Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes

Weeke-Klimp, Alida; Bax, Noortje A.M.; Bellu, Anna Rita; Winter, Elizabeth M.; Vrolijk, Johannes; Plantinga, Josée; Maas, Saskia; Brinker, Marja; Mahtab, Edris A.F.; Gittenberger-de Groot, Adriana C.; van Luyn, Marja J.A.; Harmsen, Martin C.; Lie-Venema, Heleen

doi:10.1016/j.yjmcc.2010.07.007

« Previous Next »Journal of Molecular and Cellular Cardiology
Volume 49, Issue 4 , Pages 606-616, October 2010

Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes

Alida Weeke-Klimp
- Department of Pathology and Medical Biology, Medical Biology Section, Stem Cell and Tissue Engineering Group, University Medical Center Groningen, University of Groningen, The Netherlands
- Both authors contributed equally to this study.
Noortje A.M. Bax
- Department of Anatomy and Embryology, Leiden University Medical Center, The Netherlands
- Both authors contributed equally to this study.
Anna Rita Bellu
- Department of Pathology and Medical Biology, Medical Biology Section, Stem Cell and Tissue Engineering Group, University Medical Center Groningen, University of Groningen, The Netherlands
Elizabeth M. Winter
- Department of Anatomy and Embryology, Leiden University Medical Center, The Netherlands
Johannes Vrolijk
- Department of Molecular Cell Biology, Leiden University Medical Center, The Netherlands
Josée Plantinga
- Department of Pathology and Medical Biology, Medical Biology Section, Stem Cell and Tissue Engineering Group, University Medical Center Groningen, University of Groningen, The Netherlands
Saskia Maas
- Department of Anatomy and Embryology, Leiden University Medical Center, The Netherlands
Marja Brinker
- Department of Pathology and Medical Biology, Medical Biology Section, Stem Cell and Tissue Engineering Group, University Medical Center Groningen, University of Groningen, The Netherlands
Edris A.F. Mahtab
- Department of Anatomy and Embryology, Leiden University Medical Center, The Netherlands
Adriana C. Gittenberger-de Groot
- Department of Anatomy and Embryology, Leiden University Medical Center, The Netherlands
Marja J.A. van Luyn
- Department of Pathology and Medical Biology, Medical Biology Section, Stem Cell and Tissue Engineering Group, University Medical Center Groningen, University of Groningen, The Netherlands
Martin C. Harmsen
- Department of Pathology and Medical Biology, Medical Biology Section, Stem Cell and Tissue Engineering Group, University Medical Center Groningen, University of Groningen, The Netherlands
Heleen Lie-Venema
- Department of Anatomy and Embryology, Leiden University Medical Center, The Netherlands
- Corresponding author. Department of Anatomy and Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands; P.O. Box 9600, 2300 RC Leiden, The Netherlands. Tel.: +31 71 526 9338; fax: +31 71 526 8289.

Received 15 January 2010; received in revised form 24 June 2010; accepted 15 July 2010. published online 26 July 2010.

Abstract

During heart development, cells from the proepicardial organ spread over the naked heart tube to form the epicardium. From here, epicardium-derived cells (EPDCs) migrate into the myocardium. EPDCs proved to be indispensable for the formation of the ventricular compact zone and myocardial maturation, by largely unknown mechanisms. In this study we investigated in vitro how EPDCs affect cardiomyocyte proliferation, cellular alignment and contraction, as well as the expression and cellular distribution of proteins involved in myocardial maturation. Embryonic quail EPDCs induced proliferation of neonatal mouse cardiomyocytes. This required cell–cell interactions, as proliferation was not observed in transwell cocultures. Western blot analysis showed elevated levels of electrical and mechanical junctions (connexin43, N-cadherin), sarcomeric proteins (Troponin-I, α-actinin), extracellular matrix (collagen I and periostin) in cocultures of EPDCs and cardiomyocytes. Immunohistochemistry indicated more membrane-bound expression of Cx43, N-cadherin, the mechanotransduction molecule focal adhesion kinase, and higher expression of the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a). Newly developed software for analysis of directionality in immunofluorescent stainings showed a quantitatively determined enhanced cellular alignment of cardiomyocytes. This was functionally related to increased contraction. The in vitro effects of EPDCs on cardiomyocytes were confirmed in three reciprocal in vivo models for EPDC-depletion (chicken and mice) in which downregulation of myocardial N-cadherin, Cx43, and FAK were observed. In conclusion, direct interaction of EPDCs with cardiomyocytes induced proliferation, correct mechanical and electrical coupling of cardiomyocytes, ECM-deposition and concurrent establishment of cellular array. These findings implicate that EPDCs are ideal candidates as adjuvant cells for cardiomyocyte integration during cardiac (stem) cell therapy.

Research Highlights

►Direct contact with epicardium-derived cells (EPDCs) increases proliferation and cytoarchitectural maturation of cardiomyocytes ►This is reflected by increased levels of gap junctions (Cx43), adherens junctions (N-cadherin), SERCA2a, and cellular and sarcomeric alignment of the cardiomyocytes ►Deposition of extracellular matrix and recruitment of FAK are likely to be instrumental in the EPDC-dependent cytoarchitectural maturation of cardiomyocytes and associated with an increase in their contractility.

Keywords: Epicardium-derived cell, Cardiomyocyte, Cellular maturation, Cellular coupling, Extracellular matrix, Alignment