Date of Award


Degree Type


Degree Name

Doctor of Philosophy


In heart and skeletal muscle, the sarcoplasmic reticulum (SR) plays a critical role in the contraction-relaxation cycle of the myocyte: a Ca{dollar}\sp{lcub}2+{rcub}{dollar} channel releases Ca{dollar}\sp{lcub}2+{rcub}{dollar} from SR into cytoplasm to trigger contraction, and a Ca{dollar}\sp{lcub}2+{rcub}{dollar} pump resequesters Ca{dollar}\sp{lcub}2+{rcub}{dollar} to cause relaxation. A major regulator of this cycle is {dollar}\rm Ca\sp{lcub}2+{rcub}{dollar}/calmodulin-dependent protein kinase (CaM kinase), which is known to phosphorylate and thereby activate the cardiac SR Ca{dollar}\sp{lcub}2+{rcub}{dollar} pump, Ca{dollar}\sp{lcub}2+{rcub}{dollar} channel and endogenous Ca{dollar}\sp{lcub}2+{rcub}{dollar}-pump inhibitor phospholamban. This thesis examines the Ca{dollar}\sp{lcub}2+{rcub}{dollar} pump and its regulation in different tissues (cardiac, slow-twitch and fast-twitch muscle SR: CMSR, SMSR and FMSR) and different stages in development (fetal, newborn and adult rabbits) in order to identify the features common to these contractile systems and the variations that reflect functional differences.;This study demonstrates that the Ca{dollar}\sp{lcub}2+{rcub}{dollar} pump in heart is intrinsically different from that in skeletal muscle by showing that fluoride inhibits the {dollar}\rm Ca\sp{lcub}2+{rcub}{dollar}-bound conformation of the pump in CMSR but not in FMSR. Further, the regulatory mechanism differs between tissues: the Ca{dollar}\sp{lcub}2+{rcub}{dollar} pump is phosphorylated by an SR-associated CaM kinase in CMSR and SMSR, but not in FMSR. This phosphorylation has functional significance in SMSR as in CMSR: purified SMSR Ca{dollar}\sp{lcub}2+{rcub}{dollar} pump, when phosphorylated by exogenous CaM kinase, doubles its catalytic activity without altering Ca{dollar}\sp{lcub}2+{rcub}{dollar} sensitivity. Phosphorylation is quantitatively similar in CMSR and SMSR as regards rate and dependence on pH and temperature.;Studying fetal and newborn heart shows how Ca{dollar}\sp{lcub}2+{rcub}{dollar} pump regulation develops, and is medically important because of its implications for therapy of congenital heart disease. This thesis demonstrates that the SR {dollar}\rm Ca\sp{lcub}2+{rcub}{dollar} pump and the SR-associated CaM kinase (identified here as {dollar}\delta{dollar}-CaM kinase II) are present in fetal rabbit heart by 21 days gestation (term = 31), and that CaM kinase phosphorylates the pump at this time.;The ability of the SR-associated CaM kinase to phosphorylate the CA{dollar}\sp{lcub}2+{rcub}{dollar} pump, channel and phospholamban equip it to play a major role in regulating SR Ca{dollar}\sp{lcub}2+{rcub}{dollar} cycling and thus the contraction-relaxation function of heart and slow-twitch muscle. This regulatory system may therefore be an important target for medical treatment of heart disease from fetal through to adult life.



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