• Acute ROS in Cardiac Calcium Signaling

      Bamgboye, Moradeke A.; Lederer, W. Jonathan (2014)
      Modulation of cardiac function usually involves changes in cellular Ca2+ signaling. Reactive oxygen species (ROS) have been implicated in effecting changes in cardiac Ca2+ signaling. It has been unclear, however, exactly what these changes are and if they are ultimately detrimental or beneficial. The work reported here investigates the effect of acute application of H2O2 in low concentrations on Ca2+ signaling in the heart cell. The driving hypothesis is that ROS in short bursts is a non - detrimental physiological signal that fine tunes Ca2+ signaling. To this end the effect of ROS is investigated in 3 modules. i. the effect of rapid application of 100 μM H2O2 on Ca2+ signaling in heart cells, ii. the effect of H2O2 on Ca2+ signaling during β-adrenergic activation in heart cells and finally to examine endogenously generated ROS, iii. Ca2+ signaling in a murine model of NOX2 overexpression (NOX2 is a ROS generating enzyme). Using a combination of confocal imaging of fluorescent Ca2+ dyes and electrophysiological techniques such as whole cell voltage clamp and current clamp; Ca2+ sparks, Ca2+ transients, ICa, Sarcoplasmic Reticulum (SR) Ca2+ load and Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase (SERCA) function were measured in all three paradigms listed above. The work shows that low concentrations of H2O2 for a brief period deplete SR load without affecting other parameters of Ca2+ signaling, and this depletion of SR load is prevented by β-adrenergic activation. The results further our understanding of ROS modulation of Ca2+ signaling and lays some groundwork for further exploration into the pathways by which ROS may interact with other modifiers of the Ca2+ signaling machinery of the cardiac cell.
    • Cardiac Ca2+ Signals: From Local Elevations, A Matrix of Potential

      Wescott, Andrew; Lederer, W. Jonathan; 0000-0003-4620-4343 (2018)
      The heart has frequent and robust elevations of cytosolic calcium ([Ca2+]i) that underlie each contraction. Ca2+ release originates from thousands of Ca2+ release units (CRUs) distributed throughout the cardiomyocyte which can generate localized Ca2+ elevations, Ca2+ sparks, that are synchronized during excitation-contraction (EC) coupling to produce the global [Ca2+]i transient. Positioned in close proximity (~ 100 nm) to these CRUs are the intramyofibrillar mitochondria (IFMs) which are briefly (10 - 20 ms) exposed to microdomains of high [Ca2+]i (1 - 10 μM) during Ca2+ release. Elevated [Ca2+]i coupled with the highly polarized inner mitochondrial membrane (IMM) potential (ΔΨm ≈ -180 mV) creates a powerful electrochemical driving force for Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU) complex. Low "physiological" mitochondrial matrix Ca2+ ([Ca2+]m) (~0.1 - 10 μM) is thought to regulate metabolism via oxidative phosphorylation, while "pathophysiological" [Ca2+]m overload (> 10 μM) leads to necrotic cell death. To date, the biophysical details surrounding the magnitude and regulation of mitochondrial Ca2+ uptake remain poorly understood, with the functional significance of [Ca2+]m signals providing further controversy. Three independent studies are provided in this thesis that look to improve our quantitative understanding of "local control" of [Ca2+]i signaling, the regulation and magnitude of [Ca2+]m signals, and the mechanism by which [Ca2+]m contributes to dynamic mitochondrial adenosine triphosphate (ATP) synthesis. The first study tests the hypothesis that "stable and synchronous release of local [Ca2+]i signals relies on physiological Ca2+ sensitivity of the ryanodine receptor". The second study tests the hypothesis that "mitochondrial Ca2+ uptake is under thermodynamic control to yield the small alterations in [Ca2+]m during EC coupling." The final study tests the hypothesis that "[Ca2+]m regulates ATP production through altering the thermodynamic driving force for ATP synthesis". The novel quantitative results provided herein help to clarify and constrain our understanding of EC coupling and the role of Ca2+ in the mitochondrial matrix.
    • Depletion of endoplasmic reticulum calcium triggers the loss of ER resident proteins

      Trychta, Kathleen Anne; Harvey, Brandon K. (2019)
      The endoplasmic reticulum (ER) contains proteins that carry out the diverse functions of the ER including calcium storage, protein folding, modification, and trafficking, lipid metabolism, and drug detoxification. When soluble ER resident proteins with an ER retention sequence (ERS) depart from the ER they interact with KDEL receptors in the Golgi membrane and are retrogradely transported to the ER lumen via the KDEL receptor retrieval pathway. ER calcium depletion disrupts this process resulting in the mass departure of ERS-containing proteins into the extracellular space. Such a loss of ER resident proteins has two potential consequences to an affected cell. First, there is a loss of proteins involved in the aforementioned critical ER functions. Second, the relocation of such proteins and their associated functions outside of the cell may cause changes in the extracellular environment. This dissertation describes the identification and characterization of a phenomenon whereby ER resident protein secretion is triggered by pathophysiological ER calcium depletion. By exploiting the enzymatic activity of one of the identified ERS-containing proteins we developed an assay to monitor changes in the ER proteome. We also developed a high-throughput screen that identified drugs that could prevent the release of ER resident proteins following ER calcium depletion and showed that several of these compounds have therapeutic potential in models of ER stress and ischemia. Taken together, the work described in my dissertation identifies a novel molecular mechanism of cellular dysfunction for which I have identified both endogenous biomarkers and possible therapeutics.
    • Effects of Dietary Intake on Endoplasmic Reticulum Calcium Homeostasis

      Simons Wires, Emily Marie; Harvey, Brandon K.; 0000-0002-9032-4732 (2016)
      The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 1,000-10,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for longitudinal in vivo studies. Our lab has recently developed a novel secreted ER calcium monitoring protein (GLuc-SERCaMP), to longitudinally monitor ER calcium levels in vivo by measuring small volumes of blood. Additionally, we describe a complementary tool to measure the unfolded protein response utilizing a Nano luciferase (NLuc) reporter. This work highlights the application of both reporters in vivo. Furthermore, towards the overarching goal of monitoring ER calcium homeostasis in a disease model, we describe this use of GLuc-SERCaMP in in vitro and in vivo models of diet-induced obesity. Disruption to ER calcium homeostasis has been implicated in obesity, however, the ability to directly monitor fluctuations to ER calcium has been limited with previous techniques. GLuc-SERCaMP release revealed ER calcium depletion in the presence of free fatty acid (FFA), palmitate. Consumption of a cafeteria diet or high fat pellets further demonstrated alterations to hepatic ER calcium homeostasis in rats, as evidenced by increased GLuc-SERCaMP release. Attenuation of GLuc-SERCaMP was observed during dantrolene administration. Taken together, our results further corroborate the influence of dietary intake on ER calcium homeostasis.
    • Evaluation Of Calcium Homeostasis Within The Mdx Skeletal Muscle

      Michaelson, Luke Philip; Ward, Christopher, Ph.D. (2011)
      Background: Duchenne muscular dystrophy is a terminal X-linked muscle wasting disease occurring in 1 out of every 3,500 live male births. Currently, no cure exists. However, current data implicate a relationship between reactive oxygen species (ROS), Ca2+, and membrane permeability possibly affecting the muscle degeneration associated with DMD. Design: I assayed myoplasmic ROS during field-stimulated contractions using a green fluorescent protein targeted to the mitochondria. Its fluorescence changed depending on the oxidizing or reducing conditions. My findings suggested the mitochondria are not the primary source of intracellular ROS generation during field-stimulated contractions. Instead, NADPH oxidase may provide an important source for ROS generation during unloaded contraction. I then evaluated the sensitivity of Excitation Coupled Calcium Entry (ECCE) to ROS, using manganese (Mn2+) quench (a surrogate to Ca2+ influx), after treating the muscle fibers with oxidizing or reducing solutions. Results: Exogenous H2O2 treatment of myofibers did not alter either the basal or ECCE Ca2+ permeability in the WT fibers when compared to the non-treated controls. Oxidation with H2O2 did not significantly affect the basal Ca2+ permeability in the mdx fibers, but did greatly increase ECCE permeability by almost 8 fold greater than WT basal rate with 100 μM [H2O2]. In opposition to oxidation, scavenging ROS with n-acetylcysteine decreased Mn2+ permeability during ECCE in mdx muscle. The non-specific TRP channel inhibitor BTP2, reduced the Mn2+ permeability during basal conditions and Mn2+ permeability during ECCE in mdx muscle fibers by 87% and 96%, p <.05, respectively. In WT fibers, BTP2 reduced Mn2+ permeability ECCE by 67%, p<.05, but did not inhibit the basal permeability. Conclusions: We demonstrate that the ECCE Ca2+ entry pathway is exuberant in mdx muscle. Furthermore, we determine that ECCE is ROS sensitive. We conclude that ECCE contributes to the altered Ca2+ homeostasis in mdx skeletal muscle. The increased ROS generation in mdx skeletal muscle may directly or indirectly mediate TRPC activity to generate the Ca2+ dyshomeostasis associated with dystrophin deficiency
    • Identification of Small Ankyrin 1 as a novel SERCA1 regulatory protein in Skeletal Muscle

      Desmond, Patrick Francis; Bloch, Robert J.; 0000-0002-2006-6280 (2016)
      Small Ankyrin 1 (sAnk1) is a ~20 kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum (nSR) in skeletal muscle. Previous reports from out lab show that sAnk1 knock down results in loss of network SR integrity, along with a decrease in SR Ca2+ load and Ca2+ re-uptake rates. Upon closer examination of the sAnk1 transmembrane domain, we discovered that sAnk1 shares sequence similarity with sarcolipin (SLN), a small protein that inhibits activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). The goal of the current study was to determine if sAnk1 interacts with SERCA1 or SLN directly in skeletal muscle, and elucidate the consequences such interactions pose on SERCA1 activity. Our results indicate that sAnk1 interacts specifically with SERCA1 in SR vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method (AFRET). Binding was significantly reduced by the replacement of all the TM amino acids of sAnk1 to leucines by mutagenesis. This suggests that, like SLN, sAnk1 interacts with SERCA1 via its TM domain. Assays of ATPase activity show that co-expression of sAnk1 with SERCA1 leads to a reduction of SERCA1's apparent Ca2+ affinity, but that sAnk1's effect is less than that of SLN. Interestingly, the sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM domain to regulate SERCA1 activity and thereby modulate the sequestration of Ca2+ in the lumen of the ER and SR. Additionally, we determined that sAnk1 can also interact with SLN using the same analytical methods. Unexpectedly, ATPase assays in which all three proteins were co-expressed showed that sAnk1 was able to limit SLN's ability to inhibit SERCA1 activity. Furthermore, coIP and AFRET experiments demonstrate that SLN promotes the interaction between SERCA1 and sAnk1. The identification of sAnk1 as a novel regulator of SERCA1 activity has significant implications for the physiology of muscle and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca2+ misregulation.
    • The Role and Inhibition of S100B in Melanoma Cell Signaling

      Hartman, Kira Gianni; Weber, David J., Ph.D. (2012)
      The calcium–binding protein S100B is an effective and extensively used prognostic marker for melanoma, with increasing S100B being predictive of disease stage, increased recurrence, and low survival. Establishing the mechanism by which S100B alters cell signaling provides insight into how it may facilitate the progression of melanoma and aid in developing new pharmacological drugs to inhibit cancer advancement. To evaluate the significance of S100B in melanoma, knock–down and over–expression studies were conducted, finding a positive correlation between S100B expression and cell viability, as well as ERK phosphorylation. However, phosphorylation of RSK, a downstream ERK target, was determined to have an inverse relationship with S100B. Over–expression of a calcium–binding mutant S100B yields neither effect, indicating that each response is calcium–dependent. Pull–down experiments established the direct calcium–dependent binding of S100B to the C–terminus of RSK and kinase assays demonstrated that S100B prevents RSK phosphorylation at Thr573. Over–expression of S100B in melanoma cells reduces the phosphorylation of RSK, sequestering it in the cytosol. Conversely, cells with diminished S100B expression exhibited increased staining of phosphorylated RSK within the nucleus. Together these data are consistent with a mechanism in which elevated S100B binds RSK directly in a calcium–dependent manner, preventing ERK–mediated phosphorylation and subsequent nuclear translocation. Thus, S100B uniquely affects MAPK signaling by increasing levels of phosphorylated ERK while simultaneously preventing the phosphorylation of RSK. Capitalizing on this discovery, in addition to previously known S100B interactions such as with p53, we are searching for S100B inhibitors that will prevent cancer progression. To this end, in vitro FPCA was employed to rapidly screen 2,000 compounds, establishing whether they bind Ca<super>2+</super>–loaded S100B and inhibit S100B target complex formation. Building upon this, we developed a cell–based high throughput assay capable of screening an extensive library of 14,400 compounds, in addition to 26 putative S100B inhibitors identified through FPCA, by comparing their effects on cells expressing elevated S100B to cells where S100B has been significantly knocked–down. The desired endpoint of this research is the development of a drug with therapeutic activity for the treatment of malignant melanoma and/or other cancers with elevated S100B.
    • Structure, Function, and Inhibition of S100B

      Charpentier, Thomas H.; Weber, David J., Ph.D. (2009)
      Aberrant levels of the small dimeric protein S100B have been found in malignant melanoma, renal cell cancer, and astrocytomas. S100B may aid in cancer progression via its interaction with and down regulation of the tumor suppressor p53, in a Ca²⁺ and possibly Zn²⁺ dependent manner. S100B bound to Ca²⁺ undergoes a conformational change exposing a hydrophobic cleft for the p53-S100B interaction. S100B binds to the C-terminus and tetramerization domains (319-393) of p53. Experiments reducing S100B expression via siRNA restores p53 levels in primary malignant melanoma cells. Thus, several small molecules have been identified that bind S100B and inhibit the Ca²⁺-S100B-p53 interaction. One of these small molecules is pentamidine, an FDA approved drug. We have characterized the interaction between Ca²⁺-S100B and pentamidine via nuclear magnetic resonance (NMR) and X-ray crystallography. We obtained crystal structures of pentamidine bound to Zn²⁺-Ca²⁺-S100B. The previously solved NMR structure of Zn²⁺-Ca²⁺-S100B was compared to the X-ray crystal structure solved here. We characterized the Zn²⁺ ligands in each structure to determine if Zn²⁺ binding changed the pentamidine interaction with S100B. A goal of the Weber lab has been to identify small molecules to inhibit the p53-S100B interaction and we have been moderately successful. We have identified three additional small molecules found through screens performed by our lab and the high throughput screening core (UMAB). SBi132, SBi279, and SBi523 (S100B inhibitor ###) were shown to interact with S100B through NMR and X-ray crystallography. Other small molecules derived from pentamidine or SBi132 interact with the "hinge" region of S100B, while other screening molecules were found to covalently bind to cysteine 84 on helix 4 of S100B. To characterize the S100B-p53 protein-protein interaction further, TRTK-12 a peptide derived from the CapZ protein, was used to study the effects of peptide bound to S100B. Surprisingly, the Ca²⁺ coordination for both EF-hands of S100B were not affected by TRTK-12. The X-ray structure of TRTK-12 peptide bound to S100B did show differences in temperature factor. These differences in peptide binding can aid us in identifying inhibitors of the S100B-p53 complex and restore p53 levels in malignant melanoma.
    • The regulation and role of dendritic mitochondrial fission during long-term potentiation

      Divakaruni, Sai Sachin; Blanpied, Thomas A.; 0000-0003-3478-3229
      Neurons continuously modify their synaptic strength to encode memories and to adapt to experience and the environment. Long-term potentiation (LTP) is a critical cellular mechanism of this adaptation and is the prevailing form of synaptic plasticity. Baseline synaptic function is bioenergetically demanding, and this demand is elevated during episodes of synaptic plasticity. Therefore, mitochondrial functions, such as ATP synthesis and calcium handling, are likely essential for plasticity. Furthermore, mitochondrial functions themselves are regulated by mitochondrial dynamics including fission, fusion, and motility. Although axonal mitochondria have been extensively studied, LTP induction predominantly occurs postsynaptically, where the roles of mitochondria are less well understood. Additionally, mitochondrial fission has recently garnered interest because it is necessary for development and is required for normal mitochondrial function, and because perturbed fission is associated with many neurological and psychiatric diseases. However, whether or how fission in dendrites supports ongoing synaptic transmission and plasticity is still unclear. Furthermore, although the molecular mechanisms underlying mitochondrial fission have been well described in other cell types, little is known about how mitochondrial fission is accomplished in neurons, particularly in dendrites, or how neuronal activity might modulate these mechanisms. Here I tested the hypothesis that dendritic mitochondrial fission is triggered during LTP induction, and is necessary for LTP expression. Mitochondria in dendrites at rest are stationary and rarely undergo fission. However, I found that chemical induction of LTP (cLTP) by NMDAR activation in cultured rat hippocampal neurons prompted a rapid burst of dendritic mitochondrial fission. Mitochondrial fission canonically requires actin nucleation and membrane constriction by the GTPase dynamin-related protein 1 (Drp1). Consistent with this, inhibition of actin polymerization or expression of a dominant negative (DN) mutant or knockdown of Drp1 each suppressed the cLTP fission burst. Furthermore, the GTPase Dynamin 2 (Dyn2) was recently implicated in fission in cell lines, and I found similarly that expressing DN Dyn2 abolished the fission burst. Drp1 function is also known to be regulated by phosphorylation, with CaMKII as a possible activator based on studies of non-neuronal cells. In line with this, I found that fission was triggered by cytosolic calcium elevation via glutamate photolysis at dendritic spines, and also that the fission burst was prevented by acutely inhibiting CaMKII activation or by prohibiting Drp1 phosphorylation. I then tested whether mitochondrial fission is required for LTP expression. Knocking down Drp1 or expressing DN Drp1 suppressed dendritic spine growth and synaptic AMPA receptor trafficking following LTP induction. Furthermore, NMDAR-dependent LTP induction by high-frequency stimulation (HFS) of Schaffer collaterals in acute hippocampal slices decreased dendritic mitochondrial length in area CA1. Remarkably, postsynaptic expression of DN Drp1 prevented HFS LTP at Schaffer collateral-CA1 synapses in slices, with no effect on basal transmission or intrinsic electrophysiological properties of neurons. Furthermore, I found that cLTP stimulation produced transient elevations of dendritic mitochondrial calcium (i.e. mCaTs), and that expression of DN Drp1 suppressed the frequency, amplitude, and duration of evoked mCaTs. These data illustrate a novel pathway whereby synaptic activity controls mitochondrial fission, and show that dynamic control of fission is required for LTP induction perhaps by modulating mitochondrial calcium handling. Impaired synaptic function is implicated in myriad neuropsychiatric diseases, many of which are also associated with mitochondrial dysfunction. Therefore, our findings raise the important question of whether neuronal mitochondrial dysfunction contributes to cognitive impairment in these diseases by perturbing dendritic and/or synaptic plasticity.