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Turning on stem cell cardiogenesis with extremely low frequency magnetic fields

Carlo Ventura^*,1, Margherita Maioli, Yolande Asara, Daniela Santoni, Pietro Mesirca, Daniel Remondini and Ferdinando Bersani

^* Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems, University of Bologna, Bologna, Italy;
Department of Biomedical Sciences, University of Sassari, Sassari, Italy; and
ICEmB at Department of Physics, University of Bologna, Bologna, Italy

¹Correspondence: Malpighi Hospital, Institute of Cardiology, Pavilion 21, Bologna, Italy 40138. E-mail: cvent{at}libero.it

SPECIFIC AIMS

Although magnetic fields (MF) have been shown to affect cell proliferation and growth factor expression in cultured cells, compelling evidence that MF may trigger a coordinate program of cell differentiation is still lacking. Here we explored whether MF may be able to prime the expression of genes encoding for tissue-restricted transcription factors in pluripotent mouse embryonic stem (ES) cells and whether, in the affirmative, ES cell exposure to MF may ultimately ensue into targeted cell lineage specification.

PRINCIPAL FINDINGS

1. ES cell exposure to extremely low-frequency MF triggers GATA-4 and Nkx-2.5 gene expression
A sinusoidal MF (50 Hz, 0.8 mT rms) was applied to GTR1 cells, a derivative of pluripotent mouse R1 ES cells bearing the puromycin resistance gene driven by the cardiomyocyte-specific -myosin heavy chain promoter. Treatment was performed continuously after removal of leukemia inhibitory factor (LIF) until time of collection of embryoid bodies or ES-derived cardiomyocytes (3 or 10 days from LIF withdrawal, respectively). In both groups of cells, MF remarkably increased the expression of GATA-4 and Nkx-2.5 mRNA, encoding for a zinc finger-containing transcription factor and a homeodomain that are essential for cardiogenesis in various animal species, including humans (Fig. 1 ).

View larger version (35K): [in this window] [in a new window]   Figure 1. Effect of magnetic field on the expression of cardiac lineage-promoting genes. Magnetic field (MF) was applied from the time of LIF removal. EBs (A) or puromycin-selected cardiomyocytes (P, panel B) were collected after 3 or 10 additional days, respectively, and processed for RT-PCR analysis of the indicated transcripts. LIF, undifferentiated cells; C, unexposed controls; S, sham-exposed (counter-wound coils); MF, MF-exposed cells. Ethidium bromide-stained agarose gels, representative of 4 separate experiments. C) RNase protection analysis of GATA-4 mRNA expression. GTR1 ES cells were cultured as described in the absence (–) or presence (+) of MF. *Significantly different from unexposed. D) Nuclear run-off analysis of GATA-4 gene transcription in isolated ES cell nuclei. Nuclei were isolated from undifferentiated cells (LIF), EBs or P collected 3 or 10 days after LIF removal, respectively. Each group of cells was exposed in the absence (–) or presence (+) of MF from the time of LIF withdrawal. Row a: GATA-4 gene transcription. Row b: cyclophilin gene transcription. Autoradiograms are representative of 3 separate experiments. E) Immunoreactive dynorphin B (ir-dyn B) in cells (gray bars) or medium (white bars), mean ± SE (n=6). Asterisks with brackets: significant difference (1-way ANOVA, Newman Keul’s test). Significantly different from values of gray bars.

2. MF induces the activation of an endorphinergic system
MF enhanced prodynorphin mRNA expression and levels of dynorphin B, a bioactive end product of the gene acting as a natural agonist of kappa opioid receptors, in both EBs and ES-derived cardiomyocytes, as well as in their incubation media (Fig. 1) . Prodynorphin gene and dynorphin B expression has been found to play a major role in ES cell cardiogenesis priming GATA-4 and Nkx-2.5 transcription through the activation of protein kinase C signaling and nuclear opioid receptors.

3. MF acts at the transcriptional level
Nuclear run-off transcriptional analysis revealed that the transcription rate of the GATA-4 gene was greatly enhanced in nuclei that had been isolated from EBs or ES-derived cardiomyocytes obtained from MF-exposed cells compared with nuclei from unexposed cells (Fig. 1) .

4. MF increases the yield of ES-derived cardiomyocytes
Activation of a program of cardiac lineage-restricted genes was associated with an increase in the expression of the cardiac specific transcripts -myosin heavy chain (MHC) and myosin light chain-2V (Fig. 2 ). MHC expression in cardiomyocytes from MF-exposed cells was further confirmed in immunofluorescence studies (Fig. 2) . Exposure of GTR1 ES cells to MF after LIF removal and throughout 4 days of puromycin selection consistently increased the yield of ES-derived cardiomyocytes (number of beating colonies reached 180.38±33.0% of the control value estimated in cardiomyocytes selected from unexposed cells; mean ±SE of 4 separate experiments).

View larger version (33K): [in this window] [in a new window]   Figure 2. MF primes the expression of cardiac specific genes and enhances the yield of ES-derived cardiomyocytes. MF was applied from the time of LIF removal. EBs or P were collected after 3 or 10 additional days, respectively. A, B) RT-PCR analysis of -myosin heavy chain (MHC) and myosin light chain-2V (MLC). LIF, undifferentiated cells; C, unexposed controls; S, sham-exposed (counter-wound coils); MF, MF-exposed cells. Ethidium bromide-stained agarose gels representative of 4 separate experiments. MHC immunostaining was assessed by the MF20 monoclonal antibody in undifferentiated cells (C) and in cardiomyocytes derived from cells cultured in the absence (D) or presence (E) of MF. DNA was visualized with propidium iodide (1 µg/mL). Biorad Microradians confocal microscope, x20 objective.

5. Effect of MF on the expression of genes promoting non-myocardial lineages
It is noteworthy that expression of MyoD, a gene involved in skeletal myogenesis, was not affected in EBs derived from MF-exposed cells. EBs from exposed cells exhibited a slight increase in expression of neurogenin1, a neuronal specification gene.

CONCLUSIONS AND SIGNIFICANCE

MF have been shown to elicit behavioral changes in intact organisms and cell proliferation in vitro. Our data showing the ability of MF to prime a gene expression pattern of cardiogenesis may profoundly affect our understanding of the biological consequences of MF exposure at cellular level. Failure of MF to affect the transcription of a gene promoting skeletal muscle determination and the faint effect on neuronal specification seem to exclude a generalized activation of repressed genes and suggests that coupling of MF with GATA-4, Nkx-2.5 and prodynorphin gene expression may represent a mechanism pertaining to ES cell cardiogenesis.

Stem cells were proposed recently as a renewable source of donor cells for the rescue of damaged tissues. However, such a rescuing potential is limited by the fact that differentiating cells withdraw early from the cell cycle, and development of strategies affording high throughput of targeted lineages from pluripotent cells would have obvious biomedical implications. Overexpression of tissue-specific genes by vector-mediated gene transfer is a cumbersome approach that may perturb normal homeostasis in stem cells and recipient tissues and is not readily envisionable in humans. The current finding that MF can elicit a remarkable increase in the yield of ES-derived cardiomyocytes provides evidence for the potential use of magnetic fields in modifying the gene program of cardiac differentiation in ES cells without the aid of gene transfer technologies. This may have further implications in the development of strategies modulating stem cell differentiation, an important assignment for stem cell biology and cellular engineering.

Studies are under way to shed more light on molecular events underlying the differentiating response primed by MF in ES cells.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2695fje;

This article has been cited by other articles:

				S. LEV, I. KEHAT, and L. GEPSTEIN Differentiation Pathways in Human Embryonic Stem Cell-Derived Cardiomyocytes Ann. N.Y. Acad. Sci., June 1, 2005; 1047(1): 50 - 65. [Abstract] [Full Text] [PDF]

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