Physiological conditioning by electric field stimulation promotes cardiomyogenic gene expression in human cardiomyocyte progenitor cells
© Llucià-Valldeperas et al.; licensee BioMed Central Ltd. 2014
Received: 13 February 2014
Accepted: 21 July 2014
Published: 4 August 2014
The optimal cell lineage for cardiac-regeneration approaches remains mysterious. Additionally, electrical stimulation promotes cardiomyogenic differentiation of stimulated cells. Therefore, we hypothesized that electrical conditioning of cardiomyocyte progenitor cells (CMPCs) might enrich their cardiovascular potential. CMPCs were isolated from human adult atrial appendages, characterized, and electrically stimulated for 7 and 14 days. Electrical stimulation modulated CMPCs gene and protein expression, increasing all cardiac markers. GATA-binding protein 4 (GATA4) early transcription factor was significantly overexpressed (P = 0.008), but also its coactivator myocyte enhancer factor 2A (MEF2A) was upregulated (P = 0.073) under electrical stimulation. Moreover, important structural proteins and calcium handling-related genes were enhanced. The cardioregeneration capability of CMPCs is improved by electrical field stimulation. Consequently, short-term electrical stimulation should be a valid biophysical approach to modify cardiac progenitor cells toward a cardiogenic phenotype, and can be incorporated into transdifferentiation protocols. Electrostimulated CMPCs may be best-equipped cells for myocardial integration after implantation.
Cardiovascular diseases remain the leading cause of death in Western countries. Alternative strategies beyond current guidelines are actively sought to repair injured cardiac tissue, and stem cell-based therapies provide a promising path toward achieving this goal. In the past decade, progenitors from different origins have been studied for cardiac-regeneration purposes; however, the optimal cell lineage remains elusive. Despite the existence of resident cardiac stem cells, such as human cardiomyocyte progenitor cells (CMPCs), the regenerative capacity of the heart is limited.
The therapeutic potential of CMPCs was outlined in a pivotal report by Smits et al. , who demonstrated that CMPCs exhibit a certain degree of in situ differentiation into cardiomyocytes, smooth muscle cells, and endothelial cells after intramyocardial injection in a postinfarcted model in mice. Cardiomyogenic differentiation has also been promoted in a cardiac-mimetic electrical stimulation model in vitro. Accordingly, we hypothesized that the biophysical conditioning of CMPCs by electrical stimuli might enhance their cardiovascular potential and render them (once electrostimulated) fitting candidates for cardiac cell-therapy strategies.
In this study, we reported CMPCs isolation and characterization; we designed an electrostimulation protocol based on 2-millisecond pulses of 25 mV/cm alternating current, and evidenced gene and protein modulations after electric-field stimulation.
CMPCs were precisely isolated from human adult atrial appendages after the clonogenic method, as previously described . Cell-collection procedure was approved by the local Ethics Committee (Germans Trias i Pujol University Hospital Ethics Committee), and informed consent was obtained from all patients. The study protocol conformed to the principles outlined in the Declaration of Helsinki.
The electrical-stimulation protocol consisted of submitting 30,000 seeded cells to 2-ms monophasic square-wave pulses of 25 mV/cm at 1 Hz (alternating current) for 7 and 14 days .
Electrical stimulation modulated CMPC gene and protein expression (Figure 2B,C). Figure 2B shows the fold change of the studied cardiac markers at 7 and 14 days. All cardiac markers increased their expression after 14 days of stimulation.
A statistically significant overexpression of GATA4 was observed (P = 0.008), but also MEF2A (P = 0.073) was upregulated under electrical stimulation. MEF2 proteins are recruited through their DNA-binding domains by the early transcription factor GATA4 to activate cardiac promoters. Both transcription factors are expressed in the developing heart and have similar genetic expression patterns after electrical stimulation.
The presence of early transcription factors might conceivably enhance cardiac protein expression to achieve further a cardiomyocyte-like phenotype (for example, gap junctions for electrical coupling, and sarcomeric proteins for mechanical contraction) (Figure 2Ca-f). Cx43 proteins form gap junctions, which are key elements for impulse propagation throughout the heart syncytium. Cx43 protein was observed in the cytoplasm, as well as at the plasma membrane, particularly in stimulated cells (Figure 2Ca,b), in which its expression was improved. Main structural proteins for the contractile apparatus, such as cTnI and α–actinin, were also augmented (P = 0.093 and P > 0.1, respectively), although they do not show a striated pattern (Figure 2Cc,d). The absence of sarcomeres could suggest an early stage in the cardiomyogenic differentiation .
Additionally, SERCA2 protein was expressed in both conditions (Figure 2Ce,f), and was also slightly enhanced after 14 days of electrical stimulation (P > 0.1). SERCA2 proteins are intracellular pumps, which are located in the sarcoplasmic or endoplasmic reticula of muscle cells that are involved in the regulation of the cardiac contraction/relaxation cycle.
In sum, these data demonstrate that electric-field stimulation of CMPCs enhances cardiac gene expression. Gene modulation is translated to the protein level to promote CMPC phenotype differentiation. Short-term electrical stimulation appears to be a valid biophysical method to modify cardiac progenitor cells toward a cardiogenic phenotype, and can be included in transdifferentiation protocols. Electrostimulated CMPCs may be best-equipped for myocardial integration after transplantation.
Cardiomyocyte progenitor cells
cardiac troponin I
GATA-binding protein 4
myocyte enhancer factor 2
sarcoplasmic endoplasmic reticulum Ca2+ ATPase 2.
The authors thank the patients who made this study possible and the members of the Department of Cardiac Surgery for their collaboration in obtaining human samples. This work was supported by grants from the Ministerio de Economía y Competitividad (SAF2008-05144-C02-01 and SAF2011-30067-C02-01), European Commission 7th Framework Programme (RECATABI, NMP3-SL-2009-229239), La Marató de TV3 (122232), Redes de Investigación del Instituto de Salud Carlos III (Red de Terapia Celular (RD12/0019/0029) and Red de Investigación Cardiovascular (RD12/0042/0047)). We also appreciate support from the Fundació Privada Daniel Bravo Andreu. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Smits AM, van Laake LW, den Ouden K, Schreurs C, Szuhai K, van Echteld CJ, Mummery CL, Doevendans PA, Goumans MJ: Human cardiomyocyte progenitor cell transplantation preserves long-term function of the infarcted mouse myocardium. Cardiovasc Res. 2009, 83: 527-535. 10.1093/cvr/cvp146.View ArticlePubMedGoogle Scholar
- Serena E, Figallo E, Tandon N, Cannizzaro C, Gerecht S, Elvassore N, Vunjak-Novakovic G: Electrical stimulation of human embryonic stem cells: cardiac differentiation and the generation of reactive oxygen species. Exp Cell Res. 2009, 315: 3611-3619. 10.1016/j.yexcr.2009.08.015.PubMed CentralView ArticlePubMedGoogle Scholar
- Smits AM, van Vliet P, Metz CH, Korfage T, Sluijter JP, Doevendans PA, Goumans MJ: Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology. Nat Protoc. 2009, 4: 232-243. 10.1038/nprot.2008.229.View ArticlePubMedGoogle Scholar
- Llucià-Valldeperas A, Sanchez B, Soler-Botija C, Gálvez-Montón C, Prat-Vidal C, Roura S, Rosell-Ferrer J, Bragos R, Bayes-Genis A: Electrical stimulation of cardiac adipose tissue-derived progenitor cells modulates cell phenotype and genetic machinery. J Tissue Eng Regen Med. 2013, (E-pub ahead of print), doi:10.1002/term.1710Google Scholar
- Qian L, Berry EC, Fu JD, Ieda M, Srivastava D: Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro. Nat Protoc. 2013, 8: 1204-1215. 10.1038/nprot.2013.067.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.