Citations

Rewarming patients following profound hypothermia may result in acute heart failure and high mortality (50‐80%); however underlying pathophysiological mechanisms are largely unknown. We characterized myocardial dysfunction induced by hypothermia/rewarming (H/R) by measuring intracellular Ca 2+ concentration ([Ca 2+ ] i ) and twitch force in intact electrically stimulated left ventricular (LV) rat papillary muscles loaded with Fura‐2AM. In skinned papillary muscle fibers, we assessed maximal Ca 2+ ‐activated force (F max ) and Ca 2+ sensitivity of force. Following H/R, peak twitch force decreased by 30‐40%, but [Ca 2+ ] i was not significantly altered. In skinned preparations, maximal F max was decreased by ~30%, while the pCa required for 50% of F max (pCa 50 ) was reduced by ~0.14. Interestingly, in rewarmed papillary muscle, both total cTnI phosphorylation and protein kinase A (PKA)‐mediated cTnI phosphorylation at Ser23/24 were significantly increased compared to controls. We conclude that after H/R myocardial contractility is significantly reduced as evidenced by reduced twitch force and F max . Reduced myocardial contractility is attributed to decreased Ca 2+ sensitivity of force that likely results from increased cTnI phosphorylation.

The mechanisms underlying pulmonary hypertension are still being explored. Recent studies suggest a role for caveolae (pit‐like plasma membrane structures), although it is not entirely clear whether they enhance or blunt pulmonary hypertension. The proteins caveolins ‐1, and ‐2, and polymerase I and transcription release factor (PTRF) are essential to forming caveolae. In other tissues, growth factors called neurotrophins (e.g. brain‐derived neurotrophic factor, BDNF) are thought to affect cell proliferation and structure. This study investigated caveolins and neurotrophins in human pulmonary artery. Western analysis of artery from a patient with primary pulmonary hypertension showed decreased levels of caveolin‐1, caveolin‐2, PTRF and RhoA. Exposure of human pulmonary artery to 1 nM BDNF increased protein levels of caveolin‐1 and PTRF. Isolated pulmonary artery smooth muscle cells exposed to BDNF showed decreased [Ca 2+ ] i response to bradykinin (10 nM) and histamine (10 μM), but no change in caveolin or PTRF levels. These data suggest that decreased caveolin levels may contribute to increased pulmonary vascular contractility, while neurotrophins (potentially by increasing caveolin levels) may alleviate excessive constriction. Further studies are required to determine whether neurotrophins affect caveolae in endothelial cells or smooth muscle. Supported by FAMRI, NIH grant UL1RR024150, and the Department of Anesthesiology, Mayo Clinic.

There is increasing interest in the role of hydrogen sulfide (H 2 S) as a gasotransmitter similar to nitric oxide and carbon monoxide. In this study, we examined the role of H 2 S in airway smooth muscle (ASM) inflammation. H 2 S is produced by the enzymes cystathionine beta‐synthase (CBS) and cystathionine gamma‐lysase (CTH). Both of these enzymes were detected in human ASM by Western blot analysis. In tissues exposed to the pro‐inflammatory cytokine tumor necrosis factor‐a (TNFα) both CTH and CBS expression were upregulated. In ASM strips contracted with 10 µM ACh, only exposure to 1 mM of the H 2 S donor NaHS induced relaxation. Human ASM cells loaded with fura‐2 demonstrated a dose‐dependent decrease in intracellular calcium concentration ([Ca 2+ ] i ) to NaHS (50‐250 μM), in the presence of either 1 μM ACh or 10 μM histamine. However, sodium sulfide (Na 2 S, 100 µM up to 1 mM) showed no effect on [Ca 2+ ] i . Exposure to 60 mM KCl in the presence of either NaHS or Na 2 S (50 μM‐3 mM) did not affect [Ca 2+ ] i . Relaxant effects of NaHS or Na 2 S on ACh‐induced [Ca 2+ ] i elevation were absent when experiments were conducted in zero extracellular Ca 2+ . These results suggest that H 2 S relaxes ASM and reduces [Ca 2+ ] i by affecting Ca 2+ influx, potentially via voltage‐gated channels, but does not affect sarcoplasmic reticulum Ca 2+ release. The relevance of endogenous H 2 S generation in ASM, and its role in infalmmation needs to be explored further. Supported by the Flight Attendants Medical Research Institute (FAMRI), NIH grant HL74309, and the Department of Anesthesiology, Mayo Clinic.

Sarcoplasmic reticulum (SR) Ca 2+ release and reuptake are important mechanisms in airway smooth muscle (ASM) intracellular Ca 2+ [Ca 2+ ] i regulation. In other tissues, there is increasing evidence for a role of mitochondria in [Ca 2+ ] i buffering, especially following SR Ca 2+ release. Mitochondrial buffering may also influence Ca 2+ influx. The purpose of the present study was to examine the role of mitochondria in [Ca 2+ ] i buffering in human ASM. Cells were loaded with fura‐2 and exposed to 10 µM histamine in "0" Ca 2+ HBSS containing 1mM lanthanum and 1µM nifedipine (to block Ca 2+ influx/efflux) and in the additional presence of 1µM RU360 (mitochondrial Ca 2+ uniporter inhibitor) or 1µM CGP37157 (CGP; mitochondrial Na + /Ca 2+ exchange inhibitor). Compared to controls, peak [Ca 2+ ] i levels were increased by 30% in the presence of CGP, while RU360 had not effect. Both CGP and RU360 significantly increased store‐operated Ca 2+ entry. In cells co‐loaded with fura‐2 and rhod‐2 (mitochondrial Ca 2+ indicator), exposure to 5 µM caffeine showed instantaneous SR Ca 2+ release followed by an increase in [Ca 2+ ] mito . Exposure to 1 µM ACh resulted in ACh‐induced [Ca 2+ ] i oscillations that were reflected by mitochondria (but with a time delay). In ASM cells co‐labeled for mitochondria (MitoTracker) and SR (fluorescent ryanodine) migration of mitochondria towards SR was observed upon exposure to ACh or cyclopiazonic acid. This migration was completely inhibited by phalloidin (actin inhibitor). These data demonstrate an important role of mitochondria in [Ca 2+ ] i buffering in ASM. Supported by NIH grant RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

Intracellular Ca 2+ ([Ca 2+ ] i ) levels are increased in inflamed airway smooth muscle (ASM). Sarcoplasmic reticulum (SR) Ca 2+ release and reuptake are key components of ASM [Ca 2+ ] i regulation. Ca 2+ reuptake occurs via the ATPase SERCA. We hypothesized that inflammation disrupts SERCA function, contributing to increased [Ca 2+ ] i . We examined SERCA expression and regulation in human ASM exposed to pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis showed significantly decreased SERCA expression following overnight cytokine exposure. In other tissues, SERCA is regulated by the inhibitory protein phospholamban (PLB). We found that human ASM does not express PLB protein (although mRNA is detectable). Cytokine exposure did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (potentially via CaMKII) occurs in human ASM. In fura‐2 loaded human ASM cells, we examined [Ca 2+ ] i responses to ACh or bradykinin (zero extracellular Ca 2+ ) and found significant reduction in rates of fall of [Ca 2+ ] i transients in cells exposed to TNF‐α or IL‐13. We further found that CaMKII antagonist KN‐93 significantly slowed the fall of [Ca 2+ ] i transients in both controls and cytokine‐exposed human ASM cells. These data suggest that a) SERCA in human ASM is regulated by mechanisms such as CaMKII; and b) airway inflammation decreases SR refilling by blunting SERCA expression and function. Supported by NIH grants HL74309, the Flight Attendants Medical Research Institute (FAMRI), and the Department of Anesthesiology, Mayo Clinic.

Background Uterine leiomyomas, also known as fibroids (UF), are benign neoplasms of the uterus. UF consist of smooth muscle cells and fibroblasts, and display enriched extracellular matrix (ECM). Reports estimate a cumulative incidence of 70% in premenopausal women, with annual total health care costs attributed to UF exceeding 34 billion dollars in the United States alone. In spite of their prevalence, the underlying processes that drive UF development and growth are still poorly understood. Accumulating evidence implicates the canonical Hippo signaling pathway effectors Yes‐associated protein (YAP)/transcriptional coactivator with PDZ‐binding domain (TAZ) as key drivers of fibroblast biology. Thus, we seek to assess the differences in YAP/TAZ localization, ECM deposition, and gene expression between UF and normal myometrium (Myo). Methods Matched samples of UF and Myo were obtained from premenopausal women with IRB approval. Immunohistochemistry (N=5) was performed on OCT embedded tissue with YAP/TAZ antibody. For cell culture, UF and Myo were minced and digested with collagenase type 4. Cells (passages 1–4) were plated on plastic at confluence (N=4 for both). Immunostaining for YAP/TAZ, Fibronectin (FN), Collagen Type I (COL1), and Collagen Type III (COL3) were performed (N=4–5). Transcript levels were analyzed by treating cells with control or YAP/TAZ siRNA, and performing subsequent cDNA synthesis and qPCR reactions (N=4). Kruskall‐Wallis and Mann Whitney tests were used as appropriate. Results YAP/TAZ nuclear localization was upregulated in UF when compared to Myo tissue by immunofluorescence imaging. In freshly isolated cells cultured at confluence on tissue culture plastic, baseline YAP/TAZ nuclear localization was increased in UF when compared to Myo (p=0.03). UFs also exhibited a trend toward increased FN, COL1, and COL3 deposition when compared to Myo. UF displayed increased transcript levels for several matrix and pro‐fibrotic signaling genes (COL1A1, COL3A1, CTGF, EDN1, FN1) compared to the matched Myo. Following siRNA treatment for YAP/TAZ gene expression was reduced 40% and 50% for EDN1 (encoding endothelin‐1), as well as 70% and 65% for CTGF (encoding connective tissue growth factor), in UF and Myo respectively. Conclusions UF nuclear YAP/TAZ is increased at baseline in vivo as well as in vitro . UF also display upregulated deposition of the ECM proteins. Additionally, there are increased transcript levels of profibrotic genes in UF compared to Myo. Moreover, the observed decreases in gene expression following siRNA knockdown of YAP/TAZ suggests a role for the Hippo signaling pathway in regulation of certain profibrotic genes in UF. Together these results suggest that enhanced baseline YAP/TAZ localization in UF may drive upregulated expression of pro‐fibrotic genes promoting UF progression. Support or Funding Information Center for Biomedical Discovery‐Department of Obstetrics and Gynecology Team Science Pilot Award Program This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Ovarian cancer is the eighth most common cancer and the fifth leading cause of cancer death among US women. Hepatoma‐derived growth factor (HDGF) is a heparin binding, secreted mitogen with prognostic value in multiple cancers. We previously reported that HDGF is overexpressed in ovarian cancer. Surprisingly, the mechanisms by which HDGF acts in any cell type remains to be fully elucidated. We hypothesized that overexpression of HDGF in ovarian cancer cells is functionally relevant and that HDGF confers pro‐survival and proliferative properties, promoting cancer growth. To elucidate intracellular and extracellular signaling mechanisms, we examined the effect of HDGF knockdown via siRNA, overexpression of HDGF and stimulation by full‐length recombinant protein. Immunofluorescence image analysis showed nuclear localization of HDGF in OV202 cell line. Additionally, HDGF was minimally secreted but passively released in greater amounts by necrotic cells. Extracellular HDGF stimulated phosphorylation of ERK and p38. In contrast, modulating intracellular HDGF did not affect MAPK pathways. In fact, intracellular HDGF functioned in a distinct manner by regulating cellular metabolism. This suggests that HDGF acts in ovarian cancer cells via two distinct mechanisms, both of which could be exploited for ovarian cancer therapy. Supported by the Mayo Graduate School (Giri) and the Department of Anesthesiology, Mayo Clinic (Prakash).

Introduction: Children with low Vitamin D levels are at increased risk of developing asthma. In the present study, we tested the hypothesis that the Vitamin D receptor (VDR) agonist, calcitriol, will reduce tumor necrosis factor‐α (TNF‐α)‐induced expression of chemokines in human fetal airway smooth muscle cells (fASM). Methods and Results Human fASM cells were dissociated from canalicular stage (18‐22 weeks gestation) lung tissue (de‐identified samples, in vitro use only and considered exempt by Mayo Institutional Review Board). Fetal ASM cells were pre‐treated with vehicle (0.001% DMSO) or 100 nM calcitriol for 1 h, and then exposed to 10 ng/mL TNFα for 6 or 24 h. Expression of CCL5 and CCL11 were measured by quantitative real time PCR and ELISA. Cells were transfected with VDR siRNA. Treatment with TNFα for 6 h increased mRNA expression and secretion of CCL5 and CCL11. While pre‐treatment with calcitriol reduced CCL5 and CCL11 expression in the presence of TNFα. Knockdown of the VDR expression demonstrated that calcitriol's effects are mediated through VDR. Conclusions These novel data demonstrate that calcitriol modulates cytokine enhancement of chemokines in developing ASM. Vitamin D may be a new therapeutic target in reducing inflammation in the immature airway. Supported by NIH F32 HL123075 (Britt), T32 HL105355 (Vogel), and R01s HL088029 and HL056470 (Prakash), and the Mayo Clinic Department of Anesthesiology (Pabelick, Prakash).

Background Uterine leiomyomas (fibroids; UF) are benign myometrial neoplasms that produce clinically‐relevant symptoms in >25% of all women including excessive menstrual bleeding and infertility, and represent the leading cause of hysterectomy (~600,000/year in the US). Despite their importance, the mechanisms that promote UF growth are still not well‐understood. UFs are comprised of smooth muscle cells and fibroblasts with substantial extracellular matrix (ECM). While cells within UFs produce ECM, the role of ECM per se in modulating cell properties towards overall tumor growth is not known. This study tests the hypothesis that UF cells are mechanically sensitive, and that the ECM provides a positive feedback mechanism which further promotes UF growth. Methods Cells were enzymatically isolated from UFs of patients undergoing myomectomy at Mayo Clinic Rochester (IRB approved). Fibroid cells were plated onto soft (0.2kPa) vs. stiff (64kPa) polydimethylsiloxane (PDMS) elastomer substrates or standard tissue culture plastic substrates (~100GPa) for 24 h in DMEM containing 10% FBS prior to RNA isolation and qPCR analysis. Transcript levels for known ECM and profibrotic genes were determined. Nuclear localization of YAP and TAZ was visualized using immunofluorescence and quantified using a Cytation5 imaging system. Separately, 2‐color fluorescence immunohistochemistry for YAP and TAZ was performed on formalin fixed; paraffin embedded normal myometrium and fibroid tissue sections. Transciption level and nuclear localization calculations were performed with Kruskall‐Wallis and Wilcoxon Sign‐Rank tests, respectively. Results Significant increases in transcripts for ECM gene Collagen 1 ( COL1A1 ) were noted for UF cells plated on stiff PDMS and standard plastic compared to soft PDMS. The pro‐fibrotic genes Connective Tissue Growth Factor ( CTGF ), alpha smooth muscle actin ( ACTA2 ), Endothelin 1 ( EDN1 ), and plasminogen activator inhibitor 1 ( SERPINE1 ) were progressively increased in expression going from soft PDMS substrate to plastic. The anti‐fibrotic gene, cyclooxygenase 2 ( PTGS2 ) was decreased on stiff PDMS and plastic compared to soft PDMS. In cells plated on plastic, there was a significant decrease in YAP/TAZ nuclear localization in cells treated with the Rho‐kinase inhibitor Y‐27632, consistent with a mechanobiologic feedback. Immunohistochemistry showed a two‐fold increase in YAP/TAZ nuclear localization for fibroid tissue compared to normal myometrium. Conclusions Based on the growing appreciation that ECM mechanical properties contribute to UF pathobiology and fibrotic matrix deposition, we show enhanced ECM and profibrotic gene expression by fibroid cells as a function of substrate stiffness. These results demonstrate a feedback mechanism by which UFs progress through matrix stiffness‐mediated ECM deposition. Here, the mechanosensitive pathways of YAP and TAZ may be involved in transducing ECM signals to UF growth. Support or Funding Information None This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .