In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures.

In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures.

In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "May 2002." Typescript. Vita. Includes bibliographical references (leaves 110-136). Also available online.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 110-136). Also available online. Also available on the Internet.

<p> Calcium (Ca2+) is an important signaling molecule and hence its movement across cell membranes must be tightly regulated. The intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle and endothelium controls the coronary tone. After stimulation, decreasing the [Ca2+]i back to resting levels is achieved mainly by the sodium calcium exchanger (NCX), the plasma membrane calcium pump (PMCA) or the sarcoendoplasmic reticulum calcium pump (SERCA). The present study will focus on NCX and its interactions with SERCA in the smooth muscle and endothelium of pig coronary artery.</p> <p> Aim 1 of my thesis is determination of activity levels of NCX in smooth muscle cells (SMC) and endothelial cells (EC). The NCX activity in cultured cells was approximately 5 times greater in EC than in SMC. The NCX inhibitors KB-R7943 and SEA 0400 blocked the NCX mediated Ca2+ entry, as did collapsing the Na+ gradient with monensin. NCX1 is the isoform largely responsible for NCX activity in SMC and EC. NCX activity was also assayed as the Ca2+ efflux in cultured cells and as Ca2+ uptake in plasma membrane vesicles isolated from freshly isolated smooth muscle.</p> <p> Aim 2 is to assess the existence of a functional NCX mediated Ca2+ entry linked to SERCA in SMC. In the absence of thapsigargin, BAPTA loading SMC increased the NCX mediated uptake. Thapsigargin did not affect the Ca2+ uptake in BAPTA loaded cells but it inhibited the Ca2+ uptake in cells that were not loaded with BAPTA. These data are consistent with a model in which SER acts as a sink for the NCX mediated Ca2+ entry. However, with BAPTA chelation and the resulting lower intracellular Ca2+, the need for SER to act as a sink is eliminated, and NCX is driven in full force. EC did not demonstrate a NCX-SERCA linkage.</p> <p> Arterial SMC and EC differ in their structure and function. The function of SMC is the generation of tone which is achieved by the Ca2+ dependent contractile filaments. Since these filaments are distributed throughout the cell, Ca2+ must be transported to and removed from deep within the cell. As a result, the SER may play a large role in Ca2+ regulation in the SMC. Furthermore, SMC also contain higher levels of high affinity Ca2+ pumps (SERCA and PMCA) and thus Ca2+ is more tightly regulated. Endothelial cells release nitric oxide in response to an increase in [Ca2+]i, which relaxes the smooth muscle. The endothelial nitric oxide sythase produces nitric oxide and is located adjacent to the PM in EC. The SER that removes Ca2+ from deep within the cell cytosol may play a small role in Ca2+ dependent modulation of the endothelial nitric oxide synthase activity. Based on the Western blot data, EC contain a greater amount of the high capacity NCX, thus the larger quantities of Ca2+ can be removed from the cell and the vicinity of endothelial nitric oxide synthase.</p> / Thesis / Master of Science (MSc)

The cardiac sodium-calcium exchanger (NCX) is an important membrane protein which regulates cellular calcium necessary for the optimal contractile function of the heart. NCX has become a focal point in ischemic heart disease (IHD) research as evidence suggests that reactive oxygen species (ROS) produced during IHD can cause NCX to malfunction resulting in an intracellular calcium overload leading to cardiac contractile abnormalities. Therefore, I hypothesized that NCX function is mediated by ROS increasing NCX1 activity during cardiac ischemia-reperfusion. To research this hypothesis, I investigated cellular mechanisms which may play a role in NCX dysfunction and also examined methods to correct NCX function. I found that reactive oxygen species directly and irreversibly modify NCX protein, increasing its activity, thereby worsening the calcium overload which is deleterious to cardiac function. I also elucidated the molecular means by which NCX protein modification occurs. Exploring pharmacological means by which to decrease NCX function to relieve the calcium overload and reduce the damage to the heart, I discovered that ranolazine (Ranexa), indicated for the treatment of angina pectoris inhibits NCX activity directly, thereby further reducing the calcium overload-induced injury to the heart. Furthermore, many IHD patients are also co-morbid for type-2 diabetes. These patients are prescribed sulfonylurea (SU) agents which act at the ATP sensitive K+ channel (KATP). One agent such as glibenclamide is known to have cardiotoxic side effects. Therefore, SUs devoid of any cardiac side effects would beneficial. Interestingly, patients possessing the genetic variant E23K-S1369A KATP channel have improved blood glucose levels with the use of the SU gliclazide. Therefore, I determined the functional mechanism by which gliclazide has increased inhibition at the KATP channel. These findings have implications for type-2 diabetes therapy, in which 20% of the type-2 diabetic population carries the KATP channel variant. In summary, the findings presented in this thesis have implications on treatment strategies in the clinical setting, as a NCX inhibitor can be beneficial in IHD and possibly type-2 diabetes. Moreover, a pharmacogenomic approach in treating type-2 diabetes may also provide a positive outcome when considering co-morbid cardiac complications such as atrial fibrillation and heart failure.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Huntington’s Disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder. There is no cure for HD and the existing therapies only alleviate HD symptoms without eliminating the cause of this neuropathology. HD is linked to a mutation in the huntingtin gene, which results in an elongation of the poly-glutamine stretch in the huntingtin protein (Htt). A major hypothesis is that mutant Htt (mHtt) leads to aberrant Ca2+ homeostasis in affected neurons. This may be caused by increased Ca2+ influx into the cell via the N-methyl-Daspartate (NMDA)-subtype of glutamate receptors. The contribution of two major Ca2+ removal mechanisms, mitochondria and plasmalemmal Na+/Ca2+ exchangers (NCX), in neuronal injury in HD remains unclear. We investigated Ca2+ uptake capacity in isolated synaptic (neuronal) and nonsynaptic mitochondria from the YAC128 mouse model of HD. We found that both Htt and mHtt bind to brain mitochondria and the amount of mitochondriabound mHtt correlates with increased mitochondrial Ca2+ uptake capacity. Mitochondrial Ca2+ accumulation was not impaired in striatal neurons from YAC128 mice. We also found that expression of the NCX1 isoform is increased with age in striatum from YAC128 mice compared to striatum from wild-type mice. Interestingly, mHtt and Htt bind to the NCX3 isoform but not to NCX1. NCX3 expression remains unchanged. To further investigate Ca2+ homeostasis modulation, we examined the role of collapsin response mediator protein 2 (CRMP2) in wild-type neurons. CRMP2 is viewed as an axon guidance protein, but has been found to be involved in Ca2+ signaling. We found that CRMP2 interacts with NMDA receptors (NMDAR) and disrupting this interaction decreases NMDAR activity. CRMP2 also interacts with and regulates NCX3, resulting in NCX3 internalization and decreased activity. Augmented mitochondrial Ca2+ uptake capacity and an increased expression of NCX1 in the presence of mHtt suggest a compensatory reaction in response to increased Ca2+ influx into the cell. The role of NCX warrants further investigation in HD. The novel interactions of CRMP2 with NMDAR and NCX3 provide additional insight into the complexity of Ca2+ homeostasis regulation in neurons and may also be important in HD neuropathology.

Cerebral ischaemia is a leading cause of disability and death world-wide. The only effective treatments are thrombolytic therapy (plasminogen activator; tPA) and hypothermia (33?C). However, tPA has limited clinical application due to its short therapeutic time window and its specific application in thrombo-embolic stroke. Moderate hypothermia (33?C) is only being used following cardiac arrest in comatose survivors. Hence more treatments are urgently required. The first step in developing new treatments is the identification and characterisation of a potential therapeutic target. Since brain damage following cerebral ischaemia is associated with disturbances in intracellular calcium homeostasis, the sodium-calcium exchanger (NCX) is a potential therapeutic target due to its ability to regulate intracellular calcium. Currently, however there is uncertainty as to whether the plasma membrane NCX has a neuroprotective or neurodamaging role following cerebral ischemia. To address this issue I compared hippocampal neuronal injury in NCX3 knockout mice (Ncx3-/-) and wild-type mice (Ncx3+/+) following global cerebral ischaemia. In order to perform this study I first established a bilateral common carotid occlusion (BCCAO) model of global ischaemia in wild-type C57/BlHsnD mice using controlled ventilation. After trials of several ischaemic time points, 17 minutes was established as the optimum duration of ischaemia to produce selective hippocampal CA1 neuronal loss in the wild-type mice. I then subjected NCX3 knockout and wild-type mice to 17 minutes of ischaemia. Following the 17 minute period of ischaemia, wild-type mice exhibited 80% CA1 neuronal loss and 40% CA2 neuronal loss. In contrast, NCX3 knockout mice displayed > 95% CA1 neuronal loss and 95% CA2 neuronal loss. Following experiments using a 17 minute duration of global ischaemia, a 15 minute duration of ischaemia was also evaluated. Wild-type mice exposed to a 15 minute period of ischaemia, did not exhibit any significant hippocampal neuronal loss. In contrast, NCX3 knockout mice displayed 45% CA1 neuronal loss and 25% CA2 neuronal loss. The results clearly demonstrate that mice deficient for the NCX3 protein are more susceptible to global cerebral ischaemia than wild-type mice. My findings showing a neuroprotective role for NCX3 following ischaemia, suggest that the exchanger has a positive role in maintaining neuronal intracellular calcium homeostasis. When this function is disrupted, neurons are more susceptible to calcium deregulation, with resultant cell death via calcium mediated pathways. Therefore, improving NCX activity following cerebral ischaemia may provide a therapeutic strategy to reduce neuronal death.