Human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs) for disease modeling, cardiotoxicity screening, drug discovery and cell‐based therapies. However, as shown in previous studies conducted by our lab (Poon, Kong et al. 2011), human embryonic stem cells (hESCs)‐derived CMs display immature〖Ca〗^(2+)–handing properties with smaller transient amplitudes, slower rise and decay kinetics than those of adult CMs. Although the cytosolic 〖Ca〗^(2+) signaling of hESC‐CMs has only recently been understood, there is no investigation on the nuclear 〖Ca〗^(2+) signal in hESC‐CMs, despite its importance. In this dissertation, delayed kinetics of nuclear 〖Ca〗^(2+), as compared to that of cytosol during 〖Ca〗^(2+)waves or 〖Ca〗^(2+) transients, was found in hESC‐derived ventricular (V) CMs, indicating that nuclear 〖Ca〗^(2+) was initiated by 〖Ca〗^(2+) diffusion from cytosol. Besides global 〖Ca〗^(2+) signals, local nuclear 〖Ca〗^(2+) signals were observed and identified as Ca2+ release from ryanodine receptors (RyRs), and nucleoplasmic reticulum (NR) served as their structural basis. In addition, targeted expression of 〖Ca〗^(2+) buffering protein parvalbumin (PV) in cytosol or nucleus altered 〖Ca〗^(2+) transient and stimuli‐induced apoptosis of hESC‐VCMs. For cytosolic 〖Ca〗^(2+) signaling in hESC‐VCMs, the mechanistic basis of excitation‐contraction coupling of hESC‐VCMs was studied by using 〖Ca〗^(2+) sparks, which are the unitary 〖Ca〗^(2+) ‐events. The results indicated that RyRs could be sensitized by 〖Ca〗^(2+) in permeabilized hESC‐VCMs. Increasing external 〖Ca〗^(2+) dramatically escalated the basal 〖Ca〗^(2+) and spark frequency. Furthermore, RyR‐mediated Ca2+ release sensitized nearby RyRs, leading to compound 〖Ca〗^(2+) sparks, whereas inhibition of mitochondrial 〖Ca〗^(2+) + uptake promoted Ca2+ waves. The aforementioned immature 〖Ca〗^(2+)–handing properties of hESC‐CMs can be attributed to their differential expression of crucial Ca2+–handling proteins. During diastole, SERCA and NCX sequester and extrude 〖Ca〗^(2+) ions, respectively, to return cytosolic 〖Ca〗^(2+) to the resting level. As previously published in our lab, NCX, robustly expressed in hESC‐CMs but much less so in the adult counterparts, is a functional determinant of immature 〖Ca〗^(2+) homeostasis. Unlike NCX, SERCA is expressed less in hESC‐CMs than in adult‐CMs. The present study first demonstrated the effects of lentivirus‐based genetic manipulation of SERCA2a and NCX1 in hESC‐VCMs, and the results indicated that SERCA2a overexpression shortened the decay phase of low‐frequency (0.5 Hz) electrical stimulation‐elicited Ca2+ transient. Increasing pacing frequency from 0.5 Hz to 2 Hz led to a decrease of relative transient amplitude, showing that hESC‐VCMs harbored a negative‐frequency response. At a high‐stimulation frequency of 2 Hz, it was revealed that SERCA overexpression, but not NCX1 suppression, increased the amplitude of 〖Ca〗^(2+) transient by accelerating 〖Ca〗^(2+) sequestration to sarcoplasmic reticulum (SR), indicating partial rescue of the negative‐frequency response. Taken collectively, the findings provide 1) novel information on nuclear 〖Ca〗^(2+) signaling in hESC‐VCMs, 2) the first lines of direct evidence that hESC‐VCMs have functional 〖Ca〗^(2+)‐induced‐〖Ca〗^(2+)+‐release (CICR), and 3) evidence of driving hESC‐VCMs maturation by SERCA2a overexpression, which may facilitate clinical and other applications of hESC‐VCMs. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Title page, table of contents and summary only. The complete thesis in print form is available from the University of Adelaide Library. / The results presented in this thesis show the existence of TREK-1(Twik-RElated K+ channel; KCNK2), variant A and C of TREK-2 (KCNK10) and TRAAK (Twik-Related Arachidonic Acid-stimulated K+ channel; KCNK4) in human heart; the localization of TREK-1 and variants of TREK-2 on the membrane and in cytoplasmic areas of human cardiomyocyte; the notably high-level expression of TREK-1 in diseased human heart; the reverse expression of variant A and C of TREK-2 in normal and diseased human heart. These observations strongly indicate TREK channels play important roles in arrhythmia genesis and TREK-sensitive cardiac remodelling within the development of cardiac hypertrophy, ischemia cardiomyopathy and idiopathic dilated cardiomyopathy. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1248412 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2006

Thesis (M.Sc.)--York University, 2006. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR19720

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 61-67). Also available on the World Wide Web.

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisors: John F. Rabolt and D. Bruce Chase, Dept. of Materials Science & Engineering. Includes bibliographical references.

Thesis (Ph.D.)--University of Western Australia, 2006.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 75-100) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available online.