hypertrophy and remodeling, left ventricle (LV) reconstruction surgery and electrical resynchronization therapy. Accordingly, there is a huge interest in the development of potentially curative therapies for the treatment of CHF patients.
Especially (stem) cell therapy is currently in the spotlight of cardiovascular research as a possible means to regenerate damaged hearts.
Stem cell therapy for cardiac regeneration
The development of stem cell therapy to repair myocardial damage boomed after the demonstration by Orlic and colleagues that injection of hematopoietic stem cell (HSC)-enriched bone marrow (BM) fractions into the border zone of the infarcted myocardium of mice that had been previously subjected to permanent ligation of the left main coronary artery, resulted in the improvement of LV function and the presence of transplant-derived cardiomyocytes in their hearts4. However, although the presence of donor cells and an improvement of LV function could be reproduced by other groups, significant amounts of donor-derived myocytes were not found in these studies5-8. These data strongly suggest that mechanisms other than cardiac regeneration were responsible for the observed improvement
9
Chapter 1Introduction
of LV function in damaged hearts after stem cell treatment. The exact nature of this mechanism remains unknown, but as cytokines are likely involved it is commonly referred to as the paracrine effects of stem cell therapy9. Based on these data, it may be warranted to include cell types that provide supportive functions for the myocardium in future stem cell therapy strategies. A cell type that may be an interesting candidate for such a strategy, based on their role in cardiac development during embryogenesis is the epicardium-derived cell (EPDC). During embryogenesis EPDCs migrate from the epicardial cell layer into the primitive myocardium to contribute connective tissue and smooth muscle cells to the coronary arteries and to provide essential inductive signaling for myocardial growth10. In chapter 3 of this thesis, we investigate whether EPDCs can be obtained from the epicardium of human adults, and whether these adult cells still have the multilineage differentiation potential of their embryonic counterparts.
Aside from autologous somatic stem cells, embryonic stem cells represent a potential source of new cardiomyocytes, although ethical objections and immune rejection are two major disadvantages hampering the use of these cells their ability to differentiate into cardiomyocytes is well established11. Unfortunately, recent studies now show that when embryonic stem cells are injected into either healthy or damaged hearts they do not display any specific differentiation into cardiomyocytes, and are prone to forming local teratocarcinomas12. This indicates that embryonic stem cells, which are fully capable of cardiomyocyte differentiation ex vivo, do not undergo cardiomyogenesis solely in response to local stimuli from the (injured) myocardium. Nevertheless, the promising results of the early animal studies, using autologous stem cells, have led to the initiation of many small clinical studies investigating autologous (stem) cell therapy for cardiac repair (reviewed in13). These studies have overwhelmingly shown safety and feasibility. However, due to their small scale and non-randomized setup they provide only some first putative indications of efficacy. Based on these studies, it can be tentatively concluded that (stem) cell therapy can reduce the loss of cardiac function after myocardial injury, but in its present form seems unable to achieve significant cardiac regeneration. Developing methods to stimulate differentiation into desired cell types, beyond relying on spontaneous differentiation in response to local cues, would be a logical next step for achieving cardiac regeneration.
Molecular mechanisms of differentiation
During development distinct tissues arise from the same initial cell, by a process of cellular differentiation. The distinct phenotypes cells adopt during differentiation are in turn a reflection of different gene expression programs active in these cells.
Currently it is thought that at least for some tissues these cell type-specific gene expression programs are initiated by the activation of a small set of “master regulator” genes. To date several master regulator genes have been identified. The first gene proposed to be a master regulator gene was MyoD. Forced expression of the MyoD gene was shown to initiate skeletal muscle differentiation in a wide variety of cells14, 15. Overexpression of pancreatic and duodenal homeobox gene 1 (Pdx1) has been shown to convert liver cells into pancreas cells16, 17, while a combination of the gene products C/EBP
α
and PU.1 can turn a T cell precursor10
into a functional macrophage18. Recently the power of using transcription factors to reprogram cells was exemplified by Takahashi et al. who described the conversion of skin fibroblasts from the tail tips of mice into pluripotent embryonic stem cells, by overexpression of the four genes encoding Oct3/4, Sox2, c-Myc, and Klf419. However, it should be stressed that the extent of (de)differentiation reported for these putative master switch genes varies, and that the number of cell types that respond to overexpression of the aforementioned factors has either not been extensively investigated, or is known to be restricted (as discussed for MyoD below). Current data do, however, illustrate the power of master regulator genes, in inducing specific differentiation in cells not normally known to do so.
An essential requirement for the use of master regulator genes is the ability to efficiently and specifically express them in the desired target cells. Both viral and non-viral vectors have been extensively studied for their safety and efficiency in gene transfer. Recent advances have led to significant improvement of vector systems, which are currently being tested in a number of clinical trials. In debt discussion of this subject is beyond the scope of this work, and interested readers are directed to other papers20.
MyoD the archetypical master switch gene
The concept of a master switch gene was introduced after a series of subtractive hybridization experiments performed by Lassar et al.21 who showed that a single locus, later identified as the gene MyoD14 was responsible and sufficient for the transdifferentiation of 10T1/2 fibroblasts into skeletal muscle cells. Overexpression of MyoD in a wide variety of primary cells, such as dermal fibroblasts, pigment, nerve, fat and liver cells also resulted in conversion to skeletal muscle15. These findings provided the first direct evidence that a single gene product can initiate a complex program of differentiation, acting as a master switch.
Skeletal muscle conversion by overexpression of MyoD has been observed in many different cell types, including fibroblasts, chondroblasts, smooth muscle cells, melanocytes, and adipocytes. After conversion these cells express a wide variety of skeletal muscle proteins, including: sarcomeric myosin heavy and light chain, alpha actinin, alpha actin, titin, nebulin, desmin, myosin light chain 1, muscle creatin kinase, acetylcholine receptor, myogenin, troponin I, tropomyosin, and myomedin. Furthermore, like regular myoblasts, these cells can fuse into multi-nucleated myofiber-like structures, called myotubes15, 22-27. These myotubes possess a properly organized sarcomere15, 22-24, and under certain conditions can contract spontaneously23. Importantly, after skeletal muscle conversion of chondroblasts, melanocytes, and adipocytes, these cells loose all markers of their respective original differentiation program23, 24, 28, indicating that the endogenous differentiation program is overruled by the new skeletal muscle gene expression program.
After the identification of MyoD, three other closely related basic helix-loop helix (bHLH)-family members have been identified, Myf5, myogenin and herculin, each of which is also able to induce a skeletal muscle phenotype29, 30. During embryogenesis, MyoD and Myf5 initiate the initial muscle determination. Myf5 is highly expressed in apaxial myotome, giving rise to the back, intercostal, and
11
Chapter 1Introduction
abdominal muscles, while MyoD is expressed at high levels in the hypaxial myotome, giving rise to limb, tongue, and diaphragm muscles31. In line with the similar roles that MyoD and Myf5 fulfill, they are to a large extent mutually redundant. Knock- out of either factor results in compensatory upregulation of the other factor and almost normal muscle formation, only double knockout of both MyoD and Myf5 results in the absence of skeletal muscle development32. The other two bHLH- transcription factors myogenin and herculin are thought to function downstream of MyoD and Myf5, directly facilitating the activation of many muscle structural genes30, 33. Although down stream targets of MyoD and Myf5, overexpression of myogenin and herculin still can result in skeletal muscle conversion, possibly because both myogenin and herculin induce endogenous MyoD expression30. Limitations of MyoD mediated differentiation
Not all cell types are equally responsive to forced MyoD expression, and the endogenous differentiation program of cells is not always overruled. When MyoD was overexpressed in melanoma and neuroblastoma cells, in combination with cAMP to trigger neuroblast differentiation, both markers of skeletal muscle differentiation (MHC and desmin) and formation of axon-like processes indicative of neuronal differentiation were detected in the same cells15. In these cases it would appear that both the endogenous differentiation program and the muscle differentiation program induced by MyoD were initiated and are able to coexist in a single cell. On the other hand when adipogenic cell lines were transfected with MyoD in combination with adipogenic induction, they displayed either an adipocyte phenotype distinguished by the presence of fat droplets, or a muscle phenotype in a mutually exclusive manner15, indicating that fat cell and muscle cell differentiation programs are not compatible. NIH3T3 fibroblasts could not be forced to fuse into myotubes, but did express several muscle structural genes, upon forced expression of either MyoD, Myf5, myogenin or herculin34. Interestingly, prechondrocytes and preosteoblasts were found to be responsive to MyoD mediated myogenesis, but more mature chondrocytes and osteoblasts were not35. Similarly, kidney- and liver-derived cells did not display any markers of a muscle phenotype in response to MyoD transduction15. This illustrates that not all cells are permissive to MyoD induced differentiation. The differences in permissiveness of various cell types to MyoD may be explained either by the presence of inhibitory factors or the absence of necessary co-stimulatory factors, the discussion of which lies outside the scope of the current work and has been reviewed previously36.
MyoD gene transfer for myocardial repair
Only little data exists concerning MyoD gene transfer in vivo24-27. The application most studied is using MyoD gene transfer to convert scar fibroblasts from the damaged myocardium into skeletal myocytes with the aim to improve cardiac function25-27. A major problem with this strategy is, however, that when injected into the damaged myocardium, skeletal myoblasts do not differentiate into cardiomyocytes, and remain electrically isolated from the surrounding cardiomyocytes37, 38. Concomitantly, clinical trials investigating the effects of
12
transplantation of skeletal myoblasts into the heart have been terminated, as the risk of malignant arrhythmias was found to be increased39, 40. A potential explanation for the electrically isolated nature of skeletal myoblasts is the absence of gap junctional coupling41. To solve this problem, Kizana et al.26 co-infected fibroblasts with self-inactivating (SIN) lentivirus vectors coding for gap junction protein
α
1 (Cx43) and for MYOD1. This resulted in the formation of myotubes that could propagate calcium transients and transfer a gap junction-permeable cytoplasmic fluorophore to adjacent cells. Given the differences in ion channel composition between skeletal muscle cells and cardiomyocytes42 it is however unlikely that MyoD- and Cx43-transduced fibroblasts can produce a cardiac action potential. To overcome this problem, it would be preferable to use a gene encoding a transcription factor involved in cardiomyogenesis to obtain a true cardiac muscle phenotype.Genetic control of muscle development
In C. elegans muscle differentiation is controlled by three functionally redundant genes, being the MyoD homologue hlh-1, the SRF/MEF2 homologue unc-120, and HAND homologue hnd-143. When individually overexpressed each of these genes can induce muscle differentiation, and only knockout of all three results in complete absence of muscle43. Interestingly, members of the MyoD-family regulate only skeletal muscle development in vertebrates, as demonstrated by experiments showing that double knockout of two bHlH-family members, MyoD and Myf5, is sufficient to completely block skeletal muscle development in mice32, while knockout of hlh-1, representing all MyoD-family members in C. elegans does not affect muscle development in C. elegans. In vertebrates, HAND family members have clearly documented functions only in cardiac muscle, whereas the MEF2 and SRF family of transcription factors have established functions in all three muscle lineages. This suggests that the genes which regulated muscle differentiation in the ancestral muscle cell have adopted more specific functions in the differentiation of the three muscle lineages of vertebrate organisms43. Furthermore, although overexpression of MyoD can induce skeletal muscle differentiation in mammalian cells, overexpression of HAND family members, SRF or MEF2, does not.
SRF and MEF2, which both belong to the MADS box family of transcription factors are essential for the development of all three muscle lineages44. However, these factors are expressed throughout the organism and also function in more generalized processes, such as control of cellular growth. Specific activation of muscle or growth factor induced genes, is therefore thought to be dependent on tissue specific co-factors45.
Myocardin: a cardiovascular master switch gene
Many genes have been described which are essential for the proper development of the heart46. However, none of these alone or in combinations have been found to initiate even a partial cardiomyocyte specific gene expression program in somatic cells. With the identification of the heart and smooth muscle-restricted transcription factor myocardin a key regulator of cardiac and smooth muscle
13
Chapter 1Introduction
differentiation has now been identified47, 48. Myocardin was first described in Xenopus, and is expressed at the earliest stages of cardiac and smooth muscle development48. Myocardin activates genes in a complex with SRF48 and Mef2c49. A dominant negative mutant of myocardin can prevent cardiac gene expression in Xenopus embryos48, however mice lacking myocardin do not exhibit a discernable cardiac phenotype50 which may indicate redundancy with other myocardin-family members. Nonetheless, overexpression of myocardin in various Xenopus48, human51-53 and rat (unpublished results) cells leads to the activation of an extensive cardiomyocyte gene expression program. Overexpression of myocardin also leads to the activation of smooth muscle specific genes47, 51, and in chapter 2 we will show that both cardiac and smooth muscle genes are expressed at once in the same cells51. Interestingly, recent data suggest that cardiac and smooth muscle cells can arise from a common precursor cell, which also temporarily expresses both cardiac and smooth muscle specific genes54. Furthermore, many cardiac lineage tracing studies have shown that developing cardiomyocytes temporarily express numerous smooth muscle structural genes55-57. These findings underscore the close evolutionary relationship between smooth muscle cells and cardiac muscle cells and are compatible with the hypothesis that single regulator genes exist that initiate early differentiation of both heart and smooth muscle cells.
MicroRNAs in muscle differentiation
Post transcriptional regulation at the RNA level by so called micro RNAs (miRNAs), has been recently recognized as an important regulatory mechanism of gene expression. The importance of miRNAs in the regulation of embryonic development has been established by experiments which employed complete disruption of all miRNAs, by knockout of the enzyme Dicer which is critical for miRNA processing, which resulted in embryonic lethality due to developmental defects at the early gastrulation phase58. Two miRNAs have been identified with essential functions in muscle development, miRNA1 and miRNA133. Expression of miRNA1 and miRNA133 is found specifically in the somites and the heart at the looping stage, and becomes more abundant with the further development of skeletal and cardiac muscle in mouse, Drosophila and Xenopus54, 59, 60. Overexpression of miRNA1 results in increased levels of structural muscle gene expression and reduced myoblasts proliferation54, 59. Mice overexpressing miRNA1 die at embryonic day 13.5 as a result of heart failure due to underdeveloped thin walled ventricles, again showing that miRNA1 reduces myoblast proliferation60. This cardiac phenotype is comparable to that found in Hand2 knockout mice, and indeed Hand2 mRNA was shown to be a target for miRNA1 mediated knockdown60. In the reciprocal experiment knockout or inhibition of miRNA1 was shown to results in reduced expression of heart and skeletal muscle marker genes, and increased myoblast proliferation54, 59. The function of miRNA133 is the inverse of miRNA1.
Overexpression of miRNA133 represses expression of structural muscle genes and promotes myoblast proliferation54. Intriguingly, expression of these miRNAs in skeletal muscle is regulated by MyoD in combination with MEF2C, and expression in cardiac muscle is regulated by SRF and myocardin54, 59, 60.
Combined these findings suggest that miRNA1 and miRNA133 are essential
14
regulators of the striated muscle phenotype, but that in the establishment of muscle lineages they function downstream of the myocardin and SRF in the heart and MyoD and MEF2C in skeletal muscle.
Differentiation between cardiac and smooth muscle gene expression programs
The co-expression of both cardiac and smooth muscle structural genes in single cells after forced expression of myocardin does not correspond to the normal expression profile of mature cardiomyocytes and smooth muscle cells. Therefore other mechanisms must exist which further restrict the gene expression program to that of only smooth or cardiac muscle cells. Although no definitive answer to this question has been found, several additional mechanisms of regulation for myocardin mediated gene expression have been discovered.
The SRF-myocardin complex has been found to associate with the chromatin remodeling histone acetyltransferase p300 and with class II histone deacetylases61,
62. Histone acetylation which is catalyzed by histone acetyltransferases (HATs) promotes gene transcription by destabilizing chromatin structure, facilitating access of transcriptional complexes to their target genes. Conversely histone deacetylases (HDACs) catalyze the deacetylation of histones, which leads to chromatin condensation and transcriptional silencing. This suggests that myocardin through alternate association with different HATs and HDACs can in one situation activate cardiac and repress smooth muscle gene expression and in another do the reverse. The mechanism regulating which association prevails and therefore which genes are activated and which are repressed remains to be elucidated.
Recently it has been discovered that myocardin is differentially spliced in cardiac and smooth muscle tissue49. The smooth muscle-enriched isoform of myocardin (SM-MYOCD) lacks the N-terminal 79 amino acids, which are present in the cardiac-enriched isoform (CM-MYOCD) and contains a MEF2 binding domain. SRF and MEF2 regulate distinct but overlapping sets of muscle genes. Therefore, the CM-MYOCD can regulate both SRF and MEF2 target genes, while SM-MYOCD is restricted to SRF target genes.
A third mechanism for differential regulation of myocardin activity on cardiac versus smooth muscle genes involves post transcriptional modification of myocardin by small ubiquitin-like modifiers (SUMOs)63. Post translational modification of proteins by SUMOs can alter transcription target activity by altering DNA binding activity, subcellular localization and protein stability. Murine myocardin was found to be a target for SUMO modification at the lysine residue at position 445, and this selectively enhanced cardiac gene expression63.
All of the above mechanisms hold important clues as to how myocardin mediated transcription activation can be restricted to either cardiac or smooth muscle cells;
however the factor(s) or signal(s) that regulate this fate-decision remain to be elucidated (summarized in Figure 1).
15
Chapter 1Introduction
Roles for myocardin in cardiovascular remodeling
The role of myocardin is not limited to establishing a cardiac or smooth muscle phenotype, the activity of myocardin has a direct effect on cardiovascular remodeling.
Increased levels of myocardin are associated with dilated cardiomyopathy64 and accordingly overexpression of myocardin in cardiomyocytes induces a hypertrophic phenotype, with accompanying re-expression of fetal cardiac proteins65, 66. The important negative regulator of cardiomyocyte hypertrophy glycogen synthase kinase 3ß (GSK3ß), was found to phosphorylate and thereby inhibit myocardin’s transcriptional activity66. This suggests that at least part of the hypertrophy inhibiting effect of GSK3ß is mediated by modulation of the transcriptional activity of myocardin. BMP2 and TGFß1, both members of the transforming growth factor super family, regulate growth and differentiation in development and also play important parts in the pathogenesis of cardiac and smooth muscle cells. In cardiomyocytes BMP2 signaling leads to smad1-mediated synergistic activation
SRF MEF2
SRF
TGFß1SM-MYOCD BMP2
MEF2 sumoylation CM-MYOCD
Elk1 GSK3b Herp1 Foxo4 PRDM6
proliferation myogenesis
cardiac muscle smooth muscle oestrogen (SRC3) Ca2+ influx ANG2 (prx1) IGF1 insulin
?
PDGF-BB serum Notch
Figure 1. Schematic representation of the regulation of the opposing myogenic and mitogenic gene expression programs by a network of co-factor interactions with the MADS-box transcription factors SRF and MEF2C. The prime myogenic MADs-box co-factor myocardin is depicted as a second nexus for the integration of extra- and intracellular signaling pathways, resulting in differential expression of cardiac and smooth muscle genes via alternative splicing and post transcriptional modification of myocardin, and it’s interaction with different myocardin co-factors.
16
of myocardin-dependent cardiac gene expression67. In smooth muscle TGFß1 signaling through direct interaction of smad3 with myocardin induces myocardin- target genes68.
Unlike cardiomyocytes, smooth muscle cells can readily switch between a proliferative and synthetic (contractile) phenotype in response to extracellular cues.
The proliferative and synthetic phenotypes of smooth muscle cells are correlated with low and high levels of myocardin activity, respectively69-73. General mitogenic signals such as present in serum from fetal origin were found to promote binding of the Ets-factors Elk1 to SRF, while causing displacement of myocardin from SRF, resulting in reduced smooth muscle specific contractile gene expression69. Likewise Kruppel-like factor 4 (KLF4) was identified as a strong inhibitor of myocardin, and KLF4 was shown to be rapidly upregulated in the presence of the mitogen platelet derived growth factor-BB (PDGF-BB)73. The recently identified protein PRDM6 has four Kruppel-like zink fingers and also represses myocardin activity70. The transcriptional repressor Herp1 (HES-related repressor protein 1) also inhibits SRF/myocardin-dependent smooth muscle cell gene expression. Herp1 is a known target gene of Notch, thereby linking Notch mediated signaling to myocardin activity74.
Conversely, the anti-proliferative effects of several hormonal signals have also been linked to myocardin activity in smooth muscle cells. The induction of smooth muscle contractile protein encoding genes by myocardin was shown to be greatly enhanced by a direct interaction with steroid receptor coactivator 3 (SRC3), a key factor in oestrogen receptor mediated signaling75. Angiotensin II signaling stimulates myocardin expression and activity, in part by acting through the angiotensin II responsive gene paired related homeobox gene 1 (prx1)71. Finally, the forkhead transcription factor foxo4 inhibits smooth muscle gene expression by binding to myocardin, thereby blocking myocardin’s transcriptional activity.
Foxo4 was found to be shuttled out of the nucleus (thereby releasing its repressive effects on myocardin) in response to phosphorylation of the gene Akt, which is a downstream target of insulin and insulin like growth factor 1 signaling72.
Mechanical signals such as muscle contraction also affect smooth muscle contractile gene expression. A novel mechanism for this differential gene expression was shown to act though activation of the RhoA/Rho kinase signaling cascade in response to Ca2+ signaling, resulting in induction of myocardin expression, and SRF-myocardin mediated gene expression76.
These findings all support a central role for myocardin in the maintenance and control of the contractile phenotype of cardiac and smooth muscle cells (summarized in Figure 1).
Other myocardin family members
Three other myocardin related transcription factors (MRTFs) have been described.
The first one MAL is also called MRTF-A, MKL1 and BSAC, and the second factor is called MRTF-B, MKL2 or MAL16. Unlike myocardin, MAL and MRTF-B are expressed in a wide variety of cells77. Although myocardin and the MRTFs are highly homologous, and therefore probably can functionally substitute for each other to a great extent, distinct knockout phenotypes have been described. MRTF-
17
Chapter 1Introduction
B knockout results in a failure of neural crest cell development78, 79 and vascular defects and liver hemorrhage due to defective smooth muscle gene expression in the liver sinusoids, vitelline veins, and yolk sac80. MAL mutant mice display a defect in myoepithelial differentiation and function, required for the maintenance of lactation81.The activity of the MRTFs is regulated by the state of the cytoskeleton and sarcomeric integrity. Both MRTFs contain a series of RPEL motifs near their N-terminus, which interacts with monomeric G-actin causing their sequestering in the cytoplasm, relaxing sarcomeric gene expression82, 83. The most recently identified myocardin family member is named myoskeletin. Myoskeletin is highly expressed in skeletal muscle, while no expressing was found in heart and smooth muscle tissues. Furthermore, knockdown studies in Xenopus demonstrated that myoskeletin expression is essential for the formation of hypaxial muscle, while overexpression of myoskeletin in explants of non-muscle cells results in the activation of skeletal, smooth and cardiac muscle genes. The later finding suggests that myoskeletin, together with the other myocardin family members can functionally substitute for each other84. Finally, the recently described gene MASTR shares a high degree of homology with the myocardin family of transcription factors. Like myocardin MASTR is a SAP domain containing transcription factor, and it can co-activate MEF2 like the cardiac isoform of myocardin. However, unlike all other myocardin family members MASTR does not interact with SRF and does not induce gene expression from SRF target genes. MASTR is expressed in skeletal muscle, placenta, aorta, lung and brain, and was shown to enhance skeletal muscle gene expression, when overexpressed in muscle cells49. These findings combine to paint a picture where myocardin family members in interaction with SRF and MEF2 function as central regulators of the muscle gene expression program in all three muscle lineages.
Aims and outline of the thesis
Significant myocardial regeneration cannot be achieved through currently available technology. We hypothesized that overexpression of genes involved in the regulation of cardiac development might induce differentiation of target cells into cardiomyocytes. The main aims of this thesis were to test whether and to what extent the newly identified transcription factor myocardin could induce a cardiac or smooth muscle phenotype when brought to expression in clinically relevant cells and whether this leads to improved cardiac function in a mouse model of myocardial infarction.
In chapter 2 we establish that forced expression of recombinant full length human myocardin is able to induce a wide variety of cardiac and smooth muscle genes in human mesenchymal stem cells (hMSCs). Furthermore, we show that adenovirus vectors based on serotype 50 are vastly superior to common serotype 5 adenovirus vectors for gene-transfer to hMSCs.
To achieve cardiac regeneration, blood flow through the damaged area must first be restored. Although neovascularization is readily observed in the infarct area after myocardial infarction, blood flow remains much reduced and is likely insufficient for the maintenance of cardiac muscle cells. A requirement for the further development of capillary vessels, are vascular smooth muscle cells. During
18
embryonic development the coronary smooth muscle cells derive from the cells of the epicardium. In chapter 3 we investigate whether epicardial cells from human adults can still differentiate into smooth muscle cells in vitro, and therefore are an interesting target for autologous cell transfer studies aimed at myocardial regeneration.
After myocardial infarction a fibrous scar is formed, which contributes to deleterious remodeling and increased mechanical stiffness of the heart. This will result in diminished systolic and diastolic cardiac function and may ultimately lead to heart failure. In addition, the excessive deposition of extracellular matrix components during cardiac fibrosis reduces the electrical coupling between cardiomyocytes and impairs the electrical impulse propagation across the myocardium. In chapters 4 and 5 we focus on the electrophysiological aspect of myocardin transduced cells. To this end we examined whether we can endow isolated fibroblasts from a myocardial infarction scar with the electrical properties of cardiomyocytes using myocardin gene transfer.
In chapter 6 we use a mouse model of myocardial infarction to investigate whether injection of hMSCs transduced with an adenovirus encoding myocardin can acquire a cardiac phenotype in vivo and whether they can restore cardiac function to a greater extent than mock transduced hMSCs.
Finally, to establish the extent to which myocardin alone can activate a bonafide cardiomyocyte expression program and to determine whether the cardiac and smooth muscle enriched splice variants of myocardin display distinct heart and smooth muscle gene induction, we performed microarray analysis to compare the gene expression programs activated by both myocardin splice variants, to that of cardiomyocytes derived in vitro from human cardiac progenitor cells in chapter 7.
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Chapter 1Introduction
References
1. Thom T, Haase N, Rosamond W, Howard VJ, Rumsfeld J, Manolio T, Zheng Z-J, Flegal K, O’Donnell C, Kittner S, Lloyd-Jones D, Goff DC, Jr., Hong Y, Members of the Statistics Committee and Stroke Statistics S, Adams R, Friday G, Furie K, Gorelick P, Kissela B, Marler J, Meigs J, Roger V, Sidney S, Sorlie P, Steinberger J, Wasserthiel-Smoller S, Wilson M, Wolf P. Heart Disease and Stroke Statistics--2006 Update: A report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2006;113(6):e85-151.
2. Swynghedauw B. Molecular mechanisms of myocardial remodeling. Physiol Rev. 1999;79(1):215-262.
3. Rahmani M, Cruz RP, Granville DJ, McManus BM. Allograft vasculopathy versus atherosclerosis. Circ Res. 2006;99(8):801-815.
4. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature. 2001;410(6829):701-705.
5. Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman IL, Robbins RC.
Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature. 2004;428(6983):668-673.
6. Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima HO, Rubart M, Pasumarthi KBS, Ismail Virag J, Bartelmez SH, Poppa V, Bradford G, Dowell JD, Williams DA, Field LJ. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature.
2004;428(6983):664-668.
7. Nygren JM, Jovinge S, Breitbach M, Sawen P, Roll W, Hescheler J, Taneera J, Fleischmann BK, Jacobsen SEW. Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation. Nat Med. 2004;10(5):494-501.
8. Wang JS, Shum-Tim D, Chedrawy E, Chiu RC. The coronary delivery of marrow stromal cells for myocardial regeneration: pathophysiologic and therapeutic implications. J Thorac Cardiovasc Surg. 2001;122(4):699- 705.
9. Gnecchi M, He H, Liang OD, Melo LG, Morello F, Mu H, Noiseux N, Zhang L, Pratt RE, Ingwall JS, Dzau VJ. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med. 2005;11(4):367-368.
10. Winter EM, Gittenberger-de Groot AC. Cardiovascular development:
towards biomedical applicability: Epicardium-derived cells in cardiogenesis and cardiac regeneration. Cell Mol Life Sci. 2007;64(6):692-703.
11. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein L. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest. 2001;108(3):407-414.
12. Nussbaum J, Minami E, Laflamme MA, Virag JA, Ware CB, Masino A, Muskheli V, Pabon L, Reinecke H, Murry CE. Transplantation of undifferentiated
20
murine embryonic stem cells in the heart: teratoma formation and immune response. FASEB J. 2007.
13. Boyle AJ, Schulman SP, Hare JM, Oettgen P. Is stem cell therapy ready for patients? Stem cell therapy for cardiac repair. Ready for the next step.
Circulation. 2006;114(4):339-352.
14. Davis RL, Weintraub H, Lassar AB. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987;51(6):987-1000.
15. Weintraub H, Tapscott SJ, Davis RL, Thayer MJ, Adam MA, Lassar AB, Miller AD. Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci U S A. 1989;86(14):5434-5438.
16. Ferber S, Halkin A, Cohen H, Ber I, Einav Y, Goldberg I, Barshack I, Seijffers R, Kopolovic J, Kaiser N, Karasik A. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia. Nat Med. 2000;6(5):568-572.
17. Horb ME, Shen C-N, Tosh D, Slack JMW. Experimental conversion of liver to pancreas. Current Biology. 2003;13(2):105-115.
18. Laiosa CV, Stadtfeld M, Xie H, de Andres-Aguayo L, Graf T. Reprogramming of committed T cell progenitors to macrophages and dendritic cells by C/EBP[alpha] and PU.1 transcription factors. Immunity. 2006;25(5):731- 744.
19. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell.
2006;126(4):663-676.
20. Muller OJ, Katus HA, Bekeredjian R. Targeting the heart with gene therapy- optimized gene delivery methods. Cardiovasc Res. 2007;73(3):453-462.
21. Lassar AB, Paterson BM, Weintraub H. Transfection of a DNA locus that mediates the conversion of 10T1/2 fibroblasts to myoblasts. Cell.
1986;47(5):649-656.
22. Lattanzi L, Salvatori G, Coletta M, Sonnino C, Cusella De Angelis MG, Gioglio L, Murry CE, Kelly R, Ferrari G, Molinaro M, Crescenzi M, Mavilio F, Cossu G. High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies. J Clin Invest. 1998;101(10):2119- 2128.
23. Choi J, Costa ML, Mermelstein CS, Chagas C, Holtzer S, Holtzer H. MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes. Proc Natl Acad Sci U S A. 1990;87(20):7988- 7992.
24. Kocaefe YC, Israeli D, Ozguc M, Danos O, Garcia L. Myogenic program induction in mature fat tissue (with MyoD expression). Exp Cell Res.
2005;308(2):300-308.
25. Etzion S, Barbash IM, Feinberg MS, Zarin P, Miller L, Guetta E, Holbova R, Kloner RA, Kedes LH, Leor J. Cellular cardiomyoplasty of cardiac fibroblasts by adenoviral delivery of MyoD ex vivo: an unlimited source of cells for myocardial repair. Circulation. 2002;106(12 Suppl 1):I125-130.
21
Chapter 1Introduction
26. Kizana E, Ginn SL, Allen DG, Ross DL, Alexander IE. Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling.
Circulation. 2005;111(4):394-398.
27. Murry CE, Kay MA, Bartosek T, Hauschka SD, Schwartz SM. Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD. J Clin Invest. 1996;98(10):2209-2217.
28. Rosenberg MI, Georges SA, Asawachaicharn A, Analau E, Tapscott SJ.
MyoD inhibits Fstl1 and Utrn expression by inducing transcription of miR- 206. J Cell Biol. 2006;175(1):77-85.
29. Braun T, Buschhausen-Denker G, Bober E, Tannich E, Arnold HH. A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts. Embo J. 1989;8(3):701-709.
30. Aurade F, Pinset C, Chafey P, Gros F, Montarras D. Myf5, MyoD, myogenin and MRF4 myogenic derivatives of the embryonic mesenchymal cell line C3H10T1/2 exhibit the same adult muscle phenotype. Differentiation.
1994;55(3):185-192.
31. Kablar B, Krastel K, Tajbakhsh S, Rudnicki MA. Myf5 and MyoD activation define independent myogenic compartments during embryonic development. Dev Biol. 2003;258(2):307-318.
32. Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH, Jaenisch R. MyoD or Myf-5 is required for the formation of skeletal muscle. Cell.
1993;75(7):1351-1359.
33. Hollenberg SM, Cheng PF, Weintraub H. Use of a conditional MyoD transcription factor in studies of MyoD trans-activation and muscle determination. Proc Natl Acad Sci U S A. 1993;90(17):8028-8032.
34. Russo S, Tomatis D, Collo G, Tarone G, Tato F. Myogenic conversion of NIH3T3 cells by exogenous MyoD family members: dissociation of terminal differentiation from myotube formation. J Cell Sci. 1998;111 ( Pt 6):691- 700.
35. Filvaroff EH, Derynck R. Induction of myogenesis in mesenchymal cells by MyoD depends on their degree of differentiation. Dev Biol. 1996;178(2):459- 471.
36. Berkes CA, Tapscott SJ. MyoD and the transcriptional control of myogenesis.
Semin Cell Dev Biol. 2005;16(4-5):585-595.
37. Leobon B, Garcin I, Menasche P, Vilquin JT, Audinat E, Charpak S. Myoblasts transplanted into rat infarcted myocardium are functionally isolated from their host. Proc Natl Acad Sci U S A. 2003;100(13):7808-7811.
38. Rubart M, Soonpaa MH, Nakajima H, Field LJ. Spontaneous and evoked intracellular calcium transients in donor-derived myocytes following intracardiac myoblast transplantation. J Clin Invest. 2004;114(6):775- 783.
39. Menasche P, Hagege AA, Vilquin JT, Desnos M, Abergel E, Pouzet B, Bel A, Sarateanu S, Scorsin M, Schwartz K, Bruneval P, Benbunan M, Marolleau JP, Duboc D. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol.
2003;41(7):1078-1083.
22
40. Smits PC, van Geuns RJ, Poldermans D, Bountioukos M, Onderwater EE, Lee CH, Maat AP, Serruys PW. Catheter-based intramyocardial injection of autologous skeletal myoblasts as a primary treatment of ischemic heart failure: clinical experience with six-month follow-up. J Am Coll Cardiol.
2003;42(12):2063-2069.
41. Abraham MR, Henrikson CA, Tung L, Chang MG, Aon M, Xue T, Li RA, B OR, Marban E. Antiarrhythmic engineering of skeletal myoblasts for cardiac transplantation. Circ Res. 2005;97(2):159-167.
42. Hille B. Ion channels of excitable membranes. Third ed. Massachusetts, USA: Sinauer Associates, Inc; 2001.
43. Fukushige T, Brodigan TM, Schriefer LA, Waterston RH, Krause M. Defining the transcriptional redundancy of early bodywall muscle development in C. elegans: evidence for a unified theory of animal muscle development.
Genes Dev. 2006;20(24):3395-3406.
44. Black BL, Olson EN. Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu Rev Cell Dev Biol.
1998;14:167-196.
45. Miano JM. Serum response factor: toggling between disparate programs of gene expression. J Mol Cell Cardiol. 2003;35(6):577-593.
46. Zaffran S, Frasch M. Early signals in cardiac development. Circ Res.
2002;91(6):457-469.
47. Chen J, Kitchen CM, Streb JW, Miano JM. Myocardin: a component of a molecular switch for smooth muscle differentiation. J Mol Cell Cardiol.
2002;34(10):1345-1356.
48. Wang D-Z, Chang PS, Wang Z, Sutherland L, Richardson JA, Small E, Krieg PA, Olson EN. Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell. 2001;105(7):851- 862.
49. Creemers EE, Sutherland LB, Oh J, Barbosa AC, Olson EN. Coactivation of MEF2 by the SAP domain proteins myocardin and MASTR. Mol Cell.
2006;23(1):83-96.
50. Li S, Wang DZ, Wang Z, Richardson JA, Olson EN. The serum response factor coactivator myocardin is required for vascular smooth muscle development. Proc Natl Acad Sci U S A. 2003;100(16):9366-9370.
51. van Tuyn J, Knaan-Shanzer S, van de Watering MJ, de Graaf M, van der Laarse A, Schalij MJ, van der Wall EE, de Vries AA, Atsma DE. Activation of cardiac and smooth muscle-specific genes in primary human cells after forced expression of human myocardin. Cardiovasc Res. 2005;67(2):245- 255.
52. van Tuyn J, Atsma DE, Winter EM, van der Velde-van Dijke I, Pijnappels DA, Bax NA, Knaan-Shanzer S, Gittenberger-de Groot AC, Poelmann RE, van der Laarse A, van der Wall EE, Schalij MJ, de Vries AA. Epicardial cells of human adults can undergo an epithelial-to-mesenchymal transition and obtain characteristics of smooth muscle cells in vitro. Stem Cells.
2007;25(2):271-278.
53. van Tuyn J, Pijnappels DA, de Vries AA, de Vries I, van der Velde-van Dijke I, Knaan-Shanzer S, van der Laarse A, Schalij MJ, Atsma DE.
23
Chapter 1Introduction
Fibroblasts from human postmyocardial infarction scars acquire properties of cardiomyocytes after transduction with a recombinant myocardin gene.
FASEB J. 2007;21:3369-3379.
54. Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, Hammond SM, Conlon FL, Wang DZ. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet. 2006;38(2):228-233.
55. Jain MK, Kashiki S, Hsieh CM, Layne MD, Yet SF, Sibinga NE, Chin MT, Feinberg MW, Woo I, Maas RL, Haber E, Lee ME. Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system. Circ Res. 1998;83(10):980-985.
56. Li L, Miano JM, Cserjesi P, Olson EN. SM22 alpha, a marker of adult smooth muscle, is expressed in multiple myogenic lineages during embryogenesis.
Circ Res. 1996;78(2):188-195.
57. Samaha FF, Ip HS, Morrisey EE, Seltzer J, Tang Z, Solway J, Parmacek MS. Developmental pattern of expression and genomic organization of the calponin-h1 gene. A contractile smooth muscle cell marker. J Biol Chem.
1996;271(1):395-403.
58. Bernstein E, Kim SY, Carmell MA, Murchison EP, Alcorn H, Li MZ, Mills AA, Elledge SJ, Anderson KV, Hannon GJ. Dicer is essential for mouse development. Nat Genet. 2003;35(3):215-217.
59. Kwon C, Han Z, Olson EN, Srivastava D. MicroRNA1 influences cardiac differentiation in Drosophila and regulates Notch signaling. Proc Natl Acad Sci U S A. 2005;102(52):18986-18991.
60. Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle- specific microRNA that targets Hand2 during cardiogenesis. Nature.
2005;436(7048):214-220.
61. Cao D, Wang Z, Zhang CL, Oh J, Xing W, Li S, Richardson JA, Wang DZ, Olson EN. Modulation of smooth muscle gene expression by association of histone acetyltransferases and deacetylases with myocardin. Mol Cell Biol.
2005;25(1):364-376.
62. McDonald OG, Wamhoff BR, Hoofnagle MH, Owens GK. Control of SRF binding to CArG box chromatin regulates smooth muscle gene expression in vivo. J Clin Invest. 2006;116(1):36-48.
63. Wang J, Li A, Wang Z, Feng X, Olson EN, Schwartz RJ. Myocardin sumoylation transactivates cardiogenic genes in pluripotent 10T1/2 fibroblasts. Mol Cell Biol. 2007;27(2):622-632.
64. Torrado M, Lopez E, Centeno A, Medrano C, Castro-Beiras A, Mikhailov AT. Myocardin mRNA is augmented in the failing myocardium: expression profiling in the porcine model and human dilated cardiomyopathy. J Mol Med. 2003;81(9):566-577.
65. Xing W, Zhang TC, Cao D, Wang Z, Antos CL, Li S, Wang Y, Olson EN, Wang DZ. Myocardin induces cardiomyocyte hypertrophy. Circ Res.
2006;98(8):1089-1097.
66. Badorff C, Seeger FH, Zeiher AM, Dimmeler S. Glycogen synthase kinase 3beta inhibits myocardin-dependent transcription and hypertrophy induction through site-specific phosphorylation. Circ Res. 2005;97(7):645- 654.
24
67. Callis TE, Cao D, Wang DZ. Bone morphogenetic protein signaling modulates myocardin transactivation of cardiac genes. Circ Res. 2005;97(10):992- 1000.
68. Qiu P, Ritchie RP, Fu Z, Cao D, Cumming J, Miano JM, Wang DZ, Li HJ, Li L.
Myocardin enhances Smad3-mediated transforming growth factor-beta1 signaling in a CArG box-independent manner: Smad-binding element is an important cis element for SM22alpha transcription in vivo. Circ Res.
2005;97(10):983-991.
69. Wang Z, Wang DZ, Hockemeyer D, McAnally J, Nordheim A, Olson EN.
Myocardin and ternary complex factors compete for SRF to control smooth muscle gene expression. Nature. 2004;428(6979):185-189.
70. Davis CA, Haberland M, Arnold MA, Sutherland LB, McDonald OG, Richardson JA, Childs G, Harris S, Owens GK, Olson EN. PRISM/PRDM6, a transcriptional repressor that promotes the proliferative gene program in smooth muscle cells. Mol Cell Biol. 2006;26(7):2626-2636.
71. Yoshida T, Hoofnagle MH, Owens GK. Myocardin and Prx1 contribute to angiotensin II-induced expression of smooth muscle alpha-actin. Circ Res.
2004;94(8):1075-1082.
72. Liu ZP, Wang Z, Yanagisawa H, Olson EN. Phenotypic modulation of smooth muscle cells through interaction of Foxo4 and myocardin. Dev Cell.
2005;9(2):261-270.
73. Liu Y, Sinha S, McDonald OG, Shang Y, Hoofnagle MH, Owens GK. Kruppel- like factor 4 abrogates myocardin-induced activation of smooth muscle gene expression. J Biol Chem. 2005;280(10):9719-9727.
74. Doi H, Iso T, Yamazaki M, Akiyama H, Kanai H, Sato H, Kawai-Kowase K, Tanaka T, Maeno T, Okamoto E, Arai M, Kedes L, Kurabayashi M. HERP1 inhibits myocardin-induced vascular smooth muscle cell differentiation by interfering with SRF binding to CArG box. Arterioscler Thromb Vasc Biol.
2005;25(11):2328-2334.
75. Li HJ, Haque Z, Lu Q, Li L, Karas R, Mendelsohn M. Steroid receptor coactivator 3 is a coactivator for myocardin, the regulator of smooth muscle transcription and differentiation. Proc Natl Acad Sci U S A.
2007;104(10):4065-4070.
76. Wamhoff BR, Bowles DK, McDonald OG, Sinha S, Somlyo AP, Somlyo AV, Owens GK. L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/
SRF-dependent mechanism. Circ Res. 2004;95(4):406-414.
77. Wang DZ, Li S, Hockemeyer D, Sutherland L, Wang Z, Schratt G, Richardson JA, Nordheim A, Olson EN. Potentiation of serum response factor activity by a family of myocardin-related transcription factors. Proc Natl Acad Sci U S A. 2002;99(23):14855-14860.
78. Li J, Zhu X, Chen M, Cheng L, Zhou D, Lu MM, Du K, Epstein JA, Parmacek MS. Myocardin-related transcription factor B is required in cardiac neural crest for smooth muscle differentiation and cardiovascular development.
Proc Natl Acad Sci U S A. 2005;102(25):8916-8921.
79. Oh J, Richardson JA, Olson EN. Requirement of myocardin-related transcription factor-B for remodeling of branchial arch arteries and smooth
25
Chapter 1Introduction
muscle differentiation. Proc Natl Acad Sci U S A. 2005;102(42):15122- 15127.
80. Wei K, Che N, Chen F. Myocardin-related transcription factor B is required for normal mouse vascular development and smooth muscle gene expression.
Dev Dyn. 2007;236(2):416-425.
81. Li S, Chang S, Qi X, Richardson JA, Olson EN. Requirement of a myocardin- related transcription factor for development of mammary myoepithelial cells. Mol Cell Biol. 2006;26(15):5797-5808.
82. Miralles F, Posern G, Zaromytidou AI, Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell. 2003;113(3):329- 342.
83. Posern G, Miralles F, Guettler S, Treisman R. Mutant actins that stabilise F- actin use distinct mechanisms to activate the SRF coactivator MAL. EMBO J. 2004;23(20):3973-3983.
84. Zhao H, Rebbert ML, Dawid IB. Myoskeletin, a factor related to Myocardin, is expressed in somites and required for hypaxial muscle formation in Xenopus. Int J Dev Biol. 2007;51(4):315-320.
