bluuebunny/crossref_metadata_2025 huggingface dataset:
Serotonin 5‐HT2C Receptor of the Rat Detrusor Muscle: Evaluation of the Atypical Antipsychotics Olanzapine and Ziprasidone on the Isolated Rat Detrusor Muscle
Authors: Morris Michelle , Gossell‐Williams Maxine , Nwokocha Chukwuemeka
Published: June 2021

Background

Atypical antipsychotics are preferentially prescribed over typical agents for greater efficacy in treating symptoms associated with schizophrenia and related psychoses. However, these antipsychotics have been found to reduce bladder function in patients. Antagonism of serotonin (5‐HT) receptor, 5‐HT2C has been implicated as the cause. An in‐vitro preparation using the isolated rat detrusor muscle may be used to determine the effect of atypical agents Olanzapine and Ziprasidone on bladder contraction.

Method: Bladders isolated from male Sprague Dawley rats (250–350g), were divided into longitudinal strips and immersed in Kreb's solution with carbogen (95% O2:5% CO2)at 370C. After equilibration at 0.5g tension, the strips were stimulated with electric field stimulation (EFS) while adding 5‐HT (0.01‐30 µM and 0.1 nM‐1 µM) cumulatively at half‐log concentrations. A control concentration‐response curve (CRC) was constructed after which another CRC was constructed in the presence of Olanzapine (0.3nM‐3 µM) and Ziprasidone (0.001 nM‐1 nM) during EFS.

Results: 5‐HT caused concentration dependent potentiation of EFS induced contractions and therefore may be a co‐regulator of micturition with ACh. Olanzapine (P蠄0.00) and Ziprasidone (P蠄0.00) significantly inhibited 5‐HT potentiated contractions in a concentration dependent manner.

Conclusions

The results suggest that atypical agents Olanzapine and Ziprasidone have potential to cause urinary retention. This effect may be due to affinity for 5‐HT receptors in the bladder.

Funding: UWI, Office of Graduate Studies & Research
Divergent Effects for Celecoxib and Diclofenac on Hemodynamic and Left Ventricular Actions of Cyclosporine in Female Rats
Authors: Ibrahim Karim , El‐Yazbi Ahmed , El‐Gowelli Hanan , El‐Mas Mahmoud
Published: June 2021

The combined use of cyclosporine (CSA), a potent immunosuppressant, and nonsteroidal antiinflammatory drugs (NSAIDs) in arthritis is a common clinical practice. In this study, we evaluated whether diclofenac (nonselective COX‐1/COX‐2 inhibitor) and celecoxib (selective COX‐2 inhibitor) interact variably with CSA hemodynamics in conscious female rats. Changes evoked by acutely administered CSA in blood pressure (BP), heart rate (HR), HR variability (HRV), and myocardial contractility were assessed in the absence and presence of diclofenac or celecoxib. Compared with vehicle values, CSA (10 mg/kg i.v.) caused immediate and sustained increases in BP and decreases in the pulse pressure index and HR. CSA also increased the time and frequency domain indices of HRV and shifted the cardiac sympathovagal balance towards parasympathetic dominance. The pressor effect of CSA was intensified and abolished in rats pretreated i.v. with diclofenac (10 mg/kg) and celecoxib (10 mg/kg), respectively. The autonomic and PPI effects of CSA were blunted in the presence of celecoxib in contrast to no effect for diclofenac. We also report that celecoxib, but not diclofenac, eliminated the elevated LV contractility (dP/dtmax) and isovolumic relaxation constant (Τau, τ), which reflects the cardiac diastolic function, that accompanied the CSA‐evoked pressure load. The data highlight the preferential capacity of celecoxib to mitigate the pressor effect of CSA and consequent alterations in cardiac performance and autonomic balance in female rats.

Supported by the ALEX‐REP Grant Fund, Alexandria University, Egypt (Grant HLTH‐13‐01).
Helium Postconditioning Regulates Caveolin‐1/‐3 Translocation and Gene Expression
Authors: Flick Moritz , Oei Gezina , Albrecht Martin , Hollmann Markus , Preckel Benedikt , Patel Hemal , Weber Nina
Published: June 2021

Caveolae, lipid enriched invaginations of the plasma membrane, are epicentres of cellular signal transduction. Caveolins, structural proteins localized to caveolae, have been shown to play an important role in anesthetic‐induced cardioprotection. Helium (He) postconditioning is known to reduce infarct size and induces cardioprotection. We here hypothesize that He postconditioning regulates caveolin‐1 and ‐3 (Cav‐1 and Cav‐3) in the heart. Employing coronary artery occlusion, male Wistar rats (n=8 each group) were subjected to 25 min of cardiac ischemia and reperfusion (I/R) for 5, 15 or 30 min (I/R 5/15/30), respectively. The He postconditioning groups underwent I/R plus 70% He during reperfusion (He 5/15/30 minutes, respectively). The sham group received surgical treatment without I/R (Sham). After each protocol blood and hearts were retrieved. The tissue was obtained from the area at risk (AAR) and the non‐area at risk (NAAR) and processed for Western blot analyses and qRT‐PCR. The membrane fraction (AAR) of He 15 showed increased levels of Cav‐1 (p<0.05 vs. I/R 15) and Cav‐3 (p<0.05 vs. I/R 15). Elevated levels of Cav‐3 were also found in the He 15 serum (p<0.05 vs. I/R 15). Gene expression analyses revealed decreased expression of Cav‐3 in He 15 compared to Sham in AAR (p<0.05) and NAAR (p<0.05) tissue. These data suggest that 15 min He postconditioning might induce survival pathways by regulating caveolin‐1/‐3 translocation and gene expression.

This study was funded by the SCA.
Preservation of Coronary Vasodilator Responses to the Nitroxyl Donor Angeli's Salt after Ischemia/Reperfusion of the Rat Isolated Heart
Authors: Chin Kai Yee , Michel Lisa , Qin Chengxue , Ritchie Rebecca , Woodman Owen
Published: June 2021

We previously reported that Angeli's salt (AS) simultaneously increases cardiac contractility and coronary flow (CF) in normal rat hearts. In this study, we tested the hypothesis that the cardiac contractile and vasodilator actions of AS will be preserved after myocardial ischaemia‐reperfusion (I/R). Isolated hearts from male rats were Langendorff‐perfused for 90 min (sham) or subjected to 30 min equilibration followed by 30 min global ischaemia and 30 min reperfusion. Haemodynamic responses to AS (1 nmol ‐ 10 μmol), the nitric oxide donor, diethylamine NONOate (DEA/NO, 1 nmol – 1 μmol) and the clinically used inotrope for heart failure, dobutamine (DOB, 0.1 nmol – 0.1 μmol) were determined. I/R reduced the cardiac contractile action of AS [change (Δ) in left ventricular (LV) +dP/dt (% baseline) by 10 μmol: sham 77 ± 13%, I/R‐treated hearts 17 ± 5%, p<0.0001] however, I/R had no effect on the AS‐induced increase in heart rate (HR) [Δ HR (% baseline) by 10 μmol: sham 11 ± 3%, I/R‐treated hearts 14 ± 6%] or CF [Δ CF (% baseline) by 1 μmol: sham 72 ± 13%, I/R‐treated hearts 76 ± 15%]. In contrast, the DEA/NO‐induced vasodilatation was reduced by I/R [Δ CF (% baseline) by 1 μmol: sham 78 ± 12%, I/R‐treated hearts 37 ± 8%, p<0.05]. I/R also significantly reduced the DOB‐induced increase in contractility [Δ LV+dP/dt (% baseline) by 10 nmol: sham 71 ± 13%, I/R‐treated hearts 22 ± 4%, p<0.0001] while I/R aggravated DOB‐induced tachycardia [Δ HR (% baseline) by 0.1 μmol: sham 11 ± 4%, I/R‐treated hearts 36 ± 8%, p<0.01], which increases the risk of arrhythmias. These data suggest that AS has superior coronary vasodilator capacity after I/R.
Slow Gastric Tube Infusion as an Approach for Assessment of Controlled Release in Rodents
Authors: Small Kersten , Lieber Gissela , Bateman Thomas , Ormes James , Shen Xialoan , Madwed Jeffrey
Published: June 2021

Methods in rodents to inform on oral controlled release (CR) formulations in the drug discovery process are limited. To address this problem, here we describe slow gastric infusion in rats as an approach that may allow for more informed decision‐making regarding investment in formulation based CR. To evaluate this approach, we used a novel orally bioavailable NO donor to determine whether slow compound delivery directly to the stomach would extend the duration of action of blood pressure (BP) lowering for a compound with a short T1/2 and minimize abrupt decreases in BP and increases in heart rate (HR) observed for vasorelaxants with early Tmax and high Cmax. BP and HR are particularly advantageous pharmacodynamics (PD) readouts because continuous measurements provide rich data sets for PK/PD comparisons. To inform on study design, an aqueous vehicle for compound solubilization was identified (30% HPBCD) and used to determine dose and predict plasma level concentrations following slow gastric infusion. For PD studies, Sprague‐Dawley rats were instrumented with telemetry and gastric tubes. After surgical recovery, rats were treated with L‐NAME to induce hypertension followed by a 30 mg/kg dose of NO donor compound, administered as a bolus and a slow 24‐hr infusion via gastric tube. Compared to a bolus dose, slow gastric infusion extended the duration of BP lowering, minimized the abrupt fall in blood pressure, and inhibited the increase in heart rate. In conclusion, our data support slow gastric infusion as a means to test PK/PD hypotheses which may inform on effectiveness of CR formulations.
Chronic Allergic Pulmonary Inflammation is Aggravated in Angiotensin‐(1‐7) Mas Receptor Knockout Mice
Authors: Magalhaes Giselle , Rodrigues‐Machado M Gloria , Motta‐Santos Daisy , Barcelos Luciola , Santos Robson , Campagnole‐Santos Maria
Published: June 2021

Angiotensin‐(1‐7)/Mas receptor axis of the renin‐angiotensin system is associated with attenuation of different inflammatory processes. In this study, we investigated the involvement of Mas receptor in the inflammation and airway remodelling in a model of chronic allergic pulmonary inflammation induced by ovalbumin (OVA). FVBN mice groups: Mas KO control (Mas‐/‐CTRL), Mas KO sensitized with OVA (Mas‐/‐OVA), WT control (Mas+/+CTRL) and WT sensitized with OVA (Mas+/+OVA). The allergic groups received 4 doses of OVA (20µg/mice, i.p.) 14 days apart. After the 2nd sensitization, mice were nebulized with OVA (1% for 30 min; 3 times/week for 4 weeks) or saline (CTRL). Mice were sacrificed 72h after the last nebulization. Mas+/+OVA did not develop inflammation or pulmonary remodelling, as it is observed with this protocol in BalbC mice. However, Mas‐/‐OVA showed a worse performance in the maximum load exercise test and presented a significant increase in inflammatory cell infiltrate in the lung, collagen deposition in the airways, increased thickening of the alveolar parenchyma, increased thickening of the smooth muscle layer of the pulmonary arterioles and hypertrophy of the cardiomyocyte of the right ventricle. Furthermore, Mas‐/‐OVA showed an increase in ERK1/2 and SAPK/JNK phosphorylation and an increase in pro‐inflammatory cytokine (IL13) and chemokines (MCP‐1/CCL2 and RANTES/CCL5) in the lungs. These data indicate that genetic deletion of Mas receptor aggravates lung inflammation and remodelling in mice subjected to a model of chronic asthma.
ZF143 Enhances Zebrafish Heart Regeneration and Improves Mouse Heart Function After an LAD Injury
Authors: Yin Voot , Smith Ashley , Roberts Helen , Carlisle Heather
Published: June 2021

Heart disease is the leading cause of morbidity and mortality in the Western world due to a limited regenerative capacity. In lieu of new muscle synthesis in response to injury, the human heart replaces necrotic tissue with deposition of a non‐contractile scar. While a scar provides short‐term benefits, its long‐term perdurance often culminates in heart failure. Thus, there is a tremendous unmet need for therapeutic intervention that stimulates cardiomyocyte proliferation. The adult zebrafish is endowed with a remarkable regenerative capacity, capable of de novo cardiomyocyte creation and complete recovery of lost heart function when challenged with an acute injury. In this study, we employed the zebrafish to screen a limited number of compounds in order to identify molecules that may amplify genetic networks that underscore regenerative capacity. We identified ZF143, a naturally occurring compound that greatly enhances regeneration of heart tissues. ZF143 increases cardiomyocyte proliferation indices by over 200% when compared with control animals. This enhancement in proliferation is accompanied with new muscle creation and decreases in scar tissue deposition within the wounded apex. Importantly, ZF143 is able to restore regenerative capacity in a zebrafish genetic model with repressed heart regenerative capacity. Additionally, in blinded adult mice studies, ZF143 profoundly increased survival under conditions of LAD injury. This increased survival is accompanied with improved heart function and decreased infarct size following a 1‐month treatment with ZF143. Collectively, this data suggests ZF143 could have strong therapeutic applications for stimulating heart muscle regeneration.
GATA4‐controlled HDAC1/HDAC2‐regulated Cardiogenesis of Mesenchymal Stem Cells
Authors: Wang Meijing , Wang Lina , Yu Qing , Turrentine Mark
Published: June 2021

Histone modifications correlate with gene expression and contribute to cell fate commitment. We have reported that inhibition of histone deacetylase1 (HDAC1) or HDAC2 enhanced histone acetylation at cardiac gene loci and promoted cardiogenesis of mesenchymal stem cells (MSC). MSC infected with cardiac transcription factor GATA4 gene have demonstrated increased myocardial differentiation, indicating the transcription machinery in guidance of cardiac cell fate. However it is unclear whether functions of transcription factors and histone modifications are interdependent in control of cardiac cell fate. We hypothesize that HDAC1/HDAC2 and GATA4 competitively bind to cardiac‐specific gene promoters, thereby governing cardiac gene expression.

Methods

MSC were isolated from bone marrow of C57BL mice based on our published methods. MSC were transfected with siRNA of scramble, HDAC1 or HDAC2, or infected with retrovirus vectors (GATA4 RV/Control ‐ EGFP only; GATA4 RV ‐ EGFP+GATA4). Cardiac gene expression, histone acetylation and DNA‐binding levels of GATA4 or HDAC1/HDAC2 were analyzed at cardiac specific gene promoters by Chromatin Immunoprecipitation–qPCR and RT‐qPCR.

Results

Knock‐down of HDAC1 or HDAC2 enhanced GATA4 occupancy at promoters of cardiac α‐MHC (Myh6) and troponin T (cTnT) in MSC

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with increased gene expression and local levels of acH3/acH4. GATA4 overexpression [figure2A] did not change nuclear protein levels of HDAC1 and HDAC2 in MSC, but limited HDAC1/HDAC2 recruitment and decreased their bindings to Myh6 and cTnT promoters [figure2B]. Notably, this decreased occupancy of HDAC1 and HDAC2 was associated with upregulated gene expression and enhanced acH3/acH4 enrichment at the tested gene loci. Our results represent initial evidence that the interplay between GATA4 and HDAC1/HDAC2 controls promoter accessibility of cardiac genes, thus directing MSC cardiac commitment.
Platelets attenuate endothelial cell's dynamic shear stress induced activation
Authors: Meza Daphne , Rubenstein David , Yin Wei
Published: June 2021

The interaction between endothelial cells (ECs) and circulating platelets plays a critical role in cardiovascular pathogenesis. Their responses to blood flow induced shear stress can be either anti‐ or pro‐ atherogenic. The goal of this study was to determine if platelets adhere to ECs under altered flow conditions, and how it affects EC activity. ECs were subject to physiological or pathological dynamic shear stress with the presence of platelets. Platelet adhesion to ECs was examined using immunofluorescence microscopy; EC surface PECAM‐1 expression and phosphorylation was measured using a solid phase ELISA approach. Nitric oxide (NO) release from shear stress treated ECs was measured using a commercial EIA kit. Platelet adhesion to EC junctions was observed under both flow conditions. The presence of platelets caused a noticeable increase in EC PECAM‐1 expression and a decrease in PECAM‐1 phosphorylation under both physiological and pathological shear stress. NO release increased significantly in ECs under flow, independent of the presence of platelets. Since PECAM‐1 is a major mechanotransducer highly expressed at EC junctions, platelet adhesion to ECs can potentially affect EC mechanotransduction under flow, and the presence of platelets can attenuate EC activation by inhibiting PECAM‐1 phosphorylation. NO data suggests platelets do not alter the physiological response of ECs to shear stress, supporting the hypothesis that platelet physical contact with ECs, i.e., adhesion at the cell junctions, could potentially inhibit EC activation induced by flow.
Smooth Muscle Calcium Signaling and Contraction in the Small Airways of the House Dust Mite Asthma Mouse Model
Authors: Perez‐Zoghbi Jose , Castro‐Piedras Isabel
Published: June 2021

The small intrapulmonary airways are the major contributors to reversible airflow limitation in asthma. However, the mechanisms responsible for the increased small‐airway responsiveness to bronchoconstrictors (airway hyperresponsiveness) that characterized asthma pathophysiology are not fully understood. We used precision‐cut lung slices and microscope imaging approaches to investigate the contraction and intracellular calcium signaling in the smooth muscle of small airways from mice chronically challenged with house dust mite extract (HDM) during 3 to 7 weeks. Mice challenged with HDM developed a significant airway inflammation characterized by the presence of eosinophils, lymphocytes, neutrophils, and monocytes that spread through the airway tree and reached the small airways. Superfusion of lung slices with methacholine (MCh) induced a concentration‐dependent airway contraction that was significantly increased in the small airways of the HDM‐challenge mice than in PBS‐challenged mice (controls). MCh stimulation induced intracellular Ca2+ oscillations in the smooth muscle cells on the small airways. The frequency of these Ca2+ oscillations increased with MCh concentration and was higher in airway smooth muscle cells from the HDM‐exposed mice than from controls. The results suggest that airway hyperresponsiveness in asthma is caused by an increased contractility of the airway smooth muscle cells in the small airways which is accompanied by an increased intracellular Ca2+ response to bronchoconstrictors
Pulmonary C‐Fibers (PCFs) Are Essential for Airway Inflammation (AI) and Hyperreactivity (AHR) Induced by Respiratory Syncytial Virus (RSV) Infection
Authors: Zang Na , Zhuang Jianguo , Xu Fadi , Liu Enmei
Published: June 2021

RSV infection causes acute AI and AHR with the peak response occurring at ~5th day postinfection (PID 5) in humans and animals. Owing to the critical role of PCFs in regulating pulmonary inflammation and airway resistance, we tested the contribution of PCFs to these RSV‐induced disorders. These RSV‐induced disorders are reportedly dependent on increased IFN‐γ in our previous study. IFN‐γ could activate C‐fibers and the latter affect the production of the former. Thus, we asked whether PCFs were required in IFN‐γ upregulation by RSV and the IFN‐γ‐mediated AI and AHR. RSV and mock were instilled intranasally in PCF‐intact (CON) and PCF‐degenerated (KPCF) mice. On PID 5, we measured specific airway resistance (sRaw) in awake state and collected lung tissues after euthanasia for detecting BALF inflammatory cells, the lung viral titration, substance P (SP), IFN‐γ and its transcription factor (T‐bet) gene expressions. Compared with CON, KPCF mice showed significant decreases in: (1) weight loss and RSV titer in the lungs; (2) total cells and macrophages in BALF with little change in lymphocytes; (3) inflammatory cells infiltrated in the lungs; (4) the sRaw response to aerosol of methacholine (3.125 ‐ 50 mg/ml); and (5) SP. In addition, the augmented IFN‐γ and T‐bet by RSV infection was not significantly different between the two groups of mice. However, recombinant IFN‐γ (ip) induced AHR in the CON, but not KPCF mice. Our results suggest that PCFs play an important role in RSV replication, the RSV‐induced AI and AHR. Moreover, PCFs were required for IFN‐γ‐promoted AI and AHR (Supported by HL119683).
The Golgi apparatus regulates cGMP‐dependent protein kinase I (PKGI) proteolytic cleavage in vascular smooth muscle cells (SMC)
Authors: Kato Shin , Chen Jingsi , Zhang Huili , Cornog Katherine , Roberts Jesse
Published: June 2021

cGMP regulates vascular tone and structure through stimulating PKGI. Although encoded by a single gene product, two PKGI isoforms, PKGIα and PKGIβ, form through alternate mRNA splicing. They differ in their NH2‐terminal dimerization domains. Proteolysis of the PKGI isoforms and the nuclear localization of their conserved COOH‐terminal kinase fragment (PKGIγ) play a critical role in regulating SMC gene expression and differentiation. Although PKGI has been detected in the perinuclear region of SMC, and proprotein convertases (PCs) that are resident in the Golgi apparatus (GA) have been found to stimulate PKGI proteolysis, the role of the GA in PKGI processing is not understood. Using pulmonary artery SMC and rat fetal lung fibroblast (RFL‐6) cells, which express endogenous PKGI, we determined that PKGI immunoreactivity co‐localizes with index proteins residing in the endoplasmic reticulum (ER), ER‐Golgi intermediate complex, GA cisterna, and the trans‐Golgi network, including furin a PKGI‐cleaving PC. Moreover, over‐expression of Sar1 [T39N], a GDP‐bound trans‐dominant Sar1 mutant that inhibits ER exit site budding, was found to inhibit PKGI localization to GA cisterna. Also, IP3R‐associated cGMP kinase substrate (IRAG) was found not only to tether overexpressed PKGIβ to the ER and decrease its GA localization, but also to decrease PKGI proteolysis and PKGIγ nuclear translocation. Monensin, which inhibits intra‐GA protein transport, was also found to decrease PKGI cleavage. Together these studies detail for the first time a role for ER‐GA transport in PKGI post‐translational modifications that regulate nuclear signaling. NIH R01HL096779 supported this work.
Pulmonary Slowly Adapting Sensory Units Being More Active in Large Than in Small Airways May Result From More Sensory Receptors in the Units
Authors: Liu Jun , Song Nana , Guardiola Juan , Yu Jerry
Published: June 2021

It has been recognized for a long time that sensory units of pulmonary slowly adapting receptors (SARs) are more active in larger airways than in small airways. However, there is no explanation for this phenomenon. Structure is the basis for function. Sensory structures observed in peripheral airways resemble those found in central airways. However, in larger airways the sensory structures are bigger and more complex. Possibly, larger structures provide greater surface area for depolarization, and thus have a lower activating threshold and/or a higher sensitivity to stretch, leading to more activities. Recently, a single sensory unit has been reported to contain multiple receptors. Therefore, sensory structures in the large airway may contain more SARs, which may contribute to high activities. To test this hypothesis, we used a double staining approach to examine receptor structures in large (trachea and bronchi) vs small (bronchioles <500μm) airways in the rabbit. We labeled airway tissues with Na+/K+‐ATPase antibodies to show all structures of the sensory unit and with myelin basic protein (MBP) antibodies to show the myelin sheath. We define an SAR as the end structure beyond MBP labeling. We found that even though the whole structure was bigger in large airways (5577±373 μm2) than in small airways (2531±345 μm2; P<0.0001), there was no difference in receptor sizes (583±23 μm2 vs 627±37 μm2; P>0.05). Furthermore, the receptor structure contains more SARs in the large airways (9.56±0.60) than the small airways (4.03±0.39; P<0.0001). Thus, we conclude that the higher activities in large sensory units may be due to a large number of receptors.

(Supported by a VA Merit Review award PULM‐029‐10S)
Automatic Classification of Canine Pontine Neuronal Discharge Patterns using K‐means Clustering
Authors: Zuperku Edward , Prkic Ivana , Stucke Astrid , Miller Justin , Hopp Francis , Stuth Eckehard
Published: June 2021

Respiratory‐related neurons in the parabrachial‐Kölliker‐Fuse (PB‐KF) region of the pons play a key role in the control of breathing. The neuronal activities of these pontine respiratory group (PRG) neurons exhibit a variety of inspiratory (I), expiratory (E), phase spanning and non‐respiratory related (NRM) discharge patterns. Due to the variety of patterns, it can be difficult to classify them into distinct subgroups according to their discharge contours. This report presents a method that automatically classifies neurons according to their discharge patterns and derives an average subgroup contour of each class. It is based on the K‐means clustering technique and it is implemented via SigmaPlot User‐Defined transform scripts. The discharge patterns of 135 canine PRG neurons were classified into 7 distinct subgroups. Additional methods for choosing the “optimal” number of clusters are described. Analysis of the results suggests that the k‐means clustering method offers a robust objective means of both automatically categorizing neuron patterns and establishing the underlying archetypical contours of subtypes based on the discharge patterns of group of neurons. Supported by VA grant I01BX000721.
On the Cellular Evolution of the Lung
Authors: Torday John
Published: June 2021

The water‐land transition (WLT) marked the evolution of human physiology (Torday, Evolutionary Biology Redux. Pers Biol Med, Fall 2013). Skeletal changes chronicle at least five attempts by vertebrates to breach land, but molecular ‘fossil’ evidence functionally interconnects skeletal and visceral organ evolution. During the WLT, there were three gene duplications‐ the Parathyroid Hormone‐related Protein Receptor (PTHrPR), the βAdrenergic Receptor (βAR), and the Glucocorticoid Receptor (GR). All three of these gene amplifications were necessary for the evolution of the lung from the swim bladder, functional homologs which exploited lipids for efficient gas exchange. Key to understanding the cellular‐molecular changes that mediated the evolution of the gas‐exchanger is lung surfactant, a mixture of proteins and lipids sythesized and secreted into the alveolar air space, mediated by mesenchymal‐epithelial interactions that accommodated both the phylogenetic and ontogenetic decreases in the alveolar surface area‐to‐blood volume ratio to maximize oxygenation for metabolic drive among land vertebrates. Mechanistically, shearing of microvessel walls, particularly in those tissues necessary for land adaptation, generated Radical Oxygen Species, causing context‐specific, adaptive gene mutations in response to physiologic stress‐ PTHrP is necessary for the formation of alveoli, the βARs allowed for the independent regulation of systemic and alveolar blood pressure, and the GR facilitated βAR expression. Such evolutionary connections between the pulmonary and neuroendocrine systems help solve homeostatic puzzles, and these functional genomic interrelationships provide deep insights to the First Principles of lung Physiology, referring all the way back to cholesterol, the most primitive lung surfactant, in the cell membranes of unicellular eukaryotes.
Turn (on) Your Head and “Cough”: Evolution of Lung Breathing from a Lungless Primitive Vertebrate
Authors: Harris Michael , Hoffman Megan , Taylor Barbara
Published: June 2021

Air breathing was critical to the terrestrial radiation and evolution of tetrapods and arose in fish. The origins of the vertebrate lung stem from primitive boney fish. The origin of the neural substrates, which are sensitive to metabolically produced CO2 and which rhythmically activating respiratory muscles to produce lung ventilation to match ventilation to metabolic demand, is enigmatic. We have found that a distinct periodic centrally generated rhythm, described as “cough” and occurring in lamprey in vivo and in vitro, is modulated by central sensitivity to CO2. This suggests that elements critical for the evolution of breathing in tetrapods, were present in the most basal vertebrate ancestors prior to the evolution of the lung. We propose that the evolution of breathing in all vertebrates occurred through exaptations derived from these critical basal elements.
Control of a Mechanical Ventilator Using the pre‐Bötzinger Complex
Authors: C. Howard , A. Garcia , J. Bloom , J. Ford , M. Gonzalez , E. McCuen , P. Richardson , JM. Ramirez
Published: June 2021

Chronic mechanical respiratory support is critical for patients unable to breathe spontaneously, yet current mechanical ventilators fail to synchronize to the body's physiological needs. Ventilator asynchrony often results in ventilator induced lung injury to those on respiratory support. We are developing a patient‐triggered ventilator to provide respiratory support in a more physiologically synchronous manner than existing ventilation methods. Our system utilizes neurological activity within the brainstem, particularly in the pre‐Bötzinger complex (pre‐BötC), to control a ventilator with minimal latency between brainstem electrical activity and ventilation.

In vivo experiments were performed on anesthetized mice. Extracellular recordings of the pre‐BötC neurons were made; pre‐BötC electrical activity was amplified and integrated. This integrated signal was then processed into a valve‐control signal. Constant air pressure was supplied to the valve; airway pressure (Paw) between the valve and a test lung was measured using a pressure transducer. Pre‐BötC, diaphragmatic EMG, and Paw data were visualized and recorded for analysis. System latency was determined by measuring the time between the onset of pre‐BötC inspiratory activity and the onset of resulting Paw inspiratory swings. The system was effective in using the pre‐BötC electrical activity to ventilate a test lung. The average system latency was 20 ± 10ms.

Our preliminary findings demonstrate that a mechanical ventilator can be operated using the respiratory signal produced by the pre‐BötC. Further, the system operates with latency less than the delays reported for existing patient‐triggered ventilators.

Funding source: SCRI
Ontogeny of rhythmic breathing‐related motor activity in the Zebra Finch brainstem
Authors: Vincen‐Brown Michael , Quick Forrest , Pilarski Jason
Published: June 2021

Activity in the CNS begins as a widely distributed and synchronous depolarization wave known as rhythmic spontaneous neural activity (rSNA). As growth and maturation proceed, rSNA differentiates into central pattern generators (CPGs) that produce individual rhythmic behaviors, such as breathing. While it is important to understand the mechanisms that underlie this progression, monitoring the uninterrupted development of breathing‐related motor circuits remains difficult due to the limited access of these circuits in the mammalian model and a lack of information about breathing‐related CPG behavior in other models, such as birds. Here, we use a novel brainstem spinal cord preparation from the altricial Zebra Finch embryo to study the early development and maturation of breathing‐related motor patterns conveyed in cranial nerve IX. We show that rSNA can be measured continuously from embryonic day 4 (E4) through external pipping (E14). rSNA starts at embryonic day 4 (E4) with low frequency (0.29 bursts/min) short duration (<2.5 sec) single bursts, and progresses to a more complex pattern consisting of episodes with short duration bursts coupled to slow frequency (0.2 burst/minute) long duration bursts (>80 sec) at E9. The activity returns to short duration (<2.5 sec), high frequency (6‐12 bursts/min) at hatching (E14). At E4 the activity is nicotinergic‐sensitive and in the older animals (>E7) the short duration bursts are completely blocked by NMDA and non‐NMDA antagonism. The long duration bursts only diminished by bath application of m‐type glutamate antagonists. These findings establish an extremely tractable new experimental model to study the development of central breathing behaviors in both health and disease.
Mitochondrial Fragmentation and Mitophagy Contribute to Doxorubicin‐induced Cardiomyocyte Death
Authors: Catanzaro Michael , Kobayashi Satoru , Gerdes Martin , Liang Qiangrong
Published: June 2021

Objectives

Doxorubicin (DOX) is a widely used and highly effective chemotherapeutic antineoplastic agent that can cause severe cardiotoxicity. Previous studies have demonstrated that DOX‐induced cardiomyocyte (CM) death is accompanied by excessive mitochondrial (MT) fragmentation. However, it is unclear if this MT fragmentation mediates DOX‐induced cardiomyocyte damage. This study aimed to determine whether or not inhibiting MT fragmentation can reduce DOX‐induced CM death, and whether changes in MT autophagic degradation (mitophagy) are involved.

Methods

H9c2 cardiac myoblast cells were transfected with siRNA targeting DRP‐1, a protein required for MT fission, before DOX administration. Changes in MT morphology and mitophagy were monitored with confocal microscopy after infection with the adenovirus encoding mitochondria‐targeting reporter MitoDsRed or MitoRosella. Propidium iodide (PI) staining was used to quantify the extent of DOX‐induced cell injury, while protein expression levels of apoptotic markers, cleaved Caspase‐3 and cleaved PARP, were measured with western blotting.

Results

Morphometric analysis demonstrated that DRP‐1 knockdown with siRNA markedly diminished DOX‐induced MT fragmentation as shown by form factor, aspect ratio, and mitochondrial size. This led to reduced CM death as shown by percentage of PI‐positive cells, caspase‐3 cleavage, and PARP cleavage. DRP‐1 knockdown also attenuated DOX‐induced mitophagy as assessed by the dual fluorescent mitophagy reporter MitoRosella.

Conclusion

These results suggest that DOX‐induced cardiotoxicity may be due to excessive mitochondrial fragmentation and degradation through autophagy.
Effects of Resveratrol on Mitochondrial Autophagy in Doxorubicin Treated Cardiac Myoblast Cells
Authors: Burina Lyudmila , Kobayashi Satoru , Gerdes Martin , Liang Qiangrong
Published: June 2021

Background and Objective

Doxorubicin (DOX) is a widely used chemotherapeutic agent with dose‐dependent cardiotoxic effects. It is of great clinical significance to identify a cytoprotective agent which can attenuate DOX cardiotoxicity without affecting its anti‐tumor efficacy. Resveratrol (Rev) is a plant‐derived polyphenol shown to exert cardioprotective effects in DOX‐treated cardiomyocytes, but its mechanisms of cardioprotection remain partially understood. We tested the hypothesis that Rev protects against DOX‐induced cardiomyocyte injury by inhibiting excessive degradation of mitochondria through the autophagy‐lysosome pathway (mitophagy).

Methods

H9C2 cardiac myoblasts were cultured under four conditions: 0.5 uM DOX, 5 uM Rev, 5 uM Rev + 0.5 uM DOX, and Ethanol (control). Propidium Iodide (PI) staining was used to assess necrotic cell death. Western Blotting was used to measure PARP cleavage, an apoptotic marker. Mitophagy was assessed via cellular infection with Adenovirus encoding MitoRosella, a novel dual fluorescent pH sensitive mitophagy reporter.

Results

DOX induced cardiomyocyte death as measured by PI positive cell and PARP cleavage, which was associated with increased mitophagy as assessed by the MitoRosella reporter. Pre‐treatment of cells with Rev not only attenuated DOX‐induced cell death but also inhibited DOX‐induced excessive mitophagy (Fig.1 C‐D). Rev alone had no effect on mitophagy (Fig.1 A‐B).

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Conclusion

The cardioprotective effect of Resveratrol is associated with a significant reduction in DOX‐induced mitophagy in cardiomyocytes, suggesting the excessive destruction of mitochondria as a potential cardiotoxic mechanism of DOX.
Krüppel‐like factor 6 protects the podocyte from mitochondrial injury under cell stress
Authors: Mallipattu Sandeep , Horne Sylvia
Published: June 2021

The primary etiology of chronic kidney disease is a direct consequence of initial glomerular dysfunction. Podocytes (visceral glomerular epithelial cells) in normal mature kidneys are regarded as highly differentiated and quiescent cells. Krüppel‐like factors (KLFs), zinc‐finger DNA‐binding transcription factors, play a critical role in podocyte biology. Specifically, KLF6 expression was reduced in models of podocyte injury. Podocyte‐specific loss of Klf6 (Cre+ podocin Klf6flox/flox) in mice resulted in significant albuminuria and podocyte injury with adriamycin (ADR) treatment (a model of podocyte injury). ADR‐treated Cre+ podocin Klf6flox/floxmice also exhibited dysmorphic mitochondria in the podocytes and promoter analysis revealed putative binding sites for KLF6 on cytochrome c oxidase assembly gene (SCO2).

KLF6 expression increased within 6 hours of ADR (0.4μg/μl) treatment in human podocytes (HP). Knockdown of KLF6 (shRNA‐KLF6) in HP reduced the mitochondrial membrane potential as compared to shRNA‐EV HP (control). ADR‐treated shRNA‐KLF6 HP increased mitochondrial fragmentation with reduced SCO2 expression and ATP levels. Also, ADR‐treated shRNA‐KLF6 increased cleaved‐caspase 3, 9, and cytosolic cytochrome c expression and decreased pro‐caspase 3 expression. These changes were restored after reintroduction of KLF6 in HP. Also, knockdown of SCO2 (shRNA‐SCO2) increased cytosolic cytochrome c and cleaved‐caspase 9 levels. Finally, immunostaining revealed a reduction in podocyte‐specific KLF6 expression in biopsies from patients with glomerular disease.

These findings suggest that KLF6 is an early inducible injury response gene, critical to the maintenance of mitochondrial function and preventing the activation of the intrinsic apoptotic pathway upon cell stress.
Maternal Obesity Alters Offspring Muscle Mitochondrial Function in Nonhuman Primates
Authors: Conner Blair , Hetrik Byron , Chicco Adam , McCurdy Carrie
Published: June 2021

Offspring of obese mothers have increased risk for early obesity and insulin resistance. Impairment in mitochondrial (MT) function may be a primary mechanism leading to insulin resistance. This study seeks to determine if maternal western‐style diet (WSD) or obesity alone alter fetal muscle MT function and if this persists at 3yrs. Adult Japanese Macaques were chronically (>2yr) fed a control (CON) or WSD. Some WSD‐induced obese dams were switched to CON at the start of pregnancy (REV). Fetal samples were collected at gestational day 130/165. Offspring carried to term were weaned to a CON or WSD after exposure to either gestational WSD (WSD/WSD;WSD/CON) or CON (CON/WSD;CON/CON). Fetal and juvenile gastrocnemius fibers were assayed for MT function in the presence of carbohydrate (CHO) or palmitate (PALM) using high‐resolution respirometry. Fetal respiratory control ratios (RCR) decreased in WSD and REV, demonstrating 24% increased uncoupling. CHO‐stimulated state 3 respiration was reduced by 20% (p<0.01) in WSD and REV, but neither were significantly decreased in PALM. In juveniles, RCR was doubled in CON/WSD vs. CON/CON, but this increase did not occur in WSD/WSD. Likewise, CHO coupling control ratio was doubled in CON/CON vs. CON/WSD (p<0.05), but not in WSD/WSD. Maternal obesity with or without WSD reduces MT function and metabolic flexibility in fetal muscle. Inflexibility persists in the postnatal period, but only when lean offspring are challenged with WSD. Our data suggest that exposure to maternal obesity alters cellular systems responsible for MT adaptation to WSD and may contribute to increased risk of metabolic disease.

Funding: NIH DK090964
Mitochondria Dynamics in Auditory Cells
Authors: Nuttall Alfred , Foster Sarah , Zhang Yuan , Wilson Teresa
Published: June 2021

Excessive superoxide radical production by mitochondria is one of the underlying causes of noise‐induced damage to tissues in the inner ear. Mitochondria are highly dynamic organelles that are maintained by the opposing processes of fusion and fission, events which alter mitochondrial morphology, biogenesis, and energy metabolism. The HEI‐OC1 auditory cell line possesses a highly interconnected mitochondrial network structure under normal growth conditions as revealed by immunolabeling for Tom20. Treatment with antimycin A increased fission processes resulting in a primary population of punctate and donut‐shaped mitochondria. Inhibition of dynamin related protein 1 effectively reduced mitochondrial fission and reactive oxygen species production and increased cell survival following antimycin A treatment. Super‐resolution imaging of Tom20 immunolabeled mitochondria in murine inner ear cells showed primarily punctate mitochondria in auditory outer hair cells, a mixed population of punctate and intermediate length mitochondria in inner hair cells, and elongated mitochondria in Deiters' cells. These results indicate that even under ambient noise conditions, it is likely that outer hair cells place high energy demands on their mitochondria. The punctate mitochondrial morphology in this cell type suggests that the balance of fission and fusion processes is altered, potentially due to a higher oxidant environment being present in these cells.
The LMNA Mutation R321X Associated with Dilated Cardiomyopathy Leads to Reorganization of the Endoplasmic Reticulum and Aberrant Ca2+ Signaling in HEK 293 Cells
Authors: Gerbino Andrea , Carmosino Monica , Del Vecchio Francesca , Torretta Silvia , Schena Giorgia , Procino Giuseppe , Svelto Maria
Published: June 2021

The lamin A/C (LMNA) are nuclear intermediate filament proteins composing the nuclear lamina. Mutations in LMNA are associated with a broad range of tissue‐specific laminopathies. In particular, dilated cardiomyopathy (DCM) with conduction system disease and sudden death is frequently caused by a premature termination codon mutation in LMNA resulting in an aberrant expression of a truncated LMNA on the nuclear envelope. In this study, we used a known LMNA nonsense mutation, R321X, to gain more insight into the pathophysiological mechanisms induced by LMNA‐R321X in human embryonic kidney (HEK293) cells.

Western blot analysis of homogenate from HEK293 cells transfected with LMNA‐R321X showed a 65kDa band corresponding to the truncated LMNA. Also, immunofluorescence confocal analysis demonstrated that expression of GFP‐tagged LMNA‐R321X, but not GFP‐tagged LMNA‐WT, caused (I) loss of nuclear envelope shape and integrity (II) retention of lamin A in the endoplasmic reticulum (ER) and (III) dramatic reorganization of the ER. Interestingly, LMNA‐R321X‐induced ER reorganization dramatically perturbed Ca2+ homeostasis since both cyclopiazonic acid (CPA)‐induced Ca2+ release and capacitative Ca2+ entry were perturbed in HEK293 cells loaded with the Ca2+ dye Rhod‐2.

Here, we proposed for the first time that the aberrant geometry of the ER induced by the expression of truncated LMNA may potentially lead to the development of DCM, rhythm disturbances and cardiac sudden death also via perturbance of Ca2+ homeostasis.
Targeting Mechanically‐Activated NOS Signaling in Dystrophic Cardiomyopathy
Authors: Garbincius Joanne , Michele Daniel
Published: June 2021

Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin‐glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. Our objective was to identify the mechanisms of how the DGC regulates cardiac muscle nitric oxide synthase (NOS) activity. Isolated adult wild‐type (WT) or dystrophin‐deficient mdx mouse cardiomyocytes were plated on deformable membranes, and live‐cell DAF‐FM dye fluorescence and biochemical approaches were used to investigate how the DGC regulates mechanically‐activated NO signaling. 1Hz cyclic stretch of WT cardiomyocytes significantly increased NO production and neuronal NOS (nNOS) S1412 phosphorylation. In contrast, stretch‐induced NO production and nNOS phosphorylation were impaired in mdx cardiomyocytes, suggesting that dystrophin regulates the mechanical activation of NOS. We found that inhibition of AMP‐activated protein kinase (AMPK) inhibited stretch‐induced NO production in WT cardiomyocytes, indicating that AMPK plays a critical role in mechanoactivation of NOS. Importantly, direct pharmacological activation of AMPK increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC regulates the mechanical activation of cardiac NOS, and that pharmacological AMPK activation may be a therapeutic strategy to restore NOS activity in dystrophin‐deficient cardiomyocytes. Supported by the Muscular Dystrophy Association, the University of Michigan CVRE Training Program, and NIH T32 GM‐08322.
MALIGNANT HYPERTHERMIA:ORGANON 9426 WAS EVALUATED IN 10 mhs PIGS AND 8 CONTROL PIGS
Authors: Williams Charles
Published: June 2021

Control pigs required 109.9 ug/kg/min and MHS pigs required 72.4 ug/kg/min infusion of ORG 9426 to maintain a 90% block. It appears that ORG 9426 is only one‐third to one‐half as potent as Vecuronium in pigs. The fact that MHS pigs only required 66% of the infusion dose to maintain a 90% block suggests that there is a marked difference in the neuromuscular effect of ORG 9426 in MHS vs control pigs.

A linear regression analysis of the dose response data to ORG 9426 in MHS pigs indicated that 427.033 ug/kg would be required to produce a 100% neuromuscular blockade vs 586.31 ug/kg in control pigs.

Nine of the ten MHS pigs did not develop MH or show any signs of impending MH during the halothane and succinylcholine challenge at the end of the infusion period. Organon 9426 is the first muscle relaxant to offer significant protective action at a clinical dose. This suggests that there is an allosteric site on the sodium channel (acetylcholine receptor) which regulates the flow of sodium ions through the sodium channel.

Organon 9426 may be therapeutically effective in an active MH case.
INCRETIN AMELIORATES DIABETIC MICROANGIOPATHY IN HEART BY ACTIVATION OF AUTOPHAGY VIA PKA/AMPK AXIS.
Authors: Bando Yasuko , Monji Akio
Published: June 2021

Introduction

The incretin hormone glucagon‐like peptide‐1 (GLP‐1) restores diabetic myocardial remodeling observed in type 2 diabetic mice (T2DM); however, it remains unclear whether GLP‐1 may modulates angiogenic activity in diabetic heart.

Hypothesis

We evaluated the impact of GLP‐1 receptor activation on angiogenesis and its link to endothelial autophagy under diabetic condition.

Methods

T2DM mice were treated with Ex4 (24 nmole/kg/day for 4 weeks). Cardiac capiary density was measured by CD31 immunohistological staining. Cultured ECs were used for in vitro experiments. Analyses for the changes in autophagy (LC3‐turnover assay and protein levels of p62 and Beclin1), and angiogenesis (tube formation assay and Akt/AMPK/eNOS activity), were evaluated with or without molecular‐targetted knockdown by RNA interference (siRNA).

Results

T2DM exhibited reduced cardiac capillary density in diabetic heart, which was restored by Ex‐4 with increase in myocardial cyclic AMP concentration. GLP‐1 receptor expression in ECs was confirmed by immunoblot and QPCR. Ex‐4 and PKA enhancers (10 μM forskolin and 1 mM 8‐bromo‐cyclic AMP) facilitated angiogenesis and autophagy in ECs. PKA/AMPK/eNOS phosphorylation levels in ECs were enhanced by Ex4, but each Akt activity remains unchanged. PKA inhibitors (H89, RP‐cAMP, and siRNA for PKA) abrogated the increase in AMPK/eNOS phosphorylation induced by Ex‐4. Tube formation assay revealed that Ex4 enhanced in vitro angiogenesis which were abrogated by inhibition of autophagy and AMPK activity.

Conclusions

GLP‐1 receptor activation by Ex‐4 restores impaired angioogenic activity in diabetic heart via the PKA/AMPK‐dependent endothelial autophagic activation.