title: Cardiac progenitor cells engineered with Pim-1 (CPCeP) develop cardiac phenotypic electrophysiological properties as they are co-cultured with neonatal myocytes.
abstract: Stem cell transplantation has been successfully used for amelioration of cardiomyopathic injury using adult cardiac progenitor cells (CPC). Engineering of mouse CPC with the human serine/threonine kinase Pim-1 (CPCeP) enhances regeneration and cell survival in vivo, but it is unknown if such apparent lineage commitment is associated with maturation of electrophysiological properties and excitation-contraction coupling. This study aims to determine electrophysiology and Ca(2+)-handling properties of CPCeP using neonatal rat cardiomyocyte (NRCM) co-culture to promote cardiomyocyte lineage commitment. Measurements of membrane capacitance, dye transfer, expression of connexin 43 (Cx43), and transmission of ionic currents (I(Ca), I(Na)) from one cell to the next suggest that a subset of co-cultured CPCeP and NRCM becomes connected via gap junctions. Unlike NRCM, CPCeP had no significant I(Na), but expressed nifedipine-sensitive I(Ca) that could be measured more consistently with Ba(2+) as permeant ion using ramp-clamp protocols than with Ca(2+) and step-depolarization protocols. The magnitude of I(Ca) in CPCeP increased during culture (4-7 days vs. 1-3 days) and was larger in co-cultures with NRCM and with NRCM-conditioned medium, than in mono-cultured CPCeP. I(Ca) was virtually absent in CPC without engineered expression of Pim-1. Caffeine and KCl-activated Ca(2+)-transients were significantly present in co-cultured CPCeP, but smaller than in NRCM. Conversely, ATP-induced (IP(3)-mediated) Ca(2+) transients were larger in CPCeP than in NRCM. I(NCX) and I(ATP) were expressed in equivalent densities in CPCeP and NRCM. These in vitro studies suggest that CPCeP in co-culture with NRCM: a) develop I(Ca) current and Ca(2+) signaling consistent with cardiac lineage, b) form electrical connections via Cx43 gap junctions, and c) respond to paracrine signals from NRCM. These properties may be essential for durable and functional myocardial regeneration under in vivo conditions.
authors.affiliation: Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina and Clemson University, Charleston, SC 29403, USA.
authors.forename: Hale
authors.initials: H
authors.lastname: Tufan
authors.forename: Xiao-Hua
authors.initials: XH
authors.lastname: Zhang
authors.forename: Nicole
authors.initials: N
authors.lastname: Haghshenas
authors.forename: Mark A
authors.initials: MA
authors.lastname: Sussman
authors.forename: Lars
authors.initials: L
authors.lastname: Cleemann
authors.forename: Martin
authors.initials: M
authors.lastname: Morad
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology; D053687:Adult Stem Cells / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D020013:Calcium Signaling; D002450:Cell Communication; D002454:Cell Differentiation; D048430:Cell Shape; D002478:Cells, Cultured; D018920:Coculture Techniques; D018031:Connexin 43 / Q000378:metabolism; D017077:Culture Media, Conditioned; D017629:Gap Junctions / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008564:Membrane Potentials; D051379:Mice; D008822:Mice, Transgenic; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010641:Phenotype; D051573:Proto-Oncogene Proteins c-pim-1 / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D019831:Sodium-Calcium Exchanger / Q000378:metabolism
chemical_list: D018031:Connexin 43; D017077:Culture Media, Conditioned; D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; C496634:PIM1 protein, human; D051573:Proto-Oncogene Proteins c-pim-1