Facilitated maturation of Ca2+ handling properties of human embryonic stem cell-derived cardiomyocytes by calsequestrin expression.
Cardiomyocytes (CMs) are nonregenerative. Self-renewable pluripotent human embryonic stem cells (hESCs) can differentiate into CMs for cell-based therapies. We recently reported that Ca(2+) handling, crucial to excitation-contraction coupling of hESC-derived CMs (hESC-CMs), is functional but immature. Such immature properties as smaller cytosolic Ca(2+) transient amplitudes, slower kinetics, and reduced Ca(2+) content of sarcoplasmic reticulum (SR) can be attributed to the differential developmental expression profiles of specific Ca(2+) handling and regulatory proteins in hESC-CMs and their adult counterparts. In particular, calsequestrin (CSQ), the most abundant, high-capacity but low-affinity, Ca(2+)-binding protein in the SR that is anchored to the ryanodine receptor, is robustly expressed in adult CMs but completely absent in hESC-CMs. Here we hypothesized that gene transfer of CSQ in hESC-CMs suffices to induce functional improvement of SR. Transduction of hESC-CMs by the recombinant adenovirus Ad-CMV-CSQ-IRES-GFP (Ad-CSQ) significantly increased the transient amplitude, upstroke velocity, and transient decay compared with the control Ad-CMV-GFP (Ad-GFP) and Ad-CMV-CSQDelta-IRES-GFP (Ad-CSQDelta, which mediated the expression of a nonfunctional, truncated version of CSQ) groups. Ad-CSQ increased the SR Ca(2+) content but did not alter L-type Ca(2+) current. Pharmacologically, untransduced wild-type, Ad-GFP-, Ad-CSQDelta-, and Ad-CSQ-transduced hESC-CMs behaved similarly. Whereas ryanodine significantly reduced the Ca(2+) transient amplitude and slowed the upstroke, thapsigargin slowed the decay. Neither triadin nor junctin was affected. We conclude that CSQ expression in hESC-CMs facilitates Ca(2+) handling maturation. Our results shed insights into the suitability of hESC-CMs for therapies and as certain heart disease models for drug screening.
Stem Cell Program and Department of Cell Biology and Human Anatomy, University of California Davis, Davis, California, USA.
Jing
J
Liu
Deborah K
DK
Lieu
Chung Wah
CW
Siu
Ji-Dong
JD
Fu
Hung-Fat
HF
Tse
Ronald A
RA
Li
D000256:Adenoviridae / Q000235:genetics; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002155:Calsequestrin / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014161:Transduction, Genetic
C443109:CASQ2 protein, human; D020746:Calcium Channels, L-Type; D002155:Calsequestrin; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases