Scholarly Works, School of Biomedical Engineering and Sciences

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Research articles, presentations, and other scholarship

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Connexin 43-Based Therapeutics for Dermal Wound Healing
Montgomery, Jade; Ghatnekar, Gautam S.; Grek, Christina L.; Moyer, Kurtis E.; Gourdie, Robert G. (MDPI, 2018-06-15)
The most ubiquitous gap junction protein within the body, connexin 43 (Cx43), is a target of interest for modulating the dermal wound healing response. Observational studies found associations between Cx43 at the wound edge and poor healing response, and subsequent studies utilizing local knockdown of Cx43 found improvements in wound closure rate and final scar appearance. Further preclinical work conducted using Cx43-based peptide therapeutics, including alpha connexin carboxyl terminus 1 (αCT1), a peptide mimetic of the Cx43 carboxyl terminus, reported similar improvements in wound healing and scar formation. Clinical trials and further study into the mode of action have since been conducted on αCT1, and Phase III testing for treatment of diabetic foot ulcers is currently underway. Therapeutics targeting connexin activity show promise in beneficially modulating the human body’s natural healing response for improved patient outcomes across a variety of injuries.
Diabetes Increases Cryoinjury Size with Associated Effects on Cx43 Gap Junction Function and Phosphorylation in the Mouse Heart.
Palatinus, Joseph A.; Gourdie, Robert G. (2016)
Diabetic patients develop larger myocardial infarctions and have an increased risk of death following a heart attack. The poor response to myocardial injury in the diabetic heart is likely related to the many metabolic derangements from diabetes that create a poor substrate in general for wound healing, response to injury and infection. Studies in rodents have implicated a role for the gap junction protein connexin 43 (Cx43) in regulating the injury response in diabetic skin wounds. In this study, we sought to determine whether diabetes alters Cx43 molecular interactions or intracellular communication in the cryoinjured STZ type I diabetic mouse heart. We found that epicardial cryoinjury size is increased in diabetic mice and this increase is prevented by preinjury insulin administration. Consistent with these findings, we found that intercellular coupling via gap junctions is decreased after insulin administration in diabetic and nondiabetic mice. This decrease in coupling is associated with a concomitant increase in phosphorylation of Cx43 at serine 368, a residue known to decrease channel conductance. Taken together, our results suggest that insulin regulates both gap junction-mediated intercellular communication and injury propagation in the mouse heart.
Distinguishing between overdrive excited and suppressed ventricular beats in guinea pig ventricular myocardium
Greer-Short, Amara D.; Poelzing, Steven (Frontiers, 2015-02-18)
Rapid ventricular pacing rates induces two types of beats following pacing cessation: recovery cycle length (RCL) prolongation (overdrive suppression) and RCL shortening (overdrive excitation). The goals of this study were to compare common experimental protocols for studying triggered activity in whole-heart preparations and differentiate between recovery beats using a new methodology. Post-pacing recovery beat cycle length (RCL) and QRS were normalized to pre-paced R-R and QRS intervals and analyzed using a K-means clustering algorithm. Control hearts only produced suppressed beats: RCL ratio increased with rapid pacing (25 +/- 4.0%, n = 10) without changing QRS duration. Rapid pacing during hypercalcemia + hypothermia (5.5 mM and 34 degrees C) produced significantly earlier excited beats (53 +/- 14%, n = 5) with wider QRS durations (58 +/- 6.3%, n = 5) than suppressed beats. Digoxin + hypothermia (0.75 mu M) produced the most excited beats with significantly earlier RCL (44 +/- 3.2%, n = 6) and wider QRS (60 +/- 3.1%, n = 6) ratios relative to suppressed beats. Increasing pacing further shortened RCL (30 +/- 7.8%, n = 6). In a prospective study, TTX (100 nM) increased RCL ratio (15 +/- 6.0%, n = 10) without changing the QRS duration of excited beats. The algorithm was compared to a cross-correlation analysis with 93% sensitivity and 94% specificity. This ECG based algorithm distinguishes between triggered and automatic activity.
In Cocaine Dependence, Neural Prediction Errors During Loss Avoidance Are Increased With Cocaine Deprivation and Predict Drug Use
Wang, John M.; Zhu, Lusha; Brown, Vanessa M.; De La Garza, Richard II; Newton, Thomas F.; Casas, Brooks; Chiu, Pearl H. (Elsevier, 2018-08-03)
Background: In substance-dependent individuals, drug deprivation and drug use trigger divergent behavioral responses to environmental cues. These divergent responses are consonant with data showing that short- and long-term adaptations in dopamine signaling are similarly sensitive to state of drug use. The literature suggests a drug state–dependent role of learning in maintaining substance use; evidence linking dopamine to both reinforcement learning and addiction provides a framework to test this possibility. Methods: In a randomized crossover design, 22 participants with current cocaine use disorder completed a probabilistic loss-learning task during functional magnetic resonance imaging while on and off cocaine (44 sessions). Another 54 participants without Axis I psychopathology served as a secondary reference group. Within-drug state and paired-subjects’ learning effects were assessed with computational model–derived individual learning parameters. Model-based neuroimaging analyses evaluated effects of drug use state on neural learning signals. Relationships among model-derived behavioral learning rates (α+, α−), neural prediction error signals (δ+, δ−), cocaine use, and desire to use were assessed. Results: During cocaine deprivation, cocaine-dependent individuals exhibited heightened positive learning rates (α+), heightened neural positive prediction error (δ+) responses, and heightened association of α+ with neural δ+ responses. The deprivation-enhanced neural learning signals were specific to successful loss avoidance, comparable to participants without psychiatric conditions, and mediated a relationship between chronicity of drug use and desire to use cocaine. Conclusions: Neurocomputational learning signals are sensitive to drug use status and suggest that heightened reinforcement by successful avoidance of negative outcomes may contribute to drug seeking during deprivation. More generally, attention to drug use state is important for delineating substrates of addiction. © 2018
Is sham cTBS real cTBS? The effect on EEG dynamics
Opitz, Alexander; Legon, Wynn; Mueller, Jerel K.; Barbour, Aaron J.; Paulus, Walter; Tyler, William J. (Frontiers, 2015-01-08)
Increasing sensitivity of modern evaluation tools allows for the study of weaker electric stimulation effects on neural populations. In the current study we examined the effects of sham continuous theta burst (cTBS) transcranial magnetic stimulation to the left dorsolateral prefrontal cortex (DLPFC) upon somatosensory evoked potentials (SEP) and frontal-parietal phase coupling of alpha and beta bands. Sham TMS results in an induced electric field amplitude roughly 5% that of real TMS with a similar spatial extent in cortex. Both real and sham cTBS reduced the amplitude of the frontal P14-N30 SEP and increased local phase coupling in the alpha-beta frequency bands of left frontal cortex. In addition, both sham and real cTBS increased frontal-parietal phase coupling in the alpha-beta bands concomitant with an increase in amplitude of parietal P50-N70 complex. These data suggest that weak electric fields from sham cTBS can affect both local and downstream neuronal circuits, though in a different manner than high strength TMS.
A microchip platform for structural oncology applications.
Winton, Carly E.; Gilmore, Brian L.; Demmert, Andrew C.; Karageorge, Vasilea; Sheng, Zhi; Kelly, Deborah F. (2016)
Recent advances in the development of functional materials offer new tools to dissect human health and disease mechanisms. The use of tunable surfaces is especially appealing as substrates can be tailored to fit applications involving specific cell types or tissues. Here we use tunable materials to facilitate the three-dimensional (3D) analysis of BRCA1 gene regulatory complexes derived from human cancer cells. We employed a recently developed microchip platform to isolate BRCA1 protein assemblies natively formed in breast cancer cells with and without BRCA1 mutations. The captured assemblies proved amenable to cryo-electron microscopy (EM) imaging and downstream computational analysis. Resulting 3D structures reveal the manner in which wild-type BRCA1 engages the RNA polymerase II (RNAP II) core complex that contained K63-linked ubiquitin moieties-a putative signal for DNA repair. Importantly, we also determined that molecular assemblies harboring the BRCA1(5382insC) mutation exhibited altered protein interactions and ubiquitination patterns compared to wild-type complexes. Overall, our analyses proved optimal for developing new structural oncology applications involving patient-derived cancer cells, while expanding our knowledge of BRCA1's role in gene regulatory events.
Molecular Analysis of BRCA1 in Human Breast Cancer Cells Under Oxidative Stress
Gilmore, Brian L.; Liang, Yanping; Winton, Carly E.; Patel, Kaya; Karageorge, Vasilea; Varano, A. Cameron; Dearnaley, William J.; Sheng, Zhi; Kelly, Deborah F. (Nature Publishing Group, 2017-03-06)
The precise manner in which physical changes to the breast cancer susceptibility protein (BRCA1) affect its role in DNA repair events remain unclear. Indeed, cancer cells harboring mutations in BRCA1 suffer from genomic instability and increased DNA lesions. Here, we used a combination of molecular imaging and biochemical tools to study the properties of the BRCA1 in human cancer cells. Our results reveal new information for the manner in which full-length BRCA1 engages its binding partner, the BRCA1-associated Ring Domain protein (BARD1) under oxidative stress conditions. We also show how physical differences between wild type and mutated BRCA15382insC impact the cell’s response to oxidative damage. Overall, we demonstrate how clinically relevant changes to BRCA1 affect its structure-function relationship in hereditary breast cancer.
A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of Human Disease
Gilmore, Brian L.; Winton, Carly E.; Demmert, Andrew C.; Tanner, Justin R.; Bowman, Sam; Karageorge, Vasilea; Patel, Kaya; Sheng, Zhi; Kelly, Deborah F. (Springer Nature, 2015-09-23)
We present a new molecular toolkit to investigate protein assemblies natively formed in the context of human disease. The system employs tunable microchips that can be decorated with switchable adaptor molecules to select for target proteins of interest and analyze them using molecular microscopy. Implementing our new streamlined microchip approach, we could directly visualize BRCA1 gene regulatory complexes from patient-derived cancer cells for the first time.
Neural computations underlying social risk sensitivity
Lauharatanahirun, Nina; Christopoulos, George I.; Casas, Brooks (Frontiers, 2012-08-02)
Under standard models of expected utility, preferences over stochastic events are assumed to be independent of the source of uncertainty. Thus, in decision-making, an agent should exhibit consistent preferences, regardless of whether the uncertainty derives from the unpredictability of a random process or the unpredictability of a social partner. However, when a social partner is the source of uncertainty, social preferences can influence decisions over and above pure risk attitudes (RA). Here, we compared risk-related hemodynamic activity and individual preferences for two sets of options that differ only in the social or non-social nature of the risk. Risk preferences in social and non-social contexts were systematically related to neural activity during decision and outcome phases of each choice. Individuals who were more risk averse in the social context exhibited decreased risk-related activity in the amygdala during non-social decisions, while individuals who were more risk averse in the non-social context exhibited the opposite pattern. Differential risk preferences were similarly associated with hemodynamic activity in ventral striatum at the outcome of these decisions. These findings suggest that social preferences, including aversion to betrayal or exploitation by social partners, may be associated with variability in the response of these subcortical regions to social risk.
A novel frequency analysis method for assessing K(ir)2.1 and Na (v)1.5 currents.
Rigby, J. R.; Poelzing, Steven (2012-04)
Voltage clamping is an important tool for measuring individual currents from an electrically active cell. However, it is difficult to isolate individual currents without pharmacological or voltage inhibition. Herein, we present a technique that involves inserting a noise function into a standard voltage step protocol, which allows one to characterize the unique frequency response of an ion channel at different step potentials. Specifically, we compute the fast Fourier transform for a family of current traces at different step potentials for the inward rectifying potassium channel, K(ir)2.1, and the channel encoding the cardiac fast sodium current, Na(v)1.5. Each individual frequency magnitude, as a function of voltage step, is correlated to the peak current produced by each channel. The correlation coefficient vs. frequency relationship reveals that these two channels are associated with some unique frequencies with high absolute correlation. The individual IV relationship can then be recreated using only the unique frequencies with magnitudes of high absolute correlation. Thus, this study demonstrates that ion channels may exhibit unique frequency responses.
Opponent Effects of Hyperarousal and Re-experiencing on Affective Habituation in Posttraumatic Stress Disorder
McCurry, Katherine L.; Frueh, B. Christopher; Chiu, Pearl H.; Casas, Brooks (2020-02)
BACKGROUND: Aberrant emotion processing is a hallmark of posttraumatic stress disorder (PTSD), with neurobiological models suggesting both heightened neural reactivity and diminished habituation to aversive stimuli. However, empirical work suggests that these response patterns may be specific to subsets of those with PTSD. This study investigates the unique contributions of PTSD symptom clusters (re-experiencing, avoidance and numbing, and hyperarousal) to neural reactivity and habituation to negative stimuli in combat-exposed veterans. METHODS: Ninety-five combat-exposed veterans (46 with PTSD) and 53 community volunteers underwent functional magnetic resonance imaging while viewing emotional images. This study examined the relationship between symptom cluster severity and hemodynamic responses to negative compared with neutral images (NEG>NEU). RESULTS: Veterans exhibited comparable mean and habituation-related responses for NEG>NEU, relative to civilians. However, among veterans, habituation, but not mean response, was differentially related to PTSD symptom severity. Hyperarousal symptoms were related to decreased habituation for NEG>NEU in a network of regions, including superior and inferior frontal gyri, ventromedial prefrontal cortex, superior and middle temporal gyri, and anterior insula. In contrast, re-experiencing symptoms were associated with increased habituation in a similar network. Furthermore, re-experiencing severity was positively related to amygdalar functional connectivity with the left inferior frontal gyrus and dorsal anterior cingulate cortex for NEG>NEU. CONCLUSIONS: These results indicate that hyperarousal symptoms in combat-related PTSD are associated with decreased neural habituation to aversive stimuli. These impairments are partially mitigated in the presence of re-experiencing symptoms, such that during exposure to negative stimuli, re-experiencing symptoms are positively associated with amygdalar connectivity to prefrontal regions implicated in affective suppression.
Sex Differences in beta-Adrenergic Responsiveness of Action Potentials and Intracellular Calcium Handling in Isolated Rabbit Hearts
Hoeker, Gregory S.; Hood, A. R.; Katra, R. P.; Poelzing, Steven; Pogwizd, S. M. (PLOS, 2014-10-23)
Cardioprotection in females, as observed in the setting of heart failure, has been attributed to sex differences in intracellular calcium handling and its modulation by b-adrenergic signaling. However, further studies examining sex differences in badrenergic responsiveness have yielded inconsistent results and have mostly been limited to studies of contractility, ion channel function, or calcium handling alone. Given the close interaction of the action potential (AP) and intracellular calcium transient (CaT) through the process of excitation-contraction coupling, the need for studies exploring the relationship between agonist-induced AP and calcium handling changes in female and male hearts is evident. Thus, the aim of this study was to use optical mapping to examine sex differences in ventricular APs and CaTs measured simultaneously from Langendorff-perfused hearts isolated from naı¨ve adult rabbits during b-adrenergic stimulation. The non-selective b-agonist isoproterenol (Iso) decreased AP duration (APD90), CaT duration (CaD80), and the decay constant of the CaT (t) in a dosedependent manner (1–316.2 nM), with a plateau at doses $31.6 nM. The Iso-induced changes in APD90 and t (but not CaD80) were significantly smaller in female than male hearts. These sex differences were more significant at faster (5.5 Hz) than resting rates (3 Hz). Treatment with Iso led to the development of spontaneous calcium release (SCR) with a dose threshold of 31.6 nM. While SCR occurrence was similar in female (49%) and male (53%) hearts, the associated ectopic beats had a lower frequency of occurrence (16% versus 40%) and higher threshold (100 nM versus 31.6 nM) in female than male hearts (p,0.05). In conclusion, female hearts had a decreased capacity to respond to b-adrenergic stimulation, particularly under conditions of increased demand (i.e. faster pacing rates and ‘‘maximal’’ levels of Iso effects), however this reduced badrenergic responsiveness of female hearts was associated with reduced arrhythmic activity.
Sodium channels in the Cx43 gap junction perinexus may constitute a cardiac ephapse: an experimental and modeling study
Veeraraghavan, Rengasayee; Lin, Joyce; Hoeker, Gregory S.; Keener, James P.; Gourdie, Robert G.; Poelzing, Steven (Springer, 2015-10-01)
It has long been held that electrical excitation spreads from cell-to-cell in the heart via low resistance gap junctions (GJ). However, it has also been proposed that myocytes could interact by non-GJ-mediated “ephaptic” mechanisms, facilitating propagation of action potentials in tandem with direct GJmediated coupling. We sought evidence that such mechanisms contribute to cardiac conduction. Using super-resolution microscopy, we demonstrate that Nav1.5 is localized within 200 nm of the GJ plaque (a region termed the perinexus). Electron microscopy revealed close apposition of adjacent cell membranes within perinexi suggesting that perinexal sodium channels could function as an ephapse, enabling ephaptic cell-to-cell transfer of electrical excitation. Acute interstitial edema (AIE) increased intermembrane distance at the perinexus andwas associated with preferential transverse conduction slowing and increased spontaneous arrhythmia incidence. Inhibiting sodium channels with 0.5 μM flecainide uniformly slowed conduction, but sodium channel inhibition during AIE slowed conduction anisotropically and increased arrhythmia incidence more than AIE alone. Sodium channel inhibition during GJ uncoupling with 25 μM carbenoxolone slowed conduction anisotropically and was also highly proarrhythmic. A computational model of discretized extracellular microdomains (including ephaptic coupling) revealed that conduction trends associated with altered perinexal width, sodium channel conductance, and GJ coupling can be predicted when sodium channel density in the intercalated disk is relatively high. We provide evidence that cardiac conduction depends on a mathematically predicted ephaptic mode of coupling as well as GJ coupling. These data suggest opportunities for novel anti-arrhythmic therapies targeting noncanonical conduction pathways in the heart.
Stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization
Veeraraghavan, Rengasayee; Gourdie, Robert G. (American Society for Cell Biology, 2016-11-07)
The spatial association between proteins is crucial to understanding how they function in biological systems. Colocalization analysis of fluorescence microscopy images is widely used to assess this. However, colocalization analysis performed on two-dimensional images with diffraction-limited resolution merely indicates that the proteins are within 200–300 nm of each other in the xy-plane and within 500–700 nm of each other along the z-axis. Here we demonstrate a novel three-dimensional quantitative analysis applicable to single-molecule positional data: stochastic optical reconstruction microscopy–based relative localization analysis (STORM-RLA). This method offers significant advantages: 1) STORM imaging affords 20-nm resolution in the xy-plane and <50 nm along the z-axis; 2) STORM-RLA provides a quantitative assessment of the frequency and degree of overlap between clusters of colabeled proteins; and 3) STORM-RLA also calculates the precise distances between both overlapping and nonoverlapping clusters in three dimensions. Thus STORM-RLA represents a significant advance in the high-throughput quantitative assessment of the spatial organization of proteins.
Structural analysis of BRCA1 reveals modification hotspot
Liang, Yanping; Dearnaley, William J.; Varano, A. Cameron; Winton, Carly E.; Gilmore, Brian L.; Alden, Nick A.; Sheng, Zhi; Kelly, Deborah F. (American Association for the Advancement of Science, 2017-09-01)
Temporal response of ectopic activity in guinea pig ventricular myocardium in response to isoproterenol and acetylcholine
Greer-Short, Amara D.; Poelzing, Steven (Frontiers, 2015-10-20)
Both beta adrenergic and musoarinio receptor stimulation independently potentiate arrhy-thmogenesis. However, the effect of simultaneous stimulation on arrhy-thmogenesis is not well known. The purpose of this study was to determine the temporal response of arrhythmia risk to individual and combined autonomic agonists. Guinea pig hearts were excised and Langendorff-perfused. The beta adrenergic receptor and musoarinio receptor agonists were isoproterenol (ISO, 0.6 mu M) and acetylcholine (ACh, 10 mu M), respectively. All measurements with agonists occurred over 21 min. ISO induced ectopic activity for the first 8 min. ISO also transiently shortened and then prolonged R-R interval over a similar time course. ACh added after ISO transiently induced ectopic activity for 12 min, while R-R interval invariantly prolonged. ACh alone produced few ectopic beats, while invariantly prolonging R-R interval. In contrast to ISO alone, ISO following ACh significantly increased ectopic activity and shortened R-R interval for the duration of the experiment. Animals aged 17-19 months exhibited sustained arrhythmogenesis while those aged 11-14 did not. When ACh was removed in older hearts while ISO perfused, a transient increase in ectopic activity and decreased R-R interval was observed, similar to ISO alone. These data suggest that pre-treating with and maintaining ACh perfusion can sustain ISO sensitivity, in contrast to ISO perfusion alone.
TNF alpha Modulates Cardiac Conduction by Altering Electrical Coupling between Myocytes
George, Sharon A.; Calhoun, Patrick J.; Gourdie, Robert G.; Smyth, James W.; Poelzing, Steven (Frontiers, 2017-05-23)
Background: Tumor Necrosis Factor alpha (TNF alpha) upregulation during acute inflammatory response has been associated with numerous cardiac effects including modulating Connexin43 and vascular permeability. This may in turn alter cardiac gap junctional (GJ) coupling and extracellular volume (ephaptic coupling) respectively. We hypothesized that acute exposure to pathophysiological TNF alpha levels can modulate conduction velocity (CV) in the heart by altering electrical coupling: GJ and ephaptic. Methods and Results: Hearts were optically mapped to determine CV from control, TNF alpha and TNF alpha + high calcium(2.5 vs. 1.25 mM) treated guinea pig hearts over 90 mins. Transmission electron microscopy was performed to measure changes in intercellular separation in the gap junction-adjacent extracellular nanodomain-perinexus (W-P). Cx43 expression and phosphorylation were determined by Western blotting and Cx43 distribution by confocal immunofluorescence. At 90 mins, longitudinal and transverse CV (CVL and CVT, respectively) increased with control Tyrode perfusion but TNF alpha slowed CVT alone relative to control and anisotropy of conduction increased, but not significantly. TNF alpha increased W-P relative to control at 90 mins, without significantly changing GJ coupling. Increasing extracellular calcium after 30 mins of just TNF alpha exposure increased CVT within 15 mins. TNF alpha + high calcium also restored CVT at 90 mins and reduced W-P to control values. Interestingly, TNF alpha + high calcium also improved GJ coupling at 90 mins, which along with reduced W-P may have contributed to increasing CV. Conclusions: Elevating extracellular calcium during acute TNF alpha exposure reduces perinexal expansion, increases ephaptic, and GJ coupling, improves CV and may be a novel method for preventing inflammation induced CV slowing.

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