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1.  Age and sex differences in tibia morphology in healthy adult Caucasians 
Bone  2012;50(6):1324-1331.
Variability in peripheral Quantitative Computed Tomography (pQCT) measurement sites limits direct comparisons of results between studies. Further, it is unclear what estimates of bone strength are most indicative of changes due to aging, disease, or interventions. The purpose of this study was to examine age group and sex differences in tibia morphology. Additional purposes of this study were to determine which tibia site or sites are most sensitive for detecting age and sex differences.
Methods
Self-identifying Caucasian men (n=55) and women (n=59) ages 20-59 years and separated by decades had their non-dominant tibias measured with pQCT (Stratec XCT 3000) at every 10% of the limb length from 5%-85% (distal to proximal). Volumetric BMD and BMC of the total, cortical and trabecular bone were determined, as well as periosteal (PeriC) and endosteal (EndoC) circumferences, and cortical thickness (CTh).
Results
There were significant (p<0.01) site effects for all BMC, vBMD, PeriC and EndoC measures. Large sex differences (men>women) in Tot.BMC (21-28%) were paralleled by differences in Cort.BMC (21-25%) (p<0.01). Site*sex interaction effects were significant (p<0.05) for BMC (peak sex difference: 5%, 15%, 25%, 85% sites) and circumference (peak sex difference: 65% site) variables. CTh and total vBMD were lowest (p<0.05) in 50-59 yr group, and EndoC was highest in the 50-59 yr group. Site*age interactions existed for Cort.vBMD, Tot.BMC (85% site), and EndoC (25%, 35%, 55%-85% sites). Correcting for bone free lean body mass (BFLBM) greatly reduced sex differences, eliminating sex*site interaction effects, but sex main effects remained significant. Correcting for BFLBM did not eliminate age effects.
Conclusion
The magnitude of age and sex differences in tibia variables varied by measurement site demonstrating the need for standardization of measurement sites.
doi:10.1016/j.bone.2012.03.005
PMCID: PMC4082662  PMID: 22449446
pQCT; morphology; lean mass; vBMD
3.  Sarcolemmal ATP-sensitive potassium channels modulate skeletal muscle function under low-intensity workloads 
The Journal of General Physiology  2014;143(1):119-134.
ATP-sensitive potassium (KATP) channels have the unique ability to adjust membrane excitability and functions in accordance with the metabolic status of the cell. Skeletal muscles are primary sites of activity-related energy consumption and have KATP channels expressed in very high density. Previously, we demonstrated that transgenic mice with skeletal muscle–specific disruption of KATP channel function consume more energy than wild-type littermates. However, how KATP channel activation modulates skeletal muscle resting and action potentials under physiological conditions, particularly low-intensity workloads, and how this can be translated to muscle energy expenditure are yet to be determined. Here, we developed a technique that allows evaluation of skeletal muscle excitability in situ, with minimal disruption of the physiological environment. Isometric twitching of the tibialis anterior muscle at 1 Hz was used as a model of low-intensity physical activity in mice with normal and genetically disrupted KATP channel function. This workload was sufficient to induce KATP channel opening, resulting in membrane hyperpolarization as well as reduction in action potential overshoot and duration. Loss of KATP channel function resulted in increased calcium release and aggravated activity-induced heat production. Thus, this study identifies low-intensity workload as a trigger for opening skeletal muscle KATP channels and establishes that this coupling is important for regulation of myocyte function and thermogenesis. These mechanisms may provide a foundation for novel strategies to combat metabolic derangements when energy conservation or dissipation is required.
doi:10.1085/jgp.201311063
PMCID: PMC3874572  PMID: 24344248
5.  Mitochondrial Calcium Uniporter Activity Is Dispensable for MDA-MB-231 Breast Carcinoma Cell Survival 
PLoS ONE  2014;9(5):e96866.
Calcium uptake through the mitochondrial Ca2+ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca2+ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca2+ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca2+ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca2+ uptake mechanisms.
doi:10.1371/journal.pone.0096866
PMCID: PMC4011874  PMID: 24802861
6.  Pathogenic CD4+ T cells recognizing an unstable peptide of insulin are directly recruited into islets bypassing local lymph nodes 
The Journal of Experimental Medicine  2013;210(11):2403-2414.
A diabetogenic, insulin-reactive TCR transgenic mouse shows that CD4+ T cells that escape negative selection are directly recruited to islets, bypassing initial priming in pancreatic LNs, and also induce antiinsulin antibodies.
In the nonobese diabetic mouse, a predominant component of the autoreactive CD4+ T cell repertoire is directed against the B:9-23 segment of the insulin B chain. Previous studies established that the majority of insulin-reactive T cells specifically recognize a weak peptide-MHC binding register within the B:9-23 segment, that to the 12–20 register. These T cells are uniquely stimulated when the B:9-23 peptide, but not the insulin protein, is offered to antigen presenting cells (APCs). Here, we report on a T cell receptor (TCR) transgenic mouse (8F10) that offers important new insights into the biology of these unconventional T cells. Many of the 8F10 CD4+ T cells escaped negative selection and were highly pathogenic. The T cells were directly recruited into islets of Langerhans, where they established contact with resident intra-islet APCs. Immunogenic insulin had to be presented in order for the T cells to localize and cause disease. These T cells bypassed an initial priming stage in the pancreatic lymph node thought to precede islet T cell entry. 8F10 T cells induced the production of antiinsulin antibodies and islets contained immunoglobulin (IgG) deposited on β cells and along the vessel walls.
doi:10.1084/jem.20130582
PMCID: PMC3804950  PMID: 24127484
7.  BPIFB1 IS A LUNG-SPECIFIC AUTOANTIGEN ASSOCIATED WITH INTERSTITIAL LUNG DISEASE** 
Science translational medicine  2013;5(206):10.1126/scitranslmed.3006998.
Interstitial lung disease (ILD) is a complex and heterogeneous disorder that is often associated with autoimmune syndromes (1). Despite the connection between ILD and autoimmunity, it remains unclear whether ILD can develop from an autoimmune response that specifically targets the lung parenchyma. Here, we utilized a severe form of autoimmune disease, Autoimmune Polyglandular Syndrome Type 1 (APS1), to establish a strong link between an autoimmune response to the lung-specific protein BPIFB1 and clinical ILD. Screening of a large cohort of APS1 patients revealed autoantibodies to BPIFB1 in 9.6% of APS1 subjects overall and in 100% of APS1 subjects with ILD. Further investigation of ILD outside the APS1 disorder revealed BPIFB1 autoantibodies specifically present in 14.6% of patients with connective tissue disease-associated ILD and in 12.0% of patients with idiopathic ILD. Utilizing the animal model for APS1 to examine the mechanism of ILD pathogenesis, we found that Aire−/− mice harbor autoantibodies to a similar lung antigen named BPIFB9 that are a marker for ILD, and determined that a defect in thymic tolerance is responsible for the production of BPIFB9 autoantibodies and the development of ILD. Importantly, we also found that immunoreactivity targeting BPIFB1 independent of a defect in Aire also leads to ILD, consistent with our discovery of BPIFB1 autoantibodies in non-APS1 patients. Overall, our results demonstrate that autoimmunity targeting the lung-specific antigen BPIFB1 may be important to the pathogenesis of ILD in patients with APS1 and in subsets of patients with non-APS1 ILD, demonstrating the role of lung-specific autoimmunity in the genesis of ILD.
doi:10.1126/scitranslmed.3006998
PMCID: PMC3882146  PMID: 24107778
8.  Inhibition of CaMKII Phosphorylation of RyR2 Prevents Inducible Ventricular Arrhythmias in Mice with Duchenne Muscular Dystrophy 
Background
Ventricular tachycardia (VT) is the second most common cause of death in patients with Duchenne muscular dystrophy (DMD). Recent studies have implicated enhanced sarcoplasmic reticulum (SR) Ca2+ leak via ryanodine receptors (RyR2) as a cause of VT in the mdx mouse model of DMD. However, the signaling mechanisms underlying induction of SR Ca2+ leak and VT are poorly understood.
Objective
To test whether enhanced CaMKII phosphorylation of RyR2 underlies SR Ca2+ leak and induction of VT in mdx mice.
Methods
Programmed electrical stimulation (PES) was performed on anesthetized mice, and confocal imaging of calcium release events in isolated ventricular myocytes.
Results
PES revealed inducible VT in mdx mice, which was inhibited by CaMKII inhibition or mutation S2814A in RyR2. Myocytes from mdx mice exhibited more Ca2+ sparks and Ca2+ waves compared with wild type (WT) mice, in particular at faster pacing rates. Arrhythmogenic Ca2+ waves were inhibited by CaMKII but not PKA inhibition. Moreover, mutation S2814A but not S2808A in RyR2 suppressed spontaneous Ca2+ waves in myocytes from mdx mice.
Conclusion
CaMKII blockade and genetic inhibition of RyR2-S2814 phosphorylation prevent VT induction in a mouse model of DMD. In ventricular myocytes from mdx mice, spontaneous Ca2+ sparks and Ca2+ waves can be suppressed by CaMKII inhibition or mutation S2814A in RyR2. Thus, inhibition of CaMKII-induced SR Ca2+ leak might be a new strategy to prevent arrhythmias in patients with DMD without heart failure.
doi:10.1016/j.hrthm.2012.12.016
PMCID: PMC3605194  PMID: 23246599
Cardiac arrhythmias; Ca2+/calmodulin kinase II; mouse model; ryanodine receptor; Duchenne muscular dystrophy; ventricular tachycardia
9.  CaMKII in sinoatrial node physiology and dysfunction 
The calcium and calmodulin-dependent protein kinase II (CaMKII) is present in sinoatrial node (SAN) pacemaker cells and is required for physiological “fight or flight” SAN beating rate responses. Inhibition of CaMKII in SAN does not affect baseline heart rate, but reduces heart rate increases in response to physiological stress. CaMKII senses intracellular calcium (Ca2+) changes, oxidation status, and hyperglycemia to phosphorylate substrates that regulate Ca2+-sensitive proteins, such as L-type Ca2+ channels, phospholamban, and cardiac ryanodine receptors (RyR2). All of these substrates are involved in the SAN pacemaking mechanism. Excessive CaMKII activity, as occurs under pathological conditions such as heart failure, ischemia, and diabetes, can promote intracellular Ca2+ overload and reactive oxygen species production. Oxidation of CaMKII (ox-CaMKII) locks CaMKII into a constitutively active configuration that contributes to SAN cell apoptosis and fibrosis. This ox-CaMKII-mediated loss of functional SAN cells contributes to SAN dysfunction (SND) and sudden death. Thus, CaMKII has emerged as a central regulator of physiological SAN responses and a key determinant of SND.
doi:10.3389/fphar.2014.00048
PMCID: PMC3957193  PMID: 24672485
calcium/calmodulin-dependent protein kinase II; sinoatrial node; heart rate; sinoatrial node dysfunction; calcium
10.  G Protein Inactivator RGS6 Mediates Myocardial Cell Apoptosis and Cardiomyopathy Caused by Doxorubicin 
Cancer research  2013;73(6):1662-1667.
Clinical use of the widely used chemotherapeutic agent doxorubicin is limited by life-threatening cardiotoxicity. The mechanisms underlying Dox-induced cardiomyopathy and heart failure remain unclear, but are thought to involve p53-mediated myocardial cell apoptosis. The tripartite G protein inactivating protein RGS6 has been implicated in reactive oxygen species (ROS) generation, ATM/p53 activation and apoptosis in Dox-treated cells. Thus, we hypothesized that RGS6, the expression of which is enriched in cardiac tissue, might also be responsible for the pathological effects of Dox treatment in heart. In this study, we show that RGS6 expression is induced strongly by Dox in the ventricles of mice and isolated ventricular myocytes (VCM) via a post-transcriptional mechanism. While Dox-treated wild type (WT) mice manifested severe left ventricular dysfunction, loss of heart and body mass, along with decreased survival five days after Dox administration, mice lacking RGS6 were completely protected against these pathogenic responses. Activation of ATM/p53-apoptosis signaling by Dox in ventricles of WT mice was also absent in their RGS6−/− counterparts. Dox-induced ROS generation was dramatically impaired in both the ventricles and VCM isolated from RGS6−/− mice, and the apoptotic response to Dox in VCM required RGS6-dependent ROS production. These results identify RGS6 as an essential mediator of the pathogenic responses to Dox in heart, and they argue that RGS6 inhibition offers a rational means to circumvent Dox cardiotoxicity in human cancer patients.
doi:10.1158/0008-5472.CAN-12-3453
PMCID: PMC3602152  PMID: 23338613
RGS6; doxorubicin cardiotoxicity; apoptosis; p53; reactive oxygen species
11.  Seminal Plasma Initiates a Neisseria gonorrhoeae Transmission State 
mBio  2014;5(2):e01004-13.
ABSTRACT
Niche-restricted pathogens are evolutionarily linked with the specific biological fluids that are encountered during infection. Neisseria gonorrhoeae causes the genital infection gonorrhea and is exposed to seminal fluid during sexual transmission. Treatment of N. gonorrhoeae with seminal plasma or purified semen proteins lactoferrin, serum albumin, and prostate-specific antigen each facilitated type IV pilus-mediated twitching motility of the bacterium. Motility in the presence of seminal plasma was characterized by high velocity and low directional persistence. In addition, infection of epithelial cells with N. gonorrhoeae in the presence of seminal plasma resulted in enhanced microcolony formation. Close association of multiple pili in the form of bundles was also disrupted after seminal plasma treatment leading to an increase in the number of single pilus filaments on the bacterial surface. Thus, exposure of N. gonorrhoeae to seminal plasma is proposed to alter bacterial motility and aggregation characteristics to influence the processes of transmission and colonization.
IMPORTANCE
There are greater than 100 million estimated new cases of gonorrhea annually worldwide. Research characterizing the mechanisms of pathogenesis and transmission of Neisseria gonorrhoeae is important for developing new prevention strategies, since antibiotic resistance of the organism is becoming increasingly prevalent. Our work identifies seminal plasma as a mediator of N. gonorrhoeae twitching motility and microcolony formation through functional modification of the type IV pilus. These findings provide insight into motility dynamics and epithelial cell colonization under conditions that are relevant to sexual transmission. Type IV pili are common virulence factors with diverse functions among bacterial pathogens, and this work identifies interactions between type IV pili and the host environment. Finally, this work illustrates the importance of the host environment and niche-specific fluids on microbial pathogenesis.
doi:10.1128/mBio.01004-13
PMCID: PMC3958800  PMID: 24595372
12.  CaMKII effects on inotropic but not lusitropic force frequency responses require phospholamban 
Increasing heart rate enhances cardiac contractility (force frequency relationship, FFR) and accelerates cardiac relaxation (frequency-dependent acceleration of relaxation, FDAR). The positive FFR together with FDAR promotes rapid filling and ejection of blood from the left ventricle (LV) at higher heart rates. Recent studies indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is involved in regulating FFR and FDAR. We used isolated perfused mouse hearts to study the mechanisms of FFR and FDAR in different genetic models, including transgenic myocardial CaMKII inhibition (AC3-I) and phosphalamban knockout (PLN−/−). When the rate was increased from 360 beats/min to 630 beats/min in wild type mouse hearts, the LV developed pressure (LVDP) and the maximum rate of increase in pressure (dP/dt max) increased by 37.6 ± 4.7% and 77.0 ± 8.1%, respectively. However, hearts from AC3-I littermates showed no increase of LVDP and a relatively modest (20.4 ± 3.9 %) increase in dP/dt max. PLN−/− hearts had a negative FFR, and myocardial AC3-I expression did not change the FFR in PLN−/− mice. PLN−/− mouse hearts did not exhibit FDAR, while PLN−/−mice with myocardial AC3-I expression showed further frequency dependent reductions in cardiac relaxation, suggesting CaMKII targets in addition to PLN were critical to myocardial relaxation. We incubated a constitutively active form of CaMKII with chemically-skinned myocardium and found that several myofilament proteins were phosphorylated by CaMKII. However, CaMKII did not affect myofilament calcium sensitivity. Our study shows that CaMKII plays an important role in modulating FFR and FDAR in murine hearts and suggest that PLN is a critical target for CaMKII effects on FFR, while CaMKII effects on FDAR partially require PLN-alternative targets.
doi:10.1016/j.yjmcc.2012.06.019
PMCID: PMC3936404  PMID: 22796260
CaM kinase II; force-frequency relation; frequency-dependent acceleration of relaxation; phospholamban
13.  Glial Scar Borders Are Formed by Newly Proliferated, Elongated Astrocytes That Interact to Corral Inflammatory and Fibrotic Cells via STAT3-Dependent Mechanisms after Spinal Cord Injury 
The Journal of Neuroscience  2013;33(31):12870-12886.
Astroglial scars surround damaged tissue after trauma, stroke, infection, or autoimmune inflammation in the CNS. They are essential for wound repair, but also interfere with axonal regrowth. A better understanding of the cellular mechanisms, regulation, and functions of astroglial scar formation is fundamental to developing safe interventions for many CNS disorders. We used wild-type and transgenic mice to quantify and dissect these parameters. Adjacent to crush spinal cord injury (SCI), reactive astrocytes exhibited heterogeneous phenotypes as regards proliferation, morphology, and chemistry, which all varied with distance from lesions. Mature scar borders at 14 d after SCI consisted primarily of newly proliferated astroglia with elongated cell processes that surrounded large and small clusters of inflammatory, fibrotic, and other cells. During scar formation from 5 to 14 d after SCI, cell processes deriving from different astroglia associated into overlapping bundles that quantifiably reoriented and organized into dense mesh-like arrangements. Selective deletion of STAT3 from astroglia quantifiably disrupted the organization of elongated astroglia into scar borders, and caused a failure of astroglia to surround inflammatory cells, resulting in increased spread of these cells and neuronal loss. In cocultures, wild-type astroglia spontaneously corralled inflammatory or fibromeningeal cells into segregated clusters, whereas STAT3-deficient astroglia failed to do so. These findings demonstrate heterogeneity of reactive astroglia and show that scar borders are formed by newly proliferated, elongated astroglia, which organize via STAT3-dependent mechanisms to corral inflammatory and fibrotic cells into discrete areas separated from adjacent tissue that contains viable neurons.
doi:10.1523/JNEUROSCI.2121-13.2013
PMCID: PMC3728693  PMID: 23904622
14.  Applying Circuit Theory for Corridor Expansion and Management at Regional Scales: Tiling, Pinch Points, and Omnidirectional Connectivity 
PLoS ONE  2014;9(1):e84135.
Connectivity models are useful tools that improve the ability of researchers and managers to plan land use for conservation and preservation. Most connectivity models function in a point-to-point or patch-to-patch fashion, limiting their use for assessing connectivity over very large areas. In large or highly fragmented systems, there may be so many habitat patches of interest that assessing connectivity among all possible combinations is prohibitive. To overcome these conceptual and practical limitations, we hypothesized that minor adaptation of the Circuitscape model can allow the creation of omnidirectional connectivity maps illustrating flow paths and variations in the ease of travel across a large study area. We tested this hypothesis in a 24,300 km2 study area centered on the Montérégie region near Montréal, Québec. We executed the circuit model in overlapping tiles covering the study region. Current was passed across the surface of each tile in orthogonal directions, and then the tiles were reassembled to create directional and omnidirectional maps of connectivity. The resulting mosaics provide a continuous view of connectivity in the entire study area at the full original resolution. We quantified differences between mosaics created using different tile and buffer sizes and developed a measure of the prominence of seams in mosaics formed with this approach. The mosaics clearly show variations in current flow driven by subtle aspects of landscape composition and configuration. Shown prominently in mosaics are pinch points, narrow corridors where organisms appear to be required to traverse when moving through the landscape. Using modest computational resources, these continuous, fine-scale maps of nearly unlimited size allow the identification of movement paths and barriers that affect connectivity. This effort develops a powerful new application of circuit models by pinpointing areas of importance for conservation, broadening the potential for addressing intriguing questions about resource use, animal distribution, and movement.
doi:10.1371/journal.pone.0084135
PMCID: PMC3907452  PMID: 24497918
15.  Genetic Inhibition of Na+-Ca2+ Exchanger Current Disables Fight or Flight Sinoatrial Node Activity Without Affecting Resting Heart Rate 
Circulation research  2012;112(2):309-317.
Rationale
The sodium-calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial nodal (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown.
Objective
Determine the role of Ncx1 in heart rate.
Methods and Results
We employed global myocardial and SAN-targeted conditional Ncx1 knockout (Ncx1−/−) mice to measure the effect of the NCX current (INCX) in pacemaking activity in vivo, ex vivo and in isolated SAN cells. We induced conditional Ncx1−/− using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1−/− (retaining ~20% of control level INCX) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, heart rate and SAN cell automaticity increases in response to isoproterenol or the dihydropyridine Ca2+ channel agonist BayK8644 were significantly blunted or eliminated in Ncx1−/− mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast the ‘pacemaker’ current (If) and L-type Ca2+ currents were equivalent in control and Ncx1−/− SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, an If antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1−/− mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1−/− mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of INCX in fight or flight rate increases is enhanced after If inhibition.
Conclusion
Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo and in isolated SAN cells but not for maintaining resting heart rate.
doi:10.1161/CIRCRESAHA.111.300193
PMCID: PMC3562595  PMID: 23192947
Na+-Ca2+ exchange; sinoatrial node; L-type Ca2+ channels; pacemaker current; ion channel
16.  Aldosterone and cardiovascular disease: the heart of the matter 
Aldosterone contributes to the endocrine basis of heart failure and studies on cardiac aldosterone signaling have reinforced its value as a therapeutic target. Recent focus has shifted to new roles of aldosterone that appear to depend on co-existing pathologic stimuli, cell type, and disease etiology. This review evaluates recent advances in mechanisms underlying aldosterone-induced cardiac disease and highlights the interplay between aldosterone and Ca2+ and calmodulin dependent protein kinase II, whose hyperactivity during heart failure contributes to disease progression. Increasing evidence implicates aldosterone in diastolic dysfunction, and there is need to develop more targeted therapeutics such as aldosterone synthase inhibitors and molecularly specific anti-oxidants. Despite accumulating knowledge, many questions still persist and will likely dictate areas of future research.
doi:10.1016/j.tem.2012.09.004
PMCID: PMC3532553  PMID: 23040074
aldosterone; CaMKII; heart failure; signaling
17.  Generation of Functional Thymic Epithelium from Human Embryonic Stem Cells that Supports Host T Cell Development 
Cell stem cell  2013;13(2):10.1016/j.stem.2013.04.004.
SUMMARY
Inducing immune tolerance to prevent rejection is a key step toward successful engraftment of stem-cell-derived tissue in a clinical setting. Using human pluripotent stem cells to generate thymic epithelial cells (TECs) capable of supporting T cell development represents a promising approach to reach this goal; however, progress toward generating functional TECs has been limited. Here, we describe a robust in vitro method to direct differentiation of human embryonic stem cells (hESCs) into thymic epithelial progenitors (TEPs) by precise regulation of TGFβ, BMP4, RA, Wnt, Shh, and FGF signaling. The hESC-derived TEPs further mature into functional TECs that support T cell development upon transplantation into thymus-deficient mice. Importantly, the engrafted TEPs produce T cells capable of in vitro proliferation as well as in vivo immune responses. Thus, hESC-derived TEP grafts may have broad applications for enhancing engraftment in cell-based therapies as well as restoring age-and stress-related thymic decline.
doi:10.1016/j.stem.2013.04.004
PMCID: PMC3869399  PMID: 23684540
18.  MARCH1-mediated MHCII ubiquitination promotes dendritic cell selection of natural regulatory T cells 
The Journal of Experimental Medicine  2013;210(6):1069-1077.
Ubiquitination of MHCII molecules on dendritic cells is essential for the development of natural regulatory T cells
Membrane-associated RING-CH1 (MARCH1) is an E3 ubiquitin ligase that mediates ubiquitination of MHCII in dendritic cells (DCs). MARCH1-mediated MHCII ubiquitination in DCs is known to regulate MHCII surface expression, thereby controlling DC-mediated T cell activation in vitro. However, its role at steady state or in vivo is not clearly understood. Here, we show that MARCH1 deficiency resulted in a substantial reduction in the number of thymus-derived regulatory T cells (T reg cells) in mice. A specific ablation of MHCII ubiquitination also significantly reduced the number of thymic T reg cells. Indeed, DCs deficient in MARCH1 or MHCII ubiquitination both failed to generate antigen-specific T reg cells in vivo and in vitro, although both exhibited an increased capacity for antigen presentation in parallel with the increased surface MHCII. Thus, MARCH1-mediated MHCII ubiquitination in DCs is required for proper production of naturally occurring T reg cells, suggesting a role in balancing immunogenic and regulatory T cell development.
doi:10.1084/jem.20122695
PMCID: PMC3674695  PMID: 23712430
19.  CaMKII inhibition rescues pro-arrhythmic phenotypes in model of human ankyrin-B syndrome 
Background
Cardiovascular disease is a leading cause of death worldwide. Arrhythmias are associated with significant morbidity and mortality related to cardiovascular disease. Recent work illustrates that many cardiac arrhythmias are initiated by a pathologic imbalance between kinase and phosphatase activities in excitable cardiomyocytes.
Objective
We tested the relationship between myocyte kinase/phosphatase imbalance and cellular and whole animal arrhythmia phenotypes associated with ankyrin-B cardiac syndrome.
Methods
Using a combination of biochemical, electrophysiological, and in vivo approaches, we tested the ability of CaMKII inhibition to rescue imbalance in kinase/phosphatase pathways associated with human ankyrin-B-associated cardiac arrhythmia.
Results
The cardiac ryanodine receptor (RyR2), a validated target of kinase/phosphatase regulation in myocytes, displays abnormal CaMKII-dependent phosphorylation (pS2814 hyperphosphorylation) in ankyrin-B+/− heart. Notably, RyR2 dysregulation is rescued in myocytes from ankyrin-B+/− mice overexpressing a potent CaMKII-inhibitory peptide (AC3I) and aberrant RyR2 open probability observed in ankyrin-B+/− hearts is normalized by treatment with the CaMKII inhibitor KN-93. CaMKII-inhibition is sufficient to rescue abnormalities in ankyrin-B+/− myocyte electrical dysfunction including cellular afterdepolarizations, and significantly blunts whole animal cardiac arrhythmias and sudden death in response to elevated sympathetic tone.
Conclusions
These findings illustrate the complexity of the molecular components involved in human arrhythmia and define regulatory elements of the ankyrin-B pathway in pathophysiology. Furthermore, the findings illustrate the potential impact of CaMKII-inhibition in the treatment of a congenital form of human cardiac arrhythmia.
doi:10.1016/j.hrthm.2012.08.026
PMCID: PMC3630478  PMID: 23059182
ankyrin; CaMKII; ryanodine receptor; spectrin; arrhythmia
20.  Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus 
The Journal of Clinical Investigation  2013;123(12):5135-5145.
Autoantibodies against cytokines, chemokines, and growth factors inhibit normal immunity and are implicated in inflammatory autoimmune disease and diseases of immune deficiency. In an effort to evaluate serum from autoimmune and immunodeficient patients for Abs against cytokines, chemokines, and growth factors in a high-throughput and unbiased manner, we constructed a multiplex protein microarray for detection of serum factor–binding Abs and used the microarray to detect autoantibody targets in SLE. We designed a nitrocellulose-surface microarray containing human cytokines, chemokines, and other circulating proteins and demonstrated that the array permitted specific detection of serum factor–binding probes. We used the arrays to detect previously described autoantibodies against cytokines in samples from individuals with autoimmune polyendocrine syndrome type 1 and chronic mycobacterial infection. Serum profiling from individuals with SLE revealed that among several targets, elevated IgG autoantibody reactivity to B cell–activating factor (BAFF) was associated with SLE compared with control samples. BAFF reactivity correlated with the severity of disease-associated features, including IFN-α–driven SLE pathology. Our results showed that serum factor protein microarrays facilitate detection of autoantibody reactivity to serum factors in human samples and that BAFF-reactive autoantibodies may be associated with an elevated inflammatory disease state within the spectrum of SLE.
doi:10.1172/JCI70231
PMCID: PMC3859403  PMID: 24270423
21.  CaMKII-Based Regulation of Voltage-Gated Na+ Channel in Cardiac Disease 
Circulation  2012;126(17):10.1161/CIRCULATIONAHA.112.105320.
Background
Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked with potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite over a decade of investigation. Post-translational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel post-translational modifications and disease. We recently identified a novel pathway for post-translational regulation of the primary cardiac voltage-gated Na+ channel (Nav1.5) by CaMKII. However, a role for this pathway in cardiac disease has not been evaluated.
Methods and Results
We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Nav1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Nav1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Nav1.5 resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large animal model of acquired heart disease and in failing human myocardium.
Conclusions
We identify the mechanism for two human arrhythmia variants that affect Nav1.5 channel activity through direct effects on channel post-translational modification. We propose that the CaMKII phosphorylation motif in the Nav1.5 DI-DII cytoplasmic loop is a critical nodal point for pro-arrhythmic changes to Nav1.5 in congenital and acquired cardiac disease.
doi:10.1161/CIRCULATIONAHA.112.105320
PMCID: PMC3811023  PMID: 23008441
arrhythmia (mechanisms); calmodulin dependent protein kinase II; heart failure; ion channels; long-QT syndrome; myocardial infarction
22.  New Therapeutic Targets in Cardiology: Arrhythmias and CaMKII 
Circulation  2012;126(17):2125-2139.
doi:10.1161/CIRCULATIONAHA.112.124990
PMCID: PMC3532717  PMID: 23091085
arrhythmia (mechanisms); calcium; ion channels or ion channel; structural heart disease; CaMKII
23.  CaMKII: linking heart failure and arrhythmias 
Circulation research  2012;110(12):1661-1677.
Understanding relationships between heart failure and arrhythmias, important causes of suffering and sudden death, remains an unmet goal for biomedical researchers and physicians. Evidence assembled over the last decade supports a view that activation of the multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) favors myocardial dysfunction and cell membrane electrical instability. CaMKII activation follows increases in intracellular Ca2+ or oxidation, upstream signals with the capacity to transition CaMKII into a Ca2+ and calmodulin-independeant, constitutively active enzyme. Constitutively active CaMKII appears poised to participate in disease pathways by catalyzing the phosphorylation of classes of protein targets important for excitation-contraction coupling and cell survival, including ion channels and Ca2+ homeostatic proteins, and transcription factors that drive hypertrophic and inflammatory gene expression. This rich diversity of downstream targets helps to explain the potential for CaMKII to simultaneously affect mechanical and electrical properties of heart muscle cells. Proof of concept studies from a growing number of investigators show that CaMKII inhibition is beneficial for improving myocardial performance and reducing arrhythmias. Here we review the molecular physiology of CaMKII, discuss CaMKII actions at key cellular targets and results of animal models of myocardial hypertrophy, dysfunction and arrhythmias that suggest CaMKII inhibition may benefit myocardial function while reducing arrhythmias.
doi:10.1161/CIRCRESAHA.111.243956
PMCID: PMC3789535  PMID: 22679140
CaMKII; Arrhythmias; Heart Failure; Ion channels; Remodeling
24.  Ionizing radiation regulates cardiac Ca handling via increased ROS and activated CaMKII 
Ionizing radiation (IR) is an integral part of modern multimodal anti-cancer therapies. IR involves the formation of reactive oxygen species (ROS) in targeted tissues. This is associated with subsequent cardiac dysfunction when applied during chest radiotherapy. We hypothesized that IR (i.e., ROS)-dependently impaired cardiac myocytes’ Ca handling might contribute to IR-dependent cardiocellular dysfunction. Isolated ventricular mouse myocytes and the mediastinal area of anaesthetized mice (that included the heart) were exposed to graded doses of irradiation (sham 4 and 20 Gy) and investigated acutely (after ~1 h) as well as chronically (after ~1 week). IR induced a dose-dependent effect on myocytes’ systolic function with acutely increased, but chronically decreased Ca transient amplitudes, which was associated with an acutely unaltered but chronically decreased sarcoplasmic reticulum (SR) Ca load. Likewise, in vivo echocardiography of anaesthetized mice revealed acutely enhanced left ventricular contractility (strain analysis) that declined after 1 week. Irradiated myocytes showed persistently increased diastolic SR Ca leakage, which was acutely compensated by an increase in SR Ca reuptake. This was reversed in the chronic setting in the face of slowed relaxation kinetics. As underlying cause, acutely increased ROS levels were identified to activate Ca/calmodulin-dependent protein kinase II (CaMKII). Accordingly, CaMKII-, but not PKA-dependent phosphorylation sites of the SR Ca release channels (RyR2, at Ser-2814) and phospholamban (at Thr-17) were found to be hyperphosphorylated following IR. Conversely, ROS-scavenging as well as CaMKII-inhibition significantly attenuated CaMKII-activation, disturbed Ca handling, and subsequent cellular dysfunction upon irradiation. Targeted cardiac irradiation induces a biphasic effect on cardiac myocytes Ca handling that is associated with chronic cardiocellular dysfunction. This appears to be mediated by increased oxidative stress and persistently activated CaMKII. Our findings suggest impaired cardiac myocytes Ca handling as a so far unknown mediator of IR-dependent cardiac damage that might be of relevance for radiation-induced cardiac dysfunction.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-013-0385-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00395-013-0385-6
PMCID: PMC3898380  PMID: 24068185
Irradiation; Excitation–contraction coupling; Calcium; Free radicals; CaMKII
25.  Phase Variation Leads to the Misidentification of a Neisseria Gonorrhoeae Virulence Gene 
PLoS ONE  2013;8(8):e72183.
Neisseria gonorrhoeae is the causative agent of gonorrhea and an obligate pathogen of humans. The Opa proteins of these bacteria are known to mediate attachment and internalization by host cells, including neutrophils. The Opa protein repertoire of a typical N. gonorrhoeae isolate is encoded on ∼11 genes distributed throughout the chromosome and is subject to stochastic changes in expression through phase variation. Together, these characteristics make Opa proteins a critical yet unpredictable aspect of any experimental investigation into the interaction of N. gonorrhoeae with host cells. The goal of this study was to identify novel virulence factors of N. gonorrhoeae by assessing the contribution of a set of uncharacterized hydrogen peroxide-induced genes to bacterial survival against neutrophil-mediated killing. To this end, a strain harboring an engineered mutation in the NGO0322 gene was identified that exhibited increased sensitivity to neutrophil-mediated killing, enhanced internalization by neutrophils, and the ability to induce high levels of neutrophil-generated reactive oxygen species. Each of these phenotypes reverted to near wild-type levels following genetic complementation of the NGO0322 mutation. However, after immunoblot analysis of Opa proteins expressed by the isogenic parent, mutant, and genetically complemented strains, it was determined that phase variation had resulted in a disparity between the Opa profiles of these strains. To determine whether Opa phase variation, rather than NGO0322 mutation, was the cause of the observed neutrophil-related phenotypes, NGO0322 function was investigated in N. gonorrhoeae strains lacking all Opa proteins or constitutively expressing the OpaD variant. In both cases, mutation of NGO0322 did not alter survival of gonococci in the presence of neutrophils. These results demonstrate the importance of controlling for the frequent and random variation in Opa protein production by N. gonorrhoeae when investigating host cell interactions.
doi:10.1371/journal.pone.0072183
PMCID: PMC3745409  PMID: 23977246

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