title: Absence of transverse tubules contributes to non-uniform Ca(2+) wavefronts in mouse and human embryonic stem cell-derived cardiomyocytes.
abstract: Mouse (m) and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are known to exhibit immature Ca(2+) dynamics such as small whole-cell peak amplitude and slower kinetics relative to those of adult. In this study, we examined the maturity and efficiency of Ca(2+)-induced Ca(2+) release in m and hESC-CMs, the presence of transverse (t) tubules and its effects on the regional Ca(2+) dynamics. In m and hESC-CMs, fluorescent staining and atomic force microscopy (AFM) were used to detect the presence of t-tubules, caveolin-3, amphiphysin-2 and colocalization of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs). To avoid ambiguities, regional electrically-stimulated Ca(2+) dynamics of single ESC-CMs, rather than spontaneously beating clusters, were measured using confocal microscopy. m and hESC-CMs showed absence of dyads, with neither t-tubules nor colocalization of DHPRs and RyRs. Caveolin-3 and amphiphysin-2, crucial for the biogenesis of t-tubules with robust expression in adult CMs, were also absent. Single m and hESC-CMs displayed non-uniform Ca(2+) dynamics across the cell that is typical of CMs deficient of t-tubules. Local Ca(2+) transients exhibited greater peak amplitude at the peripheral than at the central region for m (3.50 +/- 0.42 vs. 3.05 +/- 0.38) and hESC-CMs (2.96 +/- 0.25 vs. 2.72 +/- 0.25). Kinetically, both the rates of rise to peak amplitude and transient decay were faster for the peripheral relative to the central region. Immature m and hESC-CMs display unsynchronized Ca(2+) transients due to the absence of t-tubules and gene products crucial for their biogenesis. Our results provide insights for driving the maturation of ESC-CMs.
authors.affiliation: Human Embryonic Cell Consortium, Stem Cell Program, University of California, Davis, Sacramento, CA 95817, USA. dklieu@ucdavis.edu
authors.forename: Deborah K
authors.initials: DK
authors.lastname: Lieu
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Chung-Wah
authors.initials: CW
authors.lastname: Siu
authors.forename: Gregory P
authors.initials: GP
authors.lastname: McNerney
authors.forename: Hung-Fat
authors.initials: HF
authors.lastname: Tse
authors.forename: Amir
authors.initials: A
authors.lastname: Abu-Khalil
authors.forename: Thomas
authors.initials: T
authors.lastname: Huser
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Li
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling; D051244:Caveolin 3 / Q000378:metabolism; D004558:Electric Stimulation; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D021381:Protein Transport; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism
chemical_list: D020746:Calcium Channels, L-Type; D051244:Caveolin 3; D009419:Nerve Tissue Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C075637:amphiphysin