Intracellular Ca2+ mobilization via secondary messengers modulates multiple cell functions. Cyclic Adenosine 5’-Diphosphate-Ribose (cADPR) is one of the most well recognized endogenous Ca2+ mobilizing messengers. In mammalian, cADPR is mainly formed by CD38, a multi-functional enzyme, from nicotinamide adenine dinucleotide (NAD). It has previously been shown that the cADPR/CD38/Ca2+pathway mediates many cardiac functions, such as regulating the excitation-contraction coupling in cardiac myocytes and modulating the Ca2+ homeostasis during the ischemia injury of the heart. Thus it is reasonable to propose that the cADPR/CD38/Ca2+ pathway plays a role in cardiogenesis. The pluripotent mouse embryonic stem (mES) cells which can be induced to differentiate into all cell types provide an ideal model for studying cardiogenesis. The first part of this dissertation is to determine the role of CD38/cADPR/Ca2+pathwayin the cardiomyogenesis of mES cells. The data showed that CD38 expression was markedly up-regulated during the in vitro embryoid body (EB) differentiation of mouse ES cells, which indicated a regulatory role of CD38 in the differentiation process. Lentivirus mediated shRNA provides a convenient method to knockdown the expression of CD38 in mES cells. Surprisingly, beating clusters appeared earlier and more in CD38 knockdown EBs than that in control EBs. Likewise, the expressions of several cardiac markers were up regulated in CD38 knockdown EBs. In addition, more cardiomyocytes (CMs) existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs than those in control EBs. On the other hand, over-expression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, we showed that CMs derived from the CD38 knock down mES cells possessed the functional properties characteristic of CMs derived fromnormal ES cells. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2cascade during CM differentiation of mES cells, and transiently inhibition of Erk1/2 blocked the enhancive effects of CD38 knockdown on the differentiation of CM from mES cells. Taken together, our data indicate that the CD38/cADPR/Ca2+ signaling pathway suppresses the cardiac differentiation of mES cells. 　　One of the main goals of the researches on cardiac differentiation of ES cells is to enhance the production of CMs from ES cells, thereby providing sufficient amount of functional intact CMs for the treatment of severe heart disease. Nitric oxide (NO) has been found to be a powerful cardiogenesis inducer of mES cells, in that it can significantly increase the yield of ES-derived CM. The second objective of this dissertation is to explore the mechanism underlying the NO facilitated cardiomyogenesis of mES cells. We found that the NO did induce intracellular Ca2+ increases in mES cells, and this Ca2+ increase was due to internal Ca2+ release from ER through theIP3 pathway. Therefore, the expression of IP3 receptors (IP3Rs) in mES cells were knocked down by lentivirus-mediated shRNAs. Interestingly, only type 3 IP3R (IP3R3) knockdown significantly inhibited the NO induced Ca2+ release in mES cells. Moreover, NO facilitated cardiogensis of mES cells was abolished in IP3R3 knockdown EBs. In summary, our results indicate that the IP3R3-Ca2+ pathway is required for NO facilitated cardiomyogenesis of mES cells. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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

A search of the Saccharomyces genome data base revealed an open reading frame of 2039 amino acids with homology to L-type calcium channels. Northern blots probed with a 540 bp PCR product of the ORF showed a transcript of 6.1 kb. Two procedures were used to disrupt the gene: the ORF was truncated by an integrative disruption after the third pore motif, or deleted in the first three pore domains using a one-step disruption construct. In most strains tested, the disruptions gave no apparent phenotype when tested under a variety of conditions. However, conspicuous phenotypes were seen in the strain YEL161-2A, a strain super-sensitive to alpha-mating factor (sst1). In most respects, truncation gave a less severe phenotype than deletion, suggesting that the truncated gene retains partial function. Calcium uptake during normal growth, as well as the increased calcium uptake in response to mating factor, were reduced progressively by the truncation and deletion respectively. Growth rate and cell viability were reduced, cell size heterogeneity increased, and recovery from mating factor arrest was delayed and abnormal. The cells became sensitive to MnCl$ sb2.$ The phenotype resulting from gene truncation was alleviated by a high-calcium medium, and exacerbated by low calcium. Complementation of the deleted strain by a Yep13 plasmid containing BAR1 (SST1) restored normal growth and viability. However, somewhat paradoxically, deletion of the putative calcium channel gene in another sst1 strain (SY1159) showed no phenotype.

Damage to one vestibular apparatus (unilateral vestibular deafferentation, UVD) results in severe postural and ocular motor disturbances (such as spontaneous nystagmus, SN) that recover over time in a process known as vestibular compensation. However, the underlying neurochemical mechanisms of vestibular compensation are poorly understood. While UVD affects many areas in the CNS, attention has focused upon the partially deafferented second order neurons in the vestibular nuclei complex (VNC). Several converging lines of evidence suggest that Ca�⁺-permeable ion channels (N-methyl-D-aspartate receptors and L-type voltage-gated Ca�⁺-channels) and intracellular Ca�⁺-dependent protein kinases play an important role in vestibular compensation. However, the nature of this involvement and the locus of these changes are unknown. The aim of this thesis was to investigate the role of Ca�⁺ signalling pathways in the VNC during vestibular compensation in guinea pig. These issues were investigated in three separate experiments that utilised two methodological approaches: i) in vitro assays were used to determine the nature and extent of protein phosphorylation within the VNC at various stages of compensation; and ii) ion channel blockers or cell-permeable kinase inhibitors were injected directly into the VNC immediately before UVD to determine whether or not these systems were causally involved in compensation. The results of experiment 1 (Chapter 5) showed that a bolus intra-VNC injection of an uncompetitive NMDA receptor antagonist, but not an L-type voltage-gated Ca�⁺ channel antagonist, temporarily reduced SN frequency at the earliest measurement time (6 hours post-UVD). These results suggested that the initial expression of SN required, in part, the activation of NMDA receptors in the VNC on the side of the UVD, and by inference, Ca�⁺ entry through the ion channel. The results of experiment 2 (Chapter 6) revealed that the medial VNC contains abundant Ca�⁺/calmodulin-dependent and Ca�⁺/phospholipid-dependent protein kinase activities. The same VNC tissue removed from animals at various times after UVD, showed that vestibular compensation is accompanied by specific changes in the phosphorylation of several major protein kinase C substrates. These included an unidentified 46-kDa band, and a 75-kDa band with similar characteristics to the myristoylated alanine-rich C kinase substrate (MARCKS). These results suggest that protein kinase C signalling pathways may be involved in vestibular compensation. The results of experiment 3 (Chapter 7) are consistent with these results showing that intra-VNC infusion of a protein kinase C inhibitor, but not a Ca�⁺/calmodulin-dependent protein kinase II inhibitor, significantly increased SN at the earliest measurement times (6 and 8 hours), but had no effect upon the time taken to achieve compensation or on postural compensation. These results suggest that the induction of SN compensation involves protein kinase C activity in the VNC. Taken together, these findings suggest that the mechanisms underlying the expression of SN (e.g., Ca�⁺ influx via NMDA receptors) are possibly distinct from those that initiate its compensation (e.g., PKC activation). The downstream effects of raised intracellular Ca�⁺ may involve protein kinase C-dependent phosphorylation of key intracellular proteins that initiate long-lasting changes in cellular function within the VNC.

Dissertation (Ph.D.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 70-78, p. 93, p. 132-140, p. 164-168, p. 194-221.

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

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.

Thesis (Ph.D.) - University of St Andrews, May 2007.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 85-100).